Cell preservation methods

Abstract

The present disclosure provides methods and compositions for culturing, cryopreserving, and administering genetically engineered B-lineage cell populations. The methods and compositions are provided herein. Among other things, the present disclosure identifies challenges in providing engineered cell preparations for the treatment of disease because storage and transport of cell preparations can result in cell death, stress response, and / or loss of transgene expression. In some embodiments, the present disclosure provides methods for cryopreservation of B-lineage cell preparations.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 445,690, filed February 14, 2023, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Cell therapy is an emerging form of treatment that involves administering viable human cells to a subject to act as a "living drug." In particular, certain cell therapies utilize engineered immune cells to combat disease. The maintenance and cryopreservation of these engineered adaptive immune cells is important for their eventual administration in both clinical and preclinical settings.

[0003] The present disclosure provides, among other things, methods for cryopreservation, culture, and administration of engineered immune cells. Summary of the Invention [Means for solving the problem]

[0004] Among other things, the present disclosure identifies challenges in providing engineered cell preparations for the treatment of disease, as storage and transport of cell preparations can result in cell death, stress response, and / or loss of transgene expression. In some embodiments, the present disclosure provides methods for cryopreservation of B-lineage cell preparations. In some embodiments, the present disclosure provides methods for cryopreservation of engineered B-lineage cell preparations. In particular, the present disclosure provides methods for cryopreservation of engineered B-lineage cell preparations to maintain or prevent a significant decrease in cell viability and / or transgene expression. In some embodiments, the present disclosure provides pharmaceutical compositions comprising engineered B-lineage cell preparations that maintain or do not show a significant decrease in engraftment and / or transgene expression after administration to a subject.

[0005] The present disclosure encompasses recognition of problems associated with the cryopreservation and eventual thawing of B-lineage cell populations. For example, cooling of the cell population and associated aqueous solution can result in the formation of excessive intracellular ice crystals, induce cellular dehydration, potentially worsening cell morphology (e.g., adhesion, cell surface markers) and metabolism (e.g., efficacy), and reduced viability (see Meneghel, et al. 2020, incorporated herein by reference in its entirety).

[0006] In particular, in one aspect, the present disclosure provides a method for preparing a B-lineage cell population for cryopreservation, the method comprising contacting the B-lineage cell population with a cryopreservation medium comprising about 4.5% to about 5% DMSO and about 2.5% to about 10% HSA, thereby producing a cell preparation for cryopreservation.

[0007] In some embodiments, the cryopreserved cell preparation undergoes controlled cooling in a controlled cooling chamber, comprising one or more of the following steps: (i) cooling the chamber until the cryopreserved cell preparation reaches 4°C; (ii) cooling the chamber at a rate of 1°C / min until the cryopreserved cell preparation reaches -4°C; (iii) cooling the chamber at a rate of 25°C / min until the chamber reaches -40°C; (iv) heating the chamber at a rate of 10°C / min until the chamber reaches -12°C; (v) cooling the chamber at a rate of 1°C / min until the chamber reaches -40°C; and (vi) cooling the chamber at a rate of 10°C / min until the chamber reaches -90°C.

[0008] In some embodiments, the B-lineage cell population is or comprises a population of genetically modified plasma cells. In some embodiments, the B-lineage cell population is or comprises a population of plasmablasts. In some embodiments, the B-lineage cell population is or comprises a population of plasma progenitor cells.

[0009] In some embodiments, the cryopreserved cell preparation maintains at least 50% viability after being frozen for at least 24 hours. In some embodiments, the cryopreserved cell preparation maintains at least 50% viability after being frozen for 1 week. In some embodiments, the cryopreserved cell preparation maintains at least 50% viability after being frozen for 1 month. In some embodiments, the cryopreserved cell preparation maintains at least 50% viability after being frozen for 6 months.

[0010] In another aspect, the present disclosure provides a composition comprising a frozen population of genetically modified B-lineage cells and a cryopreservation medium comprising about 4.5% to about 5% DMSO and about 2.5% to about 10% HSA. In some embodiments, the cryopreservation medium comprises 5 percent DMSO. In some embodiments, the cryopreservation medium comprises 2.5% HSA.

[0011] In some embodiments, the composition of B lineage cells is genetically modified at the endogenous locus with a transgene. In some embodiments, the transgene is Factor IX. In some embodiments, the endogenous locus is CCR5 or JCHAIN.

[0012] In some embodiments, the genetically modified B lineage cells have been frozen for at least 24 hours and maintain at least 50% viability after thawing. In some embodiments, the genetically modified B lineage cells have been frozen for at least 24 hours and at least 25% of the cells secrete IgG after thawing. In some embodiments, the genetically modified B lineage cells have been frozen for at least 24 hours and at least 25% of the cells express IgM after thawing.

[0013] In some embodiments, the genetically modified B-lineage cell population has been frozen for at least 1 week. In some embodiments, the genetically modified B-lineage cell population has been frozen for at least 1 month. In some embodiments, the genetically modified B-lineage cell population has been frozen for at least 6 months.

[0014] In another aspect, the disclosure provides methods of treating a disease, comprising administering a cryopreserved population of genetically modified B-lineage cells. In some embodiments, the population of genetically modified B-lineage cells is cryopreserved in a medium comprising about 4.5% to about 5% DMSO and about 2.5% to about 10% HSA and thawed prior to administration to a patient. In some embodiments, the population of genetically modified B-lineage cells is cryopreserved in a medium comprising 5 percent DMSO. In some embodiments, the population of genetically modified B-lineage cells is cryopreserved in a medium comprising 2.5% HSA. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 shows measurements of bioluminescence in mice after engraftment of engineered B-lineage cell preparations expressing luciferase ("Luc") at the CCR5 locus. Both fresh and suboptimal cryopreserved B-lineage cell preparations were administered. Conditions tested, from left to right, were PBS only, LKP13 culture condition 1 Cryo, LKP13 culture condition 1 Fresh, LKP42 culture condition 1 Cryo, and LKP42 culture condition 1 Fresh.

[0016] [Figure 2] Whole mouse bioluminescence measurements after administration and engraftment of B-lineage cell preparations and PBS control are shown.

[0017] [Figure 3] Human IgG levels in mouse plasma after engraftment of engineered B-lineage cell preparations are shown. Conditions tested (left to right) are: PBS only, LKP13 culture condition 1 Cryo, LKP13 culture condition 1 Fresh, LKP42 culture condition 1 Cryo, and LKP42 culture condition 1 Fresh.

[0018] [Figure 4]Comparative measurements of cell viability are shown for cryopreserved B-lineage cell preparations, fresh engineered B-lineage cell preparations ("Fresh Cells"), and defective engineered cryopreserved B-lineage cell preparations transplanted into mouse models ("LKP13 Culture Condition 1 Cryo" and "LKP42 Culture Condition 1 Cryo"). Cell preparations were cultured for 13 days, then cryopreserved and thawed at the times indicated.

[0019] [Figure 5] 1 shows comparative measurements of cell viability of pre-frozen engineered B-lineage cell preparations ("LKP13 Cryo" and "LKP42 Cryo"), fresh engineered B-lineage cell preparations, and cryopreserved engineered B-lineage cell preparations prepared by the methods described herein.

[0020] [Figure 6] Shown are time-course bioluminescence measurements of individual groups of mice after administration of either the indicated fresh engineered B-lineage cell preparations or the indicated cryopreserved engineered B-lineage cell preparations (bioluminescence was measured at the times indicated). Conditions tested, from left to right, are untreated, LKP76 culture condition 1 Fresh, LKP76 culture condition 1 Cryo, LKP76 culture condition 2 Fresh, LKP76 culture condition 2 Cryo, LKP60 culture condition 1 Fresh, and LKP60 culture condition 1 Cryo.

[0021] [Figure 7] Comparative measurements of viable cell density (VCD) before freezing (paired left bars) and after thawing (paired right bars) are shown for a number of engineered B-lineage cell preparations.

[0022] [Figure 8] Comparative measurements of cell viability before freezing (paired left bars) and after thawing (paired right bars) in engineered B-lineage cell preparations are shown.

[0023] [Figure 9]Shown are the viability of engineered B-lineage cell preparations before (time 0) and during the cryopreservation methods described herein, as well as the percentage of engineered B-lineage cell preparations that are CD27 positive, CD38 positive, CD27 and CD38 positive, and Factor IX (F9) transgene insertion positive.

[0024] [Figure 10] 1 shows measurements of IgG and IgM over time in mice following administration of the indicated fresh engineered B lineage cell preparations or cryopreserved B lineage cell preparations engineered using the methods disclosed herein.

[0025] [Figure 11] 1 shows the time course of human Factor IX (huFIX) measurements in mice following administration of a B-lineage cell population engineered and preserved by the methods disclosed herein.

[0026] [Figure 12A] 1 shows the time course of human factor IX (huFIX) measurements in mice following administration of four donors (LKP23008, LKP22090, LKP22094, and LKP22091) of B-lineage cell populations engineered and preserved by the methods disclosed herein. [Figure 12B] 1 shows time course measurements of human immunoglobulin G (IgG) in mice following administration of four donors (LKP23008, LKP22090, LKP22094, and LKP22091) of B-lineage cell populations engineered and preserved by the methods disclosed herein.

[0027] [Figure 13A] 1 shows time course measurements of huFIX in mice following administration and re-administration on day 21 of an engineered B-lineage cell population preserved by the methods disclosed herein. [Figure 13B-1] 1 shows time course measurements of IgG and human immunoglobulin M (huIgM) in mice following administration and re-administration on day 21 of an engineered B-lineage cell population preserved by the methods disclosed herein. [Figure 13B-2] 1 shows time course measurements of IgG and human immunoglobulin M (huIgM) in mice following administration and re-administration on day 21 of an engineered B-lineage cell population preserved by the methods disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0028] definition About: The term "about," as used herein with respect to a value, refers to a value of the same order in the context of the stated value. Generally, a person of ordinary skill in the art familiar with the context will understand the reasonable degree of variation that "about" encompasses in that context. For example, in some embodiments, the term "about" can encompass a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated value.

[0029] Activator: As used herein, the term "activator" generally refers to an agent whose presence or level correlates with increased target levels or activity compared to that observed in the absence of the agent (or at a different level of the agent). In some embodiments, an activator is one whose presence or level correlates with a target level or activity that is equal to or greater than a particular reference level or activity (e.g., that observed under appropriate reference conditions, e.g., in the presence of a known activator, e.g., a positive control).

[0030] Administration: As used herein, the term "administration" generally refers to the administration (e.g., of a composition or treatment) to a subject or system (e.g., being or including one or more cells, tissues, organisms, etc.), e.g., to achieve delivery of an agent contained in or otherwise delivered or produced by such composition or treatment. Those of skill in the art will be aware of various routes that may be utilized for administration to a subject, e.g., a human, in appropriate circumstances. For example, in some embodiments, administration may be ophthalmic, oral, parenteral, topical, etc. In some particular embodiments, administration may be or include bronchial administration (e.g., via bronchial instillation), buccal administration, dermal administration (e.g., one or more of topical administration into the dermis, intradermal administration, interdermal administration, transdermal administration, etc.), enteral administration, intraarterial administration, intradermal administration, intragastric administration, intramedullary administration, intramuscular administration, intranasal administration, intraperitoneal administration, intrathecal administration, intravenous administration, intraventricular administration, administration within a specific organ (e.g., intrahepatic administration), mucosal administration, nasal administration, oral administration, rectal administration, subcutaneous administration, sublingual administration, topical administration, tracheal administration (e.g., via intratracheal instillation), intravaginal administration, intravitreal administration, etc. In some embodiments, administration may include only a single administration. In some embodiments, administration may include the application of a fixed number of doses. In some embodiments, administration can include administration that is intermittent administration (e.g., multiple administrations separated by a period of time) and / or periodic administration (individual administrations separated by a common period of time). In some embodiments, administration can include continuous administration (e.g., perfusion) for at least a selected period of time.

[0031] Alloantigen: As used herein, the term "alloantigen" refers to an antigen associated with non-self recognition and / or transplant rejection (e.g., an antigen to which a rejection immune response is elicited). Generally, an alloantigen is a substance present in or on tissue from one individual of a particular species (e.g., a donor individual) but not present in or on tissue from another individual of that species (e.g., a recipient individual genetically distinct from the donor individual); i.e., transplantation of tissue from a donor individual into a recipient individual risks and / or results in a rejection immune response. Generally, an antigen can be or include any chemical entity, such as a small molecule, nucleic acid, polypeptide, carbohydrate, lipid, etc. In some embodiments, an alloantigen is or includes a polypeptide. A variety of polypeptides, the amino acid sequences of which can vary between and among individuals of the same species, such that they can act as alloantigens, are known in the art.

[0032] Allo-recognition: As used herein, the term "allo-recognition" refers to an immune response typically initiated by the immune system of an individual (i.e., recipient) receiving a tissue transplant from another individual of the same species (i.e., e.g., a donor who is genetically distinct from the recipient individual), where the immune response involves recognition of alloantigens on the transplanted tissue. Usually, allo-recognition involves T cell recognition of alloantigens. In many embodiments, T cells recognize alloantigen peptides, e.g., alloantigen peptides encoded by polymorphic genes whose sequences differ between the donor and recipient individuals.

[0033] Amelioration: As used herein, refers to the prevention, reduction, or alleviation of a condition in a subject, or improvement of the condition. Amelioration includes, but does not require, complete recovery or complete prevention of a disease, disorder, or condition (e.g., radiation injury).

