GLP-1 expressing modified b cells for the treatment of metabolic disease

Abstract

The present disclosure relates to compositions and methods of making modified B cells capable of expressing at least one GLP-1 analog or GLP-1 analog fusion to treat human metabolic disease and disorder. In some embodiments, the B cell secretes a GLP-1 analog or GLP-1 analog fusion and secretes follistatin. The GLP-1 fusion may comprise a GLP-1-GLP- 1R neutral antibody fusion. The compositions and methods provided in the present disclosure provide superior and long-lasting control of glucose levels and / or subject body weight via the advantageous biodistribution and lifetime properties of B cells.

Description

Attorney Docket No: IMCO-013 / 001WO GLP-1 EXPRESSING MODIFIED B CELLS FOR THE TREATMENT OF METABOLIC DISEASE CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application 63 / 617,992, filed January 5, 2024, the contents of which are herein incorporated by reference in its entirety. SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (IMCO_013_01WO_SeqList_ST26.xml; Size: 41,493 bytes; and Date of Creation: December 11, 2024) are herein incorporated by reference in its entirety. FIELD OF THE INVENTION

[0003] The present disclosure relates to compositions and methods of making modified B cells capable of expressing at least a glucagon like peptide-1 (GLP-1) analog or GLP-1 analog fusion to treat human metabolic disease and disorder. In certain embodiments, the B cell is capable of secreting a GLP-1 analog and secreting follistatin. The compositions and methods provided in the present disclosure provide superior and long-lasting control of glucose levels and / or subject body weight via the advantageous biodistribution and lifetime properties of B cells. BACKGROUND OF THE INVENTION

[0004] Diabetes mellitus currently afflicts over 200 million people worldwide. Type 1 diabetes accounts for about 10% of this number, and results from autoimmune destruction of insulin- secreting -cells in the pancreatic islets of Langerhans. Survival depends on multiple daily insulin injections. Type 2 diabetes accounts for the remaining 90% of individuals affected, and the rate of prevalence is increasing. Type 2 diabetes is often, but not always, associated with obesity, and although previously termed late-onset or adult diabetes, is now increasingly manifest in younger individuals. It is caused by a combination of insulin resistance and inadequate insulin secretion.

[0005] Diabetes and obesity are increasing health problems globally and are associated with various other diseases, particularly cardiovascular diseases (CVD), obstructive sleep apnea, stroke, peripheral artery disease, microvascular complications and osteoarthritis. There are currently 246 million people worldwide with diabetes, and by 2025 it is estimated that 380 million will have diabetes. Many have additional cardiovascular risk factors including high / aberrant LDL and triglycerides and low HDL. Cardiovascular disease accounts for about 311376670Attorney Docket: IMCO-013 / 001WO 50% of the mortality in people with diabetes, and the morbidity and mortality rates relating to obesity and diabetes underscore the medical need for efficacious treatment options.

[0006] GLP-1 has different biological activities compared to glucagon. Its actions include stimulation of insulin synthesis and secretion, inhibition of glucagon secretion, and inhibition of food intake. GLP-1 has been shown to reduce hyperglycemia (elevated glucose levels) in diabetic patients. Exendin-4, a peptide from lizard venom that shares about 50% amino acid identity with GLP-1, activates the GLP-1 receptor and likewise has been shown to reduce hyperglycemia in diabetic patients.

[0007] Current insulin therapies can supplement or replace endogenously-produced insulin, but suffer from the need for frequent injections and demonstrate hindered pharmacodynamics and pharmacokinetics. Accordingly, there remains a need in the art for the long-term treatment and maintenance of metabolic diseases and disorders that mimic physiologic insulin pharmacokinetics and pharmacodynamics for improved control of blood glucose levels and / or subject body weight. SUMMARY OF THE INVENTION

[0008] The present invention relates to autologous and / or allogeneic B cells that have been modified through the introduction of one or more nucleic acids to produce at least one GLP-1 or GLP-1 fusion protein and also relates to methods of administering the modified B cells (e.g., to treat a disease, disorder, or condition, e.g., a metabolic disease such as diabetes and / or obesity). The disclosure further provides methods of manufacture and method of treatment using the modified B cell compositions. The treatment of disease in a human subject comprises administering to the human subject a composition of modified B cells (i.e., a population of cells). The administered modified B cell composition expresses and releases a GLP-1 or GLP- 1 fusion with pharmacokinetics and pharmacodynamics similar to physiologic insulin. Therapeutic GLP-1 is currently delivered as a bolus injection. Especially for GLP-1 analogs with short half-life, this results in large peak / trough ratios with side effects. Continuous delivery of even levels of GLP-1 prevents this pattern and also avoids the need for daily / weekly injections. In addition, the present invention provides long-lasting method of treatment through long-lasting in vivo B cell lifetimes. 311376670Attorney Docket: IMCO-013 / 001WO DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention relates to autologous and / or allogeneic B cells that have been modifed through the introduction of one or more nucleic acids to produce at least one GLP-1 or GLP-1 fusion protein and also relates to methods of administering the modified B cells (e.g., to treat a disease, disorder, or condition, e.g., a metabolic disease such as diabetes and / or obesity). In some embodiments, the terms “recombinant B cell,” “engineered B cell,” “genetically engineered B cell,” “modified B cell,” and “genetically modified B cell” are used interchangeably herein to refer to such modified B cells that comprise one or more nucleic acids, or “polynucleotide” (e.g., a polynucleotide transgene) to produce at least a GLP-1 protein or GLP-1 fusion (e.g., a transgene that enables the expression of a GLP-1 polypeptide or GLP- 1 polypeptide fusion).

[0010] In some embodiments, GLP-1 is fused to any one or more of the following: a human GLP-1 receptor (GLP-1R) neutral antibody, a human serum albumin (HSA) antibody, a human serum albumin (HSA), a human transferrin, or a human glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) antagonist antibody. This is termed a “GLP-1 fusion.” In certain embodiments, the modified B cell comprises at least one polynucleotide that is capable of expressing a GLP-1 or GLP-1 fusion and a follistatin.

[0011] In some embodiments, a single polynucleotide construct is capable of expressing one or more of the GLP-1 or GLP-1 fusion, GIPR antagonist antibody, and follistatin. In some embodiments, the therapeutic proteins are expressed from at least two individual polynucleotide constructs (e.g., two plasmids).

[0012] In typical embodiments, the modified B cells can be administered as a single dosage or multiple dosages.

[0013] Accordingly, the methods for administering modified B cell compositions described herein are useful for long-term in vivo delivery and expression of GLP-1 or GLP-1 fusions. The present disclosure relates generally to methods for achieving sufficient enrichment and the number of cells producing GLP-1 and sufficient levels of GLP-1 in vivo while ensuring product safety. Modified B cell Embodiments

[0014] In some embodiments, the present disclosure provides for a modified B cell comprising a polynucleotide capable of expressing one or more of a glucagon-like peptide-1 (GLP-1) analogs or GLP-1 analog fusions. 311376670Attorney Docket: IMCO-013 / 001WO

[0015] In some embodiments, the present disclosure provides for a modified B cell comprising a polynucleotide capable of expressing one or more of a glucagon-like peptide-1 (GLP-1) analog fusions, wherein the GLP-1 analog is fused to a peptide or protein via a peptide linker.

[0016] In some embodiments, the peptide linker is cleavable. In some embodiments, the peptide linker is non-cleavable.

[0017] In some embodiments, the modified B cell further comprises a polynucleotide capable of expressing human follistatin. In some embodiments, the modified B cell further comprises a polynucleotide capable of expressing one or more human follistatin variants. Exemplary follistatin sequences are put forth in SEQ ID NOs: 23-26.

[0018] In some embodiments, the modified B cell further comprises a polynucleotide capable of expressing one or more on a human GLP-1 receptor (GLP-1R) neutral antibodies or fragments thereof (e.g., scFv).

[0019] In some embodiments, the modified B cell further comprises a polynucleotide capable of expressing one or more human serum albumin (HSA) proteins, nanobody, or fragment thereof.

[0020] In some embodiments, the modified B cell further comprises a polynucleotide capable of expressing one or more of a human glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) antagonist antibody or fragment thereof.

[0021] In some embodiments, the modified B cell comprises a polynucleotide capable of expressing a GLP-1 fusion. In certain embodiments, the GLP-1 analog is fused to a GLP-1R neutral antibody or fragment thereof. In some embodiments, the GLP-1 analog is fused to a GLP-1R neutral antibody or fragment thereof by a linker, typically a peptide linker.

[0022] In some embodiments, the GLP-1-GLP1R fusion protein is secreted. In some embodiments, the GLP-1-GLP1R fusion protein is anchored to the B cell membrane. In some embodiments, GLP-1 is fused to transferrin and / or HSA.

[0023] In some embodiments, the GLP-1 analog N-terminus or C-terminus is fused to the N- terminus or C-terminus of the light chain of the GLP-1R neutral antibody by a linker. In certain embodiments, the C-terminus of the GLP-1 analog is fused to the N-terminus of the light chain of the GLP-1R neutral antibody by a peptide linker.

[0024] In some embodiments, the modified B cell comprises a polynucleotide capable of expressing a (GLP-1)-(HSA antibody or nanobody)-(GLP-1R) fusion (i.e., a trimolecular protein fusion). In some embodiments, at least one GLP-1 peptide analog N-terminus or C- terminus is fused to the HSA antibody N-terminus or C-terminus by a linker, wherein the HSA 311376670Attorney Docket: IMCO-013 / 001WO antibody N-terminus or C-terminus is fused to the light chain of the GLP-1R neutral antibody N-terminus of the light chain of the GLP-1R neutral antibody by a linker.

[0025] In typical embodiments, the polynucleotide comprises one or more of a promoter, an enhancer, a ribosomal slipping sequence, or an internal ribosome entry site (IRES). In some embodiments, the polynucleotide is monocistronic or bicistronic. In some embodiments, the polynucleotide is at least bicistronic.

[0026] In certain embodiments, the polynucleotide further comprises a human follistatin gene. In some embodiments, the follistatin is a human splice variant. In some embodiments, the follistatin is one or more of the following: FST-344 (SEQ ID NO: 26), FST-288 (SEQ ID NO:23), FST-317 (SEQ ID NO: 24), FST-315 (SEQ ID NO: 25), FST-315-Fc or FST-315- HBS-Fc (devoid of heparan binding function) splice site variant.

[0027] In typical embodiments, the modified B cell has been transfected, transduced, or transposed with one or more polynucleotides capable of expressing at least the GLP-1 or GLP- 1 fusion. In some embodiments, the modified B cell has been transfected, transduced, or transposed with polynucleotides capable of expressing at least the GLP-1 or GLP-1 fusion. In some embodiments, the modified B cell has been transfected, transduced, or transposed with polynucleotides capable of expressing at least the GLP-1 or GLP-1 fusion and a follistatin protein. Therapeutic Agent

[0028] As used herein “gene of interest,” “gene,” or “nucleic acid of interest” refers to a transgene to be expressed in the target transfected cell. In particular embodiments, the gene is a GLP-1 analog or GLP-1 analog fusion gene. In some embodiments, the modified B cell further comprises another therapeutic gene such as follistatin. While the term “gene” may be used, this is not to imply that this is a gene as found in genomic DNA and is used interchangeably with the term “nucleic acid.” Generally, the nucleic acid of interest provides suitable nucleic acid for encoding one or more therapeutic agents (e.g., GLP-1 and follistatin) and may comprise cDNA or DNA and may or may not include introns, but generally does not include introns. As noted elsewhere, the nucleic acid of interest is operably linked to expression control sequences to effectively express the protein of interest in the target cell. In certain embodiments, the vectors described herein may comprise one or more genes of interest, and may include 2, 3, 4, or 5 or more genes of interest, such as for example, the heavy and light chains of an immunoglobulin that may be organized using an internal promoter as described herein. 311376670Attorney Docket: IMCO-013 / 001WO

[0029] In some embodiments, any one of the embodiments disclosed herein may utilize a gene of interest that is a GLP-1 protein. Thus, in some embodiments, the therapeutic agent delivered to the modified B cell, as described herein, may be a GLP-1 or GLP-1 fusion protein. GLP-1

[0030] GLP-1 is a product of posttranslational processing of the glucagon precursor proglucagon in intestinal L cells and the brain. There are two forms of full length N-terminal GLP-1, GLP-1 (1-37) and GLP-1 (1-36) amide. Both forms are active and are produced when the GLP-1 polypeptide is cleaved to remove the first six amino acids resulting in the active peptides GLP-1 (7-37), having 31 amino acids, and GLP-1 (7-36) amide, having 30 amino acids. The majority of circulating biologically active GLP-1 is found in the amidated form, GLP-1 (7-36) amide, with lesser amounts of the bioactive non-amidated GLP-1 (7-37) also detectable. The active GLP-1 undergoes rapid degradation by N-terminal cleavage of the first two amino acids (His1-Ala2) by circulating di-peptidyl peptidase IV (DPPIV) resulting in the short half-life of GLP-1.

[0031] On Apr. 29, 2005, the U.S. Food and Drug Administration approved the first incretin mimetic, exenatide injection, as an adjunct therapy for Type 2 diabetes patients who have not achieved adequate control of blood sugar with two common oral diabetes medications, metformin and / or sulfonylurea. exenatide is also indicated as a monotherapy for patients with Type 2 diabetes. According to the manufacturer, exenatide exhibits many of the same effects as the human incretin hormone GLP-1 in regulating blood sugar. Exenatide is a synthetic version of exendin-4, a naturally occurring hormone, which is a 39-amino acid peptide amide. Exenatide's amino acid sequence partially overlaps that of the human GLP-1, but has a longer half-life than native GLP-1. Exenatide has been shown to bind and activate the known human GLP-1 receptor in vitro.

[0032] Analogs of GLP-1, such as extendin-4 or the mutated GLP-1 containing glycine (Gly) as the second amino acid residue, show potent insulinotropic effects and have a longer half life than GLP-1. However, because these molecules are foreign, they cannot be degraded rapidly in the body as native GLP-1. GLP-1 means an active human glucagon-like peptide-1 GLP-1(7- 37) (SEQ ID NO: 27) or GLP-1(7-36) (SEQ ID NO: 28) or variants thereof. Preferably the GLP-1 is GLP-1(7-37) (SEQ ID NO: 27) or a variant thereof. In another embodiment, the GLP- 1 is GLP-1 (7-36) (SEQ ID NO: 28) amide or variant thereof. A “GLP-1 variant” as used herein means an amino acid sequence in which one or more amino acids have been altered from the native sequence by one or more substitution, deletion or insertion of an amino acid residue without changing the activity of the GLP-1. A GLP-1 variant may have one or more amino 311376670Attorney Docket: IMCO-013 / 001WO acid residue deletions and thus will be shorter than GLP-1 (7-36), i.e., a fragment of GLP-1, but will the same activity as the native GLP-1 sequence, e.g., in stimulating insulin secretion by pancreatic beta cells, by virtue of having the amino acid residues which are critical for GLP- 1 activity or residues which may be substituted for one or more of these critical residues.

[0033] In some embodiments, a GLP-I (7-36) or (7- 36) (SEQ ID NO: 28) moiety may be modified by mutating A8 to S, G or V and / or mutating K34 to Q, A or N.

[0034] In some embodiments, the GLP-1 analog comprises the sequence of any one of GLP- 1(7-37) (SEQ ID NO: 27), GLP-1(7-36) (SEQ ID NO: 28), exendin-4 (SEQ ID NO: 29), lixisenatide (SEQ ID NO: 30), dulaglutide (SEQ ID NO: 31), semaglutide (SEQ ID NO: 33), liraglutide (SEQ ID NO: 34), or albiglutide (SEQ ID NO: 32). In some embodiments, as described above, the GLP-1 analog is a fusion protein. In some embodiments, the GLP-1 analog is fused to a HSA nanobody or fragment. In some embodiments, the GLP-1 analog is fused to transferrin or a transferrin fragment. In some embodiments, the GLP-1 analog is fused to a Fc domain or Fc fragment domain. Exemplary GLP-1 analogs are provided in Table 1.

