Methods for expanding B cells for use in cell therapy

Abstract

The invention disclosed herein relates to improved methods for expanding cell populations, particularly B cell populations. The invention further relates to improved B cell expansion media, compositions comprising expanded B cells, and methods of using such expanded B cells. The invention further relates to a method of treating a disease or disorder, in which a population of B cells is obtained and cultured, the B cells are engineered to express a payload and / or a chimeric receptor, and the B cells are administered to a subject.

Description

[Background technology]

[0001] B cells are immune cells that are responsible for a variety of functions, including helping the body fight infection and disease. They can secrete antibodies in response to recognized antigens, present antigens, and secrete cytokines. In cancer, B cells have been found in tertiary lymphoid structures ("TLS") that surround certain tumors. TLS contain aggregates of immune cells, including both T cells and B cells. Their presence in tumors is associated with good patient outcomes. See, for example, Helmink, BA, et al., Nature, 2020, 577(7791), 549-555; Petitprez F et al., Nature, 2020, 577(7791), 556-560.

[0002] Intratumoral injection of LPS-activated splenocytes containing B cells combined with checkpoint inhibitors has been shown to generate antitumor responses. Soldevilla et al., Oncoimmunology, 2018, 7:8, e1450711. Furthermore, given the natural ability of B cells to present antigens and secrete proteins, they have great potential as cell therapy to target certain disease cell types and secrete therapeutic payloads.

[0003] However, scaling the production of engineered B cells for cell therapy is a challenging process. For decades, in vitro B cell activation and expansion has been achieved via feeder cell layer systems expressing CD40-ligand ("CD40L"). Banchereau, J. et al. (1991) Science 251:70-72; Schultze, J Let al. (1997) J. Clin. Invest. 100:2757-2765; Liebig, TM et al. (2009) J. Vis. Exp. 32:1373. These systems share a common drawback, which is the dependency on a CD40L-expressing feeder cell layer. The use of feeder cells prevents standardization of the activation and expansion process and introduces variables into the protocol. B cell expansion has been described using CD40L with mixed results. See, e.g., Wennhold et al., 2019, Transfus Med Hemother, 46:36-46. Some methods result in low expansion yields, producing insufficient amounts of B cells for cell engineering and for using B cells as therapeutics for administration to patients. Thus, there is a need for improved B cell expansion techniques, growth media, and the like. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Helmink,BA,et al.,Nature,2020,577(7791),549-555 [Non-Patent Document 2] Petitprez F et al.,Nature,2020,577(7791),556-560 [Non-Patent Document 3] Soldevilla et al.,Oncoimmunology,2018,7:8,e1450711 [Non-Patent Document 4] Banchereau, J. et al. (1991) Science 251:70-72 [Non-Patent Document 5] Schultze, J Let al. (1997) J. Clin. Invest. 100:2757-2765 [Non-Patent Document 6] Liebig, TMet al. (2009) J.Vis.Exp.32:1373 [Non-Patent Document 7] Wennhold et al.,2019,Transfus Med Hemother,46:36-46 Summary of the Invention [Means for solving the problem]

[0005] The present invention generally provides improved methods for expanding cell populations, particularly B cell populations. The invention further relates to improved cell culture media, compositions thereof, and methods of using such expanded B cells. The novel methods incorporate the use of novel CD40L fusion proteins, cross-linking antibodies, and IL-4 and / or IL-21. Such methods are shown herein to be important for efficient activation and proliferation of engineered B cells, resulting in a 200-fold increase in the desired levels of functionally expanded B cells.

[0006] The methods provided herein for expanding B cells are superior to conventional methods in that previous conventional expansion methods resulted in undesirably low cell expansion and therefore reduced yield of engineered B cells. Proper cell function and yield are important in cell therapy for allogeneic therapy, especially in an autologous treatment environment where there is often only a single opportunity to harvest, culture, expand a patient's cells, and engineer and administer an effective dose of such cells.

[0007] In various embodiments, the present invention relates to a method of treating a disease or disorder in a subject in need thereof, comprising obtaining a population of B cells from a source, culturing the B cells in a culture medium comprising a CD40L fusion protein and a CD40L cross-linking agent, engineering the B cells to express either a payload, a chimeric receptor, or both, and administering the B cells to the subject. In various embodiments, the source is a mammal. In various embodiments, the source is a biological sample comprising peripheral mononuclear blood cells.

[0008] In various embodiments, the CD40L fusion protein comprises an amino acid sequence at least 85% identical to SEQ ID NO:3. In various embodiments, CD40L comprises an amino acid sequence at least 95% identical to SEQ ID NO:3. In various embodiments, the CD40L fusion protein comprises the amino acid sequence of the fusion protein of SEQ ID NO:3. In various embodiments, the CD40L crosslinking agent is an antibody. In various embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence at least 95% identical to SEQ ID NO:5 and a heavy chain variable region comprising an amino acid sequence at least 95% identical to SEQ ID NO:7. In various embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:5 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7.

[0009] In various embodiments, the B cells are manipulated prior to culturing the B cells in medium comprising a CD40L fusion protein and a CD40L crosslinking agent. In various embodiments, the B cells are manipulated after culturing the B cells in medium comprising a CD40L fusion protein and a CD40L crosslinking agent. In various embodiments, the method comprises further culturing the B cells in the presence of IL-4. In various embodiments, the method comprises further culturing the B cells in the presence of IL-21.

[0010] In various embodiments, the cultured B cells express at least one of the following markers: CD62L, CCR7, CD80, CD86, CD54, ICAM, CD58, or CD27. In various embodiments, the disease or disorder is selected from the group consisting of at least one of cancer, cardiac disease, inflammatory disease, muscle wasting disease, or neurological disease. In various embodiments, the cancer is at least one of breast cancer, colon cancer, rectal cancer, esophageal cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, hepatocellular carcinoma, stromal tumors such as GIST, glioblastoma, and glioma. ... about 3×10 7 The population of B cells is administered to the subject. In various embodiments, the population of B cells is cultured for at least 14 days.

[0011] In various embodiments, the invention relates to a method of treating a disease or disorder in a subject in need of such treatment, comprising obtaining a population of B cells from a source; culturing the B cells in a culture medium comprising a CD40L fusion protein comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:3 and a CD40L cross-linking antibody having a light chain variable region at least 95% identical to the amino acid sequence of SEQ ID NO:5 and a heavy chain variable region at least 95% identical to the amino acid sequence of SEQ ID NO:7; and administering the B cells to the subject.

[0012] In various embodiments, the source is a mammal. In various embodiments, the CD40L fusion protein comprises the amino acid sequence of SEQ ID NO: 3. In various embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7. In various embodiments, the invention further comprises engineering the B cells to express either a payload, a chimeric receptor, or both.

[0013] In various embodiments, the B cells are manipulated prior to culturing the B cells in medium comprising CD40L and a CD40L cross-linking antibody. In various embodiments, the B cells are manipulated after culturing the B cells in medium comprising CD40L and a CD40L cross-linking antibody. In various embodiments, the invention further comprises culturing the B cells in the presence of IL-4. In various embodiments, the invention further comprises culturing the B cells in the presence of IL-21. In various embodiments, the cultured B cells express at least one of the following markers: CD62L, CCR7, CD80, CD86, CD54, ICAM, CD58, or CD27. In various embodiments, the disease or disorder is selected from the group consisting of at least one of cancer, cardiac disease, inflammatory disease, muscle wasting disease, or neurological disease. In various embodiments, the cancer is at least one of breast cancer, colon cancer, rectal cancer, esophageal cancer, lung cancer, pancreatic cancer, gastric cancer, liver cancer, hepatocellular carcinoma, stromal tumors such as GIST, glioblastoma, and glioma. 7 The population of B cells is administered to the subject. In various embodiments, the population of B cells is cultured for at least 14 days.

[0014] In various embodiments, the invention relates to a method for producing engineered B cells, comprising obtaining a population of B cells from a source, culturing the B cells in a culture medium comprising a CD40L fusion protein and a CD40L cross-linking agent, and engineering the B cells to express either a payload, a chimeric receptor, or both. In various embodiments, the source is a mammal. In various embodiments, the source is a biological sample comprising peripheral mononuclear blood cells.

[0015] In various embodiments, the CD40L fusion protein comprises an amino acid sequence at least 85% identical to SEQ ID NO:3. In various embodiments, CD40L comprises an amino acid sequence at least 95% identical to SEQ ID NO:3. In various embodiments, the CD40L fusion protein comprises the amino acid sequence of the fusion protein of SEQ ID NO:3. In various embodiments, the CD40L crosslinking agent is an antibody. In various embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence at least 95% identical to SEQ ID NO:5 and a heavy chain variable region comprising an amino acid sequence at least 95% identical to SEQ ID NO:7. In various embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:5 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7.

[0016] In various embodiments, the B cells are manipulated prior to culturing the B cells in medium comprising CD40L and a CD40L crosslinking agent. In various embodiments, the B cells are manipulated after culturing the B cells in medium comprising CD40L and a CD40L crosslinking agent. In various embodiments, the method comprises further culturing the B cells in the presence of IL-4. In various embodiments, the method comprises further culturing the B cells in the presence of IL-21. In various embodiments, the cultured B cells express at least one of the following markers: CD62L, CCR7, CD80, CD86, CD54, ICAM, CD58, or CD27. In various embodiments, the source is a mammal. In various embodiments, the source is a biological sample comprising peripheral mononuclear blood cells.

[0017] In various embodiments, the CD40L fusion protein comprises an amino acid sequence at least 85% identical to SEQ ID NO:3. In various embodiments, CD40L comprises an amino acid sequence at least 95% identical to SEQ ID NO:3. In various embodiments, the CD40L fusion protein comprises the amino acid sequence of the fusion protein of SEQ ID NO:3. In various embodiments, the CD40L crosslinking agent is an antibody. In various embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence at least 95% identical to SEQ ID NO:5 and a heavy chain variable region comprising an amino acid sequence at least 95% identical to SEQ ID NO:7. In various embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:5 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7.

[0018] In various embodiments, the method further comprises culturing the B cells in the presence of IL-4. In various embodiments, the method further comprises culturing the B cells in the presence of IL-21. In various embodiments, the cultured B cells express at least one of the following markers: CD62L, CCR7, CD80, CD86, CD54, ICAM, CD58, or CD27. In various embodiments, at least about 3×10 7 A population of B cells is obtained. In various embodiments, the population of B cells is cultured for at least 14 days.

[0019] In various embodiments, a method of producing engineered B cells is provided, the method comprising obtaining a population of B cells from a source, culturing the B cells in a culture medium comprising CD40L, the CD40L fusion protein comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:3 and a CD40L cross-linking antibody, the light chain variable region of which is at least 95% identical to the amino acid sequence of SEQ ID NO:5 and the heavy chain variable region of which is at least 95% identical to the amino acid sequence of SEQ ID NO:7, and administering the B cells to the subject. In various embodiments, the source is a mammal. In various embodiments, the CD40L fusion protein comprises the amino acid sequence of SEQ ID NO:3. In various embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:7.

