Engineered b cells and therapeutic uses thereof

Abstract

A population of immune cells, comprising a plurality of engineered B cells, which comprise one or more exogenous nucleic acids encoding one or more immune regulatory agents, such as interleukin-12 (IL-12), CD40L, tumor necrosis factor alpha (TNF-α), OX40L, 4-1BBL, PD-L1, or fusion polypeptide(s) thereof are provided. Also provided herein are methods for treating cancer using the engineered B cells.

Description

[0001] ENGINEERED B CELLS AND THERAPEUTIC USES THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of the filing date of U.S. Provisional Application No. 63 / 516,687, filed July 31, 2023, the entire contents of which is incorporated by reference herein.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on July 23, 2024, is named “063710-501001WO_Seq_Listing_ST26.xml” and is 41,020 bytes in size.

[0006] BACKGROUND OF THE INVENTION

[0007] Cancer is a disease characterized by abnormal cells that divide uncontrollably and can infiltrate and destroy normal tissues and / or organs of a subject. Cancer is the second leading cause of death globally and is responsible for nearly 10 million deaths in 2020, in which the most common cancers include, lung cancer (about 2.21 million cases; 1.80 million deaths), breast cancer (about 2.26 million cases; 685,000 deaths), colorectal cancer (about 1.93 million cases; 916,000 deaths), prostate cancer (about 1.41 million cases; 375,000 deaths), skin cancer (about 1.20 million cases), and gastric cancer (about 1.09 million cases; 769,000 deaths).

[0008] Treatments for cancers may vary with the type of cancer and how advanced it is. Conventional treatments for cancers include surgery, radiation therapy, and chemotherapy. Such treatments usually cause a variety of complications or side effects, such as infection, blood clot, bleeding, nausea and vomiting, diarrhea, nerve or muscle damage, incontinence, and sex and fertility issues. Immunotherapy provides an alternative strategy for cancer treatment that aims at specifically stimulating immune responses of a subject against cancer cells via, for example, blocking immune checkpoints, or enhancing the ability of immune cells (e.g., T cells or B cells) to target and destroy cancer cells. Serious adverse effects associated with immunotherapy-medicated overstimulation or non-specific toxicity have been reported in cancer patients, including neurotoxicity, cytokine release syndrome (CRS), allergy, organ inflammation, and autoimmune disorders.

[0009] It is therefore of great importance to develop efficient cancer treatments specifically targeting cancer cells without affecting normal cells and / or tissues. SUMMARY OF THE INVENTION

[0010] The present disclosure is based, at least in part, on the discoveries that allogenic B cells engineered (e.g., genetically engineered) to express immune modulating agents such as IL-12-Fc fusion polypeptide successfully induced T cell infiltration into tumor tissues and the co-use of the allogenic B cells and immune checkpoint inhibitors (e.g., anti-PD-1 antibody) exhibited superior anti-tumor activity. Accordingly, provided herein are immune cells comprising engineered (e.g., genetically engineered) B cells expressing one or more immune regulatory agents or fusion polypeptides comprising such, therapeutic uses of such immune cells in cancer therapy, and methods for producing the engineered e.g., genetically engineered) B cells.

[0011] In some aspects, the present disclosure provides a population of immune cells, comprising a plurality of engineered (e.g., genetically engineered) B cells. The engineered B cells comprise one or more exogenous nucleic acids encoding one or more immune regulatory agents, for example, interleukin- 12 (IL- 12), CD40L, tumor necrosis factor alpha (TNF-a), OX40L, 4-1 BBL, PD-L1, or a fusion polypeptide comprising such. The engineered B cells express the one or more immune regulatory agents encoded by the exogenous nucleic acid(s). In some embodiments, the engineered B cells stably express the one or more immune regulatory agents encoded by the exogenous nucleic acid(s).

[0012] In some embodiments, the one or more immune regulatory agents comprise at least one fusion polypeptide of IL-12, CD40L, TNF-a, OX40L, PD-L1, and / or 4-1BBL. In some instances, the at least one fusion polypeptide comprises IL- 12, CD40L, TNF-a, OX40L, PD- Ll, or 4-1 BBL, and an Fc fragment.

[0013] In some examples, the one or more immune regulatory agents comprise IL- 12, or a fusion polypeptide comprising such. For example, the IL- 12 may comprise the amino acid sequence of any one of SEQ ID NOs: 7-10. In some instances, the IL-12 polypeptide can be an IL-12-Fc fusion polypeptide. In some specific examples, the IL-12-Fc fusion polypeptide may comprise the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0014] In some examples, the one or more immune regulatory agents may comprise CD40L and TNF-a, or fusion polypeptides thereof (e.g., in combination with the IL-12 or IL-12 fusion polypeptide). For example, the CD40L may comprise the amino acid sequence of SEQ ID NO: 24, and / or the TNF-a may comprise the amino acid sequence of SEQ ID NO: 23. In some instances, the fusion polypeptides further comprise an Fc fragment.

[0015] In some examples, the one or more immune regulatory agents comprise PD-L1 or

[0016] OX40L, or a fusion polypeptide comprising PD-L1 or OX40L (e.g., in combination with the IL-12 or IL-12 fusion polypeptide). In some instances, the PD-L1 is an extracellular domain of a naturally-occurring PD-L1 molecule (PD-L1 ECD). In some specific examples, the one or more immune regulatory agents comprise a PD-L1 ECD-Fc fusion polypeptide or an OX40L-Fc fusion polypeptide.

[0017] Any of the engineered (e.g., genetically engineered) B cells may further express an immune checkpoint inhibitor, for example, an antibody that binds PD-1 or PD-L1.

[0018] In some embodiments, the plurality of B cells are human B cells, which optionally is derived from one or more healthy human donors. In some instances, the population of immune cells provided herein may further comprise a plurality of T cells expressing a chimeric antigen receptor (CAR).

[0019] In another aspect, provided herein is a method for treating a tumor in a subject, comprising administering to a subject in need thereof an effective amount of the population of immune cells as disclosed herein. In some embodiments, the plurality of engineered (e.g. , genetically engineered) B cells in the immune cell population is allogenic to the subject. In some embodiments, the subject is a human patient having a tumor, for example, a solid tumor. In some instances, the immune cells are administered intratumorally to the subject.

[0020] In some embodiments, the method may further comprise administering to the subject an immune checkpoint inhibitor, a population of T cells expressing chimeric antigen receptor (CAR), or a combination thereof. In some instances, the method further comprises administering to the subject the immune checkpoint inhibitor, which may be an antibody specific to PD-1, or PD-L1, or a combination thereof.

[0021] Also within the scope of the present disclosure are the population of immune cells, comprising the engineered e.g., genetically engineered) B cells as disclosed herein, optionally in combination with CAR-T cells, for use in treating tumor in a subject in need thereof, as well as use of such immune cells for manufacturing a medicament for cancer treatment.

[0022] Further, the present disclosure features a method for producing a population of engineered e.g., genetically engineered) B cells, the method comprising: delivering into a population of immune cells comprising B cells one or more nucleic acids encoding one or more immune regulatory agents, thereby producing a population of engineered B cells expressing the one or more immune regulatory agents. In some embodiments the immune regulatory agents may be interleukin- 12 (IL-12), CD40L, TNF-a, OX40L, 4-1BBL, PD-L1, or a fusion polypeptide comprising such. In some embodiments, the method may further comprise delivering to the population of immune cells or the population of engineered (e.g., genetically engineered) B cells one or more nucleic acids encoding an antibody specific to PD-1 or PD-L1.

[0023] In some embodiments, the population of engineered (e.g., genetically engineered) B cells are human B cells. In some examples, the population of immune cells is from one or more healthy donors, optionally healthy human donors. In other examples, the population of immune cells is a population of human peripheral blood mononuclear cells (PBMCs).

[0024] In some embodiments, the one or more nucleic acids are located on one or more vectors. Examples include, but are not limited to, retroviral vectors, adenoviral vectors, or adeno-associated viral vectors. In other embodiments, the one or more nucleic acids may be mRNAs.

[0025] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawing in combination with the detailed description of specific embodiments presented herein.

[0028] FIG. 1 shows mouse IL- 12 expression levels following activation and retroviral transduction of unmodified mouse B cells (“mB”); mouse B cells engineered to express mouse CD40L, mouse OX40L, and mouse 41BBL (“m3LB”); and mouse B cell engineered to express IL-12-Fc (“ml2B”).

[0029] FIG. 2 shows changes in tumor volume of B16 melanoma cells implanted in C57B6 mice as a function of time following: (1) intratumoral administration of a Dulbecco's phosphate-buffered saline (“DPBS”) control solution; (2) intraperitoneal administration of a checkpoint inhibitor (a-PD-1 antibodies; “PD-1”); and combined intraperitoneal administration of a-PD-1 antibodies and intratumoral administration of IL-12-Fc engineered allogeneic mouse B cells (“ml2B+PDl”).