[0035] In some embodiments, the GLP-1 analog comprises a sequence that is 95%, 96%, 97%, 98%, 99%, or 100% similar to a GLP-1 peptide analog provided in Table 1. Table 1: Exemplary GLP-1 analogs311376670Attorney Docket: IMCO-013 / 001WOGLP-1 Fusions

[0036] In various aspects, the present disclosure relates to B cells engineered to express a GLP- 1 or GLP-1 fusion. The GLP-1 fusion can be, without limitation, a GLP-1 fused to a GLP-1 receptor (GLP-1R) antibody or antibody fragment thereof. In some embodiments, the GLP-1R antibodies are neutral (i.e., neither demonstrating antagonistic or agonistic properties). GLP- 1R antibodies have been described in the art (See, Liu et al., MAbs.2021; 13(1): 1893425, the contents of which are incorporated by reference in their entireties).

[0037] In some embodiments, the GLP-1 analog is fused to a human serum albumin protein or fragment thereof (i.e., a nanobody). In some embodiments, the GLP-1-HSA fusion is further 311376670Attorney Docket: IMCO-013 / 001WO fused to a GLP-1R antibody (See, Pan et al., Artif Cells Nanomed Biotechnol. 2020 Dec; 48 (1): 854-866, the contents of which are incorporated by reference in their entireties).

[0038] Subsets of antibodies that specifically bind to GLP-1R are known in the art (see, US 10,253,103, the contents of which are incorporated by reference in their entireties). In some embodiments, the GLP-1R antibody comprises a light chain variable region selected SEQ ID NO: 1 and a heavy chain selected from SEQ ID NO: 2.

[0039] Subsets of antibodies that specifically bind to GLP-1R are known in the art (see, US 11,492,727, the contents of which are incorporated by reference in their entireties). In some embodiments, the GLP-1R antibody comprises a heavy chain selected from SEQ ID NOs: 3- 14, and a light chain selected from SEQ ID NOs: 15-21.

[0040] In some embodiments, the GLP-1R antibody comprises a sequence that is 95% similar to any one of SEQ ID NOs: 1-21.

[0041] In some embodiments, the GLP-1 analog is fused to a transferrin (Tf) protein or a modified Tf protein. A subset of GLP-1-transferrin fusion proteins are known in the art (see, WO2006096515, the contents of which are incorporated by reference in their entireties).

[0042] Modified Tf fusions may be made with any Tf protein, fragment, domain, or engineered domain. For instance, fusion proteins may be produced using the full-length Tf sequence, with or without the native Tf signal sequence. Tf fusion proteins may also be made using a single Tf domain, such as an individual N or C domain or a modified form of Tf comprising 2N or 2C domains (see U.S. Provisional Application 60 / 406,977, filed August 30, 2002, which is herein incorporated by reference in its entirety). In some embodiments, fusions of a therapeutic protein to a single C domain may be produced, wherein the C domain is altered to reduce, inhibit or prevent glycosylation. In other embodiments, the use of a single N domain is advantageous as the Tf glycosylation sites reside in the C domain and the N domain, on its own. A preferred embodiment is the Tf fusion protein having a single N domain which is expressed at a high level. Follistatin

[0043] In various aspects, the present disclosure relates to B cells engineered to express a GLP- 1 (or GLP-1 fusion) and a follistatin (e.g., one or more follistatin polypeptides). As used herein, the term “follistatin” refers to a family of follistatin (FST) proteins and follistatin-related proteins, derived from any species. Follistatin is an autocrine glycoprotein that is expressed in nearly all tissues of higher animals. It was initially isolated from follicular fluid and was identified as a protein fraction that inhibited follicle-stimulating hormone (FSH) secretion from the anterior pituitary, and therefore was designated as FSH-suppressing protein (FSP). 311376670Attorney Docket: IMCO-013 / 001WO Subsequently, its primary function has been determined to be the binding and neutralization ofmembers of the TGF- superfamily including, for example, activin, a paracrine hormone thatenhances secretion of FSH in the anterior pituitary.

[0044] Follistatin secretion is regulated via the glucagon-to-insulin ratio. In brief, glucagon enhances, and insulin reduces, the release of follistation. Plasma follistatin levels are moderately elevated in Type 2 diabetes and are associated with an increased risk of Type 2 diabetes.

[0045] In some embodiments, the terms “follistatin polypeptide,” “follistatin protein,” or “a follistatin” are used to refer to polypeptides comprising any naturally occurring polypeptide of the follistatin family as well as any variants thereof (including mutants, fragments, fusions, splice variants, and peptidomimetic forms) that retain a useful activity, including, for example, ligand binding (e.g., myostatin, GDF-11, activin A, activin B) or heparin binding. For example, in some embodiments, follistatin polypeptides may include polypeptides comprising an amino acid sequence derived from the sequence of any known follistatin having a sequence at least about 80% identical to the sequence of a follistatin polypeptide, and preferably at least 85%, 90%, 95%, 97%, 99% or greater identity.

[0046] A “follistatin variant” is a follistatin protein that comprises one or more mutations (substitutions, deletions, or insertions) as compared to the follistatin sequences put forth in SEQ ID NOs: 23-26.

[0047] A “follistatin splice variant” is follistatin that comprises the wild-type follistatin sequence but the sequence has been rearranged via alternative splicing.

[0048] Follistatin is a single-chain polypeptide with a range of molecular weights from 31 to 49 kDa based on alternative mRNA splicing and variable glycosylation of the protein. The human gene encoding follistatin (FST) has six exons spanning 5329 bp on chromosome 5q11.2 and gives rise to two main transcripts: transcript variant FST344 (1122 bp) and transcript FST317 (1386 bp). Exon 1 in FST encodes the follistatin signal peptide, exon 2 encodes the follistatin N-terminal domain, and each of exons 3-5 encode a follistatin module. Due to alternative splicing, either one of exon 6A (which codes for an acidic region in FST344) or exon 6B (which contains two bases of the stop codon of FST317) are utilized (Shimasaki, S. et al., 1988).

[0049] These alternatively spliced mRNAs (FST344 and FST317) result in the production of two follistatin proteins of 315 amino acids (i.e., FST315) and 288 amino acids (i.e., FST288), respectively, after removal of the 29 amino acid signal peptide, and follistatin 315 can be further proteolytically degraded to follistatin 303 (FST303). Analysis of the amino acid 311376670Attorney Docket: IMCO-013 / 001WO sequence has revealed that the native human follistatin polypeptide comprises five domains (from the N-terminal side): a signal sequence peptide, an N-terminal domain (FSN), follistatin domain I (FSDI), follistatin domain II (FSDII), and follistatin domain III (FSDIII). See PNAS, U.S.A., 1988, Vol.85, No 12, pp 4218-4222.

[0050] The human follistatin-288 (FST288) precursor (i.e., FST317) has the following amino acid sequence, with the signal peptide indicated in bold, the N-terminal domain (FSN) indicated by single underlining, and the follistatin domains I-III (FSI, FSII, FSIII) indicated by double underlining. MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTEL SKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDCKEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPA SSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQC TGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEA ACSSGVLLEVKHSGSCN (SEQ ID NO: 24)

[0051] The processed (mature) human follistatin variant (FST288) has the following amino acid sequence with the N-terminal domain indicated by single underlining, and the follistatin domains I-III indicated by double underlining. Moreover, it will be appreciated that any of the initial amino acids G or N, prior to the first cysteine may be removed by processing or intentionally eliminated without any consequence, and polypeptides comprising such slightly smaller polypeptides are further included. GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWM IFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKG PVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTC VVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGR SIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKS DEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCN (SEQ ID NO: 23)

[0052] The human follistatin-315 (FST315) precursor (i.e., FST344) has the following amino acid sequence, with the signal peptide indicated in bold, the N-terminal domain (FSN) indicated by single underlining, and the follistatin domains I-III (FSI, FSII, FSIII) indicated by double underlining (NCBI Accession Number AAH04107.1; 344 amino acids). MVRARHQPGGLCLLLLLLCQFMEDRSAQAGNCWLRQAKNGRCQVLYKTEL 311376670Attorney Docket: IMCO-013 / 001WO SKEECCSTGRLSTSWTEEDVNDNTLFKWMIFNGGAPNCIPCKETCENVDC GPGKKCRMNKKNKPRCVCAPDCSNITWKGPVCGLDGKTYRNECALLKARC KEQPELEVQYQGRCKKTCRDVFCPGSSTCVVDQTNNAYCVTCNRICPEPA SSEQYLCGNDGVTYSSACHLRKATCLLGRSIGLAYEGKCIKAKSCEDIQC TGGKKCLWDFKVGRGRCSLCDELCPDSKSDEPVCASDNATYASECAMKEA ACSSGVLLEVKHSGSCNSISEDTEEEEEDEDQDYSFPISSILEW (SEQ ID NO:

[0053] The processed (mature) human FST315 has the following amino acid sequence with the N-terminal domain indicated by single underlining, and the follistatin domains I-III indicated by double underlining. Moreover, it will be appreciated that any of the initial amino acids G or N, prior to the first cysteine may be removed by processing or intentionally eliminated without any consequence, and polypeptides comprising such slightly smaller polypeptides are further included. GNCWLRQAKNGRCQVLYKTELSKEECCSTGRLSTSWTEEDVNDNTLFKWM IFNGGAPNCIPCKETCENVDCGPGKKCRMNKKNKPRCVCAPDCSNITWKG PVCGLDGKTYRNECALLKARCKEQPELEVQYQGRCKKTCRDVFCPGSSTC VVDQTNNAYCVTCNRICPEPASSEQYLCGNDGVTYSSACHLRKATCLLGR SIGLAYEGKCIKAKSCEDIQCTGGKKCLWDFKVGRGRCSLCDELCPDSKS DEPVCASDNATYASECAMKEAACSSGVLLEVKHSGSCNSISEDTEEEEED EDQDYSFPISSILEW(SEQ ID NO: 25)

[0054] Follistatin polypeptides of the disclosure may include any naturally occurring domain of a follistatin protein as well as variants thereof (e.g., mutants, fragments, and peptidomimetic forms) that retain a useful activity. For example, it is well-known that FST315 and FST288 have high affinity for both activin (activin A and activin B) and myostatin (and the closely related GDF11) and that the follistatin domains (e.g., FSN and FSD I-III) are thought to beinvolved in the binding of such TGF- ligands. However, it believed that each of these threedomains may have a different affinity for these TGF- ligands. For example, a study hasdemonstrated that polypeptide constructs comprising only the N-terminal domain (FSN) and two FSDI domains in tandem retained high affinity for myostatin, demonstrated little or no affinity for activin and promoted systemic muscle growth when introduced into a mouse by gene expression (Nakatani et al., The FASEB Journal, Vol.22477-487 (2008)). 311376670Attorney Docket: IMCO-013 / 001WO

[0055] In some embodiments, the follistatin is one or more of the following: FST-344, FST-288, FST-317, FST-315, FST-315-Fc or FST-315- HBS-Fc (devoid of heparan bindingfunction) splice site variant.

[0056] Accordingly, the present disclosure encompasses, in part, variant follistatin proteinsthat demonstrate selective binding and / or inhibition of a given TGF- ligand relative to thenaturally occurring FST protein (e.g., maintaining high-affinity for myostatin while having a significantly reduced affinity for activin).

[0057] Thus, this disclosure provides polynucleotides (isolated or purified or pure polynucleotides) encoding therapeutic agents (e.g., follistatin) of this disclosure for genetically modifying B cells, vectors (including cloning vectors and expression vectors) comprising such polynucleotides, and cells (e.g., host cells) transformed or transfected with a polynucleotide or vector according to this disclosure. In certain embodiments, any one of the embodiments disclosed in the present disclosure may utilize a follistatin (e.g., for expression in a B cell) that is selected from the above-described follistatin polypeptides. In certain embodiments, any one of the embodiments disclosed in the present disclosure may utilize a follistatin (e.g., for expression in a B cell) that is a human follistatin FST344 splice site variant. In certain embodiments, a polynucleotide (DNA or RNA) encoding a protein of interest (e.g., a follistatin) of this disclosure is contemplated. Expression cassettes encoding proteins of interest are also contemplated herein.

[0058] The present disclosure also relates to vectors that include a polynucleotide of this disclosure and, in particular, to recombinant expression constructs. In one embodiment, this disclosure contemplates a vector comprising a polynucleotide encoding a protein of this disclosure (e.g., GLP-1 or follistatin), along with other polynucleotide sequences that cause or facilitate transcription, translation, and processing of such a protein-encoding sequences. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described, for example, in Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989). Exemplary cloning / expression vectors include cloning vectors, shuttle vectors, and expression constructs, that may be based on plasmids, phagemids, phasmids, cosmids, viruses, artificial chromosomes, or any nucleic acid vehicle known in the art suitable for amplification, transfer, and / or expression of a polynucleotide contained therein.

[0059] As used herein, unless as otherwise described with regard to viral vectors, “vector” means a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Exemplary vectors include plasmids, minicircles, transposons (e.g., Sleeping Beauty 311376670Attorney Docket: IMCO-013 / 001WO transposon), yeast artificial chromosomes, self-replicating RNAs, and viral genomes. Certain vectors can autonomously replicate in a host cell, while other vectors can be integrated into the genome of a host cell and thereby are replicated with the host genome. In addition, certain vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”), which contain nucleic acid sequences that are operatively linked to an expression control sequence and, therefore, are capable of directing the expression of those sequences. In certain embodiments, expression constructs are derived from plasmid vectors. Illustrative constructs include modified pNASS vector (Clontech, Palo Alto, CA), which has nucleic acid sequences encoding an ampicillin resistance gene, a polyadenylation signal and a T7 promoter site; pDEF38 and pNEF38 (CMC ICOS Biologies, Inc.), which have a CHEF1 promoter; and pD18 (Lonza), which has a CMV promoter. Other suitable mammalian expression vectors are well known (see, e.g., Ausubel et al., 1995; Sambrook et al., supra; see also, e.g., catalogs from Invitrogen, San Diego, CA; Novagen, Madison, WI; Pharmacia, Piscataway, NJ).

[0060] Useful constructs may be prepared that include a dihydrofolate reductase (DHFR)- encoding sequence under suitable regulatory control, for promoting enhanced production levels of the fusion proteins, which levels result from gene amplification following application of an appropriate selection agent (e.g., methotrexate). In one embodiment, use of a bifunctional transposon encoding a therapeutic gene (e.g., follistatin) along with drug-resistant DHFR in combination with incubation in methotrexate (MTX) to enrich for successfully modified B cells, generates a more potent product.

[0061] Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence, as described above. A vector in operable linkage with a polynucleotide according to this disclosure yields a cloning or expression construct. Exemplary cloning / expression constructs contain at least one expression control element, e.g., a promoter, operably linked to a polynucleotide of this disclosure. Additional expression control elements, such as enhancers, factor-specific binding sites, terminators, and ribosome binding sites are also contemplated in the vectors and cloning / expression constructs according to this disclosure. The heterologous structural sequence of the polynucleotide according to this disclosure is assembled in appropriate phase with translation initiation and termination sequences. Thus, for example, encoding nucleic acids as provided herein may be included in any one of a variety of expression vector constructs (e.g., minicircles) as a recombinant expression construct for expressing such a protein in a host cell. 311376670Attorney Docket: IMCO-013 / 001WO

[0062] The appropriate DNA sequence(s) may be inserted into a vector, for example, by a variety of procedures. In general, a DNA sequence is inserted into an appropriate restriction endonuclease cleavage site(s) by procedures known in the art. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are contemplated. A number of standard techniques are described, for example, in Ausubel et al. (Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, MA, 1993); Sambrook et al. (Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory, Plainview, NY, 1989); Maniatis et al. (Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, NY, 1982); Glover (Ed.) (DNA Cloning Vol. I and II, IRL Press, Oxford, UK, 1985); Hames and Higgins (Eds.) (Nucleic Acid Hybridization, IRL Press, Oxford, UK, 1985); and elsewhere.

[0063] The DNA sequence in the expression vector is operatively linked to at least one appropriate expression control sequence (e.g., a constitutive promoter or a regulated promoter) to direct mRNA synthesis. Representative examples of such expression control sequences include promoters of eukaryotic cells or their viruses, as described above. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors, kanamycin vectors, or other vectors with selectable markers. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-l. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art, and preparation of certain particularly preferred recombinant expression constructs comprising at least one promoter or regulated promoter operably linked to a nucleic acid encoding a protein or polypeptide according to this disclosure is described herein.