[0020] In various embodiments, the method further comprises engineering the B cells to express either a payload, a chimeric receptor, or both. In various embodiments, the B cells are engineered prior to culturing the B cells in medium comprising CD40L and a CD40L crosslinking agent. In various embodiments, the B cells are engineered after culturing the B cells in medium comprising CD40L and a CD40L crosslinking agent. In various embodiments, the method further comprises culturing the B cells in the presence of IL-4. In various embodiments, the method further comprises culturing the B cells in the presence of IL-21. In various embodiments, the cultured B cells express at least one of the following markers: CD62L, CCR7, CD80, CD86, CD54, ICAM, CD58, or CD27. In various embodiments, at least about 3×10 7 A population of B cells is obtained. In various embodiments, the population of B cells is cultured for at least 14 days.

[0021] In various embodiments, the invention relates to a B cell expansion medium comprising a CD40L fusion protein comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO:3 and a CD40L cross-linking antibody having a light chain variable region at least 95% identical to the amino acid sequence of SEQ ID NO:5 and a heavy chain variable region at least 95% identical to the amino acid sequence of SEQ ID NO:7, comprising administering to the subject the B cells.

[0022] In various embodiments, the CD40L fusion protein comprises the amino acid sequence of SEQ ID NO: 3. In various embodiments, the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7. In various embodiments, the medium further comprises IL-4. In various embodiments, the medium further comprises IL-21. [Brief description of the drawings]

[0023] [Figure 1] Figure 1 shows that human B cells can grow and expand in medium containing HELA-CD40 feeder cells, but not in the presence of MEGA-CD40L (Enzo Life Sciences). Purified B cells were started at an equivalent density of approximately 10,000 cells / ml on day 0 and grown on HELA-CD40L feeder cells. On day 4, the cultures were split into two groups, one group continued to grow in the same medium as HELA-CD40L feeder cells, while the other group was grown in medium supplemented with MEGA-CD40L (0.1 μg / ml) but in the absence of HELA feeder cells. On day 8, an additional 100 ng / ml of MEGA-CD40L was added to the growth medium under MEGA-CD40L conditions. The arrows indicate the time at which MEGA-CD40L treatment was administered. This data shows that by day 11 there was significant proliferation of B cells when grown on HELA-CD40L feeder cells, but no significant proliferation was observed at day 11 when grown on MEGA-CD40L. [Diagram 2]We show that human cells can grow and expand in medium containing CD40L and CD40L crosslinker, even in the absence of HELA-CD40L feeder cells, to at least about 3 x 107 cells after only 2 weeks. Two densities (150K and 1MM) were maintained throughout the course of the study. The lower density culture was able to sustain a higher growth rate and gave a larger relative total mass. In only 2 weeks, the cells could be expanded at least 200-fold. [Diagram 3] We show that expanded engineered B cells can maintain transgene expression. Figure 3A shows that 67% of expanded engineered B cells showed GPC B cell receptor expression 72 hours after adenoviral vector transfection. Figure 3B shows the percentage of GPC-BCR expressing cells in cells transfected with an empty vector (which did not express GPC3). Figure 3C shows cells that were not transduced with any vector at all. [Figure 4] Figure 1 shows the expansion and growth kinetics of B cells after Ad5RGD-GFP transduction. Ad5RGD-GFP resulted in high efficiency of GFP expression in B cells. Expression was maintained for at least 8-10 days in approximately 60% of all B cells. GFP+ B cells continued to proliferate for more than one week after transduction. [Diagram 5] Figure 5 shows successful expansion of human B cells transduced with mouse GPC3 construct (601) via RGD. Figure 5A shows the structure of the transduced anti-GPC3 scFv chimeric receptor, which contains anti-GPC3 scFv, CD8 hinge domain, CD28 transmembrane domain, and CD79a signaling domain. Figure 5B shows the relative percentage of B cells expressing the transduced GPC3 chimeric receptor. [Figure 6] Figure 6 shows the successful expansion of human B cells transduced with mouse GPC3 construct (602) via RGD. Figure 6A shows the structure of the transduced anti-GPC3 scFv chimeric receptor, which contains anti-GPC3 scFv, CD8 hinge domain, CD28 transmembrane domain, and CD79b signaling domain. Figure 6B shows the relative percentage of B cells expressing the transduced GPC3 chimeric receptor. [Figure 7] Figure 7 shows the expansion of human B cells transduced with RGD-functionalized non-viral gene delivery vector expressing chimeric receptor targeting GPC3. Figure 7A shows the structure of this chimeric receptor, which includes anti-GPC3 scFv, CD8 hinge domain, CD28 transmembrane domain, and CD79b or CD79a signaling domain. Figure 7B shows the relative percentage of B cells expressing transduced GPC3 chimeric receptor. [Figure 8] Figure 8 shows successful expansion of human B cells transduced with mouse GPC3 construct (463) via RGD. Figure 8A shows the structure of the transduced anti-GPC3 scFv chimeric receptor, which contains anti-GPC3 scFv, CD8 hinge domain, CD28 transmembrane domain, and CD79b signaling domain. Figure 8B shows the relative percentage of B cells expressing the transduced GPC3 chimeric receptor. [Figure 9] Figure 9 shows the expansion of human B cells transduced with an RGD-functionalized non-viral gene delivery vector (394) expressing a chimeric receptor targeting sarcoglycan. Figure 9A shows the structure of this chimeric receptor, which contains an anti-sarcoglycan scFv, a mouse G2a Fc domain, a transmembrane domain, and a cytoplasmic tail. Figure 9B shows the percentage of cells shown to express the chimeric receptor after transduction and expansion of B cells. [Figure 10] 1 shows in vivo homing of engineered and expanded human B cells to tumor-draining lymph nodes ("TDLN") in HPEG2 tumor-bearing mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] It is to be understood that the descriptions herein are merely exemplary and explanatory and are not intended to limit the invention as claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise.

[0025] I. Overview It should be understood that the present invention relates to methods for producing, expanding and / or isolating a population of engineered B cells. For example, the methods can be used to produce a variety of engineered B cells, such as those described in U.S. Provisional Patent Application No. 63 / 073799, filed September 2, 2020, and U.S. Provisional Patent Application No. 63 / 003120, filed March 31, 2020. These include, but are not limited to: 1) B cells that have been modified to home to a site / target of interest using a binding domain, e.g., an scFv, antibody, ligand, receptor, or fragment thereof; 2) B cells modified with a homing domain, further comprising an activation and optionally a costimulatory domain, such that the B cells can home to and thereby become activated upon interaction with a desired target; 3) B cells engineered to produce a desired protein payload, such as an antibody, therapeutic protein, polypeptide, nucleic acid sequence (e.g., RNAi), etc. 4) engineered B cells containing a homing / binding domain, an activation domain, and optionally a co-stimulatory domain, which are further engineered to express a desired protein payload, such as an antibody, a therapeutic protein, a polypeptide, a nucleic acid sequence (such as RNAi), etc. 5) B cells that have been modified to express integrins, homing antibodies, proteins, or receptors such that the B cells are attracted to specific ligands, chemokines, or attractants at a particular site / target of interest (e.g., a homing tissue) and thereby can home to the site / target of interest, e.g., to deliver a desired payload; 6) B cells that have been modified to express immune inhibitory molecules such that inflammatory and autoimmune activity of B cells localized to the desired site / target is reduced, thereby resulting in a positive therapeutic response; 7) B cells that have been treated with a compound or a derivative thereof such that B cell trafficking is altered by expression of specific B cell integrins and / or homing receptors; 8) (i) B cells that have been treated with a Toll-like receptor (TLR) agonist, and / or (ii) B cells engineered to express a constitutively active TLR to boost B cells and / or generate potent effector B cells to increase an immune response in a subject; 9) B cells electroporated with mRNA encoding a specific antigen of interest fused to a lysosomal protein targeting signal such that the B cells can simultaneously and efficiently present the specific antigen of interest and / or antigen-derived epitopes on both HLA class I and class II molecules. 10) B cells electroporated with self-amplified RNA encoding any of the items listed in parts 1-9 above.

[0026] It will be understood that the various embodiments of the engineered or modified B cells of the present application are not mutually exclusive and can be combined with each other in any manner and without any limitation, unless expressly indicated, to achieve any of the enhancing results and / or therapeutic responses contemplated herein.

[0027] More specifically, the method can be used as a manufacturing technique to produce improved expansion of B cells in the production of the following B cell embodiments, which are described in U.S. Provisional Patent Application No. 63 / 073799, filed September 2, 2020, and U.S. Provisional Patent Application No. 63 / 003120, filed March 31, 2020, the contents of which are incorporated herein by reference in their entireties. Certain methods for making the constructs and engineered immune cells of the invention are described in PCT application PCT / US2015 / 14520, the contents of which are incorporated herein by reference in their entireties. Further methods of making constructs and cells can be found in U.S. Provisional Patent Application No. 62 / 244,036, the contents of which are further incorporated herein by reference in their entireties.

[0028] The present invention also relates to methods of treating a disease or disorder using the engineered B cells produced by the methods described herein. Examples of diseases or disorders suitable for treatment include, but are not limited to, cancer, heart disease, inflammatory diseases, muscle wasting diseases, neurological diseases, etc.

[0029] II. Definition The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated herein by reference in their entirety for any purpose. When used in accordance with this disclosure, the following terms shall be understood to have the following meanings, unless otherwise indicated:

[0030] In this application, the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "including" and other forms such as "includes" and "included" is not limiting. Also, terms such as "element" or "component" encompass both elements and components that contain one unit and elements and components that contain more than one subunit, unless otherwise specified.

[0031] The terms "polynucleotide," "nucleotide," or "nucleic acid" include both single-stranded and double-stranded nucleotide polymers. The nucleotides that comprise a polynucleotide can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. Such modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and modifications of internucleotide linkages such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphoro-diselenoates, phosphoro-anilothioates, phosphoraniladates, and the like.

[0032] The term "oligonucleotide" refers to a polynucleotide containing 200 or less nucleotides. Oligonucleotides can be single-stranded or double-stranded, for example, for use in constructing mutant genes. Oligonucleotides can be sense or antisense oligonucleotides. For detection assays, oligonucleotides can include labels, including radioactive labels, fluorescent labels, hapten or antigen labels. Oligonucleotides can be used, for example, as PCR primers, cloning primers, or hybridization probes.