[0030] FIG. 3 shows immunohistochemical (IHC) CD3+ staining for detection of T cell infiltration in C57BL / 6J mice implanted with B 16 melanoma cells that were treated on days 11 and 14 with two intratumoral injections of DPBS (control) or two intratumoral injections of IL- 12-Fc engineered allogeneic mouse B cells (“ml2B”) followed by IHC staining of tumor tissue isolated from the mice at day 20.

[0031] FIG. 4 shows microscopic pictures at 4x and lOx magnification showing the ability of activated B cells to potentiate expansion of MMP2-targeting CAR-T cells and killing of MMP2-expressing A375.Her2 target cells (human melanoma cell line). The MMP2-targeting CAR-T cells were co-cultured with the MMP2-expressing A375.Her2 target cells for 48 hours in the absence (left panels) or presence (right panels) of the activated B cells.

[0032] FIGs. 5A-5B show the ability of activated B cells to increase expression and secretion of IL-2 (FIG. 5A) and TNF-a (FIG. 5B) by Her2-targeting CAR-T cells co-cultured with Her2-expressing target cells (A375.Her2).

[0033] FIGs. 6A-6D show efficacy and safety of IL-12-engineered allogeneic B cells in C57B6 mice implanted with B 16 melanoma cells (N=5), in combination with an anti-PDl antibody. FIG. 6A: tumor growth over days post implantation. FIG. 6B: tumor growth inhibition. FIG. 6C: survival over days post implantation. FIG. 6D: weight over days post implantation.

[0034] FIGs. 7A-7E shows efficacy and safety of TNF-a and CD40L-engineered allogeneic B cells in C57B6 mice implanted with B16 melanoma cells (N=5), in combination with an anti- PDl antibody. FIG. 7A: tumor growth over days post implantation. Cured mice were rechallenged with B16 tumor at day 69. FIG. 7B: tumor growth inhibition. FIG. 7C: survival over days post implantation. FIG. 7D: weight over days post implantation. FIG. 7E: mouse CD40L (mCD40L) expression on TNF-a and CD40L-engineered allogeneic B cells confirmed by flow cytometry.

[0035] FIGs. 8A-8D shows efficacy and safety of mRNA (encoding TNF-a and CD40L) electroporated allogeneic B cells in C57B6 mice implanted with B16 melanoma cells (N=5), in combination with an anti-PDl antibody. FIG. 8A: tumor growth over days post implantation. FIG. 8B: tumor growth inhibition. FIG. 8C: survival over days post implantation. FIG. 8D: weight over days post implantation.

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037] B cells are mostly known as antibody secreting cells. One underestimated fact is that B cells can present antigens and activate T cells. Studies have shown that CD40 activated B cells are excellent antigen presenting cells comparable to dendritic cells, the most potent antigen presenting cells in human body. Like T cells and macrophages, B cells can be either pro-tumor or anti-tumor depending on how they interact with the surrounding microenvironment and differentiate.

[0038] It is reported herein that, unexpectedly, allogenic B cells engineered (e.g., genetically engineered) to express immune regulatory agents such as IL-12-Fc fusion polypeptide induced a high level of T cell infiltration into tumor issues and exhibited high anti-tumor activity when co-used with immune checkpoint inhibitors such as an anti-PD-1 antibody. Further, activated B cells were found to potentiate T cell activities, including cytokine production, expansion and anti-tumor activity. Accordingly, the engineered B cells are expected to be effective in cancer therapy (e.g., in allogenic B cell therapy of cancer), either taken alone or in combination with other anti-cancer agents such as immune checkpoint inhibitors and / or CAR-T therapy.

[0039] I. Population of Immune Cells Comprising Engineered B Cells

[0040] In one aspect, the present disclosure provides a population of immune cells comprising a plurality of genetically engineered B cells provided herein, and optionally a plurality of CAR-T cells, as further describe below.

[0041] A. Engineered B Cells

[0042] The population of immune cells provided herein comprises a plurality of engineered e.g., genetically engineered) B cells that express one or more immune regulatory agents, which can be naturally occurring cytokines or immune stimulatory ligands, functional variants thereof, or fusion polypeptides comprising such (e.g., Fc fusion polypeptides). The one or more immune regulatory agents are encoded by one or more exogenous nucleic acids introduced into the B cells. The coding sequences for the one or more immune regulatory agents are in operable linkage to one or more suitable promoters so that the one or more immune regulatory agents are expressed in the engineered B cells.

[0043] As used herein, “an exogenous nucleic acid” refers to a nucleic acid that is not native to the host cells (e.g. , the parent B cells for producing the engineered B cells). The exogenous nucleic acid carries one or more transgenes encoding the immune regulatory agents disclosed herein and can be delivered into the host cells via a conventional method to produce engineered (e.g., genetically engineered) cells comprising the exogenous nucleic acid and capable of expressing the immune regulatory agents encoded by the transgene(s). In some instances, the transgene carried by the exogenous nucleic acid may have an endogenous counterpart, i.e. , an endogenous gene encoding the same immune regulatory agent. In other instances, the transgene may be distinct from any nucleic acid naturally present in the cell. In some embodiments, the engineered (e.g., genetically engineered) B cells may express the one or more immune regulatory agents transiently. Alternatively, the engineered B cells may stably express the one or more immune regulatory agents. Such engineered B cells can be stably transformed with the exogenous nucleic acid(s) encoding the immune regulatory agent(s). In some instances, the exogenous nucleic acids encoding the immune regulatory agents may be incorporated into the genome of B cells. Alternatively, the exogenous nucleic acid(s) may exist extrachromosomally (e.g., episomally).

[0044] In some embodiments, the engineered B cells provided herein may further express one or more immune checkpoint inhibitors, such as antibodies that bind PD-1 or PD-L1.

[0045] (a) Immune Regulatory Agents

[0046] The engineered (e.g., genetically engineered) B cells provided herein express one or more immune regulatory agents, which refer to polypeptides that are capable of modulating immune responses. In some embodiments, the immune regulatory agent can be a naturally occurring cytokine such as interleukin- 12 (IL- 12) or tumor necrosis factor alpha (TNF-a) or an immune stimulatory ligand, such as CD40L, OX40L, inducible T-cell co-stimulator (OX40L), 4-1BBL, ICOSL, IGITRL, CD70, LIGHT (TNFSF14, CD258), CD80 / CD86, CD155 / CD112, or PD-L1. Amino acid sequences for human TNF-a, human CD40L, human OX40L, and human 4-1BBL can be found in NCBI Accession No: NP_000585.2, NCBI Accession No. NP_000065, UniProtKB / Swiss-Prot Accession No. P23510, and NCBI Accession NP_003802, respectively.

[0047] In other embodiments, the immune regulatory agent disclosed herein may be a variant derived from a naturally occurring cytokine or immune stimulatory ligand, for example, a subunit, a fragment (e.g., an extracellular domain), or a functional variant of the cytokine or immune stimulatory ligand. In yet other embodiments, the immune regulatory agent may be a fusion polypeptide comprising the naturally occurring cytokine or immune stimulatory ligand or the variant thereof (e.g., an Fc-fusion polypeptide).

[0048] A functional variant of a naturally occurring cytokine or immune stimulatory ligand is a polypeptide or protein having substantially similar structure and bioactivity as the native counterpart. For example, the functional variant may comprise an amino acid sequence at least 85% (e.g., at least 90%, at least 95%, at least 97%, at least 98% or above) identical to that of the native counterpart. The “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264- 68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0049] Alternatively, or in addition, the functional variant may contain one or more conservative amino acid residue substitutions relative to the native counterpart. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0050] In some embodiments, the immune regulatory agent expressed in any of the engineered (e.g., genetically engineered) B cells may be a fusion polypeptide comprising any of the immune modulating polypeptides (naturally occurring or variants thereof) and a fusion partner such as an immunoglobulin Fc fragment (Fc domain). As used herein, the terms “Fc fragment”, “Fc domain” and "Fc region” refer to the C-terminal region of the heavy chain of an antibody, which includes Ig heavy chain constant region domains CH2 and CH3 and may include all or part of the Ig hinge domain. The Fc domain can form a monomer or dimer of two polypeptide chains joined via the hinge region cysteine residues forming inter-polypeptide disulfide bonds. The Fc domain typically encodes various effector function(s) that can be modified or deleted. In some embodiments, the Fc fusion polypeptides may include Fc regions modified as described in U.S. Patent No. 5,624,821 and U.S. Patent Application No. 2015 / 0266966. Exemplary mouse and human Fc fragment sequences for fusion to IL- 12 subunits or other immune regulatory agents are set forth in SEQ ID NOs: 13 and 15, respectively. (i) Interleukin- 12 (IL- 12)

[0051] In one embodiment, the engineered (e.g., genetically engineered) B cells disclosed herein comprise an expression cassette for producing interleukin- 12 (IL-12). Naturally occurring Interleukin- 12 is a disulfide-linked heterodimeric cytokine with multiple biological effects on the immune system, including enhancement of cytotoxicity by T lymphocytes and NK cells, enhancement of IFN-y and TNF-a secretion, and inhibition of IL-4 production. In addition, IL- 12 has an anti-angiogenic activity, and is known to reduce tumor growth and reverse tumor-induced immunosuppression. Naturally occurring IL- 12 is a heterodimeric protein containing two subunits encoded by two separate genes, IL-12A (coding for the p35 subunit, IL- 12 p35) and IL-12B (coding for the p40 subunit, IL- 12 p40).