[0064] Variants of the polynucleotides of this disclosure are also contemplated. Variant polynucleotides are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, and preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical to one of the polynucleotides of defined sequence as described herein (i.e., SEQ ID NOs: 1-34), or that hybridizes to one of those polynucleotides of defined sequence under stringent hybridization conditions as described below. The polynucleotide variants retain the capacity to encode a binding domain or fusion protein thereof having the functionality described herein.

[0065] Hybridization stringency is principally determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015M sodium chloride, 0.0015M sodium citrate at about 311376670Attorney Docket: IMCO-013 / 001WO 65-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C (see Sambrook et ai, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agent) may also be used; however, the rate of hybridization will be affected. In instances wherein hybridization of deoxyoligonucleotides is concerned, additional exemplary stringent hybridization conditions include washing in 6x SSC, 0.05% sodium pyrophosphate at 37°C (for 14-base oligonucleotides), 48°C (for 17-base oligonucleotides), 55°C (for 20-base oligonucleotides), and 60°C (for 23-base oligonucleotides).

[0066] A further aspect of this disclosure provides a host cell transformed or transfected with, or otherwise containing, any of the polynucleotides or vector / expression constructs of this disclosure (e.g., a follistatin polynucleotides or vector / expression constructs). The polynucleotides or cloning / expression constructs of this disclosure are introduced into suitable cells using any method known in the art, including transformation, transfection and transduction (e.g., any one of the methods disclosed herein). Host cells include the cells of a subject undergoing ex vivo cell therapy including, for example, ex vivo gene therapy. Eukaryotic host cells contemplated as an aspect of this disclosure when harboring a polynucleotide, vector, or protein according to this disclosure include, in addition to a subject's own cells (e.g., a human patient's own cells), VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines (including modified CHO cells capable of modifying the glycosylation pattern of expressed multivalent binding molecules, see US Patent Application Publication No. 2003 / 0115614), COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562, HEK293 cells, HepG2 cells, N cells, 3T3 cells, Spodoptera frugiperda cells (e.g., Sf9 cells), Saccharomyces cerevisiae cells, Lymphoblastoid Cell Lines (LCL) and any other eukaryotic cell known in the art to be useful in expressing, and optionally isolating, a protein or peptide according to this disclosure. Also contemplated are prokaryotic cells, including Escherichia coli, Bacillus subtilis, Salmonella typhimurium, a Streptomycete, or any prokaryotic cell known in the art to be suitable for expressing, and optionally isolating, a protein or peptide according to this disclosure. In isolating protein or peptide from prokaryotic cells, in particular, it is contemplated that techniques known in the art for extracting protein from inclusion bodies may be used. The selection of an appropriate host is within the scope of those skilled in the art from the teachings herein. Host cells that glycosylate the fusion proteins of this disclosure are also contemplated. 311376670Attorney Docket: IMCO-013 / 001WO

[0067] The term “recombinant host cell” (or simply “host cell”) refers to a cell containing a recombinant expression vector. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. Recombinant host cells can be cultured in a conventional nutrient medium modified as appropriate for activating promoters, selecting transformants, or amplifying particular genes. The culture conditions for particular host cells selected for expression, such as temperature, pH and the like, will be readily apparent to the ordinarily skilled artisan. Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman (1981) Cell 23:175, and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and, optionally, enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5'-flanking nontranscribed sequences, for example, as described herein regarding the preparation of multivalent binding protein expression constructs. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Introduction of the construct into the host cell can be affected by a variety of methods with which those skilled in the art will be familiar, including calcium phosphate transfection, DEAE-Dextran-mediated transfection, or electroporation (Davis et al. (1986) Basic Methods in Molecular Biology). Manufacture of B cell compositions

[0068] B cells, such as memory B cells, can be cultured using in vitro methods to activate and differentiate the B cells into plasma cells or plasmablasts or both. As would be recognized by the skilled person, plasma cells may be identified by cell surface protein expression patterns using standard flow cytometry methods. For example, terminally differentiated plasma cells express relatively few surface antigens, and do not express common pan-B cell markers, such as CD19 and CD20. Instead, plasma cells may be identified by expression of CD38, CD78, CD138, and IL-6R and lack of expression of CD45. CD27 may also be used to identify plasma cells as naïve B cells are CD27-, memory B cells are CD27+ and plasma cells are CD27++. Plasma cells express high levels of CD38 and CD138. 311376670Attorney Docket: IMCO-013 / 001WO

[0069] In one embodiment, the B cells are CD138- memory B cells. In one embodiment, the B cells are CD138+ plasma cells. In one embodiment, the B cells are activated and have a cell surface phenotype of CD138-, CD27+.

[0070] In one embodiment, the B cells are CD20-, CD138- memory B cells. In one embodiment, the B cells are CD20-, CD138+ plasma cells. In one embodiment, the B cells are activated and have a cell surface phenotype of CD20-, CD138-, CD27+.

[0071] In one embodiment, the B cells are CD20-, CD38-, CD138- memory B cells. In one embodiment, the B cells are CD20-, CD38+, CD138+ plasma cells. In one embodiment, the B cells are activated and have a cell surface phenotype of CD20- CD38- CD138- CD27+.

[0072] In one embodiment, the B cells are contacted with one or more B cell activating factors, e.g., any of a variety of cytokines, growth factors or cell lines known to activate and / or differentiate B cells (see e.g., Fluckiger, et al. Blood 199892: 4509-4520; Luo, et al., Blood 2009113: 1422-1431 ). Such factors may be selected from the group consisting of, but not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL- 14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, and IL-35, IFN- , IFN- , IFN- , IFN- , C typechemokines XCL1 and XCL2, C-C type chemokines (to date including CCL1-CCL28) and CXC type chemokines (to date including CXCL1-CXCL17), and members of the TNFsuperfamily (e.g., TNF- , 4-1 BB ligand, B cell activating factor (BLyS), FAS ligand, sCD40L(including multimeric versions of sCD40L; e.g., histidine-tagged soluble recombinant CD40L in combination with anti-poly-histidine mAb to group multiple sCD40L molecules together), Lymphotoxin, OX40L, RANKL, TRAIL), CpG, and other toll like receptor agonists (e.g., CpG).

[0073] B cell activating factors may be added to in vitro cell cultures at various concentrations to achieve the desired outcome (e.g., expansion or differentiation). In one embodiment, a B cell activating factor is utilized in expanding the B cells in culture. In one embodiment, a B cell activating factor is utilized in differentiating the B cells in culture. In another embodiment, the B cell activating factor is utilized in both expanding and differentiating the B cells in culture. In one embodiment, the B cell activating factor is provided at the same concentration for expanding and differentiating. In another embodiment, the B cell activating factor is provided at a first concentration for expanding and at a second concentration for differentiating. It is contemplated that a B cell activating factor may be 1) utilized in expanding the B cells and not in differentiating the B cells, 2) utilized in differentiating the B cells and not in expanding the B cells, or 3) utilized in expanding and differentiating the B cells. 311376670Attorney Docket: IMCO-013 / 001WO

[0074] For example, in some embodiments, B cells are cultured with a B cell culture medium containing one or more B cell activating factors selected from CD40L, IL-2, IL-4, and IL-10 for expansion of the B cells. In one embodiment, the B cells are cultured with 0.25-5.0 μg / ml CD40L. In one embodiment, the concentration of CD40L is 0.5 μg / ml. In one embodiment, a crosslinking agent (such as an anti-HIS antibody in combination with HIS-tagged CD40L) is used to create multimers of CD40L. In one embodiment molecules of CD40L are covalently linked or are held together using protein multimerization domains (e.g., the Fc region of an IgG or a leucine zipper domain). In one embodiment CD40L is conjugated to beads. In one embodiment CD40L is expressed from feeder cells. In one embodiment, the B cells are cultured with 1-10 ng / ml IL-2. In one embodiment, the concentration of IL-2 is 5 ng / ml. In one embodiment, the B cells are cultured with 1-10 ng / ml IL-4. In one embodiment, the concentration of IL-4 is 2 ng / ml. In one embodiment, the B cells are cultured with 10-100 ng / ml IL-10. In one embodiment, the concentration of IL-10 is 40 ng / ml.

[0075] In one embodiment, B cells are cultured with a B cell culture medium containing one or more B cell activating factors selected from CD40L, IL-2, IL-4, IL-10, IL-15 and IL-21 for expansion of the B cells. In one embodiment, the B cells are cultured with 0.25-5.0 μg / ml CD40L. In one embodiment, the concentration of CD40L is 0.5 μg / ml. In one embodiment a crosslinking agent (such as an anti-HIS antibody in combination with HIS-tagged CD40L) is used to create multimers of CD40L. In one embodiment molecules of CD40L are covalently linked or are held together using protein multimerization domains (e.g., the Fc region of an IgG or a leucine zipper domain). In one embodiment CD40L is conjugated to beads. In one embodiment CD40L is expressed from feeder cells. In one embodiment, the B cells are cultured with 1-10 ng / ml IL-2. In one embodiment, the concentration of IL-2 is 5 ng / ml. In one embodiment, the B cells are cultured with 1-10 ng / ml IL-4. In one embodiment, the concentration of IL-4 is 2 ng / ml. In one embodiment, the B cells are cultured with 10-100 ng / ml IL-10. In one embodiment, the concentration of IL-10 is 40 ng / ml. In one embodiment, the B cells are cultured with 50-150 ng / ml IL-15. In one embodiment, the concentration of IL- 15 is 100 ng / ml. In one embodiment, the B cells are cultured with 50-150 ng / ml IL-21. In one embodiment, the concentration of IL-21 is 100 ng / ml. In a particular embodiment, the B cells are cultured with a B cell culture medium containing CD40L, IL-2, IL-4, IL-10, IL-15 and IL- 21 for expansion of the B cells.

[0076] For example, in one embodiment, B cells are cultured with a B cell culture medium containing the B cell activating factors CD40L, IL-2, IL-4, IL-10, IL-15 and IL-21 for expansion of the B cells, wherein the CD40L is crosslinked with a crosslinking agent to create 311376670Attorney Docket: IMCO-013 / 001WO multimers of CD40L. Such a culture system may be maintained throughout an entire culture period (e.g., a 7 day culture period), in which the B cells are transfected, or otherwise engineered, to express a transgene of interest (e.g., an exogenous polypeptide such as, e.g., follistatin). The transgene may be integrated into the B cell (e.g., via a viral or non-viral vector). The transgene may be expressed in the B cell via use of a transposon. The transgene may be expressed in the B cell due to the targeted integration of the transgene into the B cell’s genome. The targeted integration may be via homologous recombination. The homologous recombination may occur at a double strand break induced by a nuclease. The nuclease may be, e.g., a zinc finger nuclease, a TALE-nuclease (TALEN), a meganuclease (e.g., a homing endonuclease), or via a CRISPR / CAS9-nuclease system.

[0077] In another example, in one embodiment, B cells are cultured with a B cell culturemedium containing one or more B cell activating factors selected from CD40L, IFN- , IL-2,IL-6, IL-10, IL-15, IL-21, and P-class CpG oligodeoxynucleotides (p-ODN) for differentiation of the B cells. In one embodiment, the B cells are cultured with 25-75 ng / ml CD40L. In one embodiment, the concentration of CD40L is 50 ng / ml. In one embodiment, the B cells arecultured with 250-750 U / ml IFN- . In one embodiment the concentration of the IFN- is 500U / ml. In one embodiment, the B cells are cultured with 5-50 U / ml IL-2. In one embodiment the concentration of IL-2 is 20 U / ml. In one embodiment, the B cells are cultured with 25-75 ng / ml IL-6. In one embodiment, the concentration of IL-6 is 50 ng / ml. In one embodiment, the B cells are cultured with 10-100 ng / ml IL-10. In one embodiment, the concentration of IL- 10 is 50 ng / ml. In one embodiment, the B cells are cultured with 1-20 ng / ml IL-15. In one embodiment, the concentration of IL-15 is 10 ng / ml. In one embodiment, the B cells are cultured with 10-100 ng / ml IL-21. In one embodiment, the concentration of IL-21 is 50 ng / ml. In one embodiment, the B cells are cultured with 1-50 μg / ml p-ODN. In one embodiment, the concentration of p-ODN is 10 μg / ml.

[0078] In one embodiment, B cells are contacted or cultured on feeder cells. In one embodiment, the feeder cells are a stromal cell line, e.g., murine stromal cell line S17 or MS5. In another embodiment, isolated CD19+ cells are cultured with one or more B cell activating factor cytokines, such as IL-10 and IL-4, in the presence of fibroblasts expressing CD40-ligand (CD40L, CD154). In one embodiment, CD40L is provided bound to a surface such as tissue culture plate or a bead. In another embodiment, purified B cells are cultured, in the presence or absence of feeder cells, with CD40L and one or more cytokines or factors selected from IL-10,IL-4, IL-7, p-ODN, CpG DNA, IL-2, IL-15, IL6, and IFN- .311376670Attorney Docket: IMCO-013 / 001WO

[0079] In another embodiment, B cell activating factors are provided by transfection into the B cell or other feeder cell. In this context, one or more factors that promote differentiation of the B cell into an antibody secreting cell and / or one or more factors that promote the longevity of the antibody producing cell may be used. Such factors include, for example, Blimp-1, TRF4, anti-apoptotic factors like Bcl-xl or Bcl5, or constitutively active mutants of the CD40 receptor. Further, factors which promote the expression of downstream signaling molecules such as TNF receptor-associated factors (TRAFs) may also be used in the activation / differentiation of the B cells. In this regard, cell activation, cell survival, and antiapoptotic functions of the TNF receptor superfamily are mostly mediated by TRAF1-6 (see e.g., R.H. Arch, et al., Genes Dev. 12 (1998), pp. 2821-2830). Downstream effectors of TRAF signaling include transcriptionfactors in the NF- B and AP-1 family which can turn on genes involved in various aspects ofcellular and immune functions. Further, the activation of NF- B and AP-1 has been shown toprovide cells protection from apoptosis via the transcription of antiapoptotic genes.

[0080] In another embodiment, Epstein Barr virus (EBV)-derived proteins are used for the activation and / or differentiation of B cells or to promote the longevity of the antibody producing cell. EBV-derived proteins include but are not limited to, EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2, EBER, miRNAs, EBV-EA, EBV-MA, EBV-VCA and EBV-AN.

[0081] In certain embodiments, contacting the B cells with B cell activation factors using the methods provided herein leads to, among other things, cell proliferation (i.e., expansion), modulation of the IgM+ cell surface phenotype to one consistent with an activated mature B cell, secretion of Ig, and isotype switching. CD19+ B cells may be isolated using known and commercially available cell separation kits, such as the MiniMACS™ cell separation system (Miltenyi Biotech, Bergisch Gladbach, Germany). In certain embodiments, CD40L fibroblasts are irradiated before use in the methods described herein. In one embodiment, B cells are cultured in the presence of one or more of IL-3, IL-7, Flt3 ligand, thrombopoietin, SCF, IL-2, IL-10, G-CSF and CpG. In certain embodiments, the methods include culturing the B cells in the presence of one or more of the aforementioned factors in conjunction with transformed stromal cells (e.g., MS5) providing a low level of anchored CD40L and / or CD40L bound to a plate or a bead.

[0082] As discussed above, B cell activating factors induce expansion, proliferation, or differentiation of B cells. Accordingly, B cells are contacted with one or more B cell activating factors listed above to obtain an expanded cell population. A cell population may be expanded prior to transfection. Alternatively, or additionally, a cell population may be expanded following transfection. In one embodiment, expanding a B cell population comprises culturing 311376670Attorney Docket: IMCO-013 / 001WO cells with IL-2, IL-4, IL-10 and CD40L (see e.g., Neron et al. PLoS ONE, 20127(12):e51946). In one embodiment, expanding a B cell population comprises culturing cells with IL-2, IL-10, CpG, and CD40L. In one embodiment, expanding a B cell population comprises culturing cells with IL-2, IL-4, IL-10, IL-15, IL-21, and CD40L. In one embodiment, expanding a B cell population comprises culturing cells with IL-2, IL-4, IL-10, IL-15, IL-21, and multimerized CD40L.