[0033] The term "control sequence" refers to a polynucleotide sequence capable of influencing the expression and processing of coding sequences to which it is ligated. The nature of such control sequences may depend on the host organism. In certain embodiments, control sequences for prokaryotes may include a promoter, a ribosomal binding site, and a transcription termination sequence. For example, control sequences for eukaryotes may include a promoter containing one or more recognition sites for transcription factors, a transcription enhancer sequence, and a transcription termination sequence. A "control sequence" may include a leader sequence (signal peptide) and / or a fusion partner sequence.

[0034] As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to carry out their inherent functions under suitable conditions.

[0035] The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) used to transfer protein coding information to a host cell. The term "expression vector" or "expression construct" refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control (in conjunction with the host cell) the expression of one or more heterologous coding regions operably linked thereto. Expression constructs can include, but are not limited to, sequences that affect the transcription, translation, and, when introns are present, RNA splicing of the coding regions operably linked thereto.

[0036] The term "host cell" refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest. The term includes the progeny of a parent cell, whether or not the progeny is morphologically identical or identical in genetic make-up to the original parent cell, so long as the gene of interest is present.

[0037] The term "transformation" refers to a change in the genetic characteristics of a cell; a cell has been transformed if it has been modified to contain new DNA or RNA. For example, a cell is transformed when it is genetically modified from its native state by the introduction of new genetic material via transfection, transduction, or other techniques. After transfection or transduction, the transforming DNA may recombine with that of the cell by being physically integrated into the cell's chromosome, or it may be maintained transiently as an episomal element without being replicated, or it may replicate independently as a plasmid. If the transforming DNA is replicated following cell division, the cell is considered to be "stably transformed."

[0038] The term "transfection" refers to the uptake of foreign or exogenous DNA by a cell. Several transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al., 1973, Virology, 1973, 52:456; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2001, supra; Davis et al., Basic Methods in Molecular Biology, 1986, Elsevier; Chu et al., 1981, Gene, 13:197.

[0039] The term "transduction" refers to the process by which foreign DNA is introduced into a cell via a viral vector. See, e.g., Jones et al., Genetics: Principles and Analysis, 1998, Boston: Jones & Bartlett Publ.

[0040] The term "polypeptide" or "protein" refers to a macromolecule having the amino acid sequence of a protein, including deletions, additions, and / or substitutions of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically encompass antigen-binding molecules, antibodies, or sequences having deletions, additions, and / or substitutions of one or more amino acids of an antigen-binding protein. The term "polypeptide fragment" refers to a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion compared to the full-length native protein. Such fragments may also contain modified amino acids compared to the native protein. Useful polypeptide fragments include immunologically functional fragments of antigen-binding molecules.

[0041] The term "isolated" means: (i) free from at least some other proteins with which it is normally found; (ii) essentially free from other proteins from the same source, e.g., the same species; (iii) separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated; (iv) operably associated (by covalent or non-covalent interactions) with polypeptides with which it is not naturally associated; or (v) not occurring in nature.

[0042] A "variant" of a polypeptide (e.g., an antigen-binding molecule) includes an amino acid sequence in which one or more amino acid residues are inserted into, deleted from, and / or substituted into the amino acid sequence compared to another polypeptide sequence. Variants include fusion proteins.

[0043] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by alignment and comparison of the sequences. "Percent identity" refers to the percent of identical residues between amino acids or nucleotides in the compared molecules, and is calculated based on the minimum size of the molecules being compared. For these calculations, gaps in the alignment (if any) are preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm").

[0044] To calculate percent identity, the sequences to be compared are typically aligned to give the maximum match between sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., Nucl. Acid Res., 1984, 12, 387, Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align two polypeptides or polynucleotides for which percent sequence identity is to be determined. Sequences are aligned for the best match of their respective amino acids or nucleotides (the "match span" determined by the algorithm). In certain embodiments, standard comparison matrices (e.g., for the PAM 250 comparison matrix, see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure, 5:345-352; for the BLO-SUM 62 comparison matrix, see Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10915-10919) are also used by the algorithm.

[0045] As used herein, the twenty conventional (e.g., naturally occurring) amino acids and their abbreviations follow conventional usage. See, e.g., Immunology A Synthesis (2nd Edition, Golub and Green, Eds., Sinauer Assoc., Sunderland, Mass. (1991)), which is incorporated herein by reference for any purpose. Stereoisomers of the twenty conventional amino acids, non-natural amino acids such as alpha-, alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other non-conventional amino acids (e.g., D-amino acids) may also be suitable components for the polypeptides of the invention. Examples of unconventional amino acids include 4-hydroxyproline, gamma-carboxy-glutamate, epsilon-N,N,N-trimethyllysine, eN-acetyl-lysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, sigma-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0046] Conservative amino acid substitutions may include non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reverse or inverted amino acid moieties. Naturally occurring residues can be divided into classes based on common side chain properties: a) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; b) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c) Acidic: Asp, Glu; d) Basic: His, Lys, Arg; e) Residues that influence chain orientation: Gly, Pro; and f) Aromatics: Trp, Tyr, Phe.

[0047] For example, non-conservative substitutions may involve the exchange of a member of one of these classes for a member of another class.

[0048] According to certain embodiments, when making modifications to an antigen binding molecule, costimulatory or activation domain of an engineered T cell, the hydropathic index of amino acids may be considered. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamate (-3.5), glutamine (-3.5), aspartate (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5). See, e.g., Kyte et al., 1982, J. Mol. Biol., 157, 105-131. It is known that certain amino acids can be replaced with other amino acids having similar hydropathic indexes or scores and still retain similar biological activity. It is also understood in the art that substitutions of similar amino acids based on hydrophilicity can also be effectively made, especially when, as in this case, the biologically functional equivalent protein or peptide produced by the substitution is intended for use in immunological embodiments. Exemplary amino acid substitutions are shown in Table 1. [Table 1]

[0049] The term "derivative" refers to a molecule that contains a chemical modification other than an amino acid (or nucleic acid) insertion, deletion, or substitution. In certain embodiments, a derivative contains a covalent modification, including but not limited to, chemical conjugation with a polymer, lipid, or other organic or inorganic moiety. In certain embodiments, a chemically modified antigen-binding molecule can have a longer circulating half-life than an antigen-binding molecule that is not chemically modified. In some embodiments, a derivative antigen-binding molecule is covalently modified to contain one or more water-soluble polymer linkages, including but not limited to polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

[0050] Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of the template peptide. These types of non-peptide compounds are called "peptide mimetics" or "peptidomimetics." Fauchere, JL, 1986, Adv. Drug Res., 1986, 15, 29; Veber, DF & Freidinger, RM, 1985, Trends in Neuroscience, 8, 392-396; and Evans, BE, et al., 1987, J. Med. Chem., 30, 1229-1239, which are incorporated herein by reference for any purpose.

[0051] The term "therapeutically effective amount" refers to the amount of immune cells or other therapeutic agent that is determined to produce a therapeutic response in a mammal. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art.

[0052] The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects, as well as subjects with a formally diagnosed disorder, subjects without a formally recognized disorder, subjects receiving medical treatment, subjects at risk for developing a disorder, and the like.

[0053] The terms "treat" and "treatment" include therapeutic treatment, prophylactic treatment, and applications in which a subject reduces the risk of developing a disorder or other risk factor. Treatment does not require a complete cure of the disorder, but encompasses embodiments in which symptoms or underlying risk factors are reduced. The term "prevent" does not require 100% elimination of the likelihood of an event. Rather, it indicates that the likelihood of an event occurring is reduced in the presence of a compound or method.

[0054] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for any purpose.

[0055] As used herein, the term "substantially" or "essentially" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% higher than a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "essentially the same" or "substantially the same" refers to a range that is about the same as a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length, and refers to a range of amounts, levels, values, numbers, frequencies, percentages, dimensions, sizes, amounts, weights, or lengths.

[0056] As used herein, the terms "substantially free" and "essentially free" are used interchangeably and, when used to describe a composition such as a cell population or culture medium, refer to a composition that does not contain the specified substance, e.g., 95% free, 96% free, 97% free, 98% free, 99% free, or undetectable as measured by conventional means of the specified substance. A similar meaning can be applied to the term "absence of," which refers to the absence of a particular substance or component of a composition.

[0057] As used herein, the term "appreciable" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range, or an event that is readily detectable by one or more standard methods. The terms "not-appreciable" and "not appreciable" and equivalents refer to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length range, or an event that is readily detectable or undetectable by standard methods. In one embodiment, an event is not appreciable if it occurs 5%, 4%, 3%, 2%, 1%, 0.1%, 0.001% or less of the time.

[0058] Throughout this specification, unless the context otherwise requires, the words "comprise", "comprises", and "comprising" are understood to imply the inclusion of the stated step or element, or group of steps or elements, but not the exclusion of any other step or element, or group of steps or elements. In certain embodiments, the terms "include", "has", "contains", and "comprise" are used interchangeably.

[0059] As used herein, "consisting of" is meant to include, and is limited to, what 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.

[0060] "Consisting essentially of" means including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but other elements are optional and may or may not be present depending on whether the other elements materially affect the activity or function of the recited elements.

[0061] Throughout this specification, reference to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, the appearances of such phrases 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.

[0062] As used herein, the term "about" or "approximately" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In certain embodiments, the term "about" or "approximately" when preceding a numerical value indicates a range of the value plus or minus 15%, 10%, 5%, or 1%, or any intervening range therein.

[0063] As used herein, the term "introducing" refers to a process that includes contacting a cell with a polynucleotide, a polypeptide, or a small molecule. The introducing step may also include microinjecting the polynucleotide or polypeptide into the cell, using liposomes to deliver the polynucleotide or polypeptide into the cell, or fusing the polynucleotide or polypeptide to a cell-permeable moiety to introduce them into the cell.

[0064] Methods for expanding populations of B lymphocytes for cell therapy

[0065] In various embodiments, improved methods of expanding engineered B lymphocyte populations are contemplated. The methods involve cross-linking of CD40 expressed on B cells via cell culture media containing CD40 ligand and cross-linking antibodies. Combining CD40L with IL-4, a known growth factor for activated B cells, has been shown to be important for effective activation and proliferation of B cells. Banchereau, J. et al. (1991) Science 251:70-72; Schultze, J. Let al. (1997) J. Clin. Invest. 100:2757-2765. Similarly, IL-21 has also been reported to be an effective stimulus for B cell activation and proliferation. However, additionally, IL-21 drives B cell maturation toward a plasma cell phenotype. In particular, the cell culture media and methods of various embodiments of the present invention enable engineered B cells to achieve activation and proliferation results comparable to classical feeder cell-based NIH3T3 / tCD40L protocols.

[0066] 1. CD40 Ligand In various embodiments, the engineered B cells are expanded in the presence of CD40 ligand ("CD40L"). In various embodiments, CD40L is

[0067] MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL (sequence number 1).