[0052] As used herein, the term “IL- 12” used herein encompasses any naturally occurring IL- 12 (e.g., human IL- 12) or a subunit thereof (e.g., the IL-12p35 subunit and the IL-12p40 subunit). IL-12 as used herein also encompasses variants derived from the naturally occurring IL-12 as provided herein (a functional variant disclosed herein). For example, the IL-12 molecule may be a single-chain polypeptide comprising the IL-12p35 and IL-12p40 subunit, which may be connected by a peptide linker, such as a Gly / Ser-rich peptide (e.g., (GGGGSb, SEQ ID NO: 17). In other examples, the IL-12 molecule may be a fusion polypeptide comprising the IL-12p35 subunit, the IL-12p40 subunit, or both, and a fusion partner (e.g., an Fc fragment).

[0053] In some examples, the B cells are engineered (e.g., genetically engineered) to express the IL-12p35 and / or IL-12p40 subunits of IL- 12, which may be in fusion with a fusion partner such as an Fc fragment. In some instances, the subunit p35 and / or p40 expressed by the engineered B cells may be in monomeric form, in homodimeric form, or a combination thereof. In other examples, the B cells are engineered to express the IL-12p35 / IL-12p40 heterodimer (a.k.a. , IL-12p70). In some instances, the IL-12p35 and IL-12p40 subunits can be encoded by one exogenous nucleic acid, e.g., in a multi-cistronic expression cassette or in two separate expression cassettes cloned into one expression vector. Alternatively, the IL-12p35 and IL- 12p40 may be encoded by two separate exogenous nucleic acids, e.g., in two separate expression vectors. In some instances, one or both of the p35 and p40 subunit may be in fusion with an Fc fragment. In yet other examples, the engineered B cells may express a single polypeptide comprising the IL-12p35 and the IL-12p40 subunits, which may be connected by a peptide linker. In some instances, the single polypeptide may further comprise an Fc fragment.

[0054] In some embodiments, the IL-12p35 and / or IL-12p40 protein subunits may be from a naturally occurring IL- 12 protein, including those from any suitable species, e.g., from a mammal such as mouse, rat, rabbit, pig, a non-human primate, or human. Naturally occurring IL- 12 protein subunits from various species are well known in the art and their sequences can be retrieved from a public gene database, such as GenBank. In some instances, an IL- 12 protein subunit used herein may be a functional variant of a naturally occurring IL- 12 subunit ( .g., a functional variant of a human IL- 12 subunit) as disclosed herein. Such a functional variant may share a high sequence homology (e.g., at least 85%, at least 90%, at least 95%, or above) with the wild-type counterpart and has substantially similar bioactivity as the wild-type counterpart e.g., at least 80% of a bioactivity as compared with the wild-type counterpart).

[0055] Exemplary amino acid sequences corresponding to mouse and human IL- 12 polypeptides and other polypeptides described herein are provided in Table 1 below:

[0056] Table 1. Amino Acid Sequences for Exemplary Polypeptides Described Herein

[0057] *linkers underlined, italicized and bolded; exemplary signal peptide italicized

[0058] ( ii ) Other immune regulatory agents

[0059] In some embodiments, the engineered (e.g., genetically engineered) B cells may express a tumor necrosis factor alpha (TNF-a) polypeptide. The anti-tumor activity of TNF-a is well established and can be mediated through a variety of mechanisms including: (1) Cellular apoptosis by binding to tumor cell surface receptors; (2) T-effector cell activation (macrophage and NK cells) by blocking T-Reg cells that are immune suppressors; (3) Inducing tumor microvasculature collapse through endothelial cell modulation and disruption of neoangiogenesis including disruption of tumor vasculature; (4) Promoting TAM (tumor associated macrophages) to Ml anti-tumor stage; (5) Attraction and stimulation of neutrophils and monocytes to sites of activation for anti-tumor immune responses; and (6) Downregulation of IL- 13 expression by eosinophilic-like cells and inhibition of tumor induced monocyte differentiation to immunosuppressive phenotypes (reviewed in J. Transl. Med., 16, Article No. 242 (2018)).

[0060] In some embodiments, the engineered (e.g., genetically engineered) B cells may express an immune stimulatory molecule such as an immune stimulatory ligand, which may be able to activate B cells and / or T cells. Exemplary immune stimulatory molecules for expression in the engineered B cells disclosed herein include, but are not limited to, CD40L, inducible T-cell co-stimulator (OX40L), 4-1BBL, ICOSL, IGITRL, CD70, LIGHT (TNFSF14, CD258), CD80 / CD86, PD-L1, and CD155 / CD1 12. In some instances, the immune stimulatory molecule may be a functional fragment of a naturally occurring counterpart, for example, a fragment comprising an extracellular domain of the naturally occurring counterpart e.g., PD- L1 such as human PD-L1). In some specific examples, the engineered B cells may comprise an exogenous nucleic acid encoding PD-L1, which is an extracellular domain of a naturally- occurring PD-L1 molecule (e.g., human PD-L1). In some instances, the immune stimulatory molecule can be in fusion with a fusion partner such as an Fc fragment to form an Fc fusion polypeptide.

[0061] Exemplary amino acid sequences corresponding to mouse and human TNF-a and CD40L polypeptides described herein are provided in Table 1 above.

[0062] (b) Immune Checkpoint Inhibitors

[0063] In some embodiments, the population of engineered (e.g., genetically engineered) B cells may express an immune checkpoint inhibitor. In one embodiment, the immune checkpoint inhibitor is designed to specifically target programmed death- 1 (PD-1) or its ligand PD-L 1. PD- 1 is an immune checkpoint protein that limits the activity of T cells in peripheral tissues at the time of an inflammatory response to infection and to limit autoimmunity PD-1 inhibition in vitro enhances T-cell proliferation and cytokine production in response to a challenge by specific antigen targets or by allogeneic cells in mixed lymphocyte reactions. A strong correlation between PD-1 expression and response was shown with inhibition of PD-1 (Pardoll, Nature Reviews Cancer, 12: 252-264, 2012). PD-1 inhibition can be accomplished by a variety of mechanisms including antibodies against PD-1, and antibodies against its ligands, including PD-L1 and PD-L2.

[0064] In one embodiment, the PD- 1 inhibitor expressed by the engineered B cells is an anti- PD-1 antibody. In certain embodiments the anti-PD-1 antibody is an FDA-approved antibody selected from pembrolizumab, nivolumab, cemiplimab, dostarlimab, and retifanlimab. In other embodiments, the anti-PD-1 antibody is selected from the group consisting of acrixolimab, AMP-514 (MEDI0680), camrelizumab, cosibelimab, INCMGA00012, sintilimab, spartalizumab, tislelizumab, toripalimab. In another embodiment, the PD-1 inhibitor is a PD- L1 extracellular domain (PD-L1 ECD) or AMP-224, a fusion polypeptide of B7-DC.

[0065] In another embodiment, the PD-1 or PD-L1 inhibitor is an anti-PD-Ll antibody, such as atezolizumab, avelumab, or durvalumab. In other embodiments, the PD-1 or PD-L1 inhibitor is KNO35, cosibelimab, AUNP12, CA-170, BMS-936559, or BMS-986189.

[0066] Examples of PD-1 and PD-L1 inhibitors are described in U.S. Pat. Nos. 7,488,802; 7,943,743; 8,008,449; 8,168,757; 8,217,149, and W003042402, WO2008156712, W02010089411, W02010036959, WO2011066342, WO2011159877, W02011082400, WO2011161699, and W02015035606.