[0034] Antigen: As used herein, the term "antigen" or "Ag" refers to a molecule capable of eliciting an immune response. This immune response may involve either antibody production, activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen" as used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences may be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that antigens can be generated, synthesized, or derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0035] Antibody drug: As used herein, the term "antibody drug" (interchangeably referred to herein as "antibody") refers to a polypeptide that can be expressed, released, secreted, or delivered to a target by the modified cells described herein. The polypeptide comprises sufficient canonical immunoglobulin sequence elements to confer specific binding to a particular target antigen. In some embodiments, the antibody drug consists of an antibody. As is known in the art, naturally occurring antibodies are approximately 150 kD tetrameric agents comprising two identical heavy chain polypeptides (each approximately 50 kD) and two identical light chain polypeptides (each approximately 25 kD) that associate with each other into what is commonly referred to as a "Y" structure. Each heavy chain comprises at least four domains (each approximately 110 amino acids long): an amino-terminal variable (VH) domain (at the tip of the Y structure), followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 domain (at the base of the stem of the Y). A short region known as the "switch" connects the heavy chain variable region and the heavy chain constant region. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in an intact antibody. Each light chain contains two domains, an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, separated from each other by another "switch." An intact antibody drug tetramer contains two heavy-light chain dimers in which the heavy and light chains are linked to each other by a single disulfide bond. Two other disulfide bonds connect the heavy chain hinge regions to each other so that the dimers are linked to each other to form a tetramer. Antibody drugs are also typically glycosylated in the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed by two β-sheets (e.g., a three-, four-, or five-stranded sheet) packed together into a compressed antiparallel β-barrel. Each variable domain contains three hypervariable loops (CDR1, CDR2, and CDR3) known as "complementarity-determining regions" and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4).When a natural antibody folds, the FR regions form beta sheets that provide a structural framework for the domains, and the CDR loop regions of both the heavy and light chains assemble in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y-structure. The Fc region of a naturally occurring antibody binds to components of the complement system and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. The affinity and / or other binding properties of the Fc region for the Fc receptor can be modulated by glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized (e.g., as components of CARs) according to the present disclosure comprise a glycosylated Fc domain, including Fc domains with altered or engineered glycosylation. In some embodiments, any polypeptide or complex of polypeptides that contains a sufficient immunoglobulin domain sequence found in a natural antibody may be referred to as an "antibody drug" and / or may be used as an antibody, regardless of whether such polypeptide is naturally produced (e.g., produced by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, an antibody drug is polyclonal. In some embodiments, an antibody drug is monoclonal. In some embodiments, an antibody drug has constant region sequences characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, the sequence elements of an antibody drug are humanized, primatized, chimeric, etc., as known in the art. Furthermore, the term "antibody drug," as used herein, may, in appropriate embodiments (unless otherwise specified or otherwise clear from the context), refer to any of the constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative applications. In some embodiments, antibody agents may lack covalent modifications (eg, glycan attachment) that they have when produced in nature.In some embodiments, antibody drugs may contain covalent modifications (e.g., attachment of glycans, payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other pendant groups (e.g., polyethylene glycol, etc.).

[0036] Biologically active: As used herein, refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, the presence or degree of biological activity is assessed by detection of a direct or indirect product produced by the biological pathway or event of interest.

[0037] Biomarker: The term "biomarker" is used herein, consistent with usage in the art, to refer to an entity, event, or characteristic whose presence, level, degree, type, and / or form correlates with a particular biological event or condition of interest, such that it is considered a "marker" for that event or condition. To give just a few examples, in some embodiments, a biomarker can be or include a marker of a particular pathological state or a marker of the likelihood that a particular disease, disorder, or condition will develop, occur, or recur. In some embodiments, a biomarker can be or include a marker of a particular disease or treatment outcome or the likelihood thereof. Thus, in some embodiments, a biomarker predicts a relevant biological event or condition of interest; in some embodiments, a biomarker prognoses a relevant biological event or condition of interest; and in some embodiments, a biomarker diagnoses a relevant biological event or condition of interest. A biomarker can be or include any chemical class of entity, and can be or include a combination of entities. For example, in some embodiments, a biomarker may be or include a nucleic acid, a polypeptide, a lipid, a carbohydrate, a small molecule, an inorganic agent (e.g., a metal or ion), or a combination thereof. In some embodiments, a biomarker is a cell surface marker. In some embodiments, a biomarker is an intracellular biomarker. In some embodiments, a biomarker is detected extracellularly (e.g., secreted or otherwise produced extracellularly, or present extracellularly, e.g., in a bodily fluid, e.g., blood, urine, tears, saliva, cerebrospinal fluid, etc.). In some embodiments, a biomarker may be or include a genetic signature or an epigenetic signature. In some embodiments, a biomarker may be or include a gene expression signature.

[0038] Bispecific antibody: As used herein, refers to a bispecific binding agent in which at least one, typically both, of the binding moieties is or comprises an antibody component. A variety of different bispecific antibody structures are known in the art. In some embodiments, each binding moiety in a bispecific antibody that is or comprises an antibody component is a V H and / or V L In some such embodiments, such V H and / or V L The V region is one found in a particular monoclonal antibody. In some embodiments, where a bispecific antibody contains two antibody component binding moieties, each V region is derived from a different monoclonal antibody. H and / or V L In some embodiments, a bispecific antibody contains two antibody component binding moieties, where one of the two antibody component binding moieties contains a V region containing the CDRs from a first monoclonal antibody. H and / or V L and an immunoglobulin molecule having a V region, one of the two antibody component binding moieties containing a V CDR from a second monoclonal antibody. H and / or V L These include antibody fragments having regions (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.).

[0039] Cancer: The terms "cancer," "malignancy," "neoplasm," "tumor," and "carcinoma" are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, such that they exhibit an abnormal growth phenotype characterized by a marked loss of control of cell proliferation. In some embodiments, tumors can be or include cells that are pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. The present disclosure particularly identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, the relevant cancers can be characterized as solid tumors. In some embodiments, the relevant cancers can be characterized as hematological tumors. In general, examples of different types of cancer known in the art include hematopoietic cancers, including, for example, leukemia, lymphoma (Hodgkin's lymphoma and non-Hodgkin's lymphoma), myeloma, and myeloproliferative disorders; sarcoma, melanoma, adenoma, carcinoma of solid tissue, squamous cell carcinoma of the oral cavity, throat, larynx, and lung, liver cancer, genitourinary cancers, such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, and endometrial cancer, as well as renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous melanoma or intraocular melanoma, cancers of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the head and neck, breast cancer, cancer of the gastrointestinal tract, and cancer of the nervous system, benign lesions, such as papillomas, etc.

[0040] Controlled Cooling: As used herein, the term "controlled cooling" (also interchangeably referred to herein as "controlled cooling rate") may refer to a process of both freezing and heating a cell preparation. In some embodiments, controlled cooling may preserve the structural and / or functional integrity of the cell preparation. Controlled cooling may include specific steps and parameters, where the cell preparation may be exposed to different temperatures at different cooling rates and / or cooling times to maximize the viability and functionality of the cell preparation. In some embodiments, controlled cooling comprising one or more steps may be performed in a controlled cooling chamber.

[0041] Engineered: In general, the term "engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide can be considered "engineered" if it contains two or more sequences that are not naturally linked to one another in that order, and if the two or more sequences are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide, and / or if certain residues in the polynucleotide are non-natural and / or are, by the act of man, linked to entities or moieties to which they are not naturally linked. For example, in some embodiments described and / or utilized herein, an engineered polynucleotide contains a control sequence that is found in nature operably linked to a first coding sequence but not to a second coding sequence, and has been linked by the hand of man so that the control sequence is operably linked to the second coding sequence. Equivalently, a polypeptide can be considered "genetically engineered" if it is encoded or expressed by an engineered polynucleotide and / or is produced other than by natural expression in a cell. Similarly, a cell or organism is considered "engineered" if it has been subjected to a manipulation that alters its genetic identity, epigenetic identity, and / or phenotypic identity compared to an appropriate reference cell, e.g., an otherwise identical cell that has not been similarly manipulated. In some embodiments, such a manipulation is or includes a genetic manipulation that alters its genetic information (e.g., new genetic material not previously present is introduced, e.g., by transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or previously present genetic material is modified or removed, e.g., by substitution or deletion mutations or by breeding procedures). In some embodiments, an engineered cell is one that has been engineered to contain and / or express a particular agent of interest (e.g., a protein, nucleic acid, and / or a particular form thereof) in an altered amount and / or according to altered timing compared to such an appropriate reference cell.As is customary and understood by those skilled in the art, the progeny of an engineered polynucleotide or cell will usually still be referred to as "engineered," even if the actual manipulation was performed on an earlier entity.

[0042] Excipient: As used herein, refers to a non-therapeutic agent that can be included in a pharmaceutical composition, for example, to provide or contribute to the desired consistency or stabilization. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, etc.

[0043] Expression: As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product can be a transcript. In some embodiments, the gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0044] Functional: As used herein, a "functional" biomolecule is a biomolecule in a form in which it exhibits a property and / or activity by which it is characterized.

[0045] Gene product or expression product: As used herein, the term "gene product" or "expression product" generally refers to the RNA transcribed from a gene (before and / or after processing) or the polypeptide (before and / or after modification) encoded by the RNA transcribed from a gene.

[0046] Isolated: As used herein, refers to a substance and / or entity that is (1) separated from at least some of the components with which it was associated when first produced (whether in nature or in an experimental setting) and / or with which it was otherwise previously associated, and / or (2) designed, produced, prepared, and / or manufactured by the hand of man. In some embodiments, a substance may be considered "isolated" if it is free from or separated from (or rendered free from or separated from) about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components (e.g., components with which it was previously associated). In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% pure, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those of skill in the art, a substance may still be considered "isolated" or even "pure" after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.). In such embodiments, the percent isolation or purity of a substance is calculated without including such carriers or excipients. By way of example only, in some embodiments, a biological macromolecule, such as a naturally occurring polypeptide or polynucleotide, is considered to be "isolated" if: a) by virtue of its origin or source of derivation, it is not associated with some or all of the components that naturally accompany it in its natural state in nature; b) it is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; or c) it is expressed by or otherwise associated with components from a cell or other expression system other than the species that produces it in nature.Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized by a cellular machinery different from that which produces it in nature is considered to be an "isolated" polypeptide. Alternatively, or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from other components a) with which it is associated in nature and / or (b) with which it was associated when originally produced.

[0047] Marker: As used herein, marker refers to an entity or moiety whose presence or level is characteristic of a particular condition or event. In some embodiments, the presence or level of a particular marker may be characteristic of the presence or stage of a disease, disorder, or condition. By way of example only, in some embodiments, the term refers to a gene expression product that is characteristic of a particular tumor, tumor subclass, tumor stage, etc. Alternatively or additionally, in some embodiments, the presence or level of a particular marker correlates with the activity (or activity level) of a particular signaling pathway, which may be characteristic of a particular class of tumor, for example. The statistical significance of the presence or absence of a marker may vary depending on the particular marker. In some embodiments, detection of a marker is highly specific in that it reflects a high probability that such a tumor is of a particular subclass. Such specificity may be obtained at the expense of sensitivity (i.e., a negative result may occur even when the tumor is one expected to express the marker). Conversely, a marker with high sensitivity may be less specific than one with lower sensitivity. Those skilled in the art will understand that in many embodiments, a useful marker may not discriminate with 100% accuracy.

[0048] Payload: Generally, as used herein, the term "payload" refers to an agent that can be delivered or transported by association with another entity. In some embodiments, such association can be or include a covalent bond. In some embodiments, such association can be or include non-covalent interaction(s). In some embodiments, the association can be direct. In some embodiments, the association can be indirect. The term "payload" is not limited to a particular chemical identity or chemical type. For example, in some embodiments, the payload can be or include any chemical class of entity, including, for example, lipids, metals, nucleic acids (e.g., transgenes), polypeptides, saccharides (e.g., polysaccharides), small molecules, or combinations or complexes thereof. In some embodiments, the payload can be or include a biological modifier, a detectable agent (e.g., a dye, fluorophore, radiolabel, etc.), a detection reagent, a nutrient, a therapeutic agent, etc., or a combination thereof. In some embodiments, the payload can be or include a cell or organism, or a fraction, extract, or component thereof. In some embodiments, the payload may be or may include a natural product, in that it is found in nature and / or obtained from nature. Alternatively or additionally, in some embodiments, the term may be used to refer to one or more entities that are artificial, in that they are designed, engineered, and / or produced by the hand of man and / or are not found in nature. In some embodiments, the payload may be or may include a drug in isolated or pure form. In some embodiments, such a drug may be in crude form.

[0049] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a therapeutic regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to an appropriate population. In some embodiments, the pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those suitable for oral administration, e.g., liquid drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., sterile solutions or suspensions or sustained release formulations; topical application, e.g., creams, ointments, or sustained release patches, or sprays applied to the skin, lungs, or oral cavity; vaginal or rectal use, e.g., pessaries, creams, or foams; sublingual use; ocular use; transdermal use; or nasal, pulmonary, and other mucosal surfaces.

[0050] Reference: As used herein, refers to a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or measured substantially contemporaneously with the test or measurement of interest. In some embodiments, the reference or control is a historical reference or control, optionally recorded in a tangible medium. As will be understood by those of skill in the art, a reference or control is typically measured or characterized under conditions or circumstances comparable to those being evaluated. Those of skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular available reference or control.

[0051] Response: As used herein, a response to a treatment can refer to a beneficial change in a subject's condition that occurs as a result of or correlates with treatment. In some embodiments, such a change can be or can include stabilization of the condition (e.g., prevention of deterioration that would have occurred in the absence of treatment), improvement in the symptoms of the condition, and / or improved likelihood of cure of the condition. In some embodiments, the term "response" can refer to the response of a particular system or component thereof (e.g., of a particular cell, tissue, organism, or subject). Those of skill in the art will be aware of techniques available for assessing the response of a subject of interest.

[0052] Sample: As used herein, the term "sample," as described herein, typically refers to an aliquot of material obtained from or derived from a source of interest. In some embodiments, the source of interest is a biological or environmental source. In some embodiments, the source of interest can be or include a cell or organism, such as a microorganism, a plant, or an animal (e.g., a human). In some embodiments, the source of interest is or includes a biological tissue or biological fluid. In some embodiments, the biological tissue or fluid may be or include amniotic fluid, aqueous humor, peritoneal fluid, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chyme, ejaculate, endolymph, exudate, feces, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural fluid, pus, mucosal secretions, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or component(s) thereof. In some embodiments, the biological fluid may be or include intracellular fluid, extracellular fluid, intravascular fluid (plasma), interstitial fluid, lymph, and / or transcellular fluid. In some embodiments, the biological fluid may be or include plant exudates. In some embodiments, the biological tissue or biological sample may be obtained, for example, by aspiration, biopsy (e.g., fine needle biopsy or tissue biopsy), swab (e.g., oral swab, nasal swab, skin swab, or vaginal swab), scraping, surgery, lavage or washing (e.g., bronchoalveolar lavage or washing, ductal lavage or washing, nasal lavage or washing, ocular lavage or washing, oral lavage or washing, uterine lavage or washing, vaginal lavage or washing, or other lavage or washing). In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from a source of interest by any suitable means. In some embodiments, as is clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents), for example, filtration using a semipermeable membrane.Such "processed samples" can include, for example, nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to one or more techniques, such as, for example, nucleic acid amplification or reverse transcription, isolation and / or purification of certain components, etc. In some embodiments, the sample can be a "crude" sample in that it has been subjected to relatively light processing and / or is complex in that it contains components of relatively different chemical classes.