[0068] In various embodiments, the engineered B cells are expanded in the presence of a fusion protein comprising a CD40L sequence and a multimerization domain. In various embodiments, the multimerization domain is derived from tenansucin. In various embodiments, the multimerization domain is

[0069] ACGCAAAPDIKDLLSRLEELEGLVSSLREQ (SEQ ID NO: 2).

[0070] In various embodiments, the multimerization domain is SEQ ID NO: 2 and CD40L is SEQ ID NO: 1. In various embodiments, the fusion protein comprising the multimerization domain and CD40L is

[0043] VGDGSSHHHHHSSGGGRGSHHHHHHGGACGCAAAPDIKDLLSRLEELEGLVSSLREQGGGSGGGSGGGSMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL (sequence number 3).

[0071] In a preferred embodiment, the CD40L fusion protein comprises an amino acid sequence at least 85% identical to SEQ ID NO: 3. In various embodiments, the CD40L fusion protein comprises an amino acid sequence at least 95% identical to SEQ ID NO: 3. In various embodiments, CD40L comprises the amino acid sequence of SEQ ID NO: 3.

[0072] Crosslinking Agent It is contemplated that the B cells of the present invention are expanded in the presence of a CD40L cross-linking agent. In various embodiments, the CD40L cross-linking agent is an antibody. In various embodiments, the light chain region of the cross-linking antibody is DIVMTQSPSSLSVSAGEKVTMNCKSSQSLLNSGNQRNYLAWYQQKPGQPPKLLIHGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHRYPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (sequence number 4).

[0073] In various embodiments, the light chain variable region of the cross-linked antibody comprises: DIVMTQSPSSLSVSAGEKVTMNCKSSQSLLNSGNQRNYLAWYQQKPGQPPKLLIHGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDHRYPLTFGAGTKLELK (sequence number 5).

[0074] In various embodiments, the heavy chain region of the cross-linked antibody comprises EVQLQQFGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYGSTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCARDWTGAMDYWGQGTSVTVS SAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKEC HKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERRTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK (SEQ ID NO: 6).

[0075] In various embodiments, the light chain variable region of the cross-linked antibody comprises: Contains EVQLQQFGAELVKPGASVKISCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNYGSTSYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCARDWTGAMDYWGQGTSVTVSS (sequence number 7).

[0076] In various embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence at least 95% identical to SEQ ID NO:5 and a heavy chain variable region comprising an amino acid sequence at least 95% identical to SEQ ID NO:7. In various embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence of SEQ ID NO:5 and a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:7. In various embodiments, the antibody comprises a light chain of SEQ ID NO:4 and a heavy chain of SEQ ID NO:6. In various embodiments, the antibody comprises a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO:4 and a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO:6. In various embodiments, the antibody comprises a light chain comprising an amino acid sequence at least 95% identical to SEQ ID NO:4 and a heavy chain comprising an amino acid sequence at least 95% identical to SEQ ID NO:6.

[0077] III. Engineered B Cells As used herein, the term "engineered B cells" refers to B cells that have been genetically modified to express a desired protein or molecule. Such proteins or molecules can be endogenous or chimeric receptors. Such engineered B cells can be genetically modified to express "homing" receptors that target specific tissue / organ types or target tumors or specific cell types.

[0078] 1. Antigen Tumor antigens. In certain embodiments, the desired site / target of the B cells is a tumor antigen. The choice of antigen binding domain (moiety) of the present invention depends on the particular type of cancer to be treated. Some tumor antigens may be membrane bound, while other tumor antigens may be secreted. For example, tumor antigens may be secreted and accumulated in the extracellular matrix, or tumor antigens may be expressed as part of the MHC complex. Tumor antigens are well known in the art and include, for example, CD19, KRAS, HGF, CLL, glioma-associated antigen, carcinoembryonic antigen (CEA); β-human chorionic gonadotropin, alpha fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut These may include hsp70-2, M-CSF, prostase, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, protein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, mesothelin, EGFR, BCMA, KIT, and IL-13.

[0079] Infectious disease antigens. In certain embodiments, the site / target of interest is an infectious disease antigen to which an immune response may be desired. Infectious disease antigens are well known to those skilled in the art and may include, but are not limited to, viral, bacterial, protozoan, and parasitic antigens, such as parasites, fungi, yeast, mycoplasma, viral proteins, bacterial proteins and carbohydrates, and fungal proteins and carbohydrates. In addition, the infectious disease type of the infectious disease antigen is not particularly limited and may include, but is not limited to, refractory diseases among viral infections, such as AIDS, Hepatitis B, Epstein-Barr Virus (EBV) infection, HPV infection, and HCV infection. Parasitic antigens include, but are not limited to, malaria parasite sporozoite proteins.

[0080] In certain embodiments, the modified B cells express an engineered B cell receptor (CAR-B) that comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In certain embodiments, the extracellular domain comprises a binding domain and a hinge domain.

[0081] In certain embodiments, the extracellular domain comprises a binding domain, such as an scFv, ligand, antibody, receptor, or fragment thereof, and the modified B cells can target specific target cells by binding to proteins expressed on the surface of those cells. In certain embodiments, the modified tumor cells target and bind to proteins / antigens expressed on the surface of tumor cells. In certain embodiments, the modified B cells further express a payload. In certain embodiments, the payload can increase the number of cross-presenting dendritic cells (DCs) in the tumor. In certain embodiments, the payload can activate and attract T cells to the tumor. In certain embodiments, the payload can promote the formation of tertiary lymphoid structures (TLS) in the tumor. In certain embodiments of the invention, the modified B cells express both CAR-B and a payload. In certain embodiments, the CAR-B comprises a stimulatory domain that activates expression of the payload when bound to an antigen or protein expressed on the surface of the tumor cell.

[0082] Design and domain orientation of chimeric antigen receptors in B cells (CAR-B) In various embodiments, the present invention provides chimeric B cell receptors (CAR-B). It will be understood that chimeric B cell receptors (CAR-B) are genetically engineered receptors. These engineered receptors can be easily inserted and expressed in B cells according to techniques known in the art. With CAR-B, a single receptor can be programmed to recognize both specific proteins or antigens expressed on tumor cells, and upon binding to said proteins or antigens, induces an anti-tumor response. In various embodiments, CAR-B functions in part as a homing mechanism to deliver B cells to target tissues.

[0083] It will be understood that, in comparison to a receptor-bearing cell, the chimeric B cell receptor of the present invention comprises an extracellular domain (which includes an antigen-binding domain and may include an extracellular signaling domain and / or a hinge domain), a transmembrane domain, and an intracellular domain. The intracellular domain comprises at least an activation domain, and preferably consists of CD79a (immunoglobulin alpha), CD79b (immunoglobulin beta), CD40, CD19, CD137, Fcγr2a, and / or MyD88. It will be further understood that the antigen-binding domain is engineered to be located in the extracellular protein of the molecule / construct such that it is capable of recognizing and binding to its target.

[0084] Structurally, it will be understood that these domains correspond to locations on immune cells. Exemplary CAR-B constructs according to the invention are shown in Table 2. [Table 2]

[0085] In various embodiments, the chimeric B cell receptor consists of an extracellular domain, a transmembrane domain, and a cytoplasmic domain. In various embodiments, the cytoplasmic domain comprises an activation domain. In various embodiments, the cytoplasmic domain may also comprise a costimulatory domain. In various embodiments, the extracellular domain comprises an antigen binding domain. In various embodiments, the extracellular domain further comprises a hinge region between the antigen binding domain and the transmembrane domain.

[0086] Extracellular domain. Several extracellular domains can be used in the present invention. In various embodiments, the extracellular domain comprises an antigen binding domain. In various embodiments, the extracellular domain can also comprise a hinge region and / or a signaling domain. In various embodiments, the extracellular domain comprising an IgG1 constant domain can also comprise either an IgG1(hole) or IgG1(knob) to facilitate directional cBCR formation.

[0087] Antigen-binding domains and binding domains. As used herein, "antigen-binding domain", "antigen-binding domain", or "binding domain" refers to a portion of a B-CAR capable of binding an antigen or protein expressed on the surface of a cell. In some embodiments, the antigen-binding domain binds to an antigen or protein on a cell involved in a hyperproliferative disease. In a preferred embodiment, the antigen-binding domain binds to an antigen or protein expressed on the surface of a tumor cell. Antigen-binding molecules are further understood in view of the following definitions and explanations.

[0088] The antigen-binding domain has a dissociation constant (K d ) is 1×10 -7 An antigen-binding domain is said to "specifically bind" to its target antigen or protein when it is K d 1-5×10 -9 When the affinity is M, it specifically binds to the antigen with "high affinity" and when the affinity is K d 1-5×10 -10 M specifically binds to an antigen with "very high affinity." In one embodiment, the antigen-binding domain specifically binds to an antigen with a molecular weight of 10 -9 K of M d In one embodiment, the off rate is 1×10 -5 In other embodiments, the antigen-binding domain has less than about 10 -7 M~10 -13 K of M d and in yet another embodiment, the antigen-binding domain binds to an antigen or protein at 1.0-5.0× 10 K d Combine with.

[0089] An antigen-binding domain is said to be "selective" if it binds to one target more strongly than it binds to a second target.

[0090] The term "neutralizing" refers to an antigen-binding domain that binds to a ligand and prevents or reduces the biological effect of that ligand. This can be done, for example, by directly blocking the binding site on the ligand, or by binding to the ligand and altering the ligand's ability to bind through indirect means, such as a structural or energetic change in the ligand. In some embodiments, the term can also refer to an antigen-binding domain that prevents the protein to which it is bound from carrying out a biological function.

[0091] The term "target" or "antigen" refers to a molecule or a portion of a molecule that can be bound by an antigen-binding molecule. In certain embodiments, a target can have one or more epitopes.

[0092] The term "antibody" refers to what is known as an immunoglobulin, a Y-shaped protein produced by the immune system to recognize a specific antigen. The term "antibody fragment" refers to the antigen-binding fragment of an antibody and the Fc fragment. Types of antigen-binding fragments include F(ab')2, Fab, Fab', and Fv molecules. The Fc fragment is generated entirely from the heavy chain constant region of the immunoglobulin.

[0093] Extracellular signaling domain: The extracellular domain is beneficial for signal transduction and efficient response of lymphocytes to antigens.Particular extracellular domains of use in the present invention include CD28, CD28T (see, e.g., U.S. Patent Application No. US2017 / 0283500A1), OX40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, CD79a (immunoglobulin alpha), CD79b (immunoglobulin beta), DAP-10, Fc gamma receptor, MHC class 1 molecules, TNF receptor proteins, immunoglobulins, and the like. protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The antibody may be derived from (i.e., include) AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds CD83, or any combination thereof.The extracellular domain may be derived from either natural or synthetic sources.