[0067] Another immune checkpoint protein for targeting is cytotoxic T lymphocyte antigen 4 (CTLA-4), which down-regulates T-cell activation pathways. Inhibition of CTLA-4 has been shown to augment T-cell activation and proliferation. Inhibitors of CTLA-4 include anti- CTLA-4 antibodies. Anti-CTLA-4 antibodies bind to CTLA-4 and block the interaction of CTLA-4 with its ligands CD80 / CD86 expressed on antigen presenting cells and thereby block the negative down regulation of immune responses elicited by the interaction of these molecules. Examples of anti-CTLA-4 antibodies are described in U.S. Pat. Nos. 5,811,097; 5,811,097; 5,855,887; 6,051,227; 6,207,157; 6,682,736; 6,984,720; and 7,605,238. In one embodiment, the anti-CTLA-4 antibody is tremelimumab (ticilimumab, CP-675,206). In another embodiment, the anti-CTLA-4 antibody is ipilimumab (MDX-010, MDX-101, Yervoy®), a fully human monoclonal IgG antibody that binds to CTLA-4. Ipilimumab is marketed under the name Yervoy™ and has been approved for the treatment of unresec table or metastatic melanoma.

[0068] Other immune checkpoint inhibitors include T Cell Immunoreceptor With Ig And ITIM Domains (TIGIT) inhibitors, lymphocyte- activation gene 3 (LAG3) inhibitors, such as IMP321, a soluble Ig fusion polypeptide (Brignone el al., 2007, J. Immunol. 179:4202-4211); B7 inhibitors, such as B7-H3 and B7-H4 inhibitors, e.g., anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834); T-cell immunoglobulin mucin 3 (TIM3) inhibitors; A2AR inhibitors; B and T lymphocyte attenuator (BTLA) inhibitors; indoleamine 2,3-dioxygenase 1 (IDO-1) inhibitors, e.g., INCB024360, an IDO1 inhibitor; V-domain Ig suppressor of T cell activation (VISTA) inhibitors; Killer cell immunoglobulin-like receptor (KIR) inhibitors, such as lirilumab (INN), an antibody binding to KIR2DL1 / 2L3; or an antibody binding CD27, CD161, CD73, CD38,CD39, CD93, CD47, CD70, or VTCN1.

[0069] In some embodiments, monoclonal antibodies against the foregoing products can be prepared using conventional hybridoma technology, and VH and VL polynucleotide sequences can be determined and then be cloned into one or more vectors for expression in the B- or T cells of the present disclosure. General techniques for production of mouse, humanized, and human antibodies are known in the art. Further, genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bi-specific antibodies, can be produced via, e.g., conventional recombinant DNA technology.

[0070] (c) Method of Producing Engineered B Cells

[0071] The engineered (e.g., genetically engineered) B cells disclosed herein may be prepared by conventional methods (e.g., recombinant technology) or disclosures provided herein. For example, nucleic acids encoding the immune regulatory agents or immune checkpoint inhibitors as disclosed herein can be delivered into parent B cells via routine methods to produce the genetically engineered B cells that express the immune regulatory agents and optionally the immune checkpoint inhibitors.

[0072] (i) B cells sources, isolation, and culture of B cells

[0073] The parent B cells for making the engineered (e.g., genetically engineered) B cells disclosed herein may be human B cells obtained from one or more healthy donors. The B cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, tissue from a site of infection, and spleen tissue. In one example, the source of parent B cells is PBMCs. In some examples, B cells may be isolated from peripheral blood by apheresis or leukapheresis using techniques known in the art. For example, PBMCs may be isolated using FICOLL™ (Sigma- Aldrich, St Louis, Mo.) and CD19+ B cells purified by negative or positive selection using any of a variety of antibodies known in the art, such as the Rosette tetrameric complex system (StemCell Technologies, Vancouver, Canada) or MACS™ MicroBead Technology (Miltenyi Biotec, San Diego, Calif.). In certain embodiments, memory B cells are isolated as described by Jourdan et al., (Blood. 2009 Dec. 10; 114(25):5173-81). For example, after removal of CD2+ cells using anti-CD2 magnetic beads, CD19+CD27+ memory B cells can be sorted by FACS. Bone marrow plasma cells (BMPCs) can be purified using antiCD 138 magnetic microbeads sorting or other similar methods and reagents. Human B cells may be isolated, e.g., using CD19 MicroBeads, human (Miltenyi Biotec, San Diego, Calif.). Human memory B cells may be isolated, e.g., using the Memory B Cell Isolation Kit, human (Miltenyi Biotec, San Diego, Calif.).

[0074] Other isolation kits are commercially available, such as R&D Systems' MagCellect Human B Cell Isolation Kit (Minneapolis, Minn.) and Miltenyi Biotech’s Human B Cell-, Memory Cell-, Plasma Cell- and Pan B Cell Isolation Kits (Gaithersburg, MD). In certain embodiments, resting B cells may be prepared by sedimentation on discontinuous Percoll gradients, as described in (Defranco et al., (1982) J. Exp. Med. 155:1523).

[0075] In one embodiment, PBMCs are obtained from a blood sample using a gradient based purification (e.g., FICOLL™). In another embodiment, PBMCs are obtained from apheresis- based collection. In one embodiment, B cells are isolated from PBMCs by isolating pan B cells. The isolating step may utilize positive and / or negative selection. In one embodiment, the negative selection comprises depleting T cells using anti-CD3 conjugated microbeads, thereby providing a T cell depleted fraction. In a further embodiment, memory B cells may be isolated from the pan B cells or the T cell depleted fraction by positive selection for CD27.

[0076] In one particular embodiment, memory B cells are isolated by depletion of unwanted cells and subsequent positive selection with CD27 MicroBeads. Unwanted cells, for example, T cells, NK cells, monocytes, dendritic cells, granulocytes, platelets, and erythroid cells may be depleted using a cocktail of biotinylated antibodies against CD2, CD14, CD16, CD36, CD43, and CD235a (glycophorin A), and Anti-Biotin MicroBeads.

[0077] B cells, such as memory B cells, can be cultured using in vitro methods to activate and differentiate the B cells into plasma cells or plasmablasts or both. As would be recognized by the skilled person, plasma cells may be identified by cell surface protein expression patterns using standard flow cytometry methods. For example, terminally differentiated plasma cells express relatively few surface antigens, and do not express common pan-B cell markers, such as CD 19 and CD20. Instead, plasma cells may be identified by expression of CD38, CD78, CD138, and IL-6R and lack of expression of CD45. CD27 may also be used to identify plasma cells as naive B cells are CD27-, memory B cells are CD27+ and plasma cells are CD27++. Plasma cells express high levels of CD38 and CD138.

[0078] PBMCs and B lymphocytes from donors may be isolated and cultured in IMDM medium or RPMI 1640 (GibcoBRL Invitrogen, Auckland, New Zealand) or other suitable medium as described herein, either serum-free or supplemented with serum (e.g., 5-10% FCS, human AB serum, and serum substitutes) and penicillin / streptomycin and / or other suitable supplements such as transferrin and / or insulin. In one embodiment, cells are seeded at 1x105cells in 48-well plates and concentrated vector added at various doses that may be routinely optimized by the skilled person using routine methodologies. In one embodiment, B cells are transferred to an MS5 cell monolayer in RPMI supplemented with 10% AB serum, 5% FCS, 50 ng / ml rhSCF, 10 ng / ml rhlL-15 and 5 ng / ml rhlL-2 and medium refreshed periodically as needed. As would be recognized by the skilled person, other suitable media and supplements may be used as desired.

[0079] Methods and kits for activating and expanding PBMCs and B lymphocytes are well known in the art (see e.g., U.S. Patent No. 8,133,727 and Human B Cell Expansion Kit, Miltenyi Biotec, Gaithersburg, MD). In some embodiments, PBMCs and B lymphocytes may be activated and expanded before and after transfection or infection with expression cassettes or vectors comprising exogenous nucleic acids encoding the immune regulatory agents or CARs described herein. In one embodiment, PBMCs or purified B cells are activated before and after gene delivery culturing the cells in media containing IL-4 and multimerized CD40L as described in Example 1.

[0080] (ii) Delivery of Immune Regulatory Agent-Encoding Nucleic Acids into B Cells Nucleic acids encoding the immune regulatory agents described herein for expression in B cells may be derived from any suitable species, e.g., from a mammal such as mouse, rat, rabbit, pig, a non-human primate, or human. Naturally occurring sequences from various species are well known in the art and their sequences can be retrieved from a public gene database, such as GenBank.

[0081] The exogenous nucleic acid(s) encoding one or more immune regulatory agents as describe herein may be introduced into B cells by viral transduction, non-viral transduction, or by gene editing into suitable target sites of interest. In certain embodiments, engineered e.g., genetically engineered) B cells expressing the immune regulatory agents, such as TNF-a may be administered to subjects with solid tumors in view of systemic toxicides known to be associated with their use.