[0053] Source: As used herein, the term "source" generally refers to the context in which an agent of interest (e.g., which may be or may include a carbohydrate, lipid, nucleic acid, metal, polypeptide, small molecule, or combination thereof) may be found in nature or in which such agent may be present or obtained (e.g., isolated). In some embodiments, the source may be or may include a biological source (e.g., an organism, tissue, or cell, or a sample thereof). In some embodiments, the source may be an environmental source. In some embodiments, the source may be or may include a primary sample from an organism (e.g., which may be or may include tissue or bodily fluids of such an organism, and / or may be or may include cell(s) of such an organism). In some embodiments, the organism may be or may include a prokaryotic organism (e.g., a bacterium) or a eukaryotic organism (e.g., a fungus or yeast, an insect, a mammal, a plant, a reptile, etc.). In some embodiments, an infectious agent such as a virus or phage may be considered an organism for purposes of this disclosure, and particularly with respect to being a source, which in some embodiments may be or include an engineered source, e.g., a cell line or culture, an in vitro system, etc.

[0054] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including, in some embodiments, prenatal human forms). In some embodiments, the subject is afflicted with the relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some aspects, a subject is a person who possesses one or more characteristics that characterize a susceptibility to or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or who has been diagnosed and / or treated.

[0055] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting a characteristic or property of interest to a complete or nearly complete extent or degree. Those skilled in the art of biology understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to express the potential lack of completeness inherent in many biological and chemical phenomena.

[0056] Target locus: As used herein, the term "target locus" can refer to a specific site or position on a chromosome of interest. For example, a target locus can be a site that is "manipulated" or "modified" by human hands. In some embodiments described and / or utilized herein, an engineered polynucleotide contains homology to a target locus (e.g., CCR5 as a target locus, whose homologous sequence can be part of a guide RNA to effect incorporation of an edit by CRISPR / Cas-mediated gene editing) to enable further modification at a specific site. In some embodiments, target locus can interchangeably refer to a target gene of interest for human manipulation. In some embodiments, such target locus manipulation is or includes genetic manipulation such that the genetic information is altered (e.g., new genetic material not previously present is introduced, e.g., by transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or previously present genetic material is modified or removed, e.g., by substitution or deletion mutations or by mating procedures).

[0057] Therapeutic Agent: As used herein, the phrase "therapeutic agent" refers to an agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to relieve, ameliorate, alleviate, inhibit, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0058] Variant: As used herein, in the context of a molecule, e.g., a nucleic acid, protein, or small molecule, the term "variant" refers to a molecule that exhibits significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or level of one or more biological or chemical moieties compared to the reference molecule. In some embodiments, a variant also differs functionally from its reference molecule. In some embodiments, a variant is structurally different but performs the same or similar function as its reference molecule. Generally, whether a particular molecule is appropriately considered a "variant" of a reference molecule is based on the degree of structural identity with the reference molecule. As will be understood by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs from the reference molecule in at least one aspect. To give just a few examples, a polypeptide may have characteristic sequence elements composed of multiple amino acids that have designated positions relative to one another in linear or three-dimensional space and / or that contribute to a particular structural motif and / or biological function, and a nucleic acid may have characteristic sequence elements composed of multiple nucleotide residues that have designated positions relative to one another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently attached to the polypeptide or nucleic acid backbone (e.g., polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid exhibits an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one distinctive sequence element with the reference polypeptide or nucleic acid.In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid is a truncated version of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide that is a truncated version of the reference polypeptide may exhibit equivalent, identical, or higher levels of one or more biological activities compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a "variant" of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence identical to the reference sequence except for minor sequence changes at specific positions.

[0059] Vector: As used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0060] cell therapy Cell-based therapeutics (cell therapies) are a new class of medicines that utilize innate cellular mechanisms to combat disease. Unlike many conventional treatment methods, cell therapies utilize cellular localization, migration, and proliferation within the body, which can lead to improved biodistribution and targeted delivery of therapeutic agents. Cell therapies also benefit from the ability of cells to sense and respond to various exogenous signals within a subject, including, for example, small molecules, other cells, physical forces, and / or marker proteins. In vivo cellular persistence also allows cell therapies to survive, differentiate, function, and the like within a subject for extended periods of time. These inherent attributes may result in improved safety and efficacy of cell therapies compared to other biologics or pharmaceutical compounds, thereby providing long-lasting, specifically targeted, tunable, and / or disease-responsive treatments.

[0061] Cell therapy may have potential applications for a wide range of diseases, including those that have proven refractory or difficult to manage with conventional treatment options. Diseases targeted by cell-based treatments include, for example, various cancers, autoimmune diseases, central nervous system (CNS) diseases, neurodegenerative disorders, and cardiovascular diseases, among others. Cellular therapeutics offer an alternative to other treatment options for diseases where highly specific targeting (e.g., to a particular tissue type, body region, etc.) and / or longer-term treatment efficacy (e.g., allowing for lower administration frequency, single treatment option, etc.) is highly desirable or required.

[0062] Cell therapy can use a number of different cell types that are typically modified (e.g., transgene expression, reprogrammed cell targeting, etc.) to produce a therapeutic effect. While many cell types have the potential to produce some type of therapeutic effect, adaptive immune cells, such as T lymphocytes and B lymphocytes (interchangeably referred to herein as T cells and B cells, respectively), are highly desirable in recent therapies. For example, chimeric antigen receptor T (CAR-T) cells have been engineered to treat various cancers by recognizing one or more tumor cell markers, leading to cytotoxic destruction of tumor cells. CAR-T cells have also been applied to treat infectious diseases (e.g., HIV) by recognizing other target antigens. Recent engineering efforts have focused on enhancing CAR-T receptor function, reducing innate immune responses to CAR-T, and developing allogeneic therapies utilizing donor cells.

[0063] B-lineage cell therapy While cell therapy offers an exciting new means of disease treatment, numerous challenges remain, including achieving safe, specific, and long-term therapeutic changes in target cells or tissues while simultaneously reducing off-target effects. Furthermore, immune tolerance of these cell therapies is crucial to prevent any adverse side effects (see Jeske et al. 2021, incorporated herein by reference in its entirety). To address these challenges, B-lineage cell manipulation has also been a developing area of ​​cell therapy due to the body's inherent role in producing antibodies while minimizing inflammation. Antibody-based treatments are an established and well-studied form of treatment for numerous diseases, including cancer, autoimmune diseases, and infectious diseases. Monoclonal antibodies against target antigens can be produced in the body, which is useful for treating diseases in which such antibodies cannot be induced by natural processes (e.g., autoantigens in cancer and / or autoimmune diseases, antigens that cannot elicit a natural immune response through infection and / or vaccination, etc.). Current antibody therapies require frequent administration and are costly to produce. Researchers have attempted to address these problems by using gene therapy, which utilizes various technologies (e.g., viral vectors, CRISPR / Cas9 editing) to deliver antibody expression cassettes to endogenous cells within the body, thereby resulting in sustained antibody production in the subject. However, these approaches can result in low levels of antibody expression and a neutralizing response by the subject's own immune system.

[0064] Furthermore, due to their minimal impact on a subject's natural immune system and their ability to continuously produce antibodies, B-lineage cells are highly desirable cell-based targets for secreting other payloads, including, but not limited to, enzymes, complement proteins, cytokines, cytokine receptors, chimeric antigen receptors (CARs), antifibrotic molecules, antithrombotic molecules, antigens, both wild-type and variant proteins, coagulation factors, glucose response factors, and fragments of antibodies, antigens, and proteins. A recent report, for example, described the successful engineering of B-lineage cells to produce human B-cell activating factor (hBAFF) in a mouse model. Interestingly, this study demonstrated not only successful hBAFF production, but also the engraftment of these engineered B-lineage cells in the bone marrow and their ability to survive for up to 60 days after engraftment (see Cheng et al. 2022, incorporated herein by reference in its entirety). B lineage cells are an attractive option for the development of cell therapies and may potentially offer improved therapeutic efficacy (e.g., payload delivery, targeting, prolonged payload expression, reduced autoimmune responses, etc.) compared to conventional treatments (e.g., antibody-based therapeutics, other cell therapies, etc.).

[0065] In some embodiments, B lineage cells are cells that express one or more B cell receptors (BCRs) on their cell membrane. In some embodiments, B lineage cells are modified or variants of cells that express one or more B cell receptors (BCRs) on their cell membrane. In some embodiments, B lineage cells are naive or memory B cells. In some embodiments, B lineage cells are cells derived from naive B cells (e.g., activated B lineage cells, plasmablasts, plasma cells) or variants thereof. In some embodiments, B lineage cells are activated B lineage cells. In some embodiments, B lineage cells are plasmablasts. In some embodiments, B lineage cells are plasma cells.

[0066] In some embodiments, the B-lineage cell population comprises naive B cells. In some embodiments, the naive B cell population is used as a reference cell population. In some embodiments, the naive B cell population expresses CD19 (CD19 + In some embodiments, the expression of CD19 in naive B cell populations is used as a reference to aid in the characterization of other B lineage cell populations. In some embodiments, the naive B cell population expresses CD20 (CD20 + In some embodiments, the expression of CD20 in naive B cell populations is used as a reference to aid in the characterization of other B lineage cell populations. In some embodiments, naive B cell populations express low amounts of CD27 (CD27 lo In some embodiments, the expression of CD27 in naive B cell populations is used as a reference to aid in the characterization of other B lineage cell populations. In some embodiments, naive B cell populations express low amounts of CD38 (CD38 lo In some embodiments, the expression of CD38 in naive B cell populations is used as a reference to aid in the characterization of other B lineage cell populations. In some embodiments, naive B cell populations express low amounts of CD138 (CD138 lo In some embodiments, expression of CD138 on naive B cell populations is used as a reference to aid in the characterization of other B lineage cell populations.

[0067] In some embodiments, the B-lineage cell population comprises activated B-lineage cells. In some embodiments, the activated B-lineage cell population is used as a reference cell population. In some embodiments, the activated B-lineage cell population is compared to a reference cell population (e.g., a naive B-cell population). In some embodiments, the activated B-lineage cell population expresses a lower amount of CD19 (CD19) compared to the reference cell population (e.g., a naive B-cell population). loIn some embodiments, the activated B-lineage cell population expresses a different amount (e.g., a greater or lesser amount) of CD19 compared to a reference cell population (e.g., a differentiated B-cell population). In some embodiments, the activated B-lineage cell population expresses a lesser amount of CD20 (CD20) compared to a reference cell population (e.g., a naive B-cell population). lo In some embodiments, the activated B-lineage cell population expresses a different amount (e.g., a greater or lesser amount) of CD20 compared to a reference cell population (e.g., a differentiated B-cell population). In some embodiments, the activated B-lineage cell population expresses a greater amount of CD27 (CD27) compared to a reference cell population (e.g., a naive B-cell population). hi In some embodiments, the activated B-lineage cell population expresses a different amount (e.g., a greater amount or a lesser amount) of CD27 compared to a reference cell population (e.g., a differentiated B-cell population). In some embodiments, the activated B-lineage cell population expresses a lesser amount of CD38 (CD38) compared to a reference cell population (e.g., a naive B-cell population). lo In some embodiments, the activated B-lineage cell population expresses a different amount (e.g., a greater or lesser amount) of CD38 compared to a reference cell population (e.g., a differentiated B-cell population). In some embodiments, the activated B-lineage cell population expresses a lesser amount of CD138 (CD138) compared to a reference cell population (e.g., a naive B-cell population). lo In some embodiments, the activated B-lineage cell population expresses a different amount (e.g., a greater or lesser amount) of CD138 compared to a reference cell population (e.g., a differentiated B-cell population, etc.).

[0068] In some embodiments, the B-lineage cell population can include plasmablasts. In some embodiments, the plasmablast population is used as a reference cell population. In some embodiments, the plasmablast population is compared to a reference cell population (e.g., a naive B-cell population). In some embodiments, the plasmablast population expresses a lower amount of CD19 (CD19) compared to the reference cell population (e.g., a naive B-cell population). loIn some embodiments, the plasmablast population expresses a different amount (e.g., a greater or lesser amount) of CD19 compared to a reference cell population (e.g., an activated cell population, a plasma cell population, etc.). In some embodiments, the plasmablast population expresses a lesser amount of CD20 (CD20) compared to a reference cell population (e.g., a naive B cell population). lo In some embodiments, the plasmablast population expresses a different amount (e.g., a greater or lesser amount) of CD20 compared to a reference cell population (e.g., an activated cell population, a plasma cell population, etc.). In some embodiments, the plasmablast population expresses a greater amount of CD27 (CD27) compared to a reference cell population (e.g., a naive B cell population). hi In some embodiments, the plasmablast population expresses a different amount (e.g., a greater or lesser amount) of CD27 compared to a reference cell population (e.g., an activated cell population, a plasma cell population, etc.). In some embodiments, the plasmablast population expresses a greater amount of CD38 (CD38) compared to a reference cell population (e.g., a naive B cell population). hi In some embodiments, the plasmablast population expresses a different amount (e.g., a greater or lesser amount) of CD38 compared to a reference cell population (e.g., an activated cell population, a plasma cell population, etc.). In some embodiments, the plasmablast population expresses a lesser amount of CD138 (CD138) compared to a reference cell population (e.g., a naive B cell population). lo In some embodiments, the plasmablast population expresses a different amount (e.g., a greater or lesser amount) of CD138 compared to a reference cell population (e.g., an activated cell population, a plasma cell population, etc.).

[0069] In some embodiments, the B-lineage cell population comprises plasma cells. In some embodiments, a plasma cell population is used as a reference cell population. In some embodiments, the plasma cell population is compared to a reference cell population (e.g., a naive B-cell population). In some embodiments, the plasma cell population expresses a lower amount of CD19 (CD19) compared to the reference cell population (e.g., a naive B-cell population). loIn some embodiments, the plasma cell population expresses a different amount (e.g., a greater or lesser amount) of CD19 compared to a reference cell population (e.g., an activated cell population, a plasmablast population, etc.). In some embodiments, the plasma cell population expresses a lesser amount of CD20 (CD20) compared to a reference cell population (e.g., a naive B cell population). lo In some embodiments, the plasma cell population expresses a different amount (e.g., a greater or lesser amount) of CD20 compared to a reference cell population (e.g., an activated cell population, a plasmablast population, etc.). In some embodiments, the plasma cell population expresses a greater amount of CD27 (CD27) compared to a reference cell population (e.g., a naive B cell population). hi In some embodiments, the plasma cell population expresses a different amount (e.g., a greater or lesser amount) of CD27 compared to a reference cell population (e.g., an activated cell population, a plasmablast population, etc.). In some embodiments, the plasma cell population expresses a greater amount of CD38 (CD38) compared to a reference cell population (e.g., a naive B cell population). hi In some embodiments, the plasma cell population expresses a different amount (e.g., a greater or lesser amount) of CD38 compared to a reference cell population (e.g., an activated cell population, a plasmablast population, etc.). In some embodiments, the plasma cell population expresses a greater amount of CD138 (CD138) compared to a reference cell population (e.g., a naive B cell population). hi In some embodiments, the plasma cell population expresses a different amount (e.g., a greater or lesser amount) of CD138 compared to a reference cell population (e.g., an activated cell population, a plasmablast population, etc.).