[0083] In another embodiment, expansion of a B cell population is induced and / or enhanced by a transgene introduced into the B cells. For example, a B cell that contains a recombinant receptor or an engineered receptor that induces a cell signaling pathway (e.g., signaling downstream of CD40) upon binding its ligand (e.g., a soluble ligand or a cell surface expressed ligand). In one embodiment, a B cell overexpresses CD40 due to expression of a CD40 transgene. In another embodiment, a B cell expresses an engineered receptor, including, e.g., a recombinantly engineered antibody. In one embodiment, an engineered receptor is similar to a chimeric antigen receptor (CAR) and comprises a fusion protein of an scFv and an intracellular signaling portion of a B cell receptor (e.g., CD40).

[0084] In one embodiment, expansion of a B cell population is induced and / or enhanced by a small molecule compound added to the cell culture. For example, a compound that binds to and dimerizes CD40 can be used to trigger the CD40 signaling pathway.

[0085] Any of a variety of culture media may be used in the present methods as would be known to the skilled person (see e.g., Current Protocols in Cell Culture, 2000-2009 by John Wiley & Sons, Inc.). In one embodiment, media for use in the methods described herein includes, but is not limited to Iscove modified Dulbecco medium (with or without fetal bovine or other appropriate serum). Illustrative media also includes, but is not limited to, IMDM, RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20. In further embodiments, the medium may comprise a surfactant, an antibody, plasmanate or a reducing agent (e.g. N-acetyl-cysteine, 2-mercaptoethanol), one or more antibiotics, and / or additives such as insulin, transferrin, sodium selenite and cyclosporin. In some embodiments, IL-6, soluble CD40L, and a cross-linking enhancer may also be used.

[0086] B cells are cultured under conditions and for sufficient time periods to achieve differentiation and / or activation desired. In certain embodiments, the B cells are cultured under conditions and for sufficient time periods such that 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% of the B cells are differentiated and / or activated as desired. In one embodiment, the B cells are activated and differentiated into a mixed population of plasmablasts and plasma cells. As would be 311376670Attorney Docket: IMCO-013 / 001WO recognized by the skilled person, plasmablasts and plasma cells may be identified by cell surface protein expression patterns using standard flow cytometry methods as described elsewhere herein, such as expression of one or more of CD38, CD78, IL-6R, CD27high, and CD138 and / or lack of, or reduction of, expression of one or more of CD19, CD20 and CD45. As would be understood by the skilled person, memory B cells are generally CD20+ CD19+CD27+ CD38 while early plasmablasts are CD20 CD19+ CD27++ CD38++. In oneembodiment, the cells cultured using the methods described herein are CD20-, CD38+, CD138- . In another embodiment, the cells have a phenotype of CD20-, CD38+, CD138+. In certain embodiments, cells are cultured for 1-7 days. In further embodiments, cells are cultured 7, 14, 21 days or longer. Thus, cells may be cultured under appropriate conditions for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more days. Cells are re-plated, and media and supplements may be added or changed as needed using techniques known in the art.

[0087] In certain embodiments, the B cells are cultured under conditions and for sufficient time periods such that at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the cells are differentiated and activated to produce Ig and / or to express the transgene.

[0088] The induction of B cell activation may be measured by techniques such as 3H-uridine incorporation into RNA (as B cells differentiate, RNA synthesis increases), or by 3H-thymidine incorporation, which measures DNA synthesis associated with cell proliferation. In one embodiment, interleukin-4 (IL-4) may be added to the culture medium at an appropriate concentration (e.g., about 10 ng / ml) for enhancement of B cell proliferation.

[0089] Alternatively, B cell activation is measured as a function of immunoglobulin secretion. For example, CD40L is added to resting B cells together with IL-4 (e.g., 10 ng / ml) and IL-5 (e.g., 5 ng / ml) or other cytokines that activate B cells. Flow cytometry may also be used for measuring cell surface markers typical of activated B cells. See e.g., Civin CI, Loken MR, Int'l J. Cell Cloning 987; 5:1 -16; Loken, MR, et al, Flow Cytometry Characterization of Erythroid, Lymphoid and Monomyeloid Lineages in Normal Human Bone Marrow, in Flow Cytometry in Hematology, Laerum OD, Bjerksnes R. eds., Academic Press, New York 1992; pp. 31 -42; and LeBein TW, et al., Leukemia 1990; 4:354-358.

[0090] After culture for an appropriate period of time, such as, e.g., from 2, 3, 4, 5, 6, 7, 8, 9, or more days, generally around 3 days, an additional volume of culture medium may be added. Supernatant from individual cultures may be harvested at various times during culture and quantitated for IgM and IgG1 as described in Noelle et al., (1991) J. Immunol.146:1118-1124. 311376670Attorney Docket: IMCO-013 / 001WO In one embodiment, the culture is harvested and measured for expression of the transgene of interest using flow cytometry, enzyme-linked immunosorbent assay (ELISA), ELISPOT or other assay known in the art.

[0091] In another embodiment, ELISA is used to measure antibody isotype production, e.g., IgM, or a product of the transgene of interest. In certain embodiments, IgG determinations are made using commercially available antibodies, such as goat anti-human IgG, as capture antibody followed by detection using any of a variety of appropriate detection reagents such as biotinylated goat antihuman Ig, streptavidin alkaline phosphatase and substrate.

[0092] In certain embodiments, the B cells are cultured under conditions and for sufficient time periods such that the number of cells is 1, 10, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 fold or more greater than the number of B cells at the start of culture. In one embodiment, the number of cells is 10- 1000 fold greater, including consecutive integers therein, than the number of B cells at the start of culture. For example, an expanded B cell population is at least 10 fold greater than the initial isolated B cell population. In another embodiment, the expanded B cell population is at least 100 fold greater than the initial isolated B cell population. In one embodiment, the expanded B cell population is at least 500 fold greater than the initial isolated B cell population.

[0093] In various embodiments, the present disclosure provides for methods of transfecting, infecting, or otherwise incorporating into a B cell one or more transgenes (e.g., a GLP-1 fusion and follistatin transgene), such that the transgene is expressed in the B cell. Any of the integration methods described herein or known in the art may in some embodiments be utilized for expressing GLP-1 or GLP-1 and follistatin in a B cell.

[0094] In one embodiment, the modified B cells are transfected with a transgene. In particular embodiments, the modified B cells are transfected with a GLP-1 analog transgene.

[0095] Exemplary methods for transfecting B cells are provided in WO 2014 / 152832 and WO 2016 / 100932, both of which are incorporated herein by reference in their entireties. Transfection of B cells may be accomplished using any of a variety of methods available in the art to introduce DNA or RNA into a B cell. Suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, pressure-mediated transfection or “cell squeezing” (e.g., CellSqueeze microfluidic system, SQZ Biotechnologies), nano-particle- mediated or liposome-mediated transfection and transduction using retrovirus or other virus, e.g., vaccinia. See, e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratories; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197; US 5,124,259; 311376670Attorney Docket: IMCO-013 / 001WO US 5,297,983; US 5,283,185; US 5,661,018; US 6,878,548; US 7,799,555; US 8,551,780; and US 8,633,029. One example of a commercially available electroporation technique suitable for B cells is the Nucleofector™ transfection technology.

[0096] Transfection may take place prior to or during in vitro culture of the isolated B cells in the presence of one or more activating and / or differentiating factors described above. For example, cells are transfected on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 of in vitro culture. In one embodiment, cells are transfected on day 1, 2, or 3 of in vitro culture. In a particular embodiment, cells are transfected on day 2. For example, cells are electroporated on day 2 of in vitro culture for delivery of, e.g., a plasmid, a transposon, a minicircle, or a self- replicating RNA. In another embodiment, cells are transfected on day 4, 5, 6, or 7 of in vitro culture. In a particular embodiment, cells are transfected on day 6 of in vitro culture. In another embodiment, cells are transfected on day 5 of in vitro culture.

[0097] In one embodiment, cells are transfected or otherwise engineered (e.g., via a targeted integration of a GLP-1 transgene and follistatin transgene) prior to activation. In another embodiment, cells are transfected or otherwise engineered (e.g., via a targeted integration of a GLP-1 transgene) during activation. In one embodiment, cells are transfected or otherwise engineered (e.g., via a targeted integration of a GLP-1 transgene) after activation. In one embodiment, cells are transfected or otherwise engineered (e.g., via a targeted integration of a GLP-1 transgene) prior to differentiation. In another embodiment, cells are transfected or otherwise engineered (e.g., via a targeted integration of a GLP-1 transgene) during differentiation. In one embodiment, cells are transfected or otherwise engineered (e.g., via a targeted integration of a GLP-1 transgene) after differentiation.

[0098] In one embodiment, a non-viral vector is used to deliver DNA or RNA (e.g., DNA or RNA comprising a sequence encoding a GLP-1 polypeptide) to memory B cells and / or plasma cells. For example, systems that may facilitate transfection of memory B cells and / or plasma cells without the need of a viral integration system include, without limitation, transposons (e.g., Sleeping Beauty or other transposon system such as Piggybac), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPRs), meganucleases, minicircles, replicons, artificial chromosomes (e.g., bacterial artificial chromosomes, mammalian artificial chromosomes, and yeast artificial chromosomes), plasmids, cosmids, and bacteriophage.

[0099] In some embodiments, such non-viral-dependent vector systems may also be delivered via a viral vector known in the art or described below. For example, in some embodiments, a 311376670Attorney Docket: IMCO-013 / 001WO viral vector (e.g., a retrovirus, lentivirus, adenovirus, adeno-associated virus), is utilized to deliver one or more non-viral vector (such as, e.g., one or more of the above-mentioned zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPRs) meganucleases, or any other enzyme / complementary vectors, polynucleotides, and / or polypeptides capable of facilitating the targeted integration. Accordingly, in some embodiments, a cell (e.g., B cells such as a memory B cells and / or plasma cells) may be engineered to express a sequence (e.g., a sequence encoding a GLP-1 polypeptide) via a targeted integration method. Such methods are known in the art and may comprise cleaving an endogenous locus in the cell using one or more nucleases (e.g., ZFNs, TALENs, CRISPR / Cas, meganuclease) and administering the GLP-1 transgene to the cell such that it is integrated into the endogenous locus and expressed in the cell. The GLP- 1 transgene may be comprised in a donor sequence that is integrated into the host cell’s DNA at or near the point of a cleavage by the nuclease.

[0100] The integration of the sequence (e.g., a sequence encoding a GLP-1 polypeptide) may occur via recombination. As would be clear to one of skill in the art, “Recombination” refers to a process of exchange of genetic information between two polynucleotides, including but not limited to, donor capture by non-homologous end joining (NHEJ) and homologous recombination. The recombination may be homologous recombination. For the purposes of this disclosure, “homologous recombination (HR)” refers to the specialized form of such exchange that takes place, for example, during repair of double-strand breaks in cells via homology- directed repair mechanisms. This process utilizes nucleotide sequence homology, whereby a “donor” molecule (e.g., donor polynucleotide sequence or donor vector comprising such a sequence) is utilized by a cell’s DNA-repair machinery as a template to repair of a “target” molecule (i.e., the one that experienced the double-strand break), and by these means causes the transfer of genetic information from the donor to the target. In some embodiments of HR- directed integration, the donor molecule may contain at least 2 regions of homology to the genome (“homology arms”). In some embodiments, the homology arms may be, e.g., of least 50-100 base pairs in length. The homology arms may have substantial DNA homology to a region of genomic DNA flanking the cleavage site wherein the targeted integration is to occur. The homology arms of the donor molecule may flank the DNA (e.g., comprising DNA encoding a GLP-1 analog) that is to be integrated into the target genome or target DNA locus. Breakage of the chromosome followed by repair using the homologous region of the plasmid DNA as a template may results in the transfer of the intervening transgene flanked by the homology arms into the genome. See, e.g., Koller et al. (1989) Proc. Nat'l. Acad. Sci. 311376670Attorney Docket: IMCO-013 / 001WO USA 86(22):8927-8931; Thomas et al. (1986) Cell 44(3):419-428. The frequency of this type of homology-directed targeted integration can be increased by up to a factor of 105 by deliberate creation of a double-strand break in the vicinity of the target region (Hockemeyer et al. (2009) Nature Biotech.27(9):851-857; Lombardo et al. (2007) Nature Biotech.25(11):1298-1306; Moehle et al. (2007) Proc. Nat'l Acad. Sci. USA 104(9):3055- 3060; Rouet et al. (1994) Proc. Nat'l Acad. Sci. USA 91(13):6064-6068.

[0101] Any nuclease capable of mediating the targeted cleavage of a genomic locus such that a transgene (e.g., a GLP-1 transgene) may be integrated into the genome of a target cell (e.g., by recombination such as HR) may be utilized in engineering a cell (e.g., a memory B cell or plasmablast) according to the present disclosure.

[0102] A double-strand break (DSB) or nick can be created by a site-specific nuclease such as a zinc-finger nuclease (ZFN), a TAL effector domain nuclease (TALEN), a meganuclease, or using the CRISPR / Cas9 system with an engineered crRNA / tracrRNA (single guide RNA) to guide specific cleavage. See, for example, Burgess (2013) Nature Reviews Genetics 14:80-81, Urnov et al. (2010) Nature 435(7042):646-51; United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20090263900; 20090117617; 20100047805; 20110207221; 20110301073 and International Publication WO 2007 / 014275, the disclosures of which are incorporated by reference in their entireties for all purposes.

[0103] In some embodiments, the cell (e.g., a memory B cell or a plasmablast) is engineered via Zinc Finger Nuclease-mediated targeted integration of a donor construct (e.g., a follistatin donor construct). A zinc finger nuclease (ZFN) is an enzyme that is able to recognize and cleave a target nucleotide sequence with specificity due to the coupling of a “zinc finger DNA binding protein” (ZFP) (or binding domain), which binds DNA in a sequence-specific manner through one or more zinc fingers, and a nuclease enzyme. ZFNs may comprise any suitable cleavage domains (e.g., a nuclease enzyme) operatively linked to a ZFP DNA-binding domain to form a engineered ZFN that can facilitate site-specific cleavage of a target DNA sequence (see, e.g., Kim et al. (1996) Proc Nat’l Acad Sci USA 93(3):1156-1160). For example, ZFNs may comprise a target-specific ZFP linked to a FOK1 enzyme or a portion of a FOK1 enzyme. In some embodiments, ZFN used in a ZFN-mediated targeted integration approach utilize two separate molecules, each comprising a subunit of a FOK1 enzyme each bound to a ZFP, each ZFP with specificity for a DNA sequence flanking a target cleavage site, and when the two ZFPs bind to their respective target DNA sites the FOK1 enzyme subunits are brought into proximity with one another and they bind together activating the nuclease activity which cleaves the target cleavage site. ZFNs have been used for genome modification in a variety of 311376670Attorney Docket: IMCO-013 / 001WO organisms (e.g., United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; and International Publication WO 07 / 014,275, incorporated herein by reference in their entirety) Custom ZFPs and ZFNs are commercially available from, e.g., Sigma Aldrich (St. Louis, MO), and any location of DNA may be routinely targeted and cleaved using such custom ZFNs.

[0104] In some embodiments, the cell (e.g., a memory B cell or a plasmablast) is engineered via CRISPR / Cas (e.g., CRISPR Cas9) Nuclease-mediated integration of a donor construct (e.g., a GLP-1 donor construct). A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR Associated) nuclease system is an engineered nuclease system based on a bacterial system that may be used for genome engineering. It is based on part of the adaptive immune response of many bacteria and archaea. When a virus or plasmid invades a bacterium, segments of the invader's DNA are converted into CRISPR RNAs (crRNA) by the ‘immune’ response. This crRNA then associates, through a region of partial complementarity, with another type of RNA called tracrRNA to guide the Cas9 nuclease to a region homologous to the crRNA in the target DNA called a “protospacer”. Cas9 cleaves the DNA to generate blunt ends at the DSB at sites specified by a 20-nucleotide guide sequence contained within the crRNA transcript. Cas9 requires both the crRNA and the tracrRNA for site specific DNA recognition and cleavage. This system has now been engineered such that the crRNA and tracrRNA can be combined into one molecule (the “single guide RNA”), and the crRNA equivalent portion of the single guide RNA can be engineered to guide the Cas9 nuclease to target any desired sequence (see Jinek et al (2012) Science 337, p.816-821, Jinek et al, (2013), eLife 2:e00471, and David Segal, (2013) eLife 2:e00563). Thus, the CRISPR / Cas system can be engineered to create a DSB at a desired target in a genome, and repair of the DSB can be influenced by the use of repair inhibitors to cause an increase in error prone repair. As will be clear to the skill artisan, other CRISPR nucleases, in addition to Cas9, are known and are suitable for use in the present invention.