[0082] In some instances, the nucleic acids are delivered to the B cells by viral transduction using a using a viral expression vector or recombinant viral particle. A “vector”, as used herein is any nucleic acid vehicle (DNA or RNA) or expression cassette capable of facilitating the transfer of a nucleic acid molecule into B (and T) cells. In general, vectors include, but are not limited to, plasmids, phagemids, viral vectors, viral particles, and other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of a target nucleotide sequence. A vector for use herein includes a nucleic acid molecule (e.g., a DNA molecule) comprising a nucleotide sequence encoding a protein described as herein, which may optionally include one or more suitable regulatory elements operably linked to provide constitutive or tissue-specific (e.g., B cell specific) expression, including but not limited to promoters, enhancers, 5’ and 3’ untranslated regions (UTRs), insulators, polyadenylation signals, as well as internal ribosome entry sites (IRES) and / or 2A peptide for expressing more than one gene from a single promoter. In certain embodiments, a vector may include nucleic acid(s) encoding one or more immune regulatory agents, wherein the recombinant vector is designed for site specific chromosomal integration (and / or gene replacement / disruption) by gene editing (e.g., CRISPR). In some embodiments, the recombinant vector includes exogenous regulatory elements for driving expression in target cells. In other embodiments, the recombinant vector does not contain exogenous regulatory elements, but is designed for site-specific integration (and / or gene replacement / disruption) into chromosomal sites so that expression is driven by native regulatory agents present in the chromosome.

[0083] A vector for viral transduction contains elements derived from a viral genome (naturally occurring or modified) and can be used to deliver genetic materials e.g., a transgene) into suitable host cells. Viral vectors may be based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with a target nucleotide sequence. In some embodiments, a non-cytopathic virus, such as a retrovirus (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Non-limiting examples of viral vectors include, but are not limited to, retroviral vectors e.g., lentiviral vectors or gammaretroviral vectors), adeno-associated viral vectors (AAV), adenoviral vectors, and hybrid vectors (containing components from different viral genomes). Additional examples of viral vectors are provided in US Patent No. 5,698,443, US Patent No. 5,650,309, and US Patent No, 5,827,703, the relevant disclosures of each of which are herein incorporated by reference for the purpose and subject matter referenced herein.

[0084] In some embodiments, a retroviral vector (e.g., lenti virus-based vector) can be used to introduce the exogenous nucleic acid(s) into the B cells disclosed herein. Preferably, the retroviruses (RVs) are replication-defective (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Retrovirus expression systems are well established and known to those skilled in the art for high-efficiency transduction of genes in vitro, ex vivo, and in vivo. Standard protocols for producing replication-defective RVs (including the steps of incorporating exogenous genetic material into retroviral plasmid vectors, transfecting and encapsidating retroviral plasmid vectors using retroviral packaging cell lines to produce recombinant RV particles, collecting recombinant RV particles from tissue culture media, and infecting target cells with the recombinant RV particles) are known in the art. Lenti virus vectors are particularly useful for producing engineered B cells in view of the natural tropism of lentiviruses for immune cells, including B cells, which is mediated by complement-bound HIV particles binding to complement receptor CD21 on B cells. In some embodiments, a lentivirus vector is used for delivering immune regulatory agent-encoded nucleic acids into B cells (and / or CAR-encoded nucleic acids into T cells).

[0085] In other embodiments, an AAV vector may be used to introduce the exogenous nucleic acid(s) into the B cells disclosed herein. Adeno-associated viruses (AAVs) are small viruses which can, in certain instances, integrate site- specifically into the host genome. Inverted terminal repeats (ITRs) are present flanking the AAV genome and / or a transgene of interest and serve as origins of replication. Preferably, the AAVs are replication-defective (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). AAV expression systems are well established and known to those skilled in the art for high-efficiency transduction of genes in vitro, ex vivo, and in vivo. Standard protocols for producing replication-defective AAVs (including the steps of incorporating exogenous genetic material into AAV plasmid vectors, transfecting and encapsidating AAV plasmid vectors using AAV packaging cell lines (expressing rep and cap) to produce recombinant AAV particles, collecting recombinant AAV particles from tissue culture media, and infecting target cells with the recombinant AAV particles) are known in the art.

[0086] Depending on the packaging line used, surface receptors on capsids in the recombinant AAV particles define various AAV serotypes that can determine the target organs to which the capsids will primarily bind to and most efficiently infect. There are twelve currently known human AAV serotypes. In some embodiments, an AAV serotype 6 (AAV) vector is used for delivering immune regulatory agent-encoded nucleic acids into B cells (and / or CAR-encoded nucleic acids into T cells). In some embodiments, the cells are transduced with a replicationdefective AAV.

[0087] In some embodiments, the non-viral plasmid vector may be complexed with an agent, such as a liposome or poloxamer, for transfection into a B cells (and / or T cells) using standard methods known in the art, such as lipofection. In some embodiments the B cells are activated prior to and after transfection with a plasmid vector or expression vector, or infection with a recombinant virus particle. In some embodiments, the cells are transfected or infected in the presence of an apoptosis inhibitor.

[0088] In some embodiments, the vector is delivered into B cells via encapsulation into lipid nanoparticles. As used herein, the term “lipid nanoparticle” or “LNP” refers to a particle comprising one or more lipids. Lipid nanoparticles include, but are not limited to, liposomes and micelles. Any of a number of lipids may be present, including cationic lipids, ionizable lipids, anionic lipids, neutral lipids, amphipathic lipids, conjugated lipids (e.g., PEGylated lipids), and / or structural lipids e.g., sterols). Such lipids can be used alone or in combination.

[0089] In some embodiments, the nucleic acids encoding the immune regulatory agents may be mRNAs. In some examples, the mRNAs can be delivered into B cells by encapsulation into lipid nanoparticles according to methods known to those skilled in the art. Alternatively, the mRNAs may be delivered to host cells by electroporation. Messenger RNA (mRNA) electroporation is a non-viral method of genetic modification of cells, which has been widely used in immunotherapy (Campillo-Davo et al., Pharmaceutics, 2021 Mar; 13(3): 396-418). Electroporation allows fine-tuning of transfection protocols as well as introduction of more than one protein-coding mRNAs at once.

[0090] A vector may also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate transcriptional control sequences to enable expression in the host cells. Examples of selective markers include a dihydrofolate reductase gene and a neomycin resistance gene for eukaryotic cell culture, and a tetracycline resistance gene and an ampicillin resistance gene for culture of E. coli and other bacteria. By use of such selection markers, it can be confirmed whether the polynucleotide encoding the polypeptide(s) of the present invention has been transferred into the host cells and then expressed without fail.

[0091] (d) CAR-T Cells

[0092] In some embodiments, the population of immune cells comprises a plurality of engineered (e.g., genetically engineered) B cells described herein in combination with a plurality of genetically engineered T cells expressing a chimeric antigen receptor (CAR). A chimeric antigen receptor (CAR) refers to an artificial immune cell receptor that is engineered to recognize and bind to an antigen expressed by undesired cells, for example, disease cells such as cancer cells. A T cell that expresses a CAR polypeptide is referred to as a CAR-T cell. CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner. The non-MHC -restricted antigen recognition gives CAR-T cells the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape. Moreover, when expressed on T-cells, CARs advantageously do not dimerize with endogenous T-cell receptor (TCR) alpha and beta chains.

[0093] There are various generations of CARs, each of which contains different components. First generation CARs join an antibody-derived scFv to the CD3 zeta (^ or z) intracellular signaling domain of the T-cell receptor through hinge and transmembrane domains. Second generation CARs incorporate an additional co- stimulatory domain, e.g., CD28, 4-1BB (41BB), or ICOS, to supply a costimulatory signal. Third-generation CARs contain two costimulatory domains (e.g. , a combination of CD27, CD28, 4-1BB, ICOS, or 0X40) fused with the TCR CD3^ chain. Maude et al., Blood. 2015; 125(26):4017-4023 ; Kakarla and Gottschalk, Cancer J. 2014; 20(2): 151-155). Any of the various generations of CAR constructs is within the scope of the present disclosure.

[0094] In some instances, a CAR can be a fusion polypeptide comprising an extracellular antigen binding domain that recognizes a target antigen (e.g., a single chain variable fragment (scFv) of an antibody or other antibody fragment) and an intracellular domain comprising a signaling domain of the T-cell receptor (TCR) complex (e.g., CD3 and, in most cases, a costimulatory domain. (Enblad et al., Human Gene Therapy. 2015; 26(8):498-505). A CAR construct may further comprise a hinge and transmembrane domain between the extracellular domain and the intracellular domain. Methods for generating scFv expression cassettes from antibodies or antibody sequences are described above.

[0095] The CAR disclosed herein comprises an antigen binding extracellular domain, which may be a single-chain variable fragment (scFv). The antigen-binding extracellular domain may be specific to a target antigen of interest, for example, a pathologic antigen such as a tumor antigen.