[0070] cell manipulation The generation of engineered cells for various applications, including, for example, cell therapy, is an area of ​​active development. Genomic and epigenomic modifications, synthetic biology, and the application of biomaterials can be used to generate engineered cells with desirable properties for therapeutic applications. The selection of an appropriate method for generating engineered cells typically depends on the desired outcome and effect of the cell therapy and requires optimization for different cell types, transgenes of interest, etc. It is understood in the art that an engineering method that is effective in a particular cell type may be less effective (or not feasible) in another cell type. Furthermore, engineering methods may differ depending on whether the therapy requires cellular localization, expression of endogenous or exogenous proteins, removal of endogenous proteins, etc.

[0071] Genome editing Genome editing tools can modify cellular genomes to achieve desired therapeutic effects, such as the expression of therapeutic proteins. Targeted nucleases (e.g., Cas proteins, TALENs, ZFNs, etc.), viral vectors (e.g., AAV, adenovirus, lentivirus, etc.), recombinases (e.g., Cre recombinase, Flp recombinase, PhiC31 integrase, etc.), and other tools can be used to achieve gene modification. Gene editing efficiency can vary depending on, for example, cell type, desired function, and ease of delivery. Therefore, editing methods often require extensive optimization to obtain engineered cells with the intended function for therapeutic applications.

[0072] B lineage cell manipulation Various B-lineage cell engineering techniques have been described in the art, including, for example, CRISPR / Cas9, AAV, lentivirus, and recombinase-based methods. These methods are generally used to introduce a payload (e.g., an expression cassette, a transgene, etc.) into naive B cells to express a protein of interest. The payload can be designed for episomal expression, integration into a specific target locus (e.g., the locus of an endogenous target gene), or integration into a non-specific locus (e.g., an endogenous random locus or a non-target locus). The payload can be designed so that the endogenous target gene locus continues to produce a functional protein and / or perform its original function (non-disruptive integration). The payload can also be designed to intentionally disrupt the endogenous target gene locus to produce reduced or undetectable levels of functional protein and / or some amount of non-functional protein.

[0073] Methods for integration of a payload (e.g., a transgene) into an endogenous target locus can include site-specific cleavage by a targeted nuclease (e.g., a Cas protein, including Cas9), followed by integration of the payload (e.g., a transgene) via endogenous repair pathways (e.g., homologous recombination, homology-directed repair, etc.).

[0074] In some embodiments, the method of B lineage cell manipulation is or comprises administering a ribonucleoprotein (RNP) to the cell population. In some embodiments, the method of B lineage cell manipulation is or comprises administering a composition comprising a Cas protein complexed with a guide RNA (gRNA) to the cell population. In some embodiments, the method of B lineage cell manipulation is or comprises administering a composition comprising a Cas9 / guide RNA complex to the cell population. In some embodiments, the method of B lineage cell manipulation is or comprises administering a composition comprising a Cas9 / guide RNA complex to the cell population. In some embodiments, the method of B lineage cell manipulation is or comprises administering a composition comprising a payload of interest (e.g., a transgene) to the cell population. In some embodiments, the method of B lineage cell manipulation is or comprises administering a composition comprising a payload (e.g., a transgene) to the cell population using a viral vector. In some embodiments, the method of B lineage cell engineering is or comprises administering to a population of cells a composition comprising a payload (e.g., a transgene) packaged within an AAV capsid (e.g., AAV2, AAV3, AAV5, AAV6, AAV8, etc.). In some embodiments, the method of B lineage cell engineering is or comprises administering to a population of cells a composition comprising a transgene packaged within an AAV capsid (e.g., AAV2, AAV3, AAV5, AAV6, AAV8, etc.) in combination with or in addition to administering a composition comprising a Cas9 / gRNA complex.

[0075] In some embodiments, the method of B lineage cell engineering includes a step of electroporation to promote cellular uptake of one or more engineered elements. In some embodiments, the method of B lineage cell engineering includes a step of electroporation to promote cellular uptake of a Cas9 / gRNA complex. In some embodiments, the method of B lineage cell engineering includes a step of electroporation to promote cellular uptake of a Cas9 / gRNA complex. In some embodiments, the method of B lineage cell engineering includes a step of electroporation to promote cellular uptake of a Cas9 / gRNA complex and a payload. In some embodiments, the method of B lineage cell engineering can include a step of electroporation to promote cellular uptake of a Cas9 / gRNA complex and a payload (e.g., a transgene) encapsulated within an AAV capsid (e.g., AAV2, AAV3, AAV5, AAV6, AAV8, etc.). In some embodiments, the method of B lineage cell engineering includes a step of electroporation to promote cellular uptake of a Cas9 / gRNA complex and a payload (e.g., a transgene) encapsulated within an AAV6 capsid.

[0076] In some embodiments, the method of B lineage cell engineering comprises a step of viral transduction to promote cellular uptake of a payload (e.g., a transgene). In some embodiments, the method of B lineage cell engineering comprises a step of viral transduction to promote cellular uptake of a payload (e.g., a transgene) packaged within an AAV capsid (AAV2, AAV3, AAV5, AAV6, AAV8, etc.). In some embodiments, the method of B lineage cell engineering comprises a step of viral transduction to promote cellular uptake of a payload (e.g., a transgene) packaged within an AAV6 capsid. In some embodiments, the method of B lineage cell engineering comprises one or more of the following steps: (i) electroporation to promote cellular uptake of the Cas9 / gRNA complex; and (ii) viral transduction to promote cellular uptake of a payload packaged within an AAV capsid.

[0077] In some embodiments, methods of cell engineering are particularly effective against one type of cell (e.g., T cells) and less effective against another type of cell (e.g., B cells). In some embodiments, methods of B lineage cell engineering provide improved genome editing efficiency in B lineage cells compared to other cell types (e.g., T cells). In some embodiments, methods of B cell engineering provide editing efficiencies of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the method of B lineage cell engineering provides at least about 1E6, 1.5E6, 2E6, 2.5E6, 3E6, 3.5E6, 4E6, 4.5E6, 5E6, 5.5E6, 6E6, 6.5E6, 7E6, 7.5E6, 8E6, 8.5E6, 9E6, 9.5E6, 1E7, 1.5E7, 2E7, 2.5E7, 3E7, 3.5E7, 4E7, 4.5E7, or 5E7 edited B lineage cells.

[0078] In some embodiments, the method of B lineage cell manipulation comprises editing activated B cells. In some embodiments, the method of B lineage cell manipulation comprises editing B lineage cells after an activation step of about 1, 2, 3, 4, or 5 days. In some embodiments, the method of B lineage cell manipulation comprises editing B lineage cells after an activation step of 2 days. In some embodiments, the method of B lineage cell manipulation comprises editing B lineage cells after an activation step of about 1, 2, 3, 4, or 5 days, and expanding the edited B lineage cells in activation medium for an additional period of about 1, 2, 3, 4, 5, 6, 7, or 8 days. In some embodiments, the method of B lineage cell manipulation comprises editing B lineage cells after an activation step of 2 days, and expanding the edited B lineage cells in activation medium for an additional 6 days.

[0079] payload The various methods described herein can be used to generate engineered cells (e.g., B-lineage cell populations, etc.) comprising one or more payloads. In some embodiments, the engineered cells (e.g., B-lineage cell populations, etc.) comprise a polynucleotide sequence encoding one or more payloads. According to various aspects of the present disclosure, any of a variety of payloads (e.g., for therapeutic or monitoring purposes) can be used alone or in combination. In some embodiments, the payload is or comprises a polynucleotide sequence encoding a peptide or polypeptide. In some embodiments, the payload is or comprises one or more transgenes. In some embodiments, the payload is or comprises one or more homology arm sequences. In some embodiments, the payload is or comprises a transgene flanked by one or more homologous sequences.

[0080] Transgene In some embodiments, the transgene is a corrective gene selected to ameliorate one or more signs and / or symptoms of a disease, disorder, or condition. In some embodiments, the transgene is a functional version of a disease-associated gene (i.e., a gene isoform(s) associated with the onset or worsening of the disease, disorder, or condition) found in the subject. In some embodiments, the transgene is an optimized version (e.g., a codon-optimized or expression-optimized variant) of a disease-associated gene found in the subject. In some embodiments, the transgene is a variant (e.g., a functional gene fragment or variant thereof) of a disease-associated gene found in the subject. In some embodiments, the transgene is a gene that causes expression of a peptide that is normally expressed in one or more healthy tissues.

[0081] In some embodiments, the transgene is a gene that drives expression of an engineered protein having a gain-of-function or loss-of-function mutation. In some embodiments, the transgene is a gene that drives expression of a fusion protein. In some embodiments, the transgene is a gene that drives expression of an antibody drug. In some embodiments, the transgene is a gene that drives expression of a multispecific antibody. In some embodiments, the transgene is an antibody, antigen, or protein fragment. In some embodiments, the transgene is a gene that drives expression of an enzyme (e.g., for enzyme replacement therapy). In some embodiments, the transgene is a gene that drives expression of a cytokine. In some embodiments, the transgene is a gene that drives expression of a cytokine receptor. In some embodiments, the transgene is a gene that drives expression of a chimeric antigen receptor (CAR). In some embodiments, the transgene is a gene that drives expression of an antithrombotic molecule. In some embodiments, the transgene is a gene that drives expression of a coagulation factor. In some embodiments, the transgene is a gene that drives expression of a glucose response factor. In some embodiments, the transgene is a gene that drives expression of a nanobody. In some embodiments, the transgene is a gene that drives expression of factor IX (FIX) or a variant thereof. In some embodiments, the transgene is a gene that causes expression of sphingomyelin phosphodiesterase 1 (SMPD1) or a variant thereof.

[0082] In some embodiments, the transgene is or includes a gene encoding a functional nucleic acid. In some embodiments, the therapeutic agent is or includes an agent (e.g., including a ribozyme, guide RNA (gRNA), antisense oligonucleotide (ASO), miRNA, siRNA, and / or shRNA) that has a therapeutic effect on a host cell or subject. For example, in some embodiments, the therapeutic agent promotes a biological process to treat at least one symptom of a medical condition, e.g., a disease, disorder, or condition.

[0083] In some embodiments, expression of the transgene in the subject is substantially due to integration at the target locus. In some embodiments, 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, 99.5% or more) of total transgene expression in the subject is due to transgene integration at the target locus. In some embodiments, 25% or less (e.g., 20% or less, 15% or less, 10% or less, 5% or less, 1% or less, 0.5% or less, 0.1% or less) of total transgene expression in the subject is due to sources other than transgene integration at the target locus (e.g., episomal expression, integration at a non-target locus).

[0084] In some embodiments, the transgene is or comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to a corresponding wild-type reference nucleotide sequence (e.g., a wild-type gene sequence). In some embodiments, the transgene is or comprises a sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to a portion of a corresponding wild-type reference nucleotide sequence (e.g., a wild-type gene sequence).

[0085] Targeted integration In some embodiments, the compositions disclosed herein induce integration of a payload (e.g., a transgene) at a target locus (e.g., an endogenous gene). In some embodiments, the compositions and constructs provided herein induce integration of a payload at a target locus in a specific cell type (e.g., naive B cells, B-lineage cell populations, etc.).

[0086] In some embodiments, the compositions and constructs provided herein direct integration of a payload at a target locus considered a safe harbor site (e.g., CCR5, AAVS1). In some embodiments, the target locus is selected from any genomic site suitable for use with the methods and compositions provided herein. In some embodiments, the target locus encodes a polypeptide. In some embodiments, the target locus encodes a polypeptide that is highly expressed in a subject (e.g., a subject not afflicted with a disease, disorder, or condition, or a subject afflicted with a disease, disorder, or condition). In some embodiments, the target locus is selected from one or more of CD19, CD20, IGH, B2M, CCR5, JCHAIN, PAX5, IRF4, IRF8, BACH2, EZH2, XBP1, CARD11, PRDM1, and BAFF.

[0087] B lineage cell culture method Various methods for the in vitro culture and long-term maintenance of B lineage cells have been described in the art (see Rawlings et al. 1995, Rawlings et al. 1997, and Fluckinger et al. 1998, each of which is incorporated herein by reference in its entirety). B lineage cell culture conditions can significantly affect normal human B lineage development and the generation of mature Ig-secreting B cells. Without wishing to be bound by any theory, it is generally believed that ex vivo activation of B lineage cells may be required for subsequent genome editing procedures utilizing homologous recombination repair (HDR) because the required DNA repair proteins are present during the G2 / S phase of the cell cycle (see Rogers and Cannon 2021, incorporated herein by reference in its entirety).

[0088] Activation of B-lineage cells Various techniques have been described for in vitro activation of B lineage cells (also referred to interchangeably as "activated B lineage cells"). Traditionally, in vitro B lineage cell activation and expansion has utilized CD40L-expressing feeder cell layer systems. Such feeder cell systems have been described as difficult to standardize and often unreliable for providing consistent levels of B lineage cell activation and expansion. Recent advances have shifted toward protocols for in vitro activation and expansion of B lineage cells in specific culture systems containing cytokines or other components in the absence of feeder cells (see Jourdan et al., 2009 and Hartweger et al., 2019, each of which is incorporated herein by reference in its entirety).

[0089] In some embodiments, methods for B lineage cell activation include contacting cells with a medium comprising one or more components of the present disclosure. In some embodiments, methods for B lineage cell activation include contacting cells with a medium comprising one or more cytokines and / or oligonucleotides (e.g., multimeric human CD40L, IL-2, IL-10, IL-15, IL-21, and / or CpG). In some embodiments, methods for B cell activation include contacting the cells with medium containing at least about 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL, or 500 ng / mL of one or more cytokines and / or oligonucleotides (e.g., multimeric human CD40L, IL-2, IL-10, IL-15, IL-21, and / or CpG). In some embodiments, methods for B lineage cell activation include contacting cells with medium containing at least about 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.4 μg / mL, 0.5 μg / mL, 0.6 μg / mL, 0.7 μg / mL, 0.8 μg / mL, 0.9 μg / mL, 1 μg / mL, 1.5 μg / mL, 2 μg / mL, 2.5 μg / mL, 3 μg / mL, 3.5 μg / mL, 4 μg / mL, 4.5 μg / mL, or 5 μg / mL of one or more cytokines and / or oligonucleotides (e.g., multimeric human CD40L, IL-2, IL-10, IL-15, IL-21, and / or CpG).