[0094] Hinge domain. As described herein, the extracellular domain often includes a hinge portion. This is the portion of the extracellular domain that is adjacent to the cell membrane. The extracellular domain may further include a spacer region. A variety of hinges may be used according to the present invention, including costimulatory molecules as discussed above, as well as immunoglobulin (Ig) sequences, 3X strep II spacers, or other suitable molecules to achieve the desired specific distance from the target cell. In some embodiments, the entire extracellular region includes the hinge region. In some embodiments, the hinge region includes the extracellular domain of CD28 or CD8 as described herein, or a portion thereof.

[0095] Transmembrane domain. B-CAR can be designed to include a transmembrane domain that is fused or otherwise linked to the extracellular domain of B-CAR. Similarly, it can be fused to the intracellular domain of B-CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in B-CAR is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein to minimize interaction with other members of the receptor complex. The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-associated or transmembrane protein.Transmembrane regions of particular use in the present invention include CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, CD79a (immunoglobulin alpha), CD79b (immunoglobulin beta), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, insulin, insulin receptor ... Tegrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The antibody may be derived from (i.e., include) AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds CD83, or any combination thereof.

[0096] Optionally, a short linker may form a bond between any or some of the extracellular, transmembrane, and intracellular domains of the B-CAR.

[0097] In certain embodiments, the transmembrane domain in the B-CAR of the invention is the CD28 transmembrane domain. In one embodiment, the transmembrane domain in the B-CAR of the invention is the CD8 transmembrane domain.

[0098] Intracellular (cytoplasmic) domain. The intracellular (IC, or cytoplasmic) domain of the B-CAR receptor of the present invention can provide for the activation of at least one of the normal effector functions of an immune cell.

[0099] It will be understood that suitable intracellular molecules include, but are not limited to, CD79a (immunoglobulin alpha), CD79b (immunoglobulin beta), CD40, CD19, CD137, Fcγr2a, and MyD88.Intracellular molecules include CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte Activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR-B of the present invention may further comprise a ligand that specifically binds to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof. The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR-B of the present invention may be linked to each other in a random or specified order.

[0100] As used herein, the term "costimulatory" domain or molecule refers to a heterogeneous group of cell surface molecules that act to amplify or counteract the initial activation signal of a cell.

[0101] In one preferred embodiment, the cytoplasmic domain is designed to include the signaling domain of hCD19. In another embodiment, the cytoplasmic domain is designed to include the signaling domain of hCD40. In another embodiment, the cytoplasmic domain is designed to include the signaling domain of hCD40 and hCD79b. In another embodiment, the cytoplasmic domain is designed to include the signaling domain of hCD40 and hCD137. In another embodiment, the cytoplasmic domain is designed to include the signaling domain of hCD40 and hFcγr2a. In another embodiment, the cytoplasmic domain is designed to include the signaling domain of hCD40 and hMyd88. In another embodiment, the cytoplasmic domain is designed to include the signaling domain of hCD79a. In another embodiment, the cytoplasmic domain is designed to include the signaling domain of hCD79b. These embodiments are preferably of human origin, but may be derived from other species.

[0102] B. Modified B cells expressing a payload. In various embodiments of the present invention, modified B cells capable of expressing a payload are provided. As used herein, the term "payload" refers to a nucleic acid sequence encoding an amino acid sequence, peptide or protein, or an RNA molecule for use as a therapeutic agent. In certain embodiments, the payload is for delivery to a tumor or tumor microenvironment. In certain embodiments, it is desirable for B cells to deliver a payload to a tumor or tumor microenvironment that can, for example, increase the number of cross-presenting dendritic cells (DCs) in the tumor. Cross-presenting DCs allows for improved presentation of tumor antigens. In various embodiments, the payload may be capable of activating and attracting T cells to the tumor. Activating more T cells in the tumor can complement the cross-presenting DCs and reshape the tumor environment to have stronger anti-tumor immune capabilities. The payload may also promote the formation of tertiary lymphoid structures (TLS) in the tumor. Clinical studies have shown that there is a relationship between B cells, TLS, and response to immune checkpoint blockade.

[0103] Non-exclusive examples of payloads of the invention include IL-1, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, IL-18, IL-21, interferon alpha, interferon beta, interferon gamma, TSLP, CCL21, FLT3L, XCL1, LIGHT (TNFSF14), OX40L, CD137L, CD40L, ICOSL, anti-CD3 antibodies, CD47, TIM4-FC, CXCL13, CCL21, CD80, CD40L, IFNα A2, LIGHT, 4-1BBL, MDGF (C19orf10), FGF10, PDGF, agrin, TNF-α, GM-CSF, anti-FAP antibodies, anti-TGF-β antibodies; TGF-β traps, decoys, or other inhibitory molecules; anti-BMP antibodies; BMP traps, decoys, or other inhibitory molecules.

[0104] Signaling for payload expression. In various embodiments of the present invention, the payload is expressed in the modified B cells as a DNA construct under the control of an activated transcription pathway. In certain embodiments, the expression of the payload is controlled by the nuclear factor of activated T cells ("NFAT") pathway. The NFAT pathway is a transcription factor pathway that is activated during an immune response and is activated by NFκB. In various embodiments, the modified B cells express both the payload and CAR-B. In various embodiments in which the modified B cells express both the payload and CAR-B, the CAR-B may further encode a signaling molecule that induces activation of the NFκB pathway. Such molecules include, but are not limited to, CD79a (immunoglobulin alpha), CD79b (immunoglobulin beta), CD40, CD19, CD137, Fcγr2a, and MyD88.

[0105] In various embodiments, the invention relates to isolated B cells expressing at least one payload. In various embodiments, the invention relates to isolated B cells expressing more than one payload. In various embodiments, the invention relates to isolated B cells expressing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 different payloads.

[0106] Modification of B cells for homing. In various embodiments of the present invention, engineered B cells can be modified with a homing domain (e.g., as shown in FIG. 2) to enable the B cells to home to a desired site / target and become activated upon interaction with the target. Additionally, B cell homing receptors expressed on the B cell membrane that recognize addressins and ligands on target tissues, compounds or derivatives thereof that alter trafficking of B cells to specific sites, and molecules that suppress inflammatory and autoimmune activity of B cells can be involved in B cell homing and development of specialized immune responses.

[0107] Modified B cells expressing an integrin of interest. The major homing receptors expressed by lymphocytes are integrins, a large group of molecules characterized by a heterodimeric structure of α and β chains. In general, the pairing of a particular α and β chain of an integrin determines the type of homing receptor. For example, the pairing of α4 and β7 chains characterizes the major integrin molecule (α4β7) involved in lymphocyte binding to mucosal addressin cell adhesion molecule 1 (MAdCAM-1) expressed on high endothelial venules (HEV) in Peyer's patches (PP) and gastrointestinal (GI) tract lamina propria endothelial venules (LPV). Similarly, the pairing of α4 and β1 chains characterizes the homing receptor for the skin (α4β1).

[0108] In various embodiments of the present invention, the modified B cells can be preselected with certain traits that mediate preferred localization.For example, memory B cells expressing CXCR3 can be enriched and then engineered.CXCR3 cells can be attracted to ligands expressed at inflammatory sites.In this way, modified B cells can preferentially localize to such sites.

[0109] In various embodiments of the invention, modified B cells are provided that express integrin α4 and β7 chains. Desirably, expression of α4β7 integrin promotes homing of the modified B cells to the colon. In various embodiments, modified B cells are provided that express integrin α4 and β1 chains. Desirably, expression of α4β1 integrin promotes homing of the modified B cells to the skin. In various embodiments, modified B cells are provided that express a desired pairing of integrin α and β chains such that the expressed integrin promotes homing of the modified B cells to a desired site / target of interest. Thus, in various embodiments, any desired combination of integrin α and β chains is contemplated for expression in B cells such that modified B cells expressing a particular integrin will target a desired site / target of interest.

[0110] Modified B cells expressing desired homing receptors. B cells have the ability to home to inflamed tissues, and altering the expression of their homing receptors can complement their natural homing tendency. B cell localization is also driven by the expression of attractant molecules (e.g., targets such as ligands and chemokines) at the site of inflammation in a particular location or tissue. Such molecules can also include antibodies such as MECA79 antibodies that target cells to peripheral nodal addressins (PNAds). Bahmani et al., J Clin Invest. 2018; 128(11):4770-4786, Azzi et al., Cell Rep. 2016; 15(6):1202-13. Thus, B cells can be engineered to express specific antibodies, proteins, and receptors that facilitate the B cells' home to the desired site / target and the interaction of such B cells with the desired target. In certain cases, the expression of such receptors redirects the B cells to the desired tissue.

[0111] In various embodiments of the present invention, modified B cells are provided that can express homing antibodies, proteins, or receptors, or whose expression can direct B cells to a specific site / target of interest. Exemplary homing of T cells to specific homing tissues (target tissues) using specific homing receptor / ligand pairs is shown in Table 3. The same specific homing receptor / ligand pairs can also promote homing of B cells to specific homing tissues (target tissues). Thus, in various embodiments of the present invention, homing of modified B cells to exemplary homing tissues (target tissues) is promoted using the corresponding homing receptor / ligand pairs listed in Table 3. [Table 3-1] [Table 3-2]

[0112] Exemplary homing tissue (target tissue) types and ligands or chemokines that enable tissue-restricted B cell homing according to the present invention are shown in Table 4. [Table 4]

[0113] In various embodiments of the present invention, modified B cells are provided that express one or more of the antibodies, proteins, or receptors that facilitate homing of the modified B cells to exemplary target / homing tissues using specific homing receptor / ligand pairs as set forth in Table 3. In various embodiments of the present invention, modified B cells are provided that express one or more of the homing receptors that facilitate homing of the modified B cells to exemplary target / homing tissues using ligands or chemokines as set forth in Table 3 and / or Table 4. As used herein, the term "B cell homing" refers to localizing, targeting, transporting, directing, or redirecting the B cells of the present application to a desired site / target, such as a homing or target tissue, a site of inflammation within a particular location or tissue, or a tumor or tumor microenvironment, where delivery of a therapeutic payload is desired. When used in the context of B cell homing, the terms "antibody," "protein," or "receptor" refer to a nucleic acid sequence that encodes an amino acid sequence, peptide, or protein, or an RNA molecule for use as a therapeutic agent, which, when expressed in the modified B cells of the present invention, directs the B cells to a desired site / target.

[0114] In certain embodiments, the homing antibody, protein, or receptor molecule is for homing / targeting modified B cells expressing such molecules to a site / target of interest. In certain embodiments, the homing antibody, protein, or receptor molecule is for homing / targeting modified B cells expressing such molecules to a site of inflammation in a particular location or tissue. In certain embodiments, the homing antibody, protein, or receptor is for targeting B cells to a tumor or tumor microenvironment. In certain embodiments, it is desirable to target B cells to a particular location so that the engineered or modified B cells of the present invention can deliver a therapeutic payload to a desired location of interest, e.g., a homing or target tissue, a site of inflammation in a particular location or tissue, or a tumor or tumor microenvironment. Thus, in certain embodiments, it is desirable for the B cells to home to a site / target of interest, e.g., a tumor or tumor microenvironment, and deliver a payload to the site / target of interest that can, for example, increase the number of cross-presenting dendritic cells (DCs) at the site / target of interest (e.g., within a tumor).