[0096] Exemplary tumor-associated antigens include, but are not limited to, AFP, ALK, BAFF-R, BAGE proteins, P-catenin, BCMA, bcr-abl, BRCA1, BORIS, CA9, carbonic anhydrase IX, caspase-8, CCR5, CD2, CD3, CD5, CD7, CD19, CD20, CD22, CD30, CD38, CD70, CD123, CD133, CD171, CDK4, CEA, CEACAM5, CEACAM6, CS1, cyclin-Bl, CYP1B1 , desmoglein (Dsg3), EGFR, EGFRvITI, ErbB2 / Her2, ErbB3, ErbB4, ETV6-AML, EpCAM, EphA2, FAP, 5T4, F0LR1, Fra-1, FSHR, GAGE proteins (e.g., GAGE-1, -2), GD2, GD3, GloboH, glypican-3, GM3, gplOO, GPC3, HER-2, HLA / B-raf, HLA / k-ras, HLA / MAGE- A3, hTERT, Lewis Y, LMP2, MAGE proteins (e.g., MAGE-1, -2, -3, -4, -6, and -12), MART- 1, mesothelin, ML-IAP, Mucl, Muc2, Muc3, Muc4, Muc5, Mucl6 (CA-125), MUM1, NA 17, NKG2D, NY-BR1, NY-BR62, NY-BR85, NY-ES01, 0X40, pl5, p53, PAP, PAX3, PAX5, PCTA-1, PLAC1, PRLR, PRAME, PSCA, PSMA (FOLH1), RAGE proteins, Ras, RGS5, Rho, ROR1, SART-1, SART-3, Steap-1, Steap-2, STn, survivin, TAG-72, TGF-p, TMPRSS2, Tn, TRP-1, TRP-2, tyrosinase, VEGFR11, and uroplakin-3.

[0097] The CAR polypeptide disclosed herein may further contain a transmembrane domain, which can be a hydrophobic alpha helix that spans the membrane. As used herein, a “transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. The transmembrane domain can provide stability of the CAR containing such. In some embodiments, the transmembrane domain of a CAR as provided herein can be a CD 8 transmembrane domain. In other embodiments, the transmembrane domain can be a CD28 transmembrane domain.

[0098] Further, the CAR polypeptide may comprise a hinge domain located between an extracellular domain (comprising the antigen binding domain) and a transmembrane domain of a CAR, or between a cytoplasmic domain and a transmembrane domain of the CAR. A hinge domain can be any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or the cytoplasmic domain in the polypeptide chain. A hinge domain may function to provide flexibility to the CAR, or domains thereof, or to prevent steric hindrance of the CAR, or domains thereof.

[0099] Any of the CAR constructs contain one or more intracellular signaling domains (e.g., CD3^, and optionally one or more co-stimulatory domains), which are the functional end of the receptor. Following antigen recognition, receptors cluster and a signal is transmitted to the cell. CD3^ is the cytoplasmic signaling domain of the T cell receptor complex. CD3^ contains three (3) immunoreceptor tyrosine-based activation motif (ITAM)s, which transmit an activation signal to the T cell after the T cell is engaged with a cognate antigen. In many cases, CD3^ provides a primary T cell activation signal but not a fully competent activation signal, which requires a co-stimulatory signaling.

[0100] In some embodiments, the CAR polypeptides disclosed herein may further comprise one or more co-stimulatory signaling domains. For example, the co-stimulatory domains of CD28 and / or 4- IBB may be used to transmit a full proliferative / survival signal, together with the primary signaling mediated by CD3^. In some examples, the CAR disclosed herein comprises a CD28 co-stimulatory molecule. In other examples, the CAR disclosed herein comprises a 4- IBB co-stimulatory molecule.

[0101] IL Pharmaceutical Compositions and Therapeutic Applications

[0102] In another aspect, provided herein are pharmaceutical compositions comprising any of the immune cell populations disclosed herein and therapeutic applications thereof.

[0103] A. Pharmaceutical Compositions

[0104] Any of the engineered (e.g., genetically engineered) immune cells disclosed herein may be formulated in a pharmaceutical composition, which further comprises one or more pharmaceutically acceptable excipients. The pharmaceutical compositions can be used in therapeutic applications, for example, cancer treatment in human patients, which is also disclosed herein.

[0105] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and / or bodily fluids of the subject without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. As used herein, the term “pharmaceutically acceptable carrier” refers to solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents, or the like that are physiologically compatible. The compositions can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt. See, e.g., Berge et al., (1977) J. Pharm. Sci. 66:1-19.

[0106] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable salt. Non- limiting examples of pharmaceutically acceptable salts include acid addition salts (formed from a free amino group of a polypeptide with an inorganic acid, or an organic acid. In some embodiments, the salt formed with the free carboxyl groups is derived from an inorganic base, or an organic base. In some embodiments, the pharmaceutical composition disclosed herein comprises a population of the engineered (e.g. , genetically engineered) B cells comprising and expressing exogenous nucleic acids encoding one or more immune regulatory agents.

[0107] B. Therapeutic Applications

[0108] The immune cells comprising any of the engineered e.g., genetically engineered) B cells, optionally in combination of CAR-T cells, as disclosed herein, may be used for treating cancer, for example, a solid cancer or a hematopoietic cancer such as a T cell or B cell malignancy. Thus, provided herein are methods for treating cancer comprising administering to a subject in need of the treatment an effective amount of the engineered immune cells. In some examples described herein, the cells are expanded in culture prior to administration to the subject in need of the treatment.

[0109] The step of administering the immune cells may include the placement e.g. , transplantation) of cells, e.g., engineered human B cells, optionally in combination with CAR- T cells, into a subject, by a method or route that results in at least partial localization of the introduced cells at a desired site, such as tumor, such that a desired effect(s) is produced. Engineered B- and T cells can be administered by any appropriate route that results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g. , twenty-four hours, to a few days, to as long as several years, or even the lifetime of the subject, i.e., long-term engraftment. For example, in some aspects described herein, an effective amount of the engineered (e.g., genetically engineered) B cells, optionally in combination with CAR-T cells, is administered via a systemic route of administration, such as an intraperitoneal or intravenous route. In some examples, the engineered B cells may be administered intratumorally to a cancer patient.

[0110] The subject may be any subject for whom treatment or therapy is indicated or desired. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human patient. In some embodiments, the human patient having, suspected of having, or a risk for having a solid tumor. Non-limiting examples of solid tumors include, for example, pancreatic cancer, gastric cancer, ovarian cancer, cervical cancer, breast cancer, colon cancer, colorectal cancer, renal cell carcinoma, squamous cell carcinoma, stomach cancer, osteosarcoma, hepatocellular carcinoma, nasopharyngeal carcinoma, thyroid cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung (NSCLC), glioblastoma, and melanoma. In one embodiment, the solid tumor is melanoma, glioblastoma, head and neck cancer, or liver cancer.

[0111] In some examples, the solid tumor is pancreatic cancer such as pancreatic adenocarcinoma, pancreatic cystic tumor, pancreatic acinar cell cancer, pancreatic sarcoma, or pancreatic ampullary cancer. A subject suspected of having pancreatic cancer may exhibit one or more symptoms associated with pancreatic cancer, for example, upper abdomen pain, diarrhea, flushing of the skin and face, hypoglycemia or hyperglycemia, digestive problems, gallbladder proteins, change in weight, or a combination thereof. A subject at risk for pancreatic cancer can be a subject having one or more of the risk factors, e.g., age, increased body mass index, smoking, diabetes, and / or chronic inflammation.

[0112] The engineered B cells administered according to the methods described herein may be allogenic B cells obtained from one or more donors, for example, healthy human donors. “Allogeneic”, as used herein, refers to a cell, cell population, or biological sample comprising cells, obtained from one or more different donors of the same species, where the genes at one or more loci are not identical to the recipient. For example, an engineered CAR-T cell population, being administered to a subject can be derived from T cells from one or more unrelated donors, or from one or more non-identical siblings. In some embodiments, syngeneic cell populations may be used, such as those obtained from genetically identical donors, e.g., identical twins).

[0113] In some embodiments, the engineered immune cell population(s) being administered according to the methods described herein does not induce toxicity in non-cancer cells of the subject or does not induce toxicity in these cells when administered locally or intratumorally. In some embodiments, administration of the immune cell population(s), alone or in combination with anti-immune checkpoint regulator agents triggers apoptosis of the cancer cells.

[0114] “An effective amount” as used herein refers to the amount of each active agent (here the engineered B cells such as genetically engineered B cells) required to confer therapeutic effect on the subject, either alone or in combination with one or more other active agents. Effective amounts vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and co-usage with other active agents.