[0090] In some embodiments, methods for B lineage cell activation include contacting cells with medium comprising one or more components of the present disclosure for at least about 1, 2, 3, 4, or 5 days. In some embodiments, methods for B lineage cell activation include contacting cells with medium comprising one or more cytokines and / or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21, and / or CpG) for at least about 1, 2, 3, 4, or 5 days. In some embodiments, methods for B lineage cell activation include contacting cells with medium comprising one or more cytokines and / or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21, and / or CpG) for at least 2 days. In some embodiments, methods for B lineage cell activation include contacting cells with medium comprising one or more cytokines and / or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21, and / or CpG) for at least two days, followed by gene editing. In some embodiments, methods for B lineage cell activation include contacting cells with medium comprising one or more cytokines and / or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21, and / or CpG) for at least two days, followed by B lineage cell expansion. In some embodiments, methods for B lineage cell activation include contacting cells with medium comprising one or more cytokines and / or oligonucleotides (e.g., CD40L, IL-2, IL-10, IL-15, IL-21, and / or CpG) for at least two days, followed by B lineage cell expansion for at least about 1, 2, 3, 4, 5, 6, 7, or 8 days.

[0091] In some embodiments, the methods described herein result in an activated B-lineage cell population.

[0092] B cell differentiation into plasmablasts Plasmablasts are short-lived, rapidly generated effector cells that are primarily present in the early stages of antibody responses and are one possible product of B cell terminal differentiation. Plasmablasts can secrete antibodies, including IgM subtype antibodies, to initiate an immediate response to a specific antigen in the body. In vitro differentiation of B cells into plasmablasts can be promoted by using certain signaling molecules, including one or more cytokines (e.g., IL-2, IL-6, IL-10, and / or IL-15). Various methods for differentiating B cells into plasmablasts are known, including, but not limited to, those outlined in WO / 2018 / 170150 (incorporated herein by reference in its entirety). Plasmablasts generated by such methods express the CD27 + / CD38 + / CD138 - The cells may be characterized as cells that are

[0093] In some embodiments, methods for differentiating B cells into plasmablasts include contacting the cells with a medium comprising one or more components, hi some embodiments, methods for differentiating B cells into plasmablasts include contacting activated B lineage cells with a medium comprising one or more cytokines (e.g., IL-2, IL-6, IL-10, and / or IL-15). In some embodiments, a method for differentiation of B cells to plasmablasts comprises contacting activated B lineage cells with medium comprising at least about 0.5 ng / mL, 1 ng / mL, 1.5 ng / mL, 2 ng / mL, 2.5 ng / mL, 3 ng / mL, 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or ng / mL of one or more cytokines (e.g., IL-2, IL-6, IL-10, and / or IL-15).

[0094] In some embodiments, methods for differentiating B cells into plasmablasts include contacting the cells with a medium comprising one or more components of the present disclosure for at least about 1, 2, 3, or 4 days. In some embodiments, methods for differentiating B cells into plasmablasts include contacting activated B-lineage cells with a medium comprising one or more cytokines (e.g., IL-2, IL-6, IL-10, and / or IL-15) for at least about 1, 2, 3, or 4 days. In some embodiments, methods for differentiating B cells into plasmablasts include contacting activated B-lineage cells with a medium comprising one or more cytokines (e.g., IL-2, IL-6, IL-10, and / or IL-15) for at least 3 days. In some embodiments, methods for differentiating B cells into plasmablasts include contacting activated B-lineage cells with a medium comprising one or more cytokines (e.g., IL-2, IL-6, IL-10, and / or IL-15) for at least 3 days, followed by plasmablast expansion.

[0095] Differentiation of plasmablasts into plasma cells Although plasmablasts secrete more antibodies than naive B cells, they have a shorter lifespan and secrete fewer antibodies than plasma cells (PCs). Long-lived plasma cells (LLPCs; used interchangeably throughout with plasma cells) reside in the bone marrow, can secrete high levels of antibodies, and can survive for decades without proliferation (see Hammerland et al., 2017 and Khodadadi et al., 2019, both of which are incorporated herein in their entirety). Differentiation of plasmablasts into long-lived plasma cells can be triggered by certain events in the body, including, for example, the activity of the transcription factors Blimp-1 / PRDM1 and IRF4. Differentiation of plasmablasts into plasma cells in vitro can be promoted by the use of certain signaling molecules, including one or more cytokines (e.g., IL-6, IL-15, and / or IFNα-2β). Various methods for the differentiation of plasmablasts into plasma cells are known, including but not limited to those reviewed in Jourdan et al. 2019 and WO / 2018 / 170150 (each of which is incorporated by reference in its entirety). Plasma cells generated by such methods express CD27 + / CD38 + / CD138 + The cells may be characterized as cells that are

[0096] In some embodiments, a method for differentiation of plasmablasts into plasma cells comprises contacting the cells with a medium comprising one or more cytokines (e.g., IL-6, IL-15, and / or IFNα-2β). In some embodiments, a method for differentiation of plasmablasts into plasma cells comprises contacting the plasmablasts with a medium comprising one or more cytokines (e.g., IL-6, IL-15, and / or IFNα-2β). In some embodiments, a method for differentiation of plasmablasts to plasma cells includes contacting the plasmablasts with medium containing at least about 0.5 ng / mL, 1 ng / mL, 1.5 ng / mL, 2 ng / mL, 2.5 ng / mL, 3 ng / mL, 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or ng / mL of one or more cytokines (e.g., IL-6, IL-15, and / or IFNα-2β).

[0097] In some embodiments, methods for differentiating plasmablasts into plasma cells include contacting the cells with a medium comprising one or more components of the present disclosure for at least about 1, 2, 3, or 4 days. In some embodiments, methods for differentiating plasmablasts into plasma cells include contacting the plasmablasts with a medium comprising one or more cytokines (e.g., IL-6, IL-15, and / or IFNα-2β) for at least about 1, 2, 3, or 4 days. In some embodiments, methods for differentiating plasmablasts into plasma cells include contacting the plasmablasts with a medium comprising one or more cytokines (e.g., IL-6, IL-15, and / or IFNα-2β) for at least 3 days. In some embodiments, methods for differentiating plasmablasts into plasma cells include contacting the plasmablasts with a medium comprising one or more cytokines (e.g., IL-6, IL-15, and / or IFNα-2β) for at least 3 days, followed by cell isolation. In some embodiments, a method for differentiation of plasmablasts into plasma cells comprises contacting the plasmablasts with a medium comprising one or more cytokines (e.g., IL-6, IL-15, and / or IFNα-2β) for at least three days, followed by administration to a subject.

[0098] Cryopreservation of cell populations Cell therapy offers many potential advantages over conventional treatment options, due at least in part to the unique properties of viable cells within the body. However, those skilled in the art recognize that successful clinical outcomes require cell therapy to be delivered to subjects quickly and effectively. Therefore, it is important to develop preservation and storage techniques that maintain standardized efficacy until they can be transported and administered to subjects. Furthermore, because each cell population has different structural considerations for preservation, it is equally important to understand and identify conditions that enhance the viability and function of cell populations (see Meneghel, 2020, incorporated herein by reference in its entirety). Many people have worked hard to address these concerns and develop useful preservation techniques, but these are still highly cell-specific and may result in various inadequacies. For example, one report describes a significant decline in B-lineage cell subpopulations when attempting to preserve them for long periods of time (see Ticha et al., 2021, incorporated herein by reference in its entirety). The present disclosure seeks to address these challenges in the art by providing novel culture conditions, methods, and techniques that achieve effective cryopreservation of certain cell populations (e.g., engineered B-lineage cell populations).

[0099] Storage conditions are one of several considerations for cryopreservation. Short-term storage at low temperatures (e.g., 2–4 days at 4°C) does not adequately preserve cell therapies so that they maintain normal metabolic processes, viability, and / or expression of any payload of interest. Therefore, cell therapies often require stable storage at temperatures below −130°C, which prevents metabolic changes in the cells. Cryopreservation processes are well established for certain cell types, such as hematopoietic stem cells (HSCs) for transplantation, with over 47,000 procedures performed in Europe in 2018 (see Passweg et al., 2020, incorporated herein by reference in its entirety). To maintain the homogeneity and reproducibility of cell therapies, cryopreservation protocols must be clear and precise regarding the specific requirements for each step: freezing, storage, and thawing prior to patient administration. Cryopreservation can also be used multiple times at multiple points in the cell therapy process chain, for example, between cell isolation and patient administration.

[0100] Although various compositions and components for cryopreservation have been tested for many different cell types, optimal cryopreservation conditions may vary between cell therapies depending on the specific requirements and characteristics of each therapy. For example, in some embodiments, cryopreservation conditions that preserve the activity of one type of cell therapy may not preserve the activity of another type of cell therapy. Similarly, in some embodiments, cryopreservation conditions that preserve the viability of a particular cell type may not preserve the viability and / or activity of a cell therapy that uses engineered cells of the same cell type.

[0101] The present disclosure provides certain techniques for achieving effective cryopreservation of certain cell populations (eg, certain B-lineage cell populations).

[0102] Cryoprotectant (CPA) Cryopreservation can cause numerous cellular damages, including potentially deleterious changes in cell morphology, characteristics (e.g., adhesion, cell surface markers, protein expression profiles), metabolic activity (e.g., proliferation capacity, efficacy), and / or function (e.g., immunomodulation, signal responsiveness), and can even result in cell death. Temperature changes associated with cryopreservation methods can trigger adverse responses in cells, including, for example, activation of certain stress response pathways upon initial freezing and / or activation of apoptotic and necrotic pathways upon thawing. Ice crystal formation in the medium during cryopreservation freezing can also cause significant cell damage, for example, by physically disrupting cellular functions and processes, and by increasing solute exclusion and local solute concentrations, which can result in increased osmolality and osmotic stress (see Meneghel et al., 2020). Temperature changes can also result in intracellular ice crystal formation, which can cause cell damage and death.

[0103] To mitigate cell damage as a result of temperature changes, additive compounds known as cryoprotectants (also interchangeably referred to herein as cryoprotectants) can be added to cell culture media. Cryoprotectants (CPAs) have relatively low toxicity to cells, especially at lower temperatures. CPAs can also eliminate ice crystals at certain temperatures, maintaining a larger, unfrozen extracellular portion of the medium, thereby reducing osmotic effects and stress on cells (see Meneghel et al., 2020). CPAs (e.g., DMSO) can also promote cellular dehydration, thereby reducing the probability of intracellular ice crystal formation and interacting with / stabilizing the cell membrane (see Gao et al., 2000). CPAs diffusing through the outer cell membrane can also alter intracellular osmotic concentrations, thereby reducing and / or modulating intracellular ice crystal formation, dehydration, etc. (see Whaley et al., 2021). Mixtures of permeable and non-permeable CPAs can be used to achieve the desired effect and can be optimized for specific cell types (e.g., cell therapy) (see Balci and Can 2013). A particular CPA can also be optimized for exposure time, exposure temperature, loading time, loading temperature, etc. to balance cryoprotective effect with potential toxicity to cells. For example, permeable CPAs (e.g., DMSO) can have a cell membrane permeability that is approximately 100-1000 times lower than that of water, requiring minimal incubation time with cells to allow for equilibration of the protectant (see Mazur et al., 2004).

[0104] In some embodiments, a cryopreserved cell preparation (e.g., a cryopreserved B-lineage cell preparation) provided and / or utilized in accordance with the present disclosure comprises one or more CPAs disclosed herein. In some embodiments, a cryopreserved cell preparation comprises one or more permeabilizing CPAs. In some embodiments, a cryopreserved cell preparation comprises DMSO. In some embodiments, a cryopreserved cell preparation comprises glycerol. In some embodiments, a cryopreserved cell preparation comprises ethylene glycol. In some embodiments, a cryopreserved cell preparation comprises propylene glycol.

[0105] In some embodiments, the cryopreserved cell preparation comprises one or more non-permeant CPAs. In some embodiments, the cryopreserved cell preparation comprises glucose. In some embodiments, the cryopreserved cell preparation comprises sucrose. In some embodiments, the cryopreserved cell preparation comprises trehalose. In some embodiments, the cryopreserved cell preparation comprises raffinose. In some embodiments, the cryopreserved cell preparation comprises hydroxyethyl starch (HES). In some embodiments, the cryopreserved cell preparation comprises polyvinylpyrrolidone (PVP). In some embodiments, the cryopreserved cell preparation comprises polyvinyl alcohol (PVA). In some embodiments, the cryopreserved cell preparation comprises polyethylene glycol (PEG). In some embodiments, the cryopreserved cell preparation comprises dextran. In some embodiments, the cryopreserved cell preparation comprises one or more albumin proteins. In some embodiments, the cryopreserved cell preparation comprises human serum albumin (HSA). In some embodiments, the cryopreserved cell preparation comprises serum. In some embodiments, the cryopreserved cell preparation comprises a poloxamer (e.g., P188). In some embodiments, the cryopreserved cell preparation comprises mannitol. In some embodiments, the cryopreserved cell preparation comprises glutathione. In some embodiments, the cryopreserved cell preparation comprises thioredoxin. In some embodiments, the cryopreserved cell preparation comprises glutathione. In some embodiments, the cryopreserved cell preparation comprises ascorbic acid. In some embodiments, the cryopreserved cell preparation comprises mitoquinone. In some embodiments, the cryopreserved cell preparation comprises salidroside. In some embodiments, the cryopreserved cell preparation comprises reservatrol. In some embodiments, the cryopreserved cell preparation comprises N-acetyl-L-cysteine. In some embodiments, the cryopreserved cell preparation comprises catalase. In some embodiments, the cryopreserved cell preparation comprises α-tocopheryl acetate. In some embodiments, the cryopreserved cell preparation comprises lactated Ringer's solution. In some embodiments, the cryopreserved cell preparation comprises dextrose.

[0106] In some embodiments, the cryopreserved cell preparation comprises one or more permeable CPAs and one or more non-permeable CPAs. In some embodiments, the cryopreserved cell preparation comprises DMSO and HSA. In some embodiments, the cryopreserved cell preparation comprises lactated Ringer's solution and dextrose.

[0107] In some embodiments, cryoprotectants are added to cell preparations in a specific order to facilitate cryopreservation (e.g., HSA, followed by DMSO). In some embodiments, one or more non-permeable CPAs are added first, followed by a permeable CPA. In some embodiments, one or more non-permeable CPAs are added first, followed by another non-permeable CPA. In some embodiments, one or more permeable CPAs are added first, followed by a non-permeable CPA. In some embodiments, one or more permeable CPAs are added first, followed by another permeable CPA.

[0108] In some embodiments, the cryoprotectant is added to the cell preparation at a specific temperature to facilitate cryopreservation. In some embodiments, the cryoprotectant is added at a temperature of about -5°C, -4°C, -3°C, -2°C, -1°C, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C. In some embodiments, the cryoprotectant is added at a temperature of about 4°C. In some embodiments, the cryoprotectant is added at a temperature of about 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C. In some embodiments, the cryoprotectant is added at a temperature of about 20°C. In some embodiments, the cryoprotectant is added at a temperature of about 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C. In some embodiments, the cryoprotectant is added at a temperature of about 37° C.