[0115] In various embodiments, the homing antibody, protein, or receptor is expressed in the modified or engineered B cell as a DNA construct. In various embodiments, the homing antibody, protein, or receptor is expressed in the modified B cell as a DNA construct under the control of a constitutively activated transcription pathway. In various embodiments, the homing antibody, protein, or receptor involved in B cell homing / targeting is not naturally expressed in B cells or is expressed at a higher level than naturally expressed in B cells. Exemplary homing of modified B cells to specific homing / target tissues using specific homing receptor / ligand pairs according to the present invention is shown in Table 4. Notwithstanding the exemplary homing tissues, homing receptors, and ligand pairs listed in Table 4, it should be understood that the modified B cells of the present invention can be engineered to express any homing antibody, protein, or receptor (e.g., any homing receptor listed in Table 5) so that the modified B cell can be directed to a specific site / target of interest. [Table 5]

[0116] Non-exclusive examples of homing (target) tissue types for particular homing receptor / ligand pairs of the present invention include skin, gastrointestinal (intestine, colon, mesenteric lymph nodes (mLN), Peyer's patches (PP), small intestine), liver, lung, bone marrow, heart, peripheral lymph nodes (LN), CNS, thymus, and bone marrow.

[0117] Non-exclusive examples of homing receptors that can be paired with the specific or corresponding attractants / ligands / chemokines of the present invention include CLA (PSGL-1 glycoform), CLA (PSGL-1 glycoform), CCR10, CCR3, CCR4, CCR5, CCR6, CCR9, CD43E, CD44, c-Met, CXCR3, CXCR4, LFA-1, LFA-1 (αLβ2), selectin ligands, VLA-4, VLA-4 (α4β1), and α4β7.

[0118] Non-exclusive examples of ligands / chemokines that can be paired with the specific or corresponding homing receptors of the present invention include CXCL16, CCL17, CCL17(22), CCL20 (MIP-3α), CCL21, CCL25, CCL27, CCL28, CCL4, CCL5, CD62E, CD62P, CXCL10, CXCL12, CXCL13, CXCL16, CXCL9 / CXCL10, CXCR3, E / P-selectin, E-selectin, GPR15L, HGF, hyaluronate, ICAM-1, ligand for CCR1,2,5, MAdCAM, MAdCAM-1, PNAd, VAP-1, VCAM, and VCAM-1.

[0119] In certain embodiments of the invention, modified B cells are provided that express or have increased expression of exemplary B cell homing receptors (e.g., as set forth in Table 3) such that the modified B cells target corresponding homing tissues of interest that express the corresponding ligands / chemokines (e.g., as set forth in Tables 3 and / or 4). In certain embodiments of the invention, modified B cells are provided that co-express integrins with specific α and β chain pairings and specific B cell homing receptors (e.g., as set forth in Tables 3 and / or 4), the expression of which integrins and / or homing receptors promotes or facilitates homing / targeting of the modified B cells to the desired site / target. In some embodiments, modified B cells are provided that co-express α4β7 integrin and CCR9. Desirably, co-expression of α4β7 and CCR9 promotes small intestinal homing of the modified B cells of the invention. In some embodiments, modified B cells are provided that co-express α4β1 integrin and CCR4. Co-expression of α4β1 and CCR4 preferably promotes small intestinal homing of the modified B cells of the invention.

[0120] Modified B cells expressing immune inhibitory molecules. B cells are the primary cause of many autoimmune diseases. However, B cells can be used therapeutically to antagonize autoimmunity. Specifically, B cells can be engineered to express at least one or more immune inhibitory molecules, which can reduce the autoimmune activity of B cells and result in a reduction in autoimmune disease. Immune inhibitory molecules are well known in the art. Such inhibitory molecules include, but are not limited to, IL-10, TGF-β, PD-L1, PD-L2, LAG-3, and TIM-3. In certain embodiments of the present invention, modified B cells are provided that are engineered to express at least one or more inhibitory molecules selected from IL-10, TGF-β, PD-L1, PD-L2, LAG-3, and TIM-3, or any combination thereof, so that inflammation at the site and autoimmune activity of B cells localized at the site are reduced, thereby resulting in a positive therapeutic response.

[0121] Compounds that change B cell trafficking. In certain embodiments of the present invention, modified B cells are provided that are treated with at least one or more compounds or derivatives thereof that change B cell trafficking by inducing the expression of certain B cell integrins and / or homing receptors. Compounds or derivatives thereof that change B cell trafficking are well known in the art. In certain embodiments, modified B cells are provided that are treated with all-trans retinoic acid (ATRA) or derivatives thereof that promote B cell homing to the gastrointestinal tract (small intestine) by increasing the expression of α4β7 integrin and CCR9 homing receptor. As used herein, the term "compound" refers to a chemical, drug, therapeutic agent, or derivative thereof that changes B cell trafficking in a desired manner.

[0122] In various embodiments of the invention, modified B cells engineered to co-express a particular integrin (e.g., with a particular α and β chain pair) and a particular B cell homing receptor of interest are treated with at least one or more compounds or derivatives thereof that alter the trafficking of the modified B cells and promote homing of the cells to a particular site / target of interest due to increased expression of the particular integrin and homing receptor. In various embodiments, B cells modified to co-express a particular α and β chain pair and integrin, as well as the particular B cell homing receptor, further express at least one or more immune inhibitory molecules such that the autoimmune activity of the modified B cells targeted to a particular site of inflammation is reduced, leading to a reduction in autoimmune disease. In some embodiments, modified B cells engineered to express one or more immune inhibitory molecules, such as IL-10, TGF-β, PD-L1, PD-L2, LAG-3, and TIM-3, or a combination thereof, are treated with ATRA or a derivative thereof for a specified period of time to induce expression of α4β7 integrin and CCR9 homing receptor to promote B cell homing to a specific site / target of interest (e.g., gastrointestinal) while reducing autoimmune activity of B cells localized at the site, resulting in a positive therapeutic response. In one embodiment, modified B cells engineered to express one or more immune inhibitory molecules, such as IL-10, TGF-β, or a combination thereof, are treated with ATRA or a derivative thereof for a specified period of time to induce expression of α4β7 integrin and CCR9 homing receptor to promote B cell homing to a specific site / target of interest (e.g., gastrointestinal) while reducing autoimmune activity of B cells localized at the site, resulting in a positive therapeutic response.

[0123] It will be understood that any B cell of the present invention that has been modified to co-express specific B cell integrins and homing receptors that target the B cells to a particular homing / target tissue of interest can be further engineered to express one or more immune inhibitory molecules to reduce inflammatory and autoimmune activity of the B cells localized to that site, and / or treated with compounds that alter the homing / targeting of the modified B cells by inducing expression of specific B cell integrins and / or homing receptors.

[0124] TLR agonists and activation of B cells by TLRs. B cells have the natural ability to take up and present antigens that are recognized by their specific B cell receptor (BCR). B cells activated by Toll-like receptors (TLRs) give rise to potent effector B cells that defend the body in an immune response. Expression of TLRs or increased expression of TLRs on B cells can provide a mechanism to enhance B cells for intrinsic signals that regulate adaptive immune responses.

[0125] Activation of B cells by TLR agonists. In various embodiments of the present invention, B cells are provided and the B cells are treated in vitro or ex vivo with at least one TLR agonist. In various embodiments, the TLR may be TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and / or TLR13. In various embodiments, the TLR agonist preferentially binds to one or more TLRs selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. TLR agonists are well known in the art and may include, but are not limited to, CpG-rich oligonucleotides and double-stranded RNA mimicking polyinosinic:polycytidylic acid (poly-I:C). In various embodiments, the TLR agonist can be a CpG oligonucleotide.

[0126] In various embodiments, each B cell may be treated with one TLR agonist. In various embodiments, each B cell may be treated with more than one TLR agonist. For example, each B cell may be treated with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 different TLR agonists. Alternatively, a patient may be administered a heterogeneous population of B cells, with each B cell being treated with its own TLR agonist or combination of TLR agonists. In some embodiments, the B cells for use in the therapeutic agent are treated with one or more TLR agonists simultaneously with or prior to administration of the B cells to the subject or patient in need of administration. In certain embodiments, treatment with one or more TLR agonists can produce more powerful effector B cells for the immune response to defend the body. In certain embodiments, treatment with one or more TLR agonists can boost B cells for immune response. In some embodiments, treating a B cell of the invention with at least one or more TLR agonists induces expression or activation of one or more TLRs.

[0127] Activation of B cells by TLR expression. In various embodiments of the invention, modified B cells capable of expressing a constitutively active TLR are provided. In various embodiments, the TLR is expressed in the modified or engineered B cells as a DNA construct under the control of a constitutively activated transcription pathway. In various embodiments, the TLR is not naturally expressed in B cells or is expressed at a higher level than naturally expressed in B cells. In various embodiments, the TLR can be TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and / or TLR13.

[0128] In various embodiments, each B cell may express more than one constitutively active TLR. For example, each B cell may express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 different constitutively active TLRs. Alternatively, a patient may be administered a heterogeneous population of B cells, where each B cell may express and / or secrete a unique TLR or combination of TLRs that are constitutively active. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 different constitutively active TLRs may be administered to a subject or patient via a heterogeneous population of B cells.

[0129] In certain embodiments of the invention, the B cells are modified B cells expressing at least one constitutively active TLR. In certain embodiments, the modified B cells expressing at least one constitutively active TLR are treated with one or more TLR agonists. In certain embodiments, the expression of a constitutively active TLR can produce more powerful effector B cells to defend the body in an immune response. In certain embodiments, the expression of a constitutively active TLR can enhance B cells for an immune response. In certain embodiments, the modified B cells express both a constitutively active TLR and any CAR-B of the present application. In various embodiments, the modified B cells expressing a constitutively active TLR and / or CAR-B are further treated with one or more TLR agonists simultaneously with or prior to administration of the modified B cells to a subject or patient in need of administration. In certain embodiments, the B cells can be engineered to express payloads such as TLRs and modifiers in the absence of CAR-B for intratumoral administration.