[0105] In some embodiments, the CRISPR / Cas nuclease-mediated integration utilizes a Type II CRISPR. The Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to a protospacer 311376670Attorney Docket: IMCO-013 / 001WO adjacent motif (PAM), an additional requirement for target recognition. Forth, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.

[0106] The Cas9 related CRISPR / Cas system comprises two RNA non-coding components: tracrRNA and a pre-crRNA array containing nuclease guide sequences (spacers) interspaced by identical direct repeats (DRs). To use a CRISPR / Cas system to accomplish genome engineering, both functions of these RNAs must be present (see Cong et al, (2013) Sciencexpress 1 / 10.1126 / science 1231143). In some embodiments, the tracrRNA and pre- crRNAs are supplied via separate expression constructs or as separate RNAs. In other embodiments, a chimeric RNA is constructed where an engineered mature crRNA (conferring target specificity) is fused to a tracrRNA (supplying interaction with the Cas9) to create a chimeric cr-RNA-tracrRNA hybrid (also termed a single guide RNA). (see Jinek ibid and Cong, ibid).

[0107] In some embodiments, a single guide RNA containing both the crRNA and tracrRNA may be engineered to guide the Cas9 nuclease to target any desired sequence (e.g., Jinek et al (2012) Science 337, p.816-821, Jinek et al, (2013), eLife 2:e00471, David Segal, (2013) eLife 2:e00563). Thus, the CRISPR / Cas system may be engineered to create a DSB at a desired target in a genome.

[0108] Custom CRISPR / Cas systems are commercially available from, e.g., Dharmacon (Lafayette, CO), and any location of DNA may be routinely targeted and cleaved using such custom single guide RNA sequences. Single stranded DNA templates for recombination may be synthesized (e.g., via oligonucleotide synthesis methods known in the art and commercially available) or provided in a vector, e.g., a viral vector such as an AAV.

[0109] In some embodiments, the cell (e.g., a memory B cell or a plasmablast) is engineered via TALE-Nuclease (TALEN) mediated targeted integration of a donor construct (e.g., a GLP- 1 donor construct). A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains / units. The repeat domains are involved in binding of the TALE to its cognate target DNA sequence. A single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. TAL-effectors may contain a nuclear localization sequence, an acidic transcriptional activation domain and a centralized domain of tandem repeats where each repeat contains approximately 34 amino acids that are key to the DNA binding specificity of these proteins. (e.g., Schornack S, et al (2006) J Plant Physiol 163(3): 256-272). TAL effectors depend on the sequences found in the tandem repeats which comprises approximately 102 bp and the repeats are typically 91-100% 311376670Attorney Docket: IMCO-013 / 001WO homologous with each other (e.g., Bonas et al (1989) MoI Gen Genet 218: 127-136). These DNA binding repeats may be engineered into proteins with new combinations and numbers of repeats, to make artificial transcription factors that are able to interact with new sequences and activate the expression of a non-endogenous reporter gene (e.g., Bonas et al (1989) MoI Gen Genet 218: 127-136). Engineered TAL proteins may be linked to a FokI cleavage half domain to yield a TAL effector domain nuclease fusion (TALEN) to cleave target specific DNA sequence (e.g., Christian et al (2010) Genetics epub 10.1534 / genetics.110.120717).

[0110] Custom TALEN are commercially available from, e.g., Thermo Fisher Scientific (Waltham, MA), and any location of DNA may be routinely targeted and cleaved.

[0111] In some embodiments, the cell (e.g., a memory B cell or a plasmablast) is engineered via Meganuclease-mediated targeted integration of a donor construct (e.g., a GLP-1 donor construct). A Meganuclease (or “homing endonuclease”) is an endonuclease that binds and cleaves double-stranded DNA at a recognition sequence that is greater than 12 base pairs. Naturally occurring meganucleases may be monomeric (e.g., I-SceI) or dimeric (e.g., I-CreI). Naturally occurring meganucleases recognize 15-40 base-pair cleavage sites and are commonly grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family and the HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII. Their recognition sequences are known. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res.22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol.263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalogue. The term “Meganuclease” includes monomeric meganucleases, dimeric meganucleases and monomers that associate to form a dimeric meganucleases.

[0112] In certain embodiments, the methods and compositions described herein make use of a nuclease that comprises an engineered (non-naturally occurring) homing endonuclease (meganuclease). The recognition sequences of homing endonucleases and meganucleases such as I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I- TevI, I-TevII and I-TevIII are known. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res.22, 1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996) J. Mol. Biol.263:163-180; Argast et al. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalog. In addition, the DNA-binding specificity 311376670Attorney Docket: IMCO-013 / 001WO of homing endonucleases and meganucleases can be engineered to bind non-natural target sites. See, for example, Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et al. (2003) Nucleic Acids Res. 31:2952-2962; Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No.20070117128. The DNA-binding domains of the homing endonucleases and meganucleases may be altered in the context of the nuclease as a whole (i.e., such that the nuclease includes the cognate cleavage domain) or may be fused to a heterologous cleavage domain. Custom Meganuclease are commercially available from, e.g., New England Biolabs (Ipswich, MA), and any location of DNA may be routinely targeted and cleaved.

[0113] The engineering of the B cell may comprise administering one or more nucleases (e.g., ZFNs, TALENs, CRISPR / Cas, meganuclease) to a B cell, e.g., via one or more vectors encoding the nucleases, such that the vectors comprising the encoded nucleases are taken up by the B cell. The vectors may be viral vectors.

[0114] In some embodiments, the nucleases cleave a specific endogenous locus (e.g. safe harbor gene or locus of interest) in the cell (e.g., memory B cell or plasma cell) and one or more exogenous (donor) sequences (e.g., transgenes) are administered (e.g. one or more vectors comprising these sequences). In such embodiments, the donor sequence may encode GLP-1 (e.g., a GLP-1 transgene). The nuclease may induce a double-stranded (DSB) or single- stranded break (nick) in the target DNA. In some embodiments, targeted insertion of a donor transgene (e.g., a GLP-1 and / or follistatin donor transgene) may be performed via homology directed repair (HDR), non-homology repair mechanisms (e.g., NHEJ- mediated end capture), or insertions and / or deletion of nucleotides (e.g. endogenous sequence) at the site of integration of a transgene (e.g., a GLP-1 transgene) into the cell’s genome. In one embodiment, a method of transfecting a B cell comprises electroporating the B cell prior to contacting the B cell with a vector. In one embodiment, cells are electroporated on a day ranging from day 1 to day 12 of in vitro culture. In one embodiment, cells are electroporated on day 1, 2, 3, 4, 5, 6, 7, 8, or 9 of in vitro culture. In one embodiment, cells are electroporated on day 2 of in vitro culture for delivery of a plasmid.

[0115] In one embodiment, cells are transfected using a transposon. As used herein, the term “transposed” may in some embodiments refer to such a cell that is transfected with a transposon. Numerous transposon systems are known in the art and are suitable for use in the present invention. For example, the Sleeping Beauty transposon system and Piggybac transposon systems are well-known in the art and are suitable for use in the present invention. See e.g., Hackett P.B., et al., Evaluating Risks of Insertional Mutagenesis by DNA Transposons 311376670Attorney Docket: IMCO-013 / 001WO in Gene Therapy, Transl Res. 2013 April ; 161(4): 265–283; Hudecek M, et al., Going non- viral: the Sleeping Beauty transposon system breaks on through to the clinical side, Crit Rev Biochem Mol Biol. 2017 Aug;52(4):355-380, each of which are incorporated herein by reference in their entireties. In some embodiments, cells are transfected using a Sleeping Beauty transposon. The Sleeping Beauty transposon may in some embodiments be a T2 Sleeping Beauty transposon or a T4 Sleeping Beauty transposon. In some embodiments, utilization of the Sleeping Beauty transposon system may comprise transfecting (e.g., via electroporation) B cells with a DNA construct encoding the transposon system machinery and a DNA construct encoding the GLP-1 polypeptide. In some embodiments, the DNA construct encoding the transposon system machinery may be pCMV-SB100x. In some embodiments, utilization of the Sleeping Beauty transposon system may comprise transfecting (e.g., via electroporation) B cells with a DNA construct encoding the GLP-1, and optionally a follistatin polypeptide and further transfecting the B cells with mRNA encoding the transposon system machinery. In some embodiments, the mRNA encoding the transposon system machinery encodes the SB100x transposase. In some embodiments, cells are transfected using a Piggybac transposon. In one embodiment, cells are transfected using a transposon (e.g., a T2 or T4 Sleeping beauty transposon or a Piggybac transposon) on a day ranging from day 1 to day 12 of in vitro culture. In one embodiment, cells are transfected using a transposon (e.g., a T2 or T4 Sleeping beauty transposon or a Piggybac transposon) on day 1, 2, 3, 4, 5, 6, 7, 8, or 9 of in vitro culture. In one embodiment, cells are transfected using a minicircle on a day ranging from day 1 to day 12 of in vitro culture. In one embodiment, cells are transfected using a minicircle on day 1, 2, 3, 4, 5, 6, 7, 8, or 9 of in vitro culture.

[0116] In one embodiment, cells are transfected using a Sleeping Beauty transposon (e.g., a T2 or T4 Sleeping beauty transposon) on a day ranging from day 1 to day 12 of in vitro culture. In one embodiment, cells are transduced using a Sleeping Beauty transposon system (e.g., a T2 or T4 Sleeping beauty transposon) on day 2 of in vitro culture via electroporation. In one embodiment, cells are transduced using a Sleeping Beauty transposon system (e.g., a T2 or T4 Sleeping beauty transposon) on day 5 of in vitro culture via electroporation. In one embodiment, cells are transduced using a Sleeping Beauty transposon system (e.g., a T2 or T4 Sleeping beauty transposon) on day 8 of in vitro culture via electroporation. In one embodiment, cells are transduced using a Sleeping Beauty transposon (e.g., a T2 or T4 Sleeping beauty transposon) system on day 11 of in vitro culture via electroporation. In one embodiment, cells are transduced using a Sleeping Beauty transposon (e.g., a T2 or T4 Sleeping beauty transposon) system on day 14 of in vitro culture via electroporation. 311376670Attorney Docket: IMCO-013 / 001WO

[0117] In one embodiment, cells are transfected using a Piggybac transposon on a day ranging from day 1 to day 12 of in vitro culture. In one embodiment, cells are transduced using a Piggybac transposon on day 2 of in vitro culture via electroporation. In one embodiment, cells are transduced using a Piggybac transposon on day 5 of in vitro culture via electroporation. In one embodiment, cells are transduced using a Piggybac transposon on day 8 of in vitro culture via electroporation. In one embodiment, cells are transduced using a Piggybac transposon on day 11 of in vitro culture via electroporation. In one embodiment, cells are transduced using a Piggybac transposon on day 14 of in vitro culture via electroporation.

[0118] In one embodiment, the B cells are contacted with a vector comprising a nucleic acid of interest operably linked to a promoter, under conditions sufficient to transfect at least a portion of the B cells. In one embodiment the B cells are contacted with a vector comprising a nucleic acid of interest operably linked to a promoter, under conditions sufficient to transfect at least 5% of the B cells. In a further embodiment, the B cells are contacted with a vector under conditions sufficient to transfect at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% of the B cells. In one particular embodiment, the B cells, cultured in vitro as described herein, are transfected, in which case the cultured B cells are contacted with a vector as described herein under conditions sufficient to transfect at least 5%, 10% 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% of the B cells.

[0119] Viral vectors may be employed to transduce memory B cells and / or plasma cells. Examples of viral vectors include, without limitation, adenovirus-based vectors, adeno- associated virus (AAV)-based vectors, retroviral vectors, retroviral-adenoviral vectors, and vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication- defective HSV and attenuated HSV (see, e.g., Krisky, Gene Ther. 5: 1517-30, 1998; Pfeifer, Annu. Rev. Genomics Hum. Genet. 2:177-211, 2001, each of which is incorporated by reference in its entirety).

[0120] In one embodiment, cells are transduced with a viral vector (e.g., a lentiviral vector) on day 1, 2, 3, 4, 5, 6, 7, 8, or 9 of in vitro culture. In a particular embodiment, cells are transduced with a viral vector on day 5 of in vitro culture. In one embodiment, the viral vector is a lentivirus. In one embodiment, cells are transduced with a measles virus pseudo-typed lentivirus on day 1 of in vitro culture.

[0121] In one embodiment, B cells are transduced with retroviral vectors using any of a variety of known techniques in the art (see, e.g., Science 12 April 1996272: 263-267; Blood 2007, 311376670Attorney Docket: IMCO-013 / 001WO 99:2342- 2350; Blood 2009, 113:1422-1431 ; Blood 2009 Oct 8; 114(15):3173-80; Blood. 2003;101 (6):2167-2174; Current Protocols in Molecular Biology or Current Protocols in Immunology, John Wiley & Sons, New York, N.Y.(2009)). Additional description of viral transduction of B cells may be found in WO 2011 / 085247 and WO 2014 / 152832, each of which is herein incorporated by reference in its entirety.

[0122] For example, PBMCs, B- or T-lymphocytes from donors, and other B cell cancer cells such as B-CLLs may be isolated and cultured in IMDM medium or RPMI 1640 (GibcoBRL Invitrogen, Auckland, New Zealand) or other suitable medium as described herein, either serum-free or supplemented with serum (e.g., 5-10% FCS, human AB serum, and serum substitutes) and penicillin / streptomycin and / or other suitable supplements such as transferrin and / or insulin. In one embodiment, cells are seeded at 1 x 105cells per well in 48-well plates and concentrated vector added at various doses that may be routinely optimized by the skilled person using routine methodologies. In one embodiment, B cells are transferred to an MS5 cell monolayer in RPMI supplemented with 10% AB serum, 5% FCS, 50ng / ml rhSCF, 10ng / ml rhlL-15 and 5ng / ml rhlL-2 and medium refreshed periodically as needed. As would be recognized by the skilled person, other suitable media and supplements may be used as desired.

[0123] Certain embodiments relate to the use of retroviral vectors, or vectors derived from retroviruses. “Retroviruses” are enveloped RNA viruses that are capable of infecting animal cells, and that utilize the enzyme reverse transcriptase in the early stages of infection to generate a DNA copy from their RNA genome, which is then typically integrated into the host genome. Examples of retroviral vectors Moloney murine leukemia virus (MLV)-derived vectors, retroviral vectors based on a Murine Stem Cell Virus, which provides long-term stable expression in target cells such as hematopoietic precursor cells and their differentiated progeny (see, e.g., Hawley et al., PNAS USA 93:10297-10302, 1996; Keller et al., Blood 92:877-887, 1998), hybrid vectors (see, e.g., Choi, et al., Stem Cells 19:236-246, 2001), and complex retrovirus-derived vectors, such as lentiviral vectors.

[0124] In one embodiment, the B cells are contacted with a retroviral vector comprising a nucleic acid of interest operably linked to a promoter, under conditions sufficient to transduce at least a portion of the B cells. In one embodiment the B cells are contacted with a retroviral vector comprising a nucleic acid of interest operably linked to a promoter, under conditions sufficient to transduce at least 2% of the B cells. In a further embodiment, the B cells are contacted with a vector under conditions sufficient to transduce at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% of the resting B cells. In one particular embodiment, the 311376670Attorney Docket: IMCO-013 / 001WO differentiated and activated B cells, cultured in vitro as described herein, are transduced, in which case the cultured differentiated / activated B cells are contacted with a vector as described herein under conditions sufficient to transduce at least 2%, 3%, 4%, 5%, 10% 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or even 100% of the differentiated and activated B cells.