[0109] In some embodiments, a cryoprotectant is added to the cell preparation and allowed to equilibrate during the incubation period to facilitate cryopreservation. In some embodiments, the cell preparation is incubated with the cryoprotectant at a temperature of about 5°C, -4°C, -3°C, -2°C-1°C, 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C. In some embodiments, the cell preparation is incubated with the cryoprotectant at a temperature of about 4°C. In some embodiments, the cell preparation is incubated with the cryoprotectant at a temperature of about 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C. In some embodiments, the cell preparation is incubated with the cryoprotectant at a temperature of about 20°C. In some embodiments, the cell preparation is incubated with the cryoprotectant at a temperature of about 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., or 45° C. In some embodiments, the cell preparation is incubated with the cryoprotectant at a temperature of about 37° C.

[0110] In some embodiments, the cell preparation is incubated with the cryoprotectant for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. In some embodiments, the cell preparation is incubated with the cryoprotectant for at least about 10 minutes. In some embodiments, the cell preparation is incubated with the cryoprotectant for at least about 30, 31, 32, 33, 34, 35, or 36 minutes. In some embodiments, the cell preparation is incubated with the cryoprotectant for at least about 36 minutes. In some embodiments, the cell preparation is incubated with the cryoprotectant for at least about 120, 121, 122, 123, 124, 125, 126, or 127 minutes. In some embodiments, the cell preparation is incubated with the cryoprotectant for at least about 127 minutes.

[0111] In some embodiments, the cell preparation is exposed to the cryoprotectant for a specific fixation time prior to the freezing process. In some embodiments, the cell preparation is exposed to the cryoprotectant for about 20, 21, 22, 23, 24, or 25 minutes or less. In some embodiments, the cell preparation is exposed to the cryoprotectant for about 25 minutes or less. In some embodiments, the cell preparation is exposed to the cryoprotectant for about 45, 46, 47, 48, 49, 50, or 51 minutes or less. In some embodiments, the cell preparation is exposed to the cryoprotectant for about 51 minutes or less. In some embodiments, the cell preparation is exposed to the cryoprotectant for about 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, or 142 minutes or less. In some embodiments, the cell preparation is exposed to the cryoprotectant for about 142 minutes or less.

[0112] Freezing method Cooling rate is another consideration when optimizing cryopreservation protocols. Appropriate freezing rates can vary between cell types and can also be affected by media components and cryoprotectants. Cell damage during freezing can result from both extracellular and intracellular ice crystal formation, and controlled cooling rates can affect the time and location of ice crystal formation (see Meneghel et al., 2020). Relatively slow cooling rates can result in initial ice crystal formation within the extracellular medium, thereby excluding solutes from the ice crystals and resulting in an increase in osmolality and the potential for osmotic shock to the cells. These changes in osmolality can also result in partial dehydration of the cells, which can have beneficial effects by reducing the probability of intracellular ice crystal formation and / or stabilizing cell membranes. Relatively high cooling rates can prevent cellular dehydration from occurring prior to freezing, resulting in increased intracellular ice crystal levels and cell injury. The present disclosure recognizes, among other things, that differences in viability and function exist among various cell types under different cooling rates. To address these cell-to-cell variations in cryopreservation, the present disclosure uses a process that includes both heating and cooling phases in a process known as controlled cooling.

[0113] In some embodiments, controlled cooling methods may be used. In some embodiments, controlled cooling involves cooling the chamber until the cell preparation reaches approximately ±1-10°C. In some embodiments, controlled cooling involves cooling the chamber at a rate of ±1-25°C / min until the cryopreserved cell preparation reaches approximately ±1-20°C. In some embodiments, controlled cooling involves cooling the chamber at a rate of ±1-25°C / min until the chamber reaches approximately ±1-40°C. In some embodiments, controlled cooling involves heating the chamber at a rate of ±1-10°C / min until the chamber reaches approximately ±1-12°C. In some embodiments, controlled cooling involves cooling the chamber at a rate of ±1-10°C / min until the chamber reaches ±1-40°C. In some embodiments, controlled cooling involves cooling the chamber at a rate of ±1-10°C / min until the chamber reaches ±1-90°C.

[0114] In some embodiments, controlled cooling methods may be used. In some embodiments, controlled cooling comprises cooling the chamber until the cell preparation reaches 4°C. In some embodiments, controlled cooling comprises cooling the chamber at a rate of 1°C / min until the cryopreserved cell preparation reaches -4°C. In some embodiments, controlled cooling comprises cooling the chamber at a rate of 25°C / min until the chamber reaches -40°C. In some embodiments, controlled cooling comprises heating the chamber at a rate of 10°C / min until the chamber reaches -12°C. In some embodiments, controlled cooling comprises cooling the chamber at a rate of 1°C / min until the chamber reaches -40°C. In some embodiments, controlled cooling comprises cooling the chamber at a rate of 1°C / min until the chamber reaches -90°C.

[0115] How to save It will be appreciated by those skilled in the art that large volumes of cells and tissues, when cryopreserved, can be preserved until the appropriate time and location for preclinical and clinical testing and delivery (see Meneghel et al., 2020). Furthermore, the cryopreservation and preservation methods described herein offer numerous advantages over traditional short-term storage, including, but not limited to, the availability of preparations of stable viability and function, flexibility and predictability of delivery, less cell wastage, reproducibility, and minimal contamination.

[0116] In some embodiments, the B-lineage cell preparation may be stored in a sterile cryopreservation vial (e.g., 1-50 ml). In some embodiments, such a cryopreservation vial may be airtightly sealed. In some embodiments, the B-lineage cell preparation may be stored in a cryopreservation bag. In some embodiments, such a cryopreservation bag may be airtightly sealed. In some embodiments, the cryopreservation bag may be additionally stored in an outer bag. In some embodiments, the cryopreservation bag may be stored in a metal container. In some embodiments, the cryopreservation bag stored in an outer bag may be additionally stored in a metal container. In some embodiments, the B-lineage cell preparation may be stored in a syringe.

[0117] In some embodiments, the B-lineage cell preparation is stored at -170°C. In some embodiments, the B-lineage cell preparation is stored in an ultra-low temperature freezer (e.g., a vapor-based liquid nitrogen freezer). In some embodiments, the B-lineage cell preparation is stored in a liquid nitrogen-based container. In some embodiments, the B-lineage cell preparation is stored in the vapor phase above liquid nitrogen. In some embodiments, the B-lineage cell preparation is stored in cryogenic storage.

[0118] In some embodiments, the B-lineage cell preparation is shipped by one or more methods to maintain a temperature of about −170° C. In some embodiments, the B-lineage cell preparation is shipped in a vapor phase environment above liquid nitrogen. In some embodiments, the B-lineage cell preparation is shipped packed in dry ice.

[0119] How to unzip This disclosure describes, among other things, engineered cryopreserved B-lineage cell populations that can be used in both clinical and preclinical settings. For both successful cryopreservation of a cell population and its administration, an appropriate thawing rate must be considered. Because these are codependent processes, the thawing rate is closely related to the controlled cooling rate. Rapid cooling will result in a lower than expected amount of ice in the cryopreserved product. In some circumstances, a relatively rapid warming rate during thawing is required to prevent any potential cell damage caused by further ice crystallization. This will also minimize the level of hypertonic stress experienced by such cryopreserved cell populations upon thawing, resulting in optimal viability (see Meneghel et al., 2020). For example, it is recognized that in cryopreservation bags, the slower the controlled cooling rate, the greater the proportion of extracellular ice crystals. Therefore, a slower warming rate during thawing will be required to reduce the risk of re-ice crystallization and reduce osmotic stress.

[0120] In some embodiments, the cryopreserved B-lineage cell preparation is transferred from the cryogenic storage device to a thawing mechanism. In some embodiments, the thawing mechanism is a dry heater. In some embodiments, the thawing mechanism is a water bath.

[0121] composition Engineered cell preparations The present disclosure describes engineered cell preparations comprising a population of cells that have been modified to perform one or more desired functions. In some embodiments, the engineered cell preparation is a composition comprising a genetically modified immune cell population (e.g., B cells, T cells). In some embodiments, the engineered cell preparation is a composition comprising a genetically modified B-lineage cell population (e.g., B cells, plasmablasts, plasma cells). In some embodiments, the engineered cell preparation is a composition comprising a genetically modified plasmablast cell population. In some embodiments, the engineered cell preparation is a composition comprising a genetically modified plasma cell population.

[0122] In some embodiments, the engineered cell preparation comprises genetically modified cells that express a payload of interest (e.g., a transgene). In some embodiments, the engineered cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.). In some embodiments, the engineered cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus. In some embodiments, the engineered cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus under the control of an exogenous promoter. In some embodiments, the engineered cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus under the control of an exogenous promoter. In some embodiments, the engineered cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus without compromising endogenous gene expression and / or function. In some embodiments, engineered cell preparations include genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus and partially or completely inhibit endogenous gene expression and / or function.

[0123] Fresh engineered cell preparations The present disclosure provides a number of different cell preparations that are freshly engineered (interchangeably referred to herein as "fresh engineered cell preparations"). In some embodiments, the fresh engineered cell preparation is a composition comprising a genetically modified immune cell population (e.g., T cells, B cells, plasmablasts, plasma cells). In some embodiments, the fresh engineered cell preparation is a composition comprising a genetically modified B-lineage cell population (e.g., B cells, plasmablasts, and plasma cells). In some embodiments, the fresh engineered cell preparation is a composition comprising a genetically modified plasmablast cell population. In some embodiments, the fresh engineered cell preparation is a composition comprising a genetically modified plasma cell population.

[0124] In some embodiments, the fresh engineered cell preparation comprises genetically modified cells that express a payload of interest (e.g., a transgene). In some embodiments, the fresh engineered cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.). In some embodiments, the fresh engineered cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus. In some embodiments, the fresh engineered cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus under the control of an endogenous promoter. In some embodiments, the fresh engineered cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus under the control of an exogenous promoter. In some embodiments, the fresh engineered cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus without impairing endogenous gene expression and / or function. In some embodiments, the fresh engineered cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus and partially or completely inhibit endogenous gene expression and / or function.

[0125] The present disclosure provides methods and compositions for the cryopreservation of fresh engineered cell preparations as described herein.

[0126] Pre-frozen engineered cell preparations The present disclosure provides a number of different cell preparations that have been pre-engineered and frozen. In some embodiments, the pre-frozen engineered cell preparations are used as reference points for cryopreserved cell preparations. In some embodiments, the pre-frozen engineered cell preparations are compositions comprising immune cell populations (e.g., T cells, B cells, plasmablasts, and plasma cells). In some embodiments, the pre-frozen engineered cell preparations are genetically modified immune cell populations. In some embodiments, the pre-frozen engineered cell preparations are compositions comprising genetically modified B-lineage cells (e.g., B cells, plasmablasts, and plasma cells). In some embodiments, the pre-frozen engineered cell preparations are compositions comprising genetically modified plasmablast cell populations. In some embodiments, the pre-frozen engineered cell preparations are compositions comprising genetically modified plasma cell populations.

[0127] The present disclosure provides methods for thawing previously frozen genetically engineered cell preparations as described herein.

[0128] Engineered cryopreserved cell preparations The present disclosure provides a number of different cell preparations that have been subjected to manipulation and subsequent cryopreservation. In some embodiments, the manipulated cell preparation has been subjected to the cryopreservation methods described herein. In some embodiments, the manipulated cryopreserved cell preparation is a composition comprising an immune cell population (e.g., T cells, B cells, plasmablasts, plasma cells). In some embodiments, the manipulated cryopreserved cell preparation is a genetically modified immune cell population. In some embodiments, the manipulated cryopreserved cell preparation is a composition comprising a genetically modified B-lineage cell population. In some embodiments, the manipulated cryopreserved cell preparation is a composition comprising a genetically modified plasmablast population. In some embodiments, the manipulated cryopreserved cell preparation is a composition comprising a genetically modified plasma cell population.

[0129] In some embodiments, the engineered cryopreserved cell preparation comprises genetically modified cells that express a payload of interest (e.g., a transgene). In some embodiments, the engineered cryopreserved cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.). In some embodiments, the engineered cryopreserved cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus. In some embodiments, the engineered cryopreserved cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus under the control of an endogenous promoter. In some embodiments, the engineered cryopreserved cell preparation comprises genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus under the control of an exogenous promoter. In some embodiments, engineered cryopreserved cell preparations comprise genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus without impairing endogenous gene expression and / or function. In some embodiments, engineered cryopreserved cell preparations comprise genetically modified cells that express a transgene of interest (e.g., a therapeutic protein, antibody, etc.) from an endogenous locus and partially or completely inhibit endogenous gene expression and / or function.

[0130] In some embodiments, the engineered cryopreserved cell preparation comprises a cell population previously frozen at −170° C. in a medium comprising one or more cryopreservatives (e.g., HSA and DMSO). In some embodiments, the engineered cryopreserved cell preparation comprises a cell population previously frozen at −170° C. by one or more steps of controlled temperature shift in a medium comprising one or more of HSA, DMSO, Plasmalyte, and CryoStor 10 (CS10). In some embodiments, the medium comprises HSA, DMSO, Plasmalyte, and CryoStor 10 (CS10).

[0131] The present disclosure provides methods and compositions for the cryopreservation and thawing of engineered cryopreserved cell preparations as described herein.

[0132] Fabrication The present disclosure describes the production of certain engineered cryopreserved B-lineage cell preparations. As disclosed herein, in some embodiments, the cryopreserved B-lineage cell preparations comprise both engineered and non-engineered cells. In some embodiments, the engineered cryopreserved B-lineage cell population comprises plasmablasts. In some embodiments, the engineered cryopreserved B-lineage cell population comprises plasma cells. In some embodiments, the engineered cryopreserved B-lineage cell population comprises long-lived plasma cells. In some embodiments, the engineered cryopreserved B-lineage cell population comprises plasmablasts, plasma cells, and / or long-lived plasma cells, and / or any mixture or combination thereof as disclosed herein.

[0133] Plasmablast preparations In some embodiments, the engineered cryopreserved B-lineage cell preparation comprises plasmablasts. As understood in the art, plasmablasts are rapidly generated, short-lived effector cells early in an antibody response. Plasmablasts can be produced from activated B-lineage cells using the methods described herein. In some embodiments, engineered plasmablasts can be produced from engineered activated B-lineage cells.