[0115] The compositions of the invention are administered in effective amounts. An “effective amount” is that amount of a population of engineered B cells provided herein that alone, or together with further doses, produces the desired response, e.g., increases an immune response to tumor, for example, at the tumor microenvironment. In some cases, the desired response is inhibiting the progression of tumor growth. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods or can be monitored according to diagnostic methods of the invention discussed herein. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.

[0116] Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size, gender and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.

[0117] In some embodiments, a subject is administered a population of engineered human B cells, optionally in combination with human CAR-T cells, each at a dose of about IxlO7to about IxlO9engineered cells.

[0118] The clinical outcome of a treatment comprising a composition for the treatment of a medical condition can be determined by the skilled clinician. A treatment is considered "effective treatment," if any one or all of the signs or symptoms of, as but one example, levels of functional target are altered in a beneficial manner (e.g., increased by at least 10%), or other clinically accepted symptoms or markers of disease (e.g., cancer) are improved or ameliorated. Clinical outcome can also be measured by failure of a subject to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in subject and includes: (1) inhibiting the disease, e.g. , arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0119] C. Combination Therapies

[0120] In some embodiments, the population of modified immune cells may be administered in combination with one or more additional anti-cancer therapies, for example, cell therapy such as CAR-T cells, chemotherapeutics, surgery, and / or radiation therapy.

[0121] In one embodiment, a population of engineered (e.g., genetically engineered) B cells is administered together with CAR-T cells such as those described above. The engineered B cells may be administered before, after, or concurrently with the CAR-T cells.

[0122] When administered in combination with the engineered B cells described herein, an engineered human CAR-T cell population being administered according to the methods described herein may comprise autologous or allogeneic T cells. “Autologous”, as used herein, refers to a cell, cell population, or biological sample comprising cells, obtained from the recipient receiving the population of engineered immune cells.

[0123] In some embodiment, a population of immune cells comprising engineered B cells is administered with one or more anti-cancer therapeutic agents. Anti-cancer therapeutic agents useful for combination with the modified immune cells described herein include, but are not limited to, the immune checkpoint inhibitors described above (e.g. , PD-1, PD-L1, and CTLA4 inhibitors), anti-angiogenic agents (e.g. , TNP-470, platelet factor 4, thrombospondin- 1 , tissue inhibitors of metalloproteases, prolactin, angiostatin, endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, interferon gamma, soluble KDR and FLT-1 receptors, and placental proliferin-related protein); VEGF antagonists (e.g., anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments); and chemotherapeutic compounds. Exemplary chemotherapeutic compounds include pyrimidine analogs (e.g. , 5- fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine); purine analogs (<?.g., fludarabine); folate antagonists (e.g., mercaptopurine and thioguanine); antiproliferative or antimitotic agents, for example, vinca alkaloids; microtubule disruptors such as taxane (e.g., paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, and epidipodophyllotoxins; DNA damaging agents (e.g., actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, iphosphamide, melphalan, merchlorethamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide). The engineered B cells of the present disclosure may be administered before, after, or concurrently with the anti-cancer therapeutic agents.

[0124] In some embodiments, radiation or radiation and chemotherapy is used in combination with a population of engineered immune cells described herein. Additional useful agents and therapies can be found in Physician's Desk Reference, 59thedition, (2005), Thomson P D R, Montvale N.J.; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20thedition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15thedition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

[0125] IV. Kits for Use in Cancer Therapy

[0126] The present disclosure also provides kits for use of a population of the engineered (e.g., genetically engineered) B and / or T cells disclosed herein in methods for treating solid tumors. Such kits may include one or more containers comprising a pharmaceutical composition that comprises a population of the engineered B cells, a population of genetically engineered CAR- T cells, a pharmaceutically acceptable carrier, and optionally one or more pharmaceutical compositions comprising one or more lymphodepleting agents.

[0127] In some embodiments, the kit can comprise instructions for use in any of the methods described herein. The included instructions can comprise a description of administration of the engineered B and / or CAR-T cells and optionally the lymphodepletion agents to a subject to achieve the intended therapeutic effects. The kit may further comprise a description of selecting a human patient suitable for treatment based on identifying whether the human patient is in need of the treatment. In some embodiments, the instructions comprise a description of administering the pharmaceutical compositions contained in the kit to a human patient who is in need of the treatment.

[0128] The instructions relating to the use of the population of genetically engineered T cells described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the population of engineered e.g., genetically engineered) B and / or CAR-T cells is used for treating, delaying the onset, and / or alleviating a renal cell carcinoma in a subject.

[0129] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device, or an infusion device. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port. Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.

[0130] General Techniques

[0131] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D.N. Glover ed. 1985); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985; Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984; Animal Cell Culture (R.I. Freshney, ed. (1986» ; Immobilized Cells and Enzymes (IRL Press, (1986; and B. Perbal, A practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.).

[0132] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

[0133] EXAMPLES

[0134] While the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the disclosure.

[0135] Example 1: Generation and Characterization of Genetically Engineered B Cells Expressing Immune Regulatory Agents.

[0136] To evaluate the potential of B cells for use in cell therapies against cancer, B cells were genetically engineered with multiple immune regulatory agents to enhance the efficacy of genetically engineered B cells for use in immunotherapies against cancers, particularly solid tumors. Briefly, mouse B cells were isolated from Balb / c mouse spleens, activated by IL-4 and multimerized CD40L at day 0. The activated B cells were transduced with a retrovirus expressing the costimulatory ligands, mouse 4-1BBL (SEQ ID NO: 18), mouse 0X40 (SEQ ID NO: 19), and mouse CD40L (SEQ ID NO: 20), or a retrovirus carrying a transgene encoding mouse IL-12-Fc (SEQ ID NO: 5) on day 1 and day 2.

[0137] After the transduction, the B cells were cultured in a fresh medium containing IL-4 and the multimerized CD40L. Transgene expression from the CD40L / OX40L / 4-1BBL- engineered B cells (m3LB) and a control group (unmodified B cells, mB) was evaluated using flow cytometry. The results are shown in Table 2 below.

[0138] Table 2. Level of Transgene Expression in Genetically Engineered B Cells*

[0139] Further, secretion of IL-12-Fc by the engineered B cells (ml2B) was evaluated by ELISA. Briefly, activated mouse B cells were transduced with a retrovirus carrying a transgene encoding 3 tandemly linked costimulatory ligands, CD40L, OX40L, and 4-1BBL (SEQ ID NO: 21) or a retrovirus carrying a transgene encoding mouse IL-12-Fc (SEQ ID NO: 5) and activated again after transduction as described above. Supernatant samples were collected at day 4 and the levels of mouse IL-12-Fc were measured by ELISA following conventional methods. As shown in FIG. 1, a high level of IL-12-Fc was detected in the supernatant of engineered ml2B cells, while presence of the IL-12-Fc was not detected in the supernatant samples of engineered m3LB cells or the control mB cells.

[0140] Example 2: Combining Engineered Allogeneic Mouse B Cells Expressing IL-12-Fc with Checkpoint Inhibitor Remarkably Suppressed Melanoma Tumor Growth.

[0141] This example evaluates the effect of engineered allogeneic mouse B cells expressing an IL-12-Fc polypeptide, in combination with a checkpoint inhibitor (a-PD-1 Ab).

[0142] Allogeneic B cells isolated from Balb / c mouse spleens were activated as described in Example 1 above and transduced with a retroviral vector encoding the mouse IL-12-Fc of SEQ ID NO: 5 (ml2B cells).

[0143] C57B6 mice were implanted with 4e5 B 16 melanoma cells at day 0. At days 10, 13, and 16, the C57B6 mice were randomly assigned into three groups (five mice in each group), each of which received 3 injections of: (1) DPBS (intra-tumoral injection); (2) mouse anti- PD-1 antibodies (“PD-1”; intraperitoneal injection), or (3) ml2B allogenic cells injected intratumorally and the anti-PD-1 antibody injected intraperitoneally (“ml2B+PD-l”).

[0144] Tumor growth was monitored over time. As shown in FIG. 2, combining IL-12-Fc- expressing allogeneic mouse B cells with the checkpoint inhibitor (a-PD-1 Ab) greatly suppressed the growth of the B16 melanoma cells. Similar results were obtained when the melanoma tumor-bearing mice received injections of ml2B allogenic cells alone.

[0145] Example 3: Allogeneic Engineered B Cells Expressing IL-12-Fc Induced Heavy T Cell Infiltration in Melanoma

[0146] Immunotherapies, including those involving immune checkpoint inhibitors, have often been shown to be ineffective against non-inflamed, “cold” tumors (tumors that are not likely to trigger a strong immune response). This study evaluates the efficacy of IL-12-Fc- expressing engineered allogeneic mouse B cells in inducing T cell activation and infiltration.