[0130] Modified B cells that simultaneously present antigens in HLA class I and class II molecules. In addition to their function in antibody production, B cells also express high levels of human leukocyte antigen (HLA) class II molecules and can present antigens to CD4+ T cells. Hong et al., 2018, Immunity 49, 695-708. In various embodiments of the present invention, modified B cells are provided that can simultaneously present a specific antigen and / or an epitope derived from an antigen of interest, such as a tumor antigen or an infectious disease antigen, in both HLA class I and class II molecules. Tumor antigens and infectious disease antigens are well known in the art and described in the preceding sections. In certain embodiments, the specific antigen of interest, e.g., a tumor antigen or an infectious disease antigen, is fused to a targeting signal of a lysosomal protein that targets the antigen to lysosomes and simultaneously and efficiently presents the antigen on both HLA class I and class II molecules. In some embodiments, the targeting signal is a targeting signal of lysosomal associated membrane protein-1 (LAMP1). In some embodiments, the targeting signal is capable of entering the endosomal recycling compartment. The c-terminal sequence of Clec9A is such a targeting moiety. As used herein, a specific tumor or infectious disease antigen fused to a targeting signal refers to an amino acid sequence, a nucleic acid sequence encoding a peptide or protein, or an RNA molecule (e.g., an mRNA molecule) for use as a therapeutic. In one embodiment, a specific tumor or infectious disease antigen fused to a targeting signal refers to an mRNA molecule for use as a therapeutic. In certain embodiments, it is desirable for a specific tumor and / or infectious disease antigen fused to a targeting signal, such as a targeting signal of LAMP1 or Clec9A, to be targeted to lysosomes or endosomes and presented simultaneously and efficiently by both HLA class I and class II molecules.In certain embodiments, it is desirable to electroporate B cells (e.g., human B cells) before or after maturation with mRNA encoding a specific tumor and / or infectious disease antigen of interest fused to a targeting signal, such as a LAMP1 or Clec9A targeting signal, such that the specific antigen and / or antigen-derived epitope can be simultaneously and efficiently presented in both HLA class I and class II molecules. In various embodiments, the specific tumor and / or infectious disease antigen of interest is either not naturally presented by B cells, is not simultaneously presented by B cells in both HLA class I and class II molecules, or is not naturally presented by B cells with high efficiency in both HLA class I and class II molecules. It is contemplated that introduction of such electroporated B cells into a subject, e.g., a human host, will promote or enhance the development of an antigen-specific immune response by simultaneously and efficiently presenting the specific antigen and / or antigen-derived epitope of interest in both HLA class I and class II molecules.

[0131] In various embodiments, the present invention relates to nucleic acid sequences, e.g., mRNA sequences, encoding at least one specific antigen of interest, e.g., a tumor or infectious disease antigen, fused to a targeting signal, such as the targeting signal of LAMP1, for use as a therapeutic agent in electroporation of B cells for simultaneous and efficient presentation of specific antigens and / or antigen-derived epitopes in both HLA class I and class II molecules. In various embodiments, the present invention relates to nucleic acid sequences, e.g., mRNA sequences, encoding more than one (e.g., 1, 2, 3, 4, 5, or more) specific tumor and / or infectious disease antigens of interest fused to a targeting signal. In various embodiments, the present invention relates to pools of different nucleic acid sequences, e.g., pools of different mRNA sequences, each pool encoding at least one specific antigen of interest, e.g., a tumor or infectious disease antigen, fused to a targeting signal that is different from the other pools of mRNA sequences, for use as a therapeutic agent in electroporation of B cells as described above. Thus, in some embodiments, a subject may be administered a homogenous population of B cells, where each B cell is electroporated with an mRNA encoding at least one specific antigen of interest fused to a targeting signal. In some embodiments, a subject may be administered a homogenous population of B cells, where each B cell is electroporated with an mRNA encoding more than one specific antigen of interest fused to a targeting signal. In some embodiments, a subject may be administered a homogenous population of B cells, where each B cell is electroporated with a combination of mRNA encoding at least one specific antigen of interest fused to a different targeting signal.

[0132] In some embodiments, the B cells for use in the electroporation described above are any of the modified B cells of the present application. In some embodiments, the modified B cells comprise a chimeric antigen receptor for B cells (CAR-B). In various embodiments, the modified B cells express CAR-B and can simultaneously and efficiently present a specific antigen and / or antigen-derived epitope of interest in both HLA class I and class II molecules.

[0133] In various embodiments, the present invention relates to a method of administering isolated B cells to a patient in need thereof. In various embodiments, a population of B cells can be administered to a patient. In various embodiments, each B cell can express more than one payload peptide or protein. For example, each B cell can express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 different payloads. Alternatively, a patient can be administered a heterogeneous population of B cells, where each B cell can express and / or secrete a unique payload or combination of payloads. In various embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 different payloads can be administered to a patient via a heterogeneous population of B cells.

[0134] IV. Treatment Methods In various aspects of the invention, the expanded population of B cells is delivered as a therapeutic agent to a patient in need thereof, in various embodiments, the expanded B cell population may be capable of treating or preventing a variety of diseases or disorders, including cancer.

[0135] In some embodiments, the present invention relates to generating a B cell-mediated immune response in a subject, comprising administering to the subject an effective amount of the expanded and / or engineered B cells of the present application. In some embodiments, the B cell-mediated immune response is directed against a target cell. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor for B cells (B-CAR). In some embodiments, the target cell is a tumor cell. In some aspects, the present invention includes a method for treating or preventing a malignant tumor, comprising administering to a subject in need of treatment or prevention of a malignant tumor an effective amount of at least one isolated antigen binding molecule described herein. In some aspects, the present invention includes a method for treating or preventing a malignant tumor, comprising administering to a subject in need of treatment or prevention of a malignant tumor an effective amount of at least one immune cell, the immune cell comprising at least one chimeric antigen receptor.

[0136] In some aspects, the invention includes pharmaceutical compositions comprising an expanded population of engineered B cells comprising at least one antigen binding molecule described herein and a pharma- ceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

[0137] In some embodiments, the subject is diagnosed with metastatic disease localized to the liver. In other embodiments, the metastatic disease is cancer. In still other embodiments, the cancer has metastasized from a primary tumor in the breast, colon, rectum, esophagus, lung, pancreas, and / or stomach. In still other embodiments, the subject is diagnosed with an unresectable metastatic liver tumor. In still other embodiments, the subject is diagnosed with an unresectable metastatic liver tumor from a primary colorectal cancer. In some embodiments, the subject is diagnosed with hepatocellular carcinoma.

[0138] The target dose of modified B cells is 1 × 10 6 ~2×10 10 Cells / kg, preferably 2 x 10 6It is understood that the dose may be in the range of 100-200 mg / kg of cells / kg, more preferably 100-200 mg / kg of cells / kg. It is understood that doses above and below this range may be appropriate for a particular subject, and appropriate dose levels may be determined by a medical provider as appropriate. Additionally, multiple doses of cells may be provided in accordance with the present invention.

[0139] Also provided is a method for reducing the size of a tumor in a subject, comprising administering to the subject a modified B cell of the invention, the cell comprising a CAR-B receptor comprising an antigen binding domain that binds to an antigen on the tumor, a payload, or both the CAR-B and the payload. In some embodiments, the subject has a solid tumor or a hematological malignancy such as lymphoma or leukemia. In some embodiments, the modified B cells are delivered to the tumor bed. In some embodiments, the cancer is present in the bone marrow of the subject.

[0140] Also provided are methods for homing B cells to a site / target of interest in a subject, comprising administering to the subject modified B cells of the invention, wherein the cells comprise an integrin, homing antibody, protein, or receptor that is attracted to a ligand, chemokine, or attractant at the site / target of interest. In some embodiments, the site / target of interest is, for example, a homing or target tissue, a site of inflammation within a particular location or tissue, or a tumor or tumor microenvironment, where delivery of a therapeutic payload is desired.

[0141] Also provided is a method for reducing inflammation and autoimmune activity of B cells at a site / target of interest in a subject, comprising administering to said subject modified B cells of the invention, wherein the cells comprise an immune inhibitory molecule. In some embodiments, the site / target of interest is, for example, a homing or target tissue, a site of inflammation within a particular location or tissue, or a tumor or tumor microenvironment, where delivery of a therapeutic payload is desired.

[0142] In some embodiments, the expanded population of engineered B cells are autologous B cells. In some embodiments, the modified B cells are allogeneic B cells. In some embodiments, the modified B cells are xenogeneic B cells. In some embodiments, the modified B cells of the present application are transfected or transduced in vivo. In other embodiments, the engineered cells are transfected or transduced ex vivo.

[0143] As used herein, the term "subject" or "patient" refers to an individual. In some embodiments, the subject is a mammal, such as a human. In some embodiments, the subject may be a non-human primate. Non-human primates include marmosets, monkeys, chimpanzees, gorillas, orangutans, and gibbons, to name a few. The term "subject" also includes domestic animals, such as cats and dogs, farm animals (e.g., llamas, horses, cows), wild animals (e.g., deer, elk, moose, etc.), laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, etc.), and avian species (e.g., chickens, turkeys, ducks, etc.). Preferably, the subject is a human subject. More preferably, the subject is a human patient.

[0144] In certain embodiments, compositions comprising the CAR-expressing immune effector cells disclosed herein may be administered in conjunction with any number of chemotherapeutic agents. Exemplary chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaolamide, and trimethylolmelamine regimes; chlorambucil, chlornaphazine. , colofsphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, nitrogen mustard, such as uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, calicheamicin, and ca Lavicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinos Antibiotics such as tatins and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmophor, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU;Androgens such as calcineurin, dromostanol propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatraxate; deformamine; demecolcine; diaziquone; elformitin; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamonate nitracrine; pentostatin; phenameth; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS2000; difluoromethylorutin (DMFO); retinoic acid derivatives such as TARGRETIN™ (bexarotene), PANRETIN™ (alitretinoin); ONTAK™ (denileukin diftitox);esperamycin;capecitabine;and pharmacokinetically acceptable salts, acids, or derivatives of any of the above. Also included in this definition are antiestrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazole, 4-hydroxytamoxifen, trioxyphene, keoxyphene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and antihormonal agents that act to regulate or inhibit hormone action on tumors, such as pharmacokinetically acceptable salts, acids, or derivatives of any of the above. Optionally, a combination of chemotherapy agents is also administered, including, but not limited to, CHOP, i.e., cyclophosphamide (CYTOXAN®), doxorubicin (hydroxydoxorubicin), fludarabine, vincristine (ONCOVIN®), and prednisone.

[0145] A variety of additional therapeutic agents may be used in conjunction with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 (or PD-L1) inhibitors such as nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®), pembrolizumab, pidilizumab, and atezolizumab (TECENTRIQ®).

[0146] Suitable additional therapeutic agents for use in combination with the present invention include ibrutinib (IMBRUVICA®), ofatumumab (ARZERRA®), rituximab (RITUXAN®), bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), trastuzumab emtansine (KADCYLA®), Imatinib (GLEEVEC®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), catemaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, Sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pazopanib, regorafenib, semaxanib ...sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, pazopanib, regorafenib, semaxanib, sunitinib, tivozanib, These include, but are not limited to, mTOR inhibitors such as critinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denyluiquin difititox, everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, and CDK inhibitors such as the CDK inhibitor (palbociclib).