[0134] In some embodiments, the plasmablast population may be contacted with a medium comprising one or more components of the present disclosure (e.g., IL-6, IL-15, and / or IFNα-2β) to initiate differentiation into a plasma cell population. In some embodiments, a method for differentiation of a plasmablast population into a plasma cell population comprises contacting the plasmablast population with a medium comprising one or more cytokines (e.g., IL-6, IL-15, and / or IFNα-2β). In some embodiments, a method for differentiation of a plasmablast population to a plasma cell population comprises contacting the plasmablast population with medium comprising at least about 0.5 ng / mL, 1 ng / mL, 1.5 ng / mL, 2 ng / mL, 2.5 ng / mL, 3 ng / mL, 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or ng / mL of one or more cytokines (e.g., IL-6, IL-15, and / or IFNα-2β).

[0135] In some embodiments, methods for differentiating a plasmablast population to a plasma cell population include contacting the cells with a medium comprising one or more components of the present disclosure for at least about 1, 2, 3, or 4 days. In some embodiments, methods for differentiating a plasmablast population to a plasma cell population include contacting the plasmablast population with a medium comprising one or more cytokines (e.g., IL-6, IL-15, and / or IFNα-2β) for at least about 1, 2, 3, or 4 days. In some embodiments, methods for differentiating a plasmablast population to a plasma cell population include contacting the plasmablast population with a medium comprising one or more cytokines (e.g., IL-6, IL-15, and / or IFNα-2β) for at least 3 days. In some embodiments, methods for differentiating a plasmablast population to a plasma cell population include contacting the plasmablast population with a medium comprising one or more cytokines (e.g., IL-6, IL-15, and / or IFNα-2β) for at least 3 days, followed by cell isolation. In some embodiments, a method for differentiation of a plasmablast population into a plasma cell population comprises contacting the plasmablasts with a medium comprising one or more cytokines (e.g., IL-6, IL-15, and / or IFNα-2β) for at least three days, followed by administration to a subject.

[0136] Plasma cell preparations In some embodiments, the engineered cryopreserved B-lineage cell preparation comprises a plasma cell population. In some embodiments, the plasma cell population comprises engineered plasma progenitor cells (e.g., plasmablasts) that, upon administration to a subject, further differentiate into a mature plasma cell population. As understood in the art, a mature plasma cell population comprises quiescent, non-dividing cells of the humoral immune response that are capable of secreting large amounts of antibodies. In some embodiments, the mature plasma cell population may comprise short-lived plasma cells and / or long-lived plasma cells (LLPCs) and / or any combination thereof.

[0137] Characterization The present disclosure provides various methods for characterizing engineered cell populations (e.g., B-lineage cell populations). In some embodiments, the methods disclosed herein are used to isolate and / or characterize cryopreserved naive B cell subpopulations within a cryopreserved B-lineage cell population. In some embodiments, the naive B cell subpopulations are characterized by one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or affinity chromatography. In some embodiments, one or more methods for characterizing naive B cell subpopulations are used between each step of the controlled cooling method. In some embodiments, characterization methods are used to isolate and / or characterize engineered naive B cell subpopulations. In some embodiments, the engineered naive B cell subpopulations are characterized by one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or affinity chromatography. In some embodiments, one or more methods for characterizing the engineered naive B cell subpopulation are used during each step of the controlled cooling method.

[0138] In some embodiments, the methods disclosed herein are used to separate and / or characterize cryopreserved activated B-lineage cell subpopulations within a cryopreserved B-lineage cell population. In some embodiments, the activated B-lineage cell subpopulations are characterized by one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or affinity chromatography. In some embodiments, one or more methods for characterizing activated B-lineage cell subpopulations are used during each step of the controlled cooling method. In some embodiments, the methods disclosed herein are used to separate and / or characterize engineered cryopreserved activated B-lineage cell subpopulations. In some embodiments, the engineered activated B-lineage cell subpopulations are characterized by one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or affinity chromatography. In some embodiments, one or more methods for characterizing engineered activated B-lineage cell subpopulations are used during each step of the controlled cooling method.

[0139] In some embodiments, the methods disclosed herein are used to isolate and / or characterize cryopreserved plasmablast subpopulations within a cryopreserved B-lineage cell population. In some embodiments, the plasmablast subpopulations are characterized by one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or affinity chromatography. In some embodiments, one or more methods for characterizing plasmablast subpopulations are used during each step of the controlled cooling method. In some embodiments, the methods disclosed herein are used to isolate and / or characterize engineered cryopreserved plasmablast subpopulations. In some embodiments, the engineered plasmablast subpopulations are characterized by one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or affinity chromatography. In some embodiments, one or more methods for characterizing engineered plasmablast subpopulations are used during each step of the controlled cooling method.

[0140] In some embodiments, the methods disclosed herein are used to isolate and / or characterize cryopreserved plasmatic progenitor cell subpopulations within a cryopreserved B-lineage cell population. In some embodiments, the plasmatic progenitor cell subpopulations are characterized by one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or affinity chromatography. In some embodiments, one or more methods for characterizing plasmatic progenitor cell subpopulations are used during each step of the controlled cooling method. In some embodiments, the methods disclosed herein are used to isolate and / or characterize engineered cryopreserved plasmatic progenitor cell subpopulations. In some embodiments, the engineered plasmatic progenitor cell subpopulations are characterized by one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or affinity chromatography. In some embodiments, one or more methods for characterizing engineered plasmatic progenitor cell subpopulations are used during each step of the controlled cooling method.

[0141] In some embodiments, the methods disclosed herein are used to isolate and / or characterize cryopreserved plasma cell subpopulations within a cryopreserved B-lineage cell population. In some embodiments, the plasma cell subpopulations may be characterized by one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or affinity chromatography. In some embodiments, one or more methods for characterizing plasma cell subpopulations are used during each step of the controlled cooling method. In some embodiments, the methods disclosed herein are used to isolate and / or characterize engineered cryopreserved plasma cell subpopulations. In some embodiments, the engineered plasma cell subpopulations are characterized by one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or affinity chromatography. In some embodiments, one or more methods for characterizing engineered plasma cell subpopulations are used during each step of the controlled cooling method.

[0142] In some embodiments, the methods disclosed herein are used to separate and / or characterize a cryopreserved short-lived plasma cell population within a cryopreserved B-lineage cell population. In some embodiments, the short-lived plasma cell subpopulation is characterized by one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or affinity chromatography. In some embodiments, one or more methods for characterizing the short-lived plasma cell subpopulation are used during each step of the controlled cooling method. In some embodiments, the methods disclosed herein are used to separate and / or characterize an engineered cryopreserved short-lived plasma cell subpopulation. In some embodiments, the engineered short-lived plasma cell subpopulation is characterized by one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or affinity chromatography. In some embodiments, one or more methods for characterizing the engineered short-lived plasma cell subpopulation are used during each step of the controlled cooling method.

[0143] In some embodiments, the methods disclosed herein can be used to isolate and / or characterize cryopreserved long-lived plasma cell subpopulations within a B-lineage cell population. In some embodiments, the long-lived plasma cell subpopulations are characterized by one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or affinity chromatography. In some embodiments, one or more methods for characterizing long-lived plasma cell subpopulations are used during each step of the controlled cooling method. In some embodiments, the methods disclosed herein can be used to isolate and / or characterize engineered cryopreserved long-lived plasma cell subpopulations. In some embodiments, the engineered long-lived plasma cell subpopulations are characterized by one or more of flow cytometry, fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), and / or affinity chromatography. In some embodiments, one or more methods for characterizing engineered long-lived plasma cell subpopulations are used during each step of the controlled cooling method. [Example]

[0144] Example 1: Materials and Methods This example demonstrates a cryopreservation method that can be performed to cryopreserve a particular cell population, including, but not limited to, a B-lineage cell population. First, a particular cell population (e.g., a B-lineage cell population) is cultured to approximately 500 x 10 6 The cell population can be expanded to individual cells. The cell population can include a B-lineage cell population or can be a B-lineage cell population. The B-lineage cell population can include naive B cells or be naive B cells. The B-lineage cell population can include activated B-lineage cells or be activated B-lineage cells. The B-lineage cell population can include plasmablasts or can be plasmablasts. The B-lineage cell population can include plasma cells or can be plasma cells.

[0145] The B-lineage cell population is transferred to a culture bag (e.g., a VueLife bag). The B-lineage cell population is then subjected to a culture wash using Sepax CultureWash. The B-lineage cell population is then subjected to Ficoll cleanup and isolation. Such Ficoll cleanup and isolation can be performed using the NeatCell process.

[0146] The B-lineage cell population is transferred from the culture bag (e.g., VueLife bag) to a centrifuge tube (e.g., a 50 mL centrifuge tube). The B-lineage cell population is then transferred to a vial and then centrifuged. After centrifugation, the post-Ficoll product supernatant is removed. The B-lineage cell population is then resuspended in 5% HSA in PlasmasLyte-A. The viable cell concentration of the B-lineage cell population suspension is then obtained and calculated using a cell counter. Once the cell count is obtained, the B-lineage cell population is then further diluted to approximately 30 x 10 by adding HSA in PlasmasLyte-A. 6 CryoStor 10 (CS10) is then added to the B-lineage cell population to prepare a final concentration of 15 x 10 cells / mL. 6 Obtain a final cryopreservation preparation containing a target concentration of 1000 cells / mL.

[0147] In some embodiments, the B-lineage cell population is about 15×10 6 The cells are resuspended in cryopreservation medium to a target concentration of 100 cells / mL.

[0148] A sample of B-lineage cells is then prepared by adding 2 mL of cryopreservation medium (e.g., 2.5% HSA in PlasmaLyte-A containing 50% CS10). The sample of B-lineage cells is then placed in a controlled rate freezer and subjected to the following controlled cooling steps to create a cryopreserved B-lineage cell preparation: (i) cooling the chamber until the cryopreservation cell preparation reaches 4°C; (ii) cooling the chamber at a rate of 1°C / min until the cryopreservation cell preparation reaches -4°C; (iii) cooling the chamber at a rate of 25°C / min until the chamber reaches -40°C; (iv) heating the chamber at a rate of 10°C / min until the chamber reaches -12°C; (v) cooling the chamber at a rate of 1°C / min until the chamber reaches -40°C; (vi) cooling the chamber at a rate of 10°C / min until the chamber reaches -90°C.

[0149] The B-lineage cell preparation is then transferred to a liquid nitrogen storage transfer box (eg, a cryopod) before being stored in an ultra-low temperature freezer (eg, liquid nitrogen storage).

[0150] The above method can be performed on an engineered B-lineage cell population to produce an engineered cryopreserved B-lineage cell preparation. The cryopreservation medium can contain approximately 0.5-5% HSA, 2-5% DMSO, 25-100% PlasmaLyte-A, 25-100% CS10, and / or any combination thereof. The engineered cryopreserved B-lineage cell preparation can include a cell population previously frozen at -170°C in a medium containing one or more cryopreservation agents, including HSA, PlasmaLyte, and CryoStor 10 (CS10). The concentration of HSA was 2.5%, the concentration of PlasmaLyte was 50%, and the concentration of CS10 was 50%. The engineered cryopreserved B-lineage cell preparation may comprise an engineered B-lineage cell population that has been previously frozen at -170°C by one or more steps of controlled temperature shifting in a medium comprising 2.5% HSA, 50% Plasmalyte, and 50% CS10.

[0151] ELISA for detecting human FIX from engineered B-lineage cell populations

[0152] Supernatants from engineered B-lineage cell populations were collected from relevant experiments and frozen at -80°C before testing. Samples were then tested for human factor IX (hFIX) concentrations in a 96-well FIX ELISA (Abcam-ab108831). Briefly, all kit components, samples, and standards were thawed and allowed to equilibrate to room temperature. 50 μL of sample or standard was added per well of the pre-coated plate and then incubated at room temperature for 2 hours. After incubation, the plate was washed five times with 200 μL of 1x wash buffer. Next, 50 μL of 1x biotinylated FIX detection antibody was added to each well and incubated at room temperature for 1 hour. The plate was then washed, and 50 μL of 1x SP conjugate was added to each well. After addition of the 1x SP conjugate, the plate was then incubated at room temperature for 30 minutes. After washing the plate once more, 50 μL of chromogenic substrate was added per well and incubated at room temperature for 10 minutes. After the wells developed, 50 μL of stop solution was added to quench the color. The absorbance of the plate was read at 450 nm and 570 nm using a Cytation 5 plate reader. Sample values ​​were interpolated using standards of known hFIX concentrations.

[0153] Flow cytometry analysis After resuspension of the cell sample, 2 × 10 5Cells were set aside for flow cytometry analysis. Samples were washed and stained with antibody cocktails focused on immune cell purity (markers: CD19, CD20, CD16, CD56, CD3, CD14, CD45), B-lineage cell phenotyping (markers: CD19, CD20, CXCR4, CD27, CD38, CD138), or Ig profiling (markers: CD38, CD138, IgM, IgG, IgE, IgA). Cells were washed once with PBS and then stained with LIVE / DEAD Fixable Near-IR Dead Cell Stain (Life Technologies) for 10 minutes at room temperature. Cells were washed with Flow Cytometry Staining Buffer (eBioscience) and resuspended in Human TruStain FcX (BioLegend). After 5 minutes of Fc blocking at 4°C, samples were then resuspended in 100 μL of antibody cocktail master mix. Samples were incubated in the dark at 4°C for 20 minutes, then washed twice and resuspended in 400 μL of staining buffer for acquisition on the flow cytometer. Single-color controls for voltage, gating, and compensation were also prepared. After compensation guided by prompts in the NovoCyte software, over 10,000 cell events were acquired per sample. Files were saved and imported into FlowJo. Live cell singlets were further gated in the software, and population percentages, total event counts, and geometric mean fluorescence intensity values ​​were then exported, formatted, and statistically analyzed.

[0154] For IgM / IgG profiling, intracellular staining was performed using cell fixation and permeabilization. After the final wash from viability staining, cells were suspended in fixation buffer (4% paraformaldehyde) and incubated at room temperature for 20 minutes. Cells were then washed three times with permeabilization buffer (eBioscience) and then resuspended in Ig profiling antibody master mix (the dilution used was that recommended by the manufacturer). Samples were incubated at room temperature for 20 minutes protected from light and then washed twice with permeabilization buffer. Pellets were suspended in staining buffer for fresh cell staining and analyzed by forward / side scatter gating, adjusted for cell shrinkage after fixation.

[0155] Example 2: In vivo comparison of fresh and previously frozen cell preparations This example demonstrates that fresh engineered cell preparations exhibit stronger transgene expression compared to previously frozen engineered cell preparations frozen in identical media conditions.

[0156] Naive B cells from two different donors (LKP13 and LKP42) were isolated, activated, engineered, expanded, and differentiated as described above in Example 1. B lineage cells were engineered to express luciferase from the endogenous CCR5 locus. After Ficoll cleanup and isolation, the cell populations were transferred to basal cell culture medium and divided into two groups. The first group (fresh engineered cell preparation) was immediately administered to mice. The second group (frozen B lineage preparation) was cultured at 15 x 10 cells in medium containing 100% CS10. 6 The cells were transferred at a concentration of 100 cells / mL and passively frozen in an ethanol-based freezing container at -80°C for 24 hours, followed by freezing in liquid nitrogen at -170°C. These cells were then administered to mice after thawing.