[0125] In certain embodiments, prior to transduction, the cells are prestimulated with Staphylococcus Aureus Cowan (SAC; Calbiochem, San Diego, CA) and / or IL-2 at appropriate concentrations known to the skilled person and routinely optimized. Other B cell activating factors (e.g., PMA), as are known to the skilled artisan and described herein may be used.

[0126] As noted above, certain embodiments employ lentiviral vectors. The term “lentivirus” refers to a genus of complex retroviruses that are capable of infecting both dividing and non- dividing cells. Examples of lentiviruses include HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2), visna-maedi, the caprine arthritis-encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian immunodeficiency virus (SIV). Lentiviral vectors can be derived from any one or more of these lentiviruses (see, e.g., Evans et al., Hum Gene Ther. 10:1479-1489, 1999; Case et al., PNAS USA 96:2988-2993, 1999; Uchida et al., PNAS USA 95:1 1939-1 1944, 1998; Miyoshi et al., Science 283:682-686, 1999; Sutton et al., J Virol 72:5781 -5788, 1998; and Frecha et al., Blood. 1 12:4843-52, 2008, each of which is incorporated by reference in its entirety).

[0127] It has been documented that resting T and B cells can be transduced by a VSVG-coated LV carrying most of the HIV accessory proteins (vif, vpr, vpu, and nef) (see e.g., Frecha et al., 2010 Mol. Therapy 18:1748). In certain embodiments the retroviral vector comprises certain minimal sequences from a lentivirus genome, such as the HIV genome or the SIV genome. The genome of a lentivirus is typically organized into a 5' long terminal repeat (LTR) region, the gag gene, the pol gene, the env gene, the accessory genes (e.g., nef, vif, vpr, vpu, tat, rev) and a 3' LTR region. The viral LTR is divided into three regions referred to as U3, R (repeat) and U5. The U3 region contains the enhancer and promoter elements, the U5 region contains the polyadenylation signals, and the R region separates the U3 and U5 regions. The transcribed sequences of the R region appear at both the 5' and 3' ends of the viral RNA (see, e.g., “RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., Oxford University Press, 2000); O Narayan, J. Gen. Virology. 70:1617-1639, 1989; Fields et al., Fundamental Virology Raven Press., 1990; Miyoshi et al., J Virol. 72:8150-7,1998; and U.S. Pat. No. 6,013,516, each of which is incorporated by reference in its entirety). Lentiviral vectors may comprise any one or 311376670Attorney Docket: IMCO-013 / 001WO more of these elements of the lentiviral genome, to regulate the activity of the vector as desired, or, they may contain deletions, insertions, substitutions, or mutations in one or more of these elements, such as to reduce the pathological effects of lentiviral replication, or to limit the lentiviral vector to a single round of infection.

[0128] Typically, a minimal retroviral vector comprises certain 5'LTR and 3'LTR sequences, one or more genes of interest (to be expressed in the target cell), one or more promoters, and a cis-acting sequence for packaging of the RNA. Other regulatory sequences can be included, as described herein and known in the art. The viral vector is typically cloned into a plasmid that may be transfected into a packaging cell line, such as a eukaryotic cell (e.g., 293-HEK), and also typically comprises sequences useful for replication of the plasmid in bacteria.

[0129] In certain embodiments, the viral vector comprises sequences from the 5' and / or the 3' LTRs of a retrovirus such as a lentivirus. The LTR sequences may be LTR sequences from any lentivirus from any species. For example, they may be LTR sequences from HIV, SIV, FIV or BIV. Preferably the LTR sequences are HIV LTR sequences.

[0130] In certain embodiments, the viral vector comprises the R and U5 sequences from the 5' LTR of a lentivirus and an inactivated or “self-inactivating” 3' LTR from a lentivirus. A “self- inactivating 3' LTR” is a 3' long terminal repeat (LTR) that contains a mutation, substitution or deletion that prevents the LTR sequences from driving expression of a downstream gene. A copy of the U3 region from the 3' LTR acts as a template for the generation of both LTR's in the integrated provirus. Thus, when the 3' LTR with an inactivating deletion or mutation integrates as the 5' LTR of the provirus, no transcription from the 5' LTR is possible. This eliminates competition between the viral enhancer / promoter and any internal enhancer / promoter. Self-inactivating 3' LTRs are described, for example, in Zufferey et al., J Virol.72:9873-9880, 1998; Miyoshi et al., J Virol.72:8150-8157, 1998; and Iwakuma et al., J Virology 261: 120-132, 1999, each of which is incorporated by reference in its entirety. Self- inactivating 3' LTRs may be generated by any method known in the art. In certain embodiments, the U3 element of the 3' LTR contains a deletion of its enhancer sequence, preferably the TATA box, Spl and / or NF-kappa B sites. As a result of the self-inactivating 3' LTR, the provirus that is integrated into the host cell genome will comprise an inactivated 5' LTR.

[0131] The vectors provided herein typically comprise a gene that encodes a protein, e.g., GLP- 1, that is desirably expressed in one or more target cell. The vectors provided herein may also comprise genes that encode other molecules, (such as, e.g., siRNA) that are desirably expressed in one or more target cells. In some embodiments, in a viral vector, the gene of interest (e.g., 311376670Attorney Docket: IMCO-013 / 001WO GLP-1) is preferably located between the 5' LTR and 3' LTR sequences. Further, in some embodiments, the gene of interest (e.g., GLP-1) is preferably in a functional relationship with other genetic elements, for example, transcription regulatory sequences such as promoters and / or enhancers, to regulate expression of the gene of interest (e.g., GLP-1) in a particular manner once the gene is incorporated into the target cell. In certain embodiments, the useful transcriptional regulatory sequences are those that are highly regulated with respect to activity, both temporally and spatially.

[0132] In certain embodiments, one or more additional genes may be incorporated as a safety measure, e.g., to allow for the selective killing of or depleting transfected target cells within a heterogeneous population, such as within a human patient. In one non-limiting exemplary embodiment, the gene is a thymidine kinase gene (TK), the expression of which renders a target cell susceptible to the action of the drug gancyclovir. In some embodiments, the additional gene is a cell surface protein tag. In some embodiments, the gene is a suicide gene. In some embodiments, the suicide gene is a caspase 9 suicide gene activated by a dimerizing drug (see, e.g., Tey et al., Biology of Blood and Marrow Transplantation 13:913-924, 2007).

[0133] In certain embodiments, one or more additional genes encoding a marker protein may be placed before or after the primary gene (e.g., a GLP-1 gene) in a viral or non-viral vector to allow for identification and / or selection of cells that are expressing the desired protein (e.g., GLP-1). Certain embodiments incorporate an additional cell surface protein that may facilitate identification and / or selection of cells that are expressing the desired protein (e.g., GLP-1). Certain embodiments incorporate a fluorescent marker protein, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), along with the primary gene of interest (e.g., a GLP-1 gene). If one or more additional reporter genes are included, IRES sequences or 2A elements may also be included, separating the primary gene of interest (e.g., a GLP-1 gene) from a reporter gene and / or any other gene of interest.

[0134] Certain embodiments may employ genes that encode one or more selectable markers. Examples include selectable markers that are effective in a eukaryotic cell or a prokaryotic cell, such as a gene for a drug resistance that encodes a factor necessary for the survival or growth of transformed host cells grown in a selective culture medium. Exemplary selection genes encode proteins that confer resistance to antibiotics or other toxins, e.g., G418, hygromycin B, puromycin, zeocin, ouabain, blasticidin, ampicillin, neomycin, methotrexate, or tetracycline, complement auxotrophic deficiencies, or supply may be present on a separate plasmid and introduced by co-transfection with the viral vector. In one embodiment, the gene encodes for a mutant dihydrofolate reductase (DHFR) that confers methotrexate resistance. Certain other 311376670Attorney Docket: IMCO-013 / 001WO embodiments may employ genes that encode one or cell surface receptors that can be used for tagging and detection or purification of transfected cells (e.g., low-affinity nerve growth factor receptor (LNGFR) or other such receptors useful as transduction tag systems. See e.g., Lauer et al., Cancer Gene Ther.2000 Mar;7(3):430-7.

[0135] Certain viral vectors such as retroviral vectors employ one or more heterologous promoters, enhancers, or both. In certain embodiments, the U3 sequence from a retroviral or lentiviral 5' LTR may be replaced with a promoter or enhancer sequence in the viral construct. Certain embodiments employ an “internal” promoter / enhancer that is located between the 5' LTR and 3' LTR sequences of the viral vector, and is operably linked to the gene of interest (e.g., a GLP-1 gene).

[0136] A “functional relationship” and “operably linked” mean, without limitation, that the gene (e.g., a GLP-1 gene) is in the correct location and orientation with respect to the promoter and / or enhancer, such that expression of the gene (e.g., a GLP-1 gene) will be affected when the promoter and / or enhancer is contacted with the appropriate regulatory molecules. Any enhancer / promoter combination may be used that either regulates (e.g., increases, decreases) expression of the viral RNA genome in the packaging cell line, regulates expression of the selected gene of interest in an infected target cell, or both.

[0137] A promoter is an expression control element formed by a DNA sequence that permits polymerase binding and transcription to occur. Promoters are untranslated sequences that are located upstream (5') of the start codon of a selected gene of interest (typically within about 100 to 1000 bp) and control the transcription and translation of the coding polynucleotide sequence to which they are operably linked. Promoters may be inducible or constitutive. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as a change in temperature. Promoters may be unidirectional or bidirectional. Bidirectional promoters can be used to co-express two genes, e.g., a gene of interest such as GLP-1 and a selection marker. Alternatively, a bidirectional promoter configuration comprising two promoters, each controlling expression of a different gene, in opposite orientation in the same vector may be utilized.

[0138] A variety of promoters are known in the art, as are methods for operably linking the promoter to the polynucleotide coding sequence. Both native promoter sequences and many heterologous promoters may be used to direct expression of the selected gene of interest. Certain embodiments employ heterologous promoters, because they generally permit greater transcription and higher yields of the desired protein as compared to the native promoter. 311376670Attorney Docket: IMCO-013 / 001WO

[0139] Certain embodiments may employ heterologous viral promoters. Examples of such promoters include those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40). Certain embodiments may employ heterologous mammalian promoter, such as the actin promoter, an immunoglobulin promoter, a heat-shock promoter, or a promoter that is associated with the native sequence of the gene of interest (e.g., a GLP-1 and follistatin gene). Typically, the promoter is compatible with the target cell, such as an activated B-lymphocyte, a plasma B cell, a memory B cell or other lymphocyte target cell.

[0140] Certain embodiments may employ one or more of the RNA polymerase II and III promoters. A suitable selection of RNA polymerase III promoters can be found, for example, in Paule and White. Nucleic Acids Research., Vol. 28, pp 1283-1298, 2000, which is incorporated by reference in its entirety. RNA polymerase II and III promoters also include any synthetic or engineered DNA fragments that can direct RNA polymerase II or III, respectively, to transcribe its downstream RNA coding sequences. Further, the RNA polymerase II or III (Pol II or III) promoter or promoters used as part of the viral vector can be inducible. Any suitable inducible Pol II or III promoter can be used with the methods described herein. Exemplary Pol II or III promoters include the tetracycline responsive promoters provided in Ohkawa and Taira, Human Gene Therapy, Vol. 11, pp 577-585, 2000; and Meissner et al., Nucleic Acids Research, Vol. 29, pp 1672-1682, 2001, each of which is incorporated by reference in its entirety.

[0141] Non-limiting examples of constitutive promoters that may be used include the promoter for ubiquitin, the CMV promoter (see, e.g., Karasuyama et al., J. Exp. Med.169:13, 1989), the -actin (see, e.g., Gunning et al., PNAS USA 84:4831 -4835, 1987), the elongation factor-1 alpha (EF-1 alpha) promoter, the CAG promoter, and the pgk promoter (see, e.g., Adra et al., Gene 60:65-74, 1987); Singer-Sam et al., Gene 32:409-417, 1984; and Dobson et al., Nucleic Acids Res. 10:2635-2637, 1982, each of which is incorporated by reference). Non-limiting examples of tissue specific promoters include the lck promoter (see, e.g., Garvin et al., Mol. Cell Biol. 8:3058-3064, 1988; and Takadera et al., Mol. Cell Biol. 9:2173-2180, 1989), the myogenin promoter (Yee et al., Genes and Development 7:1277-1289. 1993), and the thy1 promoter (see, e.g., Gundersen et al., Gene 113:207-214, 1992).

[0142] Additional examples of promoters include the ubiquitin-C promoter, the human μ heavy chain promoter or the Ig heavy chain promoter (e.g., MH), and the human light chain promoter or the Ig light chain promoter (e.g., EEK), which are functional in B-lymphocytes. 311376670Attorney Docket: IMCO-013 / 001WO The MH promoter contains the human μ heavy chain promoter preceded by the iEμ enhancer flanked by matrix association regions, and the EEK promoter contains the light chainpromoter preceded an intronic enhancer (iE ), a matrix associated region, and a 3' enhancer(3E ) (see, e.g., Luo et al., Blood. 1 13:1422-1431, 2009, and U.S. Patent ApplicationPublication No. 2010 / 0203630). Accordingly, certain embodiments may employ one or more of these promoter or enhancer elements.

[0143] In one embodiment, one promoter drives expression of a selectable marker and a second promoter drives expression of the gene of interest (e.g., a GLP-1 gene). For example, in one embodiment, the EF-1 alpha promoter drives the production of a selection marker (e.g., DHFR) and a miniature CAG promoter (see, e.g., Fan et al. Human Gene Therapy 10:2273–2285, 1999) drives the expression of the gene of interest (e.g., GLP-1 or GLP-1 fusion).

[0144] As noted above, certain embodiments employ enhancer elements, such as an internal enhancer, to increase the expression of the gene of interest. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp in length, that act on a promoter to increase its transcription. Enhancer sequences may be derived from mammalian genes (e.g., globin, elastase, albumin, -fetoprotein, insulin), such as the enhancer, the intronic enhancer, and the 3' enhancer. Also included are enhancers from a eukaryotic virus, including the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Enhancers may be spliced into the vector at a position 5' or 3' to the antigen-specific polynucleotide sequence, but are preferably located at a site 5' from the promoter. Persons of skill in the art will select the appropriate enhancer based on the desired expression pattern.

[0145] In certain embodiments, promoters are selected to allow for inducible expression of the gene of interest (e.g., a GLP-1 gene). Several systems for inducible expression are known in the art, including the tetracycline responsive system and the lac operator-repressor system. It is also contemplated that a combination of promoters may be used to obtain the desired expression of the gene of interest (e.g., a GLP-1 gene). The skilled artisan will be able to select a promoter based on the desired expression pattern of the gene in the organism and / or the target cell of interest.

[0146] Certain viral vectors contain cis-acting packaging sequences to promote the incorporation of the genomic viral RNA into the viral particle. Examples include psi- sequences. Such cis-acting sequences are known in the art. In certain embodiments, the viral vectors described herein may express two or more genes, which may be accomplished, for 311376670Attorney Docket: IMCO-013 / 001WO example, by incorporating an internal promoter that is operably linked to each separate gene beyond the first gene, by incorporating an element that facilitates co-expression such as an internal ribosomal entry sequence (IRES) element (U.S. Pat. No. 4,937,190, incorporated herein by reference) or a 2A element, or both. Merely by way of illustration, IRES or 2A elements may be used when a single vector comprises sequences encoding each chain of an immunoglobulin molecule with a desired specificity. For instance, the first coding region (encoding either the heavy or light chain) may be located immediately downstream from the promoter, and the second coding region (encoding the other chain) may be located downstream from the first coding region, with an IRES or 2A element located between the first and second coding regions, preferably immediately preceding the second coding region. In other embodiments, an IRES or 2A element is used to co-express an unrelated gene, such as a reporter gene, a selectable marker, a cell surface protein, or a gene that enhances immune function. Examples of IRES sequences that can be used include, without limitation, the IRES elements of encephalomyelitis virus (EMCV), foot-and- mouth disease virus (FMDV), Theiler's murine encephalomyelitis virus (TMEV), human rhinovirus (HRV), coxsackievirus (CSV), poliovirus (POLIO), Hepatitis A virus (HAV), Hepatitis C virus (HCV), and Pestiviruses (e.g., hog cholera virus (HOCV) and bovine viral diarrhea virus (BVDV)) (see, e.g., Le et al., Virus Genes 12:135-147, 1996; and Le et al., Nuc. Acids Res. 25:362-369, 1997, each of which is incorporated by reference in their entirety). One example of a 2A element includes the F2A sequence from foot-and-mouth disease virus.