[0147] In further embodiments, the composition comprising the CAR-containing immunogen can be administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylate salts. Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporoxifene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), TNF antagonists (e.g., cytokine inhibitors such as etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®)), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies, as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular), and minocycline.

[0148] In certain embodiments, the compositions described herein are administered in conjunction with a cytokine. As used herein, "cytokine" is meant to refer to a protein released by one cell population that acts on another cell as an intercellular mediator. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Among the cytokines are growth hormones, such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones, such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; Müllerian inhibitory substance; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve growth factor (NGF), such as NGF-beta; platelet growth factor; transforming growth factor, such as TGF-alpha and TGF-beta. Examples of cytokines include transforming growth factors (TGFs); insulin-like growth factors I and II; erythropoietin (EPO); bone morphogenetic factors; interferons such as interferon-alpha, beta, and gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, tumor necrosis factors such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines. EXAMPLES

[0149] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that, in light of the teachings of the present invention, certain changes and modifications may be made without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only, and not by way of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially similar results.

[0150] Example 1: Purification and enrichment of human B cells Human B cells were collected and enriched from PBMCs according to the following protocol. PBMCs were prepared from buffy coats as follows: The volume per donor (approximately 25 ml) was brought up to 120 ml with PBS. 30 ml was then layered onto 15 ml of Ficoll in four 50 ml tubes. This was then spun at 450 g for 20 minutes. The top layer was then removed and discarded. The PBMC interface just below the first layer was then isolated (neutrophils are most dense and increase in number towards the bottom of the layer). The interface of each of the four tubes was taken from each donor and transferred to two 50 ml conical tubes. Each was brought up to 50 ml per tube with PBS. Each of the two tubes was then spun at 500 g for 5 minutes.

[0151] RBC lysis was performed by aspirating the pellet and drawing up a total volume of 10 ml combined for both pellets in ACK lysis buffer. The solution was then held at room temperature for 5 minutes. 40 ml of PBS was then added, the solution was mixed and then centrifuged at 500 g for 5 minutes. The mixture was then brought to a volume of 40 ml in PBS for counting the PBMC yield. For each B cell enrichment preparation, 15 million PBMCs were used.

[0152] B cell enrichment was performed according to the instructions for product ID 17954 EASYSEP® purification of human B cells (Stem Cell Technologies). The solution was spun down and 150 million PBMCs were resuspended in 3 ml of EASYSEP® isolation buffer. The pellet was resuspended in 3 ml of EASYSEP® buffer in a 15 ml tube. 150 μl of cocktail enhancer was added. 150 μl of isolation cocktail was added, the tube was mixed and incubated at room temperature for 5 minutes. The quickball was vortexed for 30 seconds and 150 μl was added to the tube, then mixed and transferred to the magnet. After 3 minutes, it was poured into a new tube and the magnet step was repeated.

[0153] Example 2: Expansion of B cells using HELA-CD40L feeder cells or MEGA-CD40L Due to the low frequency of B cells in human peripheral blood, it is difficult to obtain sufficient numbers of B cells for clinical cell therapy purposes. Therefore, B cell expansion is a critical step in producing clinical grade B cells. Human B cells require CD40L for growth. It has been established that B cells can survive and expand when grown on a monolayer of HEL cells expressing CD40L. However, a method for feeder cell-independent B cell growth needs to be established. With the aim of determining whether a commercially available toolore reagent exists for this purpose, Enzo MEGA-CD40L was tested in comparison to growth on HELA-CD40L feeder cells.

[0154] Growth medium conditions on HeLa-CD40L feeder cells. 5 x 10 cells per plate on a 24-well plate 6 Feeder cell plates were prepared by irradiating CD40L HeLa cells with 5% CO. Base medium consisted of RPMI-1640 + 10% FCS, Penn / strep (100u / ml, 100ug / ml), sodium selenite (100nM), and IL-4 (2ng / ml) (R&D 204-IL). Feeder cells were grown for at least 24 hours before plating B cells.

[0155] Growth media conditions with MEGA-CD40L. Media conditions were the same as above, except there were no feeder cells. Instead, B cells were plated in media containing RPMI-1640 + 10% FCS, Penn / strep (100u / ml, 100ug / ml), sodium selenite (100nM), IL-4 (2ng / ml) (R&D 204-IL), and MEGA-CD40L (100ng / ml).

[0156] B cell growth kinetics in HeLa-CD40L and MEGA-CD40L conditions are shown in Figure 1. This study demonstrated that human B cells can grow and expand in culture with HELA-CD40L feeder cells, but do not expand in the presence of MEGA-CD40L.

[0157] Purified B cells were started at an equivalent density of approximately 10,000 cells / ml on day 0 and grown on HELA-CD40L feeder cells. On day 4, the cultures were split into two groups, one group continued to grow in the same medium as the HELA-CD40L feeder cells, while the other group was grown in medium supplemented with MEGA-CD40L (0.1 μg / mL) but in the absence of HELA feeder cells. On day 8, an additional 100 ng / mL of MEGA-CD40L was added to the growth medium under MEGA-CD40L conditions. The arrows in FIG. 1 indicate the times at which MEGA-CD40L treatment was administered.

[0158] This data shows that by day 11 there was significant proliferation of B cells when grown on HELA-CD40L feeder cells, but no significant proliferation was observed at day 11 when grown on MEGA-CD40L.

[0159] Example 3: Feeder-free B cell expansion In this example, B cells were expanded in the absence of feeder cells expressing HELA-CD40L. B cells were cultured at 2.5 × 105 B cells were plated in expansion medium containing: CD40L (SEQ ID NO: 140, 500 μg resuspended in 500 μL); CD40L cross-linking antibody (SEQ ID NO: 143 and 144); human AB serum (IC092938249 VWR); and 10 μL of penicillin streptomycin. B cells were expanded for at least 15 days by plating at a density of either (i) 100,000-150,000 / ml, or (ii) 1,000,000 / mL and replenishing with fresh medium every 7 days.

[0160] Cells maintained at a density of 100,000-150,000 cells / mL were able to expand over 200-fold in two weeks, while cells maintained at a density of 1,000,000 cells / ml showed significantly less cell growth. See Figure 2.

[0161] Example 4: Manipulation of expanded B cells Expanded human B cells were engineered to express various chimeric receptors using adenoviral vectors. B cells were first purified, enriched, and expanded using the techniques described in Examples 1 and 3. B cells were cultured in Expansion Medium of Example 3 for 10 days and then transduced with the adenoviruses listed in Table 6. [Table 6]

[0162] Either Ad5 adenovirus loaded B cells were then cultured in vitro for 10 days using the B cell expansion medium described herein. The expanded B cells were then transduced with adenovirus according to the following protocol.

[0163] B cells were grown for 10 days, achieving a yield of approximately 14 million cells. Several adenovirus F35 virus constructs encoding human BCR were tested. An example is as follows: An adenovirus encoding GPC3-specific BCR was used. An adenovirus empty vector control was also used. No viral sham control was used.

[0164] 20 μl 10 9 / ml of viral particles were added to 0.5 million B cells under the following conditions:

[0165] B cells were plated on 24-well plates in OPTIMEM® containing 0.2% BSA and CD40L+X linker (Miltenyi Product #130-098-775, same concentration as described in the expansion medium above), IL-4 (described in the expansion medium), and 200 μl of 0.5 μg / mL polybrene. After adding the virus, the plates were spun at 1100 g for 1 hour, then transferred to the incubator for 2.5 hours and fed with 2 mL of fresh expansion medium. After 3 days, the cells were stained with GPC3-BV to detect B cells expressing the BCR. The results are shown in Figures 2A-2C and show that at least 67% of the cells expressed the GPC3 BCR 72 hours after transduction.

[0166] Example 5: Expression of Ad5RGD-GFP in human B cells B cells from PBMC were cultured for 10 days. B cells were transduced with an Ad5-RGD construct encoding GFP and placed in OPTIMEM® containing 0.2% BSA, CD40L+X linker, 5% human AB serum, and IL-4. Virus was then added and spun at 1100g for 1 hour, then incubated at 37°C for 3 hours. Media was then switched to normal human B cell growth media until analysis.

[0167] The results of the GFP expression test at multiple time points were as follows: [Table 7]

[0168] To address the effect of the virus on cell viability, the total cell number and the percentage of GFP positive counts are tested by VICELL® and FACS on days 6, 8, and 10. The results are shown in Figure 4. Ad5-RGD modified GFP transduction results in high efficiency of GFP expression in B cells. For total B cells, approximately 60% expression was maintained for at least 10 days. Transduced B cells continued to proliferate for at least one week after transduction.

[0169] Example 6: Expression of mouse BCR constructs or IL-10 in human B cells by adenoviral transduction B cells derived from PBMC were cultured for 10 days. B cells were then transduced with mouse IL-10 or Ad5-RGD constructs encoding mouse BCR in several formats. (Mice were used while waiting for human formats). B cells were then cultured in conditions containing OPTIMEM® with 0.2% BSA, CD40L+X linker, 5% human AB serum, and IL-4. Virus was added, spun at 1100g for 1 hour, then incubated at 37°C for 3 hours, then switched to normal human B cell growth media until analysis. Virus was tested at two ratios (20μl:0.5 million B cells and 2μl:0.5 million B cells).

[0170] Expression studies were performed on day 4, 96 hours after transduction. IL-10 supernatants were stored at -80°C. GPC3 constructs were detected by binding to human GPC3-BV. Sarcoglycan constructs were detected by binding to the FITCstrep tag. The results are shown in Figures 5-9.

[0171] Example 7: Monitoring in vivo homing and expression of human BCR in human B cells after adenoviral delivery B cells from PBMC were cultured for 10 days. B cells were then transduced with Ad5f35 constructs encoding luciferase + / - GPC3-BCR. PWF-524 / PWWF-684 (Luc+GPC3 CAR) and PWF-684 (Luc) were transduced and then cultured overnight in Wennhold medium to allow time for expression of BCR and Luc. Approximately 900,000 cells were then delivered IV into HEPG2 tumor-bearing mice. Luc was monitored by bioluminescence.

[0172] The data are shown in Figure 10. We show that GPC3 CAR(524) enriches homing of human B cells 100-fold to the TDLN of SHORN mice bearing HEPG2 tumors. Specifically, SHORN mice are deficient in T cells, B cells, and NK cells that allow human B cells not to be rejected. A 100-fold enrichment was observed in the TDLN compared to the lung. A 2-5-fold higher signal was observed in the lung, which may be due to HEPG2 metastasis. Figure 10 shows that engineered GPC3 CARS can help instruct B cells to home to inflamed lymphoid organs.

Claims

[Claim 1] The invention described in the present specification.

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