[0157] The present disclosure demonstrates, inter alia, that subjects administered a pre-frozen cell preparation may exhibit reduced transgene and / or antibody expression compared to subjects administered a fresh engineered cell preparation. Pre-frozen engineered cell preparations may exhibit lower post-transplant engraftment rates compared to fresh engineered cell preparations containing identical media conditions. The present disclosure seeks to address these challenges and concerns by the methods described herein for cryopreservation of engineered cell preparations (e.g., engineered B-lineage cell preparations) described herein.

[0158] Example 3: Improved cell viability after using the cryopreservation methods described herein This example demonstrates that engineered cryopreserved cell preparations (e.g., engineered B-lineage cell preparations) can exhibit improved cell viability and / or density when cryopreserved in medium containing certain cryoprotectants with a controlled temperature shift step.

[0159] Naive B cells from one donor (LKP22030) were isolated, activated, engineered, expanded, and differentiated as described above in Example 1. B lineage cells were engineered to express luciferase from the endogenous CCR5 locus. Cryopreserved B lineage cell preparations from two donors (LKP13 and LKP42), as described in Example 2 above, were used as references. After Ficoll cleanup and isolation, the cell population was divided into groups, and each group was placed in medium containing the cryoprotectant described in Figure 4. The cell preparations were then cooled to -170°C using the method described in Example 1 above. The engineered cryopreserved B lineage cell preparations were then thawed and further cultured in the same medium for 1 to 5 days (days 14 to 18 of culture in Figure 4).

[0160] Additional B-lineage cell preparations (BTD23073, BTD23074, BTD23084, BTD23087, BTD23092, BTD23093, BTD23099, BTD23118, BTD23119, ENG Runs 1-3) were isolated, activated, manipulated, expanded, and differentiated as described herein. Cell number and viability were measured in various B-lineage cell preparations by acridine orange (AO) and DAPI staining before and after cryopreservation as described herein. The average post-thaw cell recovery and viability were approximately 92% and 88%, respectively (Figures 7 and 8).

[0161] B-lineage cell populations were isolated, activated, engineered with a human Factor IX expression cassette, expanded, and differentiated as described above in Example 1. The engineered B-lineage cell populations were then subjected to the cryopreservation method described above in Example 1. Before cryopreservation (time 0), and at various time points after 42 days, 4 months, 6 months, and 8 months ( FIG. 9 ), viability and cell concentration were measured using AO / DAPI staining, while percentages of CD27, CD38, and CD27 / CD38 expression were measured by flow cytometry.

[0162] The present disclosure demonstrates, inter alia, that a controlled temperature shift step can enable engineered cell populations (e.g., engineered B-lineage cell populations) cryopreserved in medium comprising one or more cryoprotectants to exhibit improved cell viability and / or cell density after thawing compared to a reference condition (e.g., medium lacking one or more cryoprotectants). A controlled temperature shift step can also enable engineered B-lineage cell populations cryopreserved in medium comprising one or more cryoprotectants to exhibit improved cell viability of the engineered B-lineage cell population compared to a reference condition (e.g., medium lacking one or more cryoprotectants). A controlled temperature shift step can enable engineered B-lineage cell populations cryopreserved in medium comprising 2.5%-10% HSA and 4.5%-5% DMSO to exhibit improved cell viability after thawing and further culture compared to a reference condition (e.g., medium lacking one or more cryoprotectants). Genetically engineered B-lineage cell populations cryopreserved in medium comprising 2.5%-10% HSA and 4.5%-5% DMSO via a controlled temperature shift step may result in improved cell engraftment following administration to a subject compared to a reference condition (e.g., medium lacking one or more cryoprotectants). Populations cryopreserved in medium comprising one or more cryoprotectants via a controlled temperature shift step may also exhibit improved cell numbers of the engineered B-lineage cell population compared to a reference condition (e.g., medium lacking one or more cryoprotectants).

[0163] Example 4: In vivo comparison of fresh engineered and cryopreserved engineered cell preparations This example demonstrates that engineered cryopreserved cell preparations (e.g., engineered cryopreserved B-lineage cell populations) can exhibit comparable and / or not significantly reduced engraftment and / or transgene expression compared to fresh engineered cell preparations after administration to a subject. Subjects administered an engineered cryopreserved cell preparation (e.g., engineered cryopreserved B-lineage cell population) described herein can exhibit sustained transgene expression levels over an extended period of time (e.g., one week or more, one month or more, etc.).

[0164] Naive B cells were isolated from different donors, activated, engineered, expanded, and differentiated as described above in Example 1. B lineage cells were engineered to express luciferase from the endogenous CCR5 locus. After Ficoll cleanup and isolation, the B lineage cell populations were placed in a medium containing basal cell culture medium and then directly administered to mice (fresh engineered cell preparations) or placed in a medium containing 2.5% HSA, 50% PlasmaLyte, and 50% CS10 and frozen at -170°C (engineered cryopreserved cell preparations). The first group (fresh engineered B lineage cell preparations) was immediately administered to mice. The second group (frozen B lineage cell preparations) was frozen at -170°C and then thawed before being administered to mice.

[0165] Whole-body imaging was performed on mice at 1, 2, and 3 weeks after administration of fresh and frozen engineered cell preparations to measure bioluminescence levels (FIG. 6). Cell viability was assessed and measured among these donors, including those described in Example 2 above (FIG. 5).

[0166] Additional naive B cell populations from different donors were isolated, activated, manipulated, expanded, and differentiated as described above in Example 1. B lineage cells were engineered to express sphingomyelin phosphodiesterase 1 (SMPD1) from the endogenous CCR5 locus. After Ficoll cleanup and isolation, the cell populations were transferred to basal cell culture medium and divided into six groups. The first four groups (fresh engineered cell preparations, "Fresh BCM-SMPD1") were immediately administered to mice. The second set of three groups (frozen B lineage preparations, "Cryo BCM-SMPD1") were cultured at 15 x 10 cells / ml in medium containing 50% CS10 and 50% PlasmaLyte supplemented with HSA. 6 The cells were transferred at a concentration of 100 cells / mL, frozen in a controlled-rate freezer, and then stored in liquid nitrogen at -170°C. These cells were then thawed and administered to mice. Plasma samples were collected, and the levels of secreted IgG and IgM were quantified (Figure 10).

[0167] Further in vivo experiments were performed using B-lineage cell populations isolated from different donors, activated, manipulated, expanded, and differentiated as described above in Example 1. B-lineage cells were engineered to express either human Factor IX (huFIX) from the endogenous CCR5 locus, stored at −170°C using the methods described above in Example 1, and then administered to mice after thawing. huFIX and human immunoglobulin G (huIgG) were then measured from mouse plasma collected at various time points after administration (FIGS. 11, 12A, and 12B).

[0168] Additional in vivo experiments in a mouse model were performed using B-lineage cells that had been isolated, activated, engineered to express huFIX, expanded, differentiated, and cryopreserved as described above in Example 1. The huFIX-engineered B-lineage cells were then administered to a mouse model in which human interleukin-6 was knocked out (BNDG-hIL6 KO), and the engineered B-lineage cell population was re-administered on day 21. huFIX, huIgG, and human immunoglobulin M were measured after the first administration and after re-administration (Figures 13A and 13B).

[0169] The present disclosure demonstrates, inter alia, that the engineered cryopreserved cell preparations described herein (e.g., engineered cryopreserved B-lineage cell populations) can exhibit comparable or not significantly reduced engraftment and / or transgene expression after administration to a subject compared to a reference condition (e.g., a fresh engineered cell preparation). In some embodiments, the engineered cryopreserved cell preparations described herein (e.g., engineered cryopreserved B-lineage cell populations) can exhibit improved engraftment and / or transgene expression compared to a reference condition (e.g., a previously frozen engineered cell preparation lacking one or more cryoprotectants). In some embodiments, the engineered cryopreserved cell preparations described herein (e.g., engineered cryopreserved B-lineage cell populations) can provide transgene expression and stable cell populations for up to at least 22 weeks after administration compared to a reference condition (e.g., a previously frozen engineered cell preparation lacking one or more cryoprotectants). In some embodiments, the engineered cryopreserved cell preparations described herein (e.g., engineered cryopreserved B-lineage cell populations) can provide transgene expression and stable cell populations upon one or more administrations of the engineered cryopreserved cell preparation (e.g., a previously frozen engineered cell preparation lacking one or more cryoprotectants). References Balci and Can 2013 Cheng et al.2022, “Ex Vivo Engineered Human Plasma Cells Exhibit Robust Protein Secretion and Long-Term Engraftment In Vivo,” Nature Communications. Fluckinger et al. 1998 Hammerland et al. 2017 Jeske et al.2021, “Vector Strategies to Actualize B Cell-Based Gene Therapies,” Journal of Immunology. Jourdan et al.2009,“An In Vitro Model of Differentiation of Memory B Cells into Plasmablasts and Plasma Cells Including Detailed Phenotypic and Molecular Characterization,” Immunobiology. Khodadadi et al.2019,“The Maintenance of Memory Plasma Cells,” Frontiers in Immunology. Mazur et al.2004 Meneghel et al.2020,“Cryopreservation as a Key Element in the Successful Delivery of Cell-Based Therapies-A Review,” Frontiers in Medicine. Murray et al.2022,“Chemical Approaches to Cryopreservation,” Nature Reviews. Nguyen et al 2019,“Factors Affecting Early Antibody Secreting Cell Maturation Into Long-Lived Plasma Cells,” Frontiers in Immunology. Nutt et al.2015,“The generation of antibody-secreting plasma cells,” Nature Reviews Immunology. Rawlings et al.1995 Rawlings et al.1997 Radbruch et al., 2006, “Competence and competition: the challenge of becoming a long-lived plasma cell,” Nature Reviews Immunology. Sanza et al.2019, “Challenges and Opportunities for Consistent Classification of Human B Cell and Plasma Cell Populations,” Frontiers in Immunology. Slifka et al.2018, “Humoral Immunity Due to Long-Lived Plasma Cells,” Immunity. Ticha et al.2021, “Effects of Long-Term Cryopreservation of PBMC on Recovery of B Cell Subpopulations,” Journal of Immunological Methods Whaley et al. 2021 WO2018 / 170150, “Engraftable Cell-Based Immunotherapy for Long-Term Delivery of Therapeutic Proteins.”

[0170] equivalent It will be appreciated by those skilled in the art that various changes, modifications, and improvements to the present disclosure will readily occur to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only and are not intended to be limiting unless otherwise specified by the following claims.

[0171] Those of ordinary skill in the art will understand the typical standard deviation or error attributed to values ​​obtained from the assays or other processes described herein. Publications, websites, and other reference materials referred to herein to describe the background of the invention and to provide additional details regarding its practice are hereby incorporated by reference in their entireties.

Claims

1. 1. A method for preparing a population of B-lineage cells for cryopreservation, comprising: the B-lineage cell population, about 4.5% to about 5% DMSO; about 2.5% to about 10% HSA; contacting the mixture with a cryopreservation medium containing This method includes the step of producing a cell preparation for cryopreservation.

2. the cryopreservation cell preparation is placed in a controlled cooling chamber, (i) said freezing; cooling the chamber until the storage cell preparation reaches 4°C; (ii) cooling the chamber at a rate of 1°C / min until the cryopreservation cell preparation reaches -4°C; (iii) cooling the chamber at a rate of 25°C / min until the chamber reaches -40°C; (iv) heating the chamber at a rate of 10°C / min until the chamber reaches -12°C; (v) cooling the chamber at a rate of 1°C / min until the chamber reaches -40°C; (vi) cooling the chamber at a rate of 10°C / min until the chamber reaches -90°C. The method of claim 1 , wherein the controlled cooling comprises one or more of the steps:

3. 3. The method of claim 1 or 2, wherein the B-lineage cell population is or comprises a population of genetically modified plasma cells.

4. 3. The method of claim 1 or 2, wherein the B-lineage cell population is or comprises a population of plasmablasts.

5. 5. The method of claim 4, wherein the B-lineage cell population is or comprises a population of plasma progenitor cells.

6. The method of any one of claims 1 to 5, wherein the cryopreservation medium comprises 5% DMSO.

7. The method of any one of claims 1 to 6, wherein the cryopreservation medium comprises 2.5% HSA.

8. 8. The method of any one of claims 1 to 7, wherein the cryopreservation cell preparation maintains at least 50% viability after being frozen for at least 24 hours.

9. 9. The method of claim 8, wherein the cryopreserved cell preparation maintains at least 50% viability after being frozen for one week.

10. 9. The method of claim 8, wherein the cryopreserved cell preparation maintains at least 50% viability after being frozen for one month.

11. 9. The method of claim 8, wherein the cryopreserved cell preparation maintains at least 50% viability after being frozen for 6 months.

12. (i) a frozen population of genetically modified B-lineage cells; (ii) about 4.5% to about 5% DMSO; about 2.5% to about 10% HSA; a cryopreservation medium comprising: A composition comprising:

13. 13. The composition of claim 12, wherein the cryopreservation medium comprises 5% DMSO.

14. 14. The composition of claim 12 or 13, wherein the cryopreservation medium comprises 2.5% HSA.

15. The composition according to any one of claims 12 to 14, wherein the transgene is Factor IX.

16. The composition according to any one of claims 12 to 15, wherein the endogenous locus is CCR5 or JCHAIN.

17. 17. The composition of any one of claims 12 to 16, wherein the genetically modified B-lineage cells have been frozen for at least 24 hours and maintain at least 50% viability after thawing.

18. 18. The composition of any one of claims 12 to 17, wherein the genetically modified B-lineage cells have been frozen for at least 24 hours and, after thawing, at least 25% of the cells secrete IgG.

19. 19. The composition of any one of claims 12 to 18, wherein the genetically modified B-lineage cells have been frozen for at least 24 hours and, after thawing, at least 25% of the cells express IgM.

20. The composition of any one of claims 12 to 19, wherein the population has been frozen for at least one week.

21. The composition of any one of claims 12 to 20, wherein the population has been frozen for at least one month.

22. The composition of any one of claims 12 to 21, wherein the population has been frozen for at least 6 months.

23. 1. A method of treating a patient with a disease, comprising administering a population of cryopreserved genetically modified B-lineage cells.

24. 24. The method of claim 23, wherein the population of genetically modified B-lineage cells is cryopreserved in a medium comprising about 4.5% to about 5% DMSO and about 2.5% to about 10% HSA and is thawed prior to administration to the patient.

25. 25. The method of claim 24, wherein the medium comprises 5% DMSO.

26. The method according to any one of claims 23 to 25, wherein the medium contains 2.5% HSA.

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