[0147] In certain embodiments, the vectors provided herein also contain additional genetic elements to achieve a desired result. For example, certain viral vectors may include a signal that facilitates nuclear entry of the viral genome in the target cell, such as an HIV-1 flap signal. As a further example, certain viral vectors may include elements that facilitate the characterization of the provirus integration site in the target cell, such as a tRNA amber suppressor sequence. Certain viral vectors may contain one or more genetic elements designed to enhance expression of the gene of interest (e.g., a GLP-1 analog gene). For example, a woodchuck hepatitis virus responsive element (WRE) may be placed into the construct (see, e.g., Zufferey et al., J. Virol.74:3668-3681, 1999; and Deglon et al., Hum. Gene Ther.11:179- 190, 2000, each of which is incorporated by reference in its entirety). As another example, a chicken -globin insulator may also be included in the construct. This element has been shown to reduce the chance of silencing the integrated DNA in the target cell due to methylation and heterochromatinization effects. In addition, the insulator may shield the internal enhancer, promoter and gene from positive or negative positional effects from surrounding DNA at the 311376670Attorney Docket: IMCO-013 / 001WO integration site on the chromosome. Certain embodiments employ each of these genetic elements. In another embodiment, the viral vectors provided herein may also contain a Ubiquitous Chromatin Opening Element (UCOE) to increase expression (see e.g., Zhang F, et al., Molecular Therapy: The journal of the American Society of Gene Therapy 2010 Sep;18(9):1640–9).

[0148] In certain embodiments, the viral vectors (e.g., retroviral, lentiviral) provided herein are “pseudo-typed” with one or more selected viral glycoproteins or envelope proteins, mainly to target selected cell types. Pseudo-typing refers to generally to the incorporation of one or more heterologous viral glycoproteins onto the cell-surface virus particle, often allowing the virus particle to infect a selected cell that differs from its normal target cells. A “heterologous” element is derived from a virus other than the virus from which the RNA genome of the viral vector is derived. Typically, the glycoprotein-coding regions of the viral vector have been genetically altered such as by deletion to prevent expression of its own glycoprotein. Merely by way of illustration, the envelope glycoproteins gp41 and / or gp120 from an HIV-derived lentiviral vector are typically deleted prior to pseudo-typing with a heterologous viral glycoprotein.

[0149] In certain embodiments, the viral vector is pseudo-typed with a heterologous viral glycoprotein that targets B lymphocytes. In certain embodiments, the viral glycoprotein allows selective infection or transduction of resting or quiescent B lymphocytes. In certain embodiments, the viral glycoprotein allows selective infection of B lymphocyte plasma cells, plasmablasts, and activated B cells. In certain embodiments, the viral glycoprotein allows infection or transduction of quiescent B lymphocytes, plasmablasts, plasma cells, and activated B cells. In certain embodiments, viral glycoprotein allows infection of B cell chronic lymphocyte leukemia cells. In one embodiment, the viral vector is pseudo-typed with VSV-G. In another embodiment, the heterologous viral glycoprotein is derived from the glycoprotein of the measles virus, such as the Edmonton measles virus. Certain embodiments pseudo-type the measles virus glycoproteins hemagglutinin (H), fusion protein (F), or both (see, e.g., Frecha et al., Blood.112:4843-52, 2008; and Frecha et al., Blood.114:3173-80, 2009, each of which is incorporated by reference in its entirety). In one embodiment, the viral vector is pseudo- typed with gibbon ape leukemia virus (GALV). In one embodiment, the viral vector is pseudo- typed with cat endogenous retrovirus (RD114). In one embodiment, the viral vector is pseudo- typed with baboon endogenous retrovirus (BaEV). In one embodiment, the viral vector is pseudo-typed with murine leukemia virus (MLV). In one embodiment, the viral vector is pseudo-typed with gibbon ape leukemia virus (GALV). In further embodiments, the viral 311376670Attorney Docket: IMCO-013 / 001WO vector comprises an embedded antibody binding domain, such as one or more variable regions (e.g., heavy and light chain variable regions) which serves to target the vector to a particular cell type.

[0150] Generation of viral vectors can be accomplished using any suitable genetic engineering techniques known in the art, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, PCR amplification, and DNA sequencing, for example as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (1989)), Coffin et al. (Retroviruses. Cold Spring Harbor Laboratory Press, N.Y. (1997)) and “RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., Oxford University Press, (2000)).

[0151] Any variety of methods known in the art may be used to produce suitable retroviral particles whose genome comprises an RNA copy of the viral vector. As one method, the viral vector may be introduced into a packaging cell line that packages the viral genomic RNA based on the viral vector into viral particles with a desired target cell specificity. The packaging cell line typically provides in trans the viral proteins that are required for packaging the viral genomic RNA into viral particles and infecting the target cell, including the structural gag proteins, the enzymatic pol proteins, and the envelope glycoproteins.

[0152] In certain embodiments, the packaging cell line stably expresses certain necessary or desired viral proteins (e.g., gag, pol) (see, e.g., U.S. Pat. No.6,218,181, herein incorporated by reference). In certain embodiments, the packaging cell line is transiently transfected with plasmids that encode certain of the necessary or desired viral proteins (e.g., gag, pol, glycoprotein), including the measles virus glycoprotein sequences described herein. In one exemplary embodiment, the packaging cell line stably expresses the gag and pol sequences, and the cell line is then transfected with a plasmid encoding the viral vector and a plasmid encoding the glycoprotein. Following introduction of the desired plasmids, viral particles are collected and processed accordingly, such as by ultracentrifugation to achieve a concentrated stock of viral particles. Exemplary packaging cell lines include 293 (ATCC CCL X), HeLa (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL 1430) cell lines. Methods of Use

[0153] One aspect of the present invention is directed to the in vivo delivery of a therapeutic agent (e.g., GLP-1 or GLP-1 fusion) via delivery of a modified B cell engineered to express the therapeutic agent (e.g., GLP-1 or GLP-1 fusion). In some embodiments, the modified B cell further expresses follistatin. In particular embodiments, the modified B cells express GLP- 311376670Attorney Docket: IMCO-013 / 001WO 1 or GLP-1 fusion and are used in methods of treating and / or preventing metabolic disease (e.g., diabetes and / or obesity) in a human subject.

[0154] As used herein, the phrases “long-term in vivo survival” and “long-term survival” refer to the survival of the modified B cells described herein for 10 or more days post administration in a subject. Long-term survival may be measured in days, weeks, or even years. In one embodiment, a majority of the modified B cells survive in vivo for 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more days post-administration. In one embodiment, a majority of the modified B cells survive in vivo for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or more weeks post-administration. In another embodiment, the modified B cells survive in vivo for 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more years. Additionally, while the modified B cells described herein may survive in vivo for 10 or more days, it is understood that a majority of the modified B cells survive in vivo for 1, 2, 3, 4, 5, 6, 7, 8, 9 or more days post- administration. Accordingly, it is contemplated that modified B cells described herein are useful for short-term treatment (e.g., 4 days) and long-term treatment (e.g., 30 or more days) methods.

[0155] In some embodiments, the human subject is an adult. In some embodiments, the human subject is a juvenile.

[0156] Modified B cells described herein may be administered in a manner appropriate to the disease or disorder to be treated or prevented.

[0157] The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0158] In one embodiment, a single dose of modified B cells is administered to a subject. In one embodiment, two or more doses of modified B cells are administered sequentially to a subject. In one embodiment, three doses of modified B cells are administered sequentially to a subject. In one embodiment, a dose of modified B cells is administered weekly, biweekly, monthly, bimonthly, quarterly, semiannually, annually, or biannually to a subject. In one embodiment, a second or subsequent dose of modified B cells is administered to a subject when an amount of a therapeutic agent produced by the modified B cells decreases.

[0159] In one embodiment, a dose of modified B cells is administered to a subject at a certain frequency (e.g., weekly, biweekly, monthly, bimonthly, or quarterly) until a desired amount 311376670Attorney Docket: IMCO-013 / 001WO (e.g., an effective amount) of a therapeutic agent (e.g., GLP-1 or GLP-1 fusion) is detected in the subject. In one embodiment, an amount of the therapeutic agent (e.g., GLP-1 or GLP-1 fusion) is monitored in the subject. In one embodiment, a subsequent dose of modified B cells is administered to the subject when the amount of the therapeutic agent produced by the modified B cells decreases below the desired amount.

[0160] In one embodiment, the desired amount is a range that produces the desired effect. For example, in a method for treating diabetes a desired amount of GLP-1 or GLP-1 fusion may be reflected in the blood glucose levels or H1Ac levels of a subject. In some embodiments, the desired amount may be an amount of GLP-1 or GLP-1 fusion that results in a certain level of weight loss by the subject.

[0161] When “an effective amount,” “therapeutic amount,” a “therapeutically effective amount,” or a “therapeutically effective dose” is indicated, these terms refer to an amount of a compound of the invention that, when administered to a subject, (e.g., preferably a mammal, more preferably a human), is sufficient to effect treatment, as defined herein, of a disease or condition in the subject. The precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). B cell compositions may also be administered multiple times at an appropriate dosage(s). The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988).

[0162] The optimal dosage and treatment regime for a particular patient can be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. The treatment may also be adjusted after measuring the levels of a therapeutic agent (e.g., GLP-1 or GLP-1 fusion) in a biological sample (e.g., body fluid such as plasma or a tissue sample) can also be used to assess the treatment efficacy, and the treatment may be adjusted accordingly to increase or decrease.

[0163] In some aspects of the present disclosure, an optimal dosage of the modified B cells for a multi-dose regime may be determined by first determining an optimal single-dose concentration of the B cells for a subject, decreasing the number of B cells present in the optimal single-dose concentration to provide a sub-optimal single-dose concentration of the modified B cells, and administering two or more dosages of the sub-optimal single-dose concentration of modified B cells to the subject. In some aspects, 2, 3, or more dosages of a sub-optimal single-dose concentration of modified B cells are administered to the subject. In some aspects, the administration of 2, 3, or more dosages of a sub-optimal single-dose 311376670Attorney Docket: IMCO-013 / 001WO concentration of modified B cells to a subject results in synergistic in vivo production of a therapeutic polypeptide that the modified B cells are engineered to express. In some aspects, the sub-optimal single-dose concentration comprises 1 / 2 or 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10 fold, or less than the optimal single-dose concentration. In some aspects, the therapeutic polypeptide is GLP-1 or GLP-1 fusion.

[0164] In some aspects of the present disclosure, lower numbers of the transfected B cells of the present disclosure, in the range of 106 / kilogram (106-1011per patient) may be administered. In certain embodiments, the B cells are administered at 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x107, 5 x 107, 1 x 108, 5 x 108, 5 x 109, 1 x 1010, 5 x 1010, 1 x 1011, 5 x 1011, or 1 x 1012cells to the subject. B cell compositions may be administered multiple times at dosages within these ranges. The cells may be autologous or heterologous (e.g., allogeneic) to the patient undergoing therapy. If desired, the treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines, and / or chemokines (e.g., GM-CSF, IL-4, IL-6, IL-13, IL-21, Flt3-L, RANTES, MIP1 , BAFF, etc.) as described herein to enhanceinduction of an immune response and engraftment of the infused B cells.

[0165] The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intrathecally, intramuscularly, intravenously, or intraperitoneally. The compositions described herein may be administered to a patient directly into the nervous system. In one embodiment, the B cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the B cell compositions as described herein are preferably administered by i.v. injection. The compositions of B cells may be injected directly into a tumor, lymph node, bone marrow or site of infection.

[0166] In yet another embodiment, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, 1990, Science 249:1527-1533; Sefton 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980; Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321 :574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, 1974, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.; Controlled Drug Bioavailability, Drug Product Design and Performance, 1984, Smolen and Ball (eds.), Wiley, New York; Ranger and Peppas, 1983; J. Macromol. Sci. Rev. Macromol. Chem. 23:61 ; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol.25:351 ; Howard et al., 1989, J. Neurosurg. 311376670Attorney Docket: IMCO-013 / 001WO 71 :105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Medical Applications of Controlled Release, 1984, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla., vol.2, pp.115-138).

[0167] The B cell compositions of the present disclosure may also be administered using any number of matrices. Matrices have been utilized for a number of years within the context of tissue engineering (see, e.g., Principles of Tissue Engineering (Lanza, Langer, and Chick (eds.)), 1997). The present disclosure utilizes such matrices within the novel context of acting as an artificial lymphoid organ to support and maintain the B cells. Accordingly, the present disclosure can utilize those matrix compositions and formulations which have demonstrated utility in tissue engineering. Accordingly, the type of matrix that may be used in the compositions, devices and methods of the disclosure is virtually limitless and may include both biological and synthetic matrices. In one particular example, the compositions and devices set forth by U.S. Patent Nos: 5,980,889; 5,913,998; 5,902,745; 5,843,069; 5,787,900; or 5,626,561 are utilized. Matrices comprise features commonly associated with being biocompatible when administered to a mammalian host. Matrices may be formed from natural and / or synthetic materials. The matrices may be nonbiodegradable in instances where it is desirable to leave permanent structures or removable structures in the body of an animal, such as an implant; or biodegradable. The matrices may take the form of sponges, implants, tubes, telfa pads, fibers, hollow fibers, lyophilized components, gels, powders, porous compositions, or nanoparticles. In addition, matrices can be designed to allow for sustained release seeded cells or produced cytokine or other active agent. In certain embodiments, the matrix of the present disclosure is flexible and elastic, and may be described as a semisolid scaffold that is permeable to substances such as inorganic salts, aqueous fluids and dissolved gaseous agents including oxygen.

[0168] A matrix is used herein as an example of a biocompatible substance. However, the current disclosure is not limited to matrices and thus, wherever the term matrix or matrices appears these terms should be read to include devices and other substances which allow for cellular retention or cellular traversal, are biocompatible, and are capable of allowing traversal of macromolecules either directly through the substance such that the substance itself is a semi- permeable membrane or used in conjunction with a particular semi-permeable substance.

[0169] In certain embodiments of the present disclosure, B cells transfected and activated using the methods described herein, or other methods known in the art, are administered to a patient in conjunction with (e.g. before, simultaneously or following) any number of relevant treatment 311376670Attorney Docket: IMCO-013 / 001WO modalities, including but not limited to treatment with agents such as antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, busulfan, bortezomib, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506), the proteasome (bortezomib), or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun.5:763-773, 1993; Isoniemi (supra)). In a further embodiment, the cell compositions of the present disclosure are administered to a patient in conjunction with (e.g. before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In one embodiment, the cell compositions of the present disclosure are administered following B-cell ablative therapy such as agents that react with CD20, e.g. Rituxan®. In one embodiment, the cell compositions of the present disclosure are administered following B cell ablative therapy using an agent such as bortezomib. For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present disclosure. In an additional embodiment, expanded cells are administered before or following surgery.

[0170] The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices.

[0171] The modified B cells can be used in the treatment or prevention of various metabolic diseases and disorders. In some embodiments, B cells modified to express GLP-1 or GLP-1 fusion are used in methods of treating diabetes (e.g., lowering or controlling blood glucose levels). In some embodiments, the disease is Type 1 diabetes. In some embodiments, the disease is Type 2 diabetes. In some embodiments, B cells modified to express GLP-1 or GLP- 1 fusion are used in methods of treating obesity (e.g., lowering or controlling body weight). In some embodiments, B cells modified to express GLP-1 or GLP-1 fusion and follistatin are used in methods of treating diabetes (e.g., lowering or controlling blood glucose levels). In some 311376670Attorney Docket: IMCO-013 / 001WO embodiments, B cells modified to express GLP-1 or GLP-1 fusion and follistatin are used in methods of treating obesity (e.g., lowering or controlling body weight). DEFINITIONS

[0172] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated. With regard to this specification, any time a definition of a term as defined herein, differs from a definition given for that same term in an incorporated reference, the definition explicitly defined herein is the correct definition of the term.

[0173] The words “a” and “an” denote one or more, unless specifically noted.

[0174] By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In any embodiment discussed in the context of a numerical value used in conjunction with the term “about,” it is specifically contemplated that the term about can be omitted.

[0175] A “composition” can comprise an active agent and a carrier, inert or active, e.g., a pharmaceutically acceptable carrier, diluent or excipient. In particular embodiments, the compositions are sterile, substantially free of endotoxins or non-toxic to recipients at the dosage or concentration employed.

[0176] Unless the context requires otherwise, throughout the present specification and claims, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open and inclusive sense, that is, as “including, but not limited to”.

[0177] By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0178] Reference throughout this specification to “biological activity” or “bioactivity” refers to any response induced in an in vitro assay or in a cell, tissue, organ, or organism, (e.g., an 311376670Attorney Docket: IMCO-013 / 001WO animal, or a mammal, or a human) as the result of administering any compound, agent, polypeptide, conjugate, pharmaceutical composition contemplated herein. Biological activity may refer to agonistic actions or antagonistic actions. The biological activity may be a beneficial effect; or the biological activity may not be beneficial, i.e. a toxicity. In some embodiments, biological activity will refer to the positive or negative effects that a drug or pharmaceutical composition has on a living subject, e.g., a mammal such as a human. Accordingly, the term “biologically active” is meant to describe any compound possessing biological activity, as herein described. Biological activity may be assessed by any appropriate means currently known to the skilled artisan. Such assays may be qualitative or quantitative. The skilled artisan will readily appreciate the need to employ different assays to assess the activity of different polypeptides; a task that is routine for the average researcher. Such assays are often easily implemented in a laboratory setting with little optimization requirements, and more often than not, commercial kits are available that provide simple, reliable, and reproducible readouts of biological activity for a wide range of polypeptides using various technologies common to most labs. When no such kits are available, ordinarily skilled researchers can easily design and optimize in-house bioactivity assays for target polypeptides without undue experimentation; as this is a routine aspect of the scientific process.

[0179] Reference to the term “e.g.” is intended to mean “e.g., but not limited to” and thus it should be understood that whatever follows is merely an example of a particular embodiment, but should in no way be construed as being a limiting example. Unless otherwise indicated, use of “e.g.” is intended to explicitly indicate that other embodiments have been contemplated and are encompassed by the present invention.

[0180] Reference throughout this specification to “embodiment” or “one embodiment” or “an embodiment” or “some embodiments” or “certain embodiments” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” or “in certain embodiments” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0181] An “increased” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 2.1, 2.2, 2.3, 2.4, etc.) an amount or 311376670Attorney Docket: IMCO-013 / 001WO level described herein. Similarly, a “decreased” or “reduced” or “lesser” amount is typically a “statistically significant” amount, and may include a decrease that is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.61.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 or more times (e.g., 100, 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7.1.8, etc.) an amount or level described herein.

[0182] The terms “in vitro”, “ex vivo”, and “in vivo” are intended herein to have their normal scientific meanings. Accordingly, e.g., “in vitro” is meant to refer to experiments or reactions that occur with isolated cellular components, such as, e.g., an enzymatic reaction performed in a test tube using an appropriate substrate, enzyme, donor, and optionally buffers / cofactors. “Ex vivo” is meant to refer to experiments or reactions carried out using functional organs or cells that have been removed from or propagated independently of an organism. “In vivo” is meant to refer to experiments or reactions that occur within a living organism in its normal intact state.

[0183] “Mammal” includes humans and both domestic animals such as laboratory animals and household pets, (e.g., cats, dogs, swine, cattle, sheep, goats, horses, and rabbits), and non- domestic animals such as wildlife and the like.

[0184] “Optional” or “optionally” means that the subsequently described event, or circumstances, may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.

[0185] “Pharmaceutical composition” refers to a formulation of a compound (e.g. a therapeutically useful polypeptide) and a medium generally accepted in the art for the delivery of the compound to an animal, e.g., humans. Such a medium may include any pharmaceutically acceptable carriers, diluents, or excipients therefore.

[0186] “Pharmaceutically effective excipients” and “pharmaceutically effective carriers” are well known to those of skill in the art, and methods for their preparation are also readily apparent to the skilled artisan. Such compositions, and methods for their preparation, may be found, e.g., in Remington’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995, incorporated herein).

[0187] The terms “polynucleotide,” “nucleotide,” “nucleotide sequence,” and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), 311376670Attorney Docket: IMCO-013 / 001WO transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single- and double-stranded forms of DNA and RNA. The nucleic acid or gene of interest may be any nucleic acid encoding a protein of interest.

[0188] The sequence of nucleotides may include non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0189] A “subject,” as used herein, includes any animal that exhibits a disease or symptom, or is at risk for exhibiting a disease or symptom, which can be treated with an agent of the invention. Suitable subjects include laboratory animals (such as mouse, rat, rabbit, or guinea pig), farm animals, and domestic animals or pets (such as a cat or dog). Non-human primates and, preferably, human patients, are included.

[0190] “Substantially” or “essentially” means of ample or considerable amount, quantity, size; nearly totally or completely; for instance, 95% or greater of some given quantity.

[0191] “Therapeutic agent” refers to any compound that, when administered to a subject, (e.g., preferably a mammal, more preferably a human), in a therapeutically effective amount is capable of effecting treatment of a disease or condition as defined below.

[0192] “Treating” or “treatment” as used herein covers the treatment of the disease or condition of interest in a subject, preferably a human, having the disease or condition of interest, and includes: (i) preventing or inhibiting the disease or condition from occurring in a subject, in particular, when such subject is predisposed to the condition but has not yet been diagnosed as having it; (ii) inhibiting the disease or condition, i.e., arresting its development; (iii) relieving the disease or condition, i.e., causing regression of the disease or condition; or (iv) relieving the symptoms resulting from the disease or condition. As used herein, the terms “disease,” “disorder,” and “condition” may be used interchangeably or may be different in that the particular malady, injury or condition may not have a known causative agent (so that etiology has not yet been worked out), and it is, therefore, not yet recognized as an injury or disease but only as an undesirable condition or syndrome, wherein a more or less specific set of symptoms have been identified by clinicians 311376670Attorney Docket: IMCO-013 / 001WO EXAMPLES

[0193] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations can be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); nt, nucleotide(s); and the like.

[0194] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art EXAMPLE 1: PRODUCTION OF MODIFIED B CELLS EXPRESSING GLP-1 OR GLP-1 FUSION TO GLP-1R NEUTRAL ANTIBODY

[0195] Sleeping Beauty transposon and transposase constructs for transposition and expression of human GLP-1 analog or human GLP-1 analog fusion to GLP-1R neutral antibody are generated. Transposons are assembled to achieve GLP-1 or GLP-1-GLP-1R (i.e., fusion protein) gene integration and expression in B cells. The EEK promoter, consisting of promoter and enhancer elements from the human immunoglobulin gene as well as other regulatory elements previously described, is used to achieve high level expression in B cells. To test for GLP-1 or GLP-1 fusion transposition and expression, human B cells are isolated from two separate donors and expanded in culture in B cell culture medium and incubated at 37° C. incubator with 5% CO2, electroporating on day 3 with pKT2 / EEK-FST344 plus mRNA encoding SB100x transposase. Cell lysates prepared post-electroporation contain significant increases in both the GLP-1 or GLP-1-GLP1R antibody fusion, compared to wild-type non- transfected cells, demonstrating the effectiveness of the SB transposon system to achieve high- level GLP-1 expression in expanded human B cells. 311376670Attorney Docket: IMCO-013 / 001WO EXAMPLE 2: PRODUCTION OF MODIFIED B CELLS EXPRESSING GLP-1 AND FOLLISTATIN OR GLP-1 FUSION TO GLP-1R NEUTRAL ANTIBODY AND FOLLISTATIN

[0196] Sleeping Beauty transposon and transposase constructs for transposition and expression of human GLP-1 analog and follistatin or human GLP-1 analog fusion to GLP-1R neutral antibody and follistatin are generated. Transposons are assembled to achieve GLP-1 or GLP- 1-GLP-1R and follistatin gene integration and expression in B cells. The EEK promoter, consisting of promoter and enhancer elements from the human immunoglobulin gene as well as other regulatory elements previously described, is used to achieve high level expression in B cells. In some embodiments, a ribosomal slipping sequence between the GLP-1 or GLP-1 fusions and the follistatin gene is used. In some embodiments, two separate transposons comprising the GLP-1 or GLP-1 fusion and follistatin, respectively was used. In some embodiments, a single transposon, wherein the single transposon comprises dual promoters to drive the expression of at least two genes (e.g., GLP-1 analog or GLP-1-fusion and follistatin) is used. To test for GLP-1 or GLP-1 fusion and follistatin transposition and expression, human B cells are isolated from two separate donors and expanded in culture in B cell culture medium and incubated at 37° C. incubator with 5% CO2, electroporating on day 3 with pKT2 / EEK- FST344 plus mRNA encoding SB100x transposase. Cell lysates prepared post-electroporation contain significant increases of GLP-1 and follistatin in one case, and in the other case, GLP- 1 fusion and follistatin, compared to wild-type non-transfected cells, demonstrating the effectiveness of the SB transposon system to achieve high-level GLP-1 or GLP-1 fusion and follistatin expression in expanded human B cells. EXAMPLE 3: IN VIVO PRODUCTION OF GLP-1 OR GLP-1 FUSION AND FOLLISTATIN

[0197] To determine whether B cells engineered according to the present disclosure can facilitate in vivo increases in GLP-1 or GLP-1 fusion and follistatin production to treat diabetic symptoms, wild-type mice and / or in mice that replicate the diabetes phenotype are injected with either: 1. GLP-1, or 2. GLP-1 fusion, or 3. GLP-1 and follistatin, or 311376670Attorney Docket: IMCO-013 / 001WO 4. GLP-1 fusion and follistatin modified B cells produced as described in Example 1 or Example 2.

[0198] Specifically, mice receive intravenous (tail vein) injections on day 0 of vehicle (500 l phosphate-buffered saline (PBS)) or human modified B cells (listed as 1-4 above) diluted in the vehicle (PBS) to 500 l. Additionally, mice are infused intraperitoneally (i.p.) on day 7 with primary autologous peripheral blood cells enriched for CD4+ T cells to provide support for the pKT2 / EEK-FST plus mRNA encoding SB100x transposase transposed B cells. An increase in all therapeutic proteins (i.e., proteins 1-4) in the plasma of wild-type mice and / or diabetic model mice treated with the B cells (listed as 1-4 above) is observed providing strong evidence for successful human B cell adoptive transfer.

[0199] To determine whether the modified B cells had any effect on treated mice, the blood glucose and / or weight of control or modified B cell-treated mice is monitored over time. Modified B cell mice demonstrate prolonged blood glucose level and / or body weight control. INCORPORATION BY REFERENCE

[0200] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. 311376670

Claims

Attorney Docket: IMCO-013 / 001WO WHAT IS CLAIMED IS:

1. A modified B cell comprising a polynucleotide capable of expressing one or more glucagon-like peptide-1(GLP-1) analogs. The modified B cell of claim 1, wherein the GLP-1 analogs comprise a sequence that is at least 95% similar to any one of SEQ ID NOs: 27-34.

3. The modified B cell of any one of claims 1-2, further comprising a polynucleotide capable of expressing human follistatin.

4. The modified B cell of any one of claims 1-3, further comprising a polynucleotide capable of expressing one or more of a human GLP-1 receptor (GLP-1R) neutral antibodies.

5. The modified B cell of claim 4, wherein the GLP-1R neutral antibody comprises at least one sequence that is at least 95% similar to any one of SEQ ID NOs: 1-21.

6. The modified B cell of any one of claims 1-5, further comprising a polynucleotide capable of expressing one or more of a human glucose-dependent insulinotropic polypeptide receptor (GIPR) antagonist antibody.

7. The modified B cell of any one of claims 1-6, further comprising a polynucleotide capable of expressing one or more of a human serum albumin (HSA) antibody or human serum albumin protein.

8. The modified B cell of any one of claims 4-7, wherein the at least one GLP-1 peptide analog is fused to the GLP-1R neutral antibody by a linker.

9. The modified B cell of claim 8, further comprising one or more of an HSA antibody, HSA, or transferrin. 311376670Attorney Docket: IMCO-013 / 001WO 10. The modified B cell of claim 8, wherein the at least one GLP-1 peptide analog is additionally fused to the N-terminus or C-terminus of the light chain of the GLP-1R neutral antibody by a linker.

11. The modified B cell of claim 10, wherein the at least one GLP-1 peptide analog is fused to the N-terminus of the light chain of the GLP-1R neutral antibody by a linker.

12. The modified B cell of claim 9, wherein the at least one GLP-1 peptide analog N- terminus or C-terminus is fused to the HSA antibody N-terminus or C-terminus by a linker or HSA N-terminus or C-terminus by a linker, wherein the HSA antibody N-terminus or C- terminus or HSA N-terminus or C-terminus is fused to the light chain of the GLP-1R neutral antibody N-terminus or C-terminus by a linker.

13. The modified B cell of any one of claims 1-12, wherein the polynucleotide comprises at least one promoter.

14. The modified B cell of any one of claims 3-13, wherein the follistatin is FST-344,FST-288, FST-317, FST-315- HBS-Fc splice site variant.

15. The modified B cell of claim 14, wherein the follistatin comprises any one of SEQ ID NOs: 23-26.

16. The modified B cell of any one of claims 8-14, wherein the linker is a peptide linker.

17. The modified B cell of any one of the proceeding claims, wherein the modified B cell has been transfected, transduced, or transposed with the polynucleotide.

18. The modified B cell of claim 17, wherein the modified B cell has been transposed using a transposon system.

19. The modified B cell of claim 18, wherein the transposon system is a Sleeping Beauty transposon system or a Piggyback transposon system.

20. The modified B cell of claim 17, wherein the polynucleotide is integrated into the B cell genome by one or more zinc finger nuclease, transcription activator-like effector 311376670Attorney Docket: IMCO-013 / 001WO nucleases (TALENs), and / or CRISPR / Cas systems including, but not limited to CRISPR / Cas9 systems.

21. The modified B cell of claim 17, wherein the polynucleotide is delivered by using a method selected from the group consisting of retroviral vectors, lentiviral vectors, adeno- associated virus vectors, adenovirus vectors, any other RNA or DNA virus vectors, non-viral DNA and / or RNA introduced using chemical or physical means such and lipofection, polycation complexation, and electroporation. The modified B cell of any one of the proceeding claims, wherein any one of the GLP-1 or fusion thereof, the follistatin, or the GIPR is secreted by the modified B cell.

23. A method of manufacturing a modified B cell of any one of claims 1-22, the method comprising: 1) collecting and isolating immune cells from the blood of the subject; 2) transfecting, transducing, or transposing the cells with DNA; 3) expanding selected cells ex vivo; and 4) differentiating the expanded cells ex vivo into plasma cells and / or plasmablasts.

24. A method treating a human disease or disorder comprising administering a therapeutically effective amount to a subject in need thereof the modified B cells of any one of claims 1-22.

25. The method of claim 24, wherein the disease or disorder is a metabolic disease or disorder.

26. The method of claim 25, wherein the metabolic disease or disorder is one or more of diabetes or obesity.

27. The method of any one of claims 24-26, wherein the method comprises administering two or more sequential doses of modified B cells to a subject.

28. The method of any claim 27, wherein the method comprises two or more doses of the modified B cells at sub-optimal single-dose concentrations. 311376670Attorney Docket: IMCO-013 / 001WO 29. The method of any one of claims 24-28, wherein the administering comprises intravenous, intraperitoneal, subcutaneous, intrathecal, intracameral or intramuscular injection.

30. The method of any one of claims 24-29 wherein the subject is an adult.

31. The method of any one of claims 24-29 , wherein the subject is a juvenile. 311376670