[0147] Briefly, allogeneic B cells were isolated from BALB / c mouse spleens and engineered to express with the mouse IL-12-Fc following the procedures described in Examples 1 and 2 above. C57BL / 6J mice were subcutaneously implanted with B16 melanoma cells at day 0. At days 11 and 14, the C57BL / 6J mice were intratumorally injected with two doses of the allogeneic engineered B cells (ml2B) or DPBS. At day 20, tumor tissue from the treated mice were isolated and subjected to IHC staining. FIG. 3 shows representative slides stained with an anti-CD3 antibody. A high level of CD3+ T cell infiltration was observed in tissues from mice treated with the ml2B cell, but not in tissues from mice treated with DPBS. Compare the left panels with the right panels in FIG. 3. The results of this experiment indicate that allogenic B cells expressing an IL- 12 molecule successfully induced infiltration of T cells into the poorly immunogenic melanoma tissues. Similar results were obtained when the melanoma tumor-bearing mice received injections of ml2B allogenic cells alone.

[0148] Example 4: Impact of Activated B cells on CAR-T Cell Activities

[0149] This example investigates the impact of activated B cells on CAR-T cell features, such as cytokine production, expansion, and anti-tumor cell activity.

[0150] Briefly, frozen human PBMCs were thawed and activated with IL4 and multimerized CD40L to selectively expand B cells for 4 days. MMP2-targeting CAR-T cells were cocultured with MMP2 expressing target cells (A375.Her2), with or without the activated B cells, for 48 hrs.

[0151] Microscopic pictures taken at 4x and lOx showed extensive cell killing of the MMP- expressing human melanoma cells in the presence of the activated B cells as compared with the co-culture without the activated B cells. FIG. 4.

[0152] Further, Her2-targeting CAR-T cells were co-cultured with Her2 expressing target cells (A375.Her2), with or without activated B cells, for 72 hrs. Supernatant samples from coculturing were collected and analyzed to measure the levels of IL-2 and TNF-a. As shown in FIGs. 5A and 5B, co-culture with the activated B cells significantly increased IL-2 and TNF- a production by the CAR-T cells.

[0153] Example 5: Long-Term Efficacy and Safety of IL-12-Engineered Allogeneic B Cells

[0154] This example investigates the efficacy and safety of B cells engineered to expressing the IL-12-Fc fusion polypeptide of SEQ ID NO: 5 (IL-12-engineered allogeneic B cells).

[0155] C57B6 mice were implanted with 5 x 105B16 melanoma cells at day 0. Allogeneic B cells were isolated from Balb / c mouse spleen, activated and retro virally transduced with a viral vector carrying a transgene that expresses the IL-12-Fc fusion polypeptide. At day 10, 11, 12, and 13, C57B6 mice received four treatments as indicated. Each treatment includes an intratumoral injection of the IL-12-engineered allogeneic B cells and an i.p. injection of a mouse PD1 antibody. Tumor growths, mouse weights, and survival were monitored over time.

[0156] As shown in FIGs. 6A-6C, the IL-12-engineered allogeneic B cells, in combination with the anti-PDl antibody, achieved 100% response rate and 40% cure rate (FIG. 6A), remarkable tumor growth inhibition (FIG. 6B), and better survival (FIG. 6C) compared to the control mice, which were treated with DPBS. No significant weight loss was observed in the IL-12-engineered allogeneic B cells (FIG. 6D), indicating safety of the treatment. Tt was observed in prior studies that anti-PDl treatment alone did not exhibit anti-cancer effect in this melanoma mouse model (see, e.g., FIG. 2).

[0157] Overall, intra-tumoral delivery of IL-12-engineered allogeneic B cells exhibited promising anti-cancer activities on the B 16 melanoma model.

[0158] Example 6: Efficacy and Safety of TNF-a and CD40L-Engineered Allogeneic B Cells

[0159] This example investigates the efficacy and safety of TNF-a and CD40L-engineered allogeneic B cells, which are B cells engineered to express TNF-a and CD40L.

[0160] C57B6 mice were implanted with 5 x 105B16 melanoma cells at day 0. Allogeneic B cells were isolated from Balb / c mouse spleen. Allogeneic B cells were activated and retrovirally transduced with viral vectors comprising transgenes for expressing TNF-a (SEQ ID NO:22) and CD40L(SEQ ID NO: 20). At day 8 and 9, C57B6 mice received two treatments as indicated. Each treatment includes an intratumoral injection of TNF-a and CD40L-engineered allogeneic B cells and an i.p. injection of a mouse PD1 antibody. Tumor growths, mouse weights, and survival were monitored over time. For rechallenge study, the cured two mice in the TNF-a and CD40L-engineered allogeneic B cells treatment group were injected with 5 x 105B16 melanoma cells for a second time at day 69, with 5 naive mice receiving the same dose of tumor cells as the control group.

[0161] As shown in FIGs. 7A-7E, TNF-a and CD40L-engineered allogeneic B cells, in combination with the anti-PDl antibody, achieved 100% response rate and 40% cure rate (FIG. 7A), remarkable tumor growth inhibition (FIG. 7B), and better survival (FIG. 7C), as compared with control mice, which were treated with DPBS. Cured mice show resistance to tumor rechallenge (FIG. 7A). No significant weight loss was observed in the treated mice (FIG. 7D), indicating safety of the treatment. Expression of mouse CD40L (mCD40L) on TNF-a and CD40L-engineered allogeneic B cells was confirmed by flow cytometry (FIG. 7E). It was observed in prior studies that anti-PDl treatment alone did not exhibit anti-cancer effect in this melanoma mouse model (see, e.g., FIG. 2).

[0162] Overall, intra-tumoral delivery of TNF-a and CD40L-engineered allogeneic B cells exhibited promising anti-cancer activities on B16 melanoma model.

[0163] Example 7: Efficacy and Safety of mRNA Electroporated Allogeneic B Cells

[0164] This example investigates the efficacy and safety of mRNA electroporated allogeneic B cells.

[0165] C57B6 mice were implanted with 5 x 105B16 melanoma cells at day 0. Allogeneic B cells were isolated from Balb / c mouse spleen. Allogeneic B cells were activated and electroporated with mRNA encoding mouse TNF-a (SEQ ID NO: 22) and CD40L (SEQ ID NO: 20). At day 10, 11, 12, and 13, C57B6 mice received four treatments as indicated. Each treatment includes an intratumoral injection of mRNA electroporated allogeneic B cells and an i.p. injection of mouse PD1 antibody. Tumor growths, mouse weights, and survival were monitored over time.

[0166] As shown in FIGs. 8A-8D, the mRNA electroporated allogeneic B cells, in combination with the anti-PDl antibody, achieved 100% response rate (FIG. 8A), remarkable tumor growth inhibition (FIG. 8B), and better survival (FIG. 8C) relative to control mice treated with DPBS. No significant weight loss was observed, indicating safety of the treatment. FIG. 8D. It was observed in prior studies that anti-PDl treatment alone did not exhibit anti-cancer effect in this melanoma mouse model (see, e.g., FIG. 2).

[0167] Overall, intra-tumoral delivery of mRNA electroporated allogeneic B cells exhibited promising anti-cancer activities on B 16 melanoma model.

[0168] OTHER EMBODIMENTS

[0169] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0170] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims. EQUIVALENTS

[0171] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of examples only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0172] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0173] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0174] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. Tn general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. , “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0175] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0176] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0177] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,”

[0178] “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

What Is Claimed Is:

1. A population of immune cells, comprising a plurality of engineered B cells, wherein the engineered B cells comprise one or more exogenous nucleic acids encoding one or more immune regulatory agents, which optionally are interleukin- 12 (IL- 12), CD40L, tumor necrosis factor alpha (TNF-a), OX40L, 4-1BBL, PD-L1, or a fusion polypeptide comprising such; and wherein the engineered B cells express the one or more immune regulatory agents encoded by the exogenous nucleic acid(s).

2. The population of immune cells of claim 1, wherein the engineered B cells stably express the one or more immune regulatory agents encoded by the exogenous nucleic acid(s).

3. The population of immune cells of claim 1 or claim 2, wherein the one or more immune regulatory agents comprise at least one fusion polypeptide of IL- 12, CD40L, TNF-a, OX40L, PD-L1, and / or 4-1BBL; and wherein the at least one fusion polypeptide further comprises an Fc fragment.

4. The population of immune cells of any one of claims 1-3, wherein the one or more immune regulatory agents comprise an IL- 12 polypeptide, which is an IL- 12 molecule or a fusion polypeptide comprising such.

5. The population of immune cells of claim 4, wherein the IL- 12 polypeptide, comprises the amino acid sequence of any one of SEQ ID NOs: 7-10.

6. The population of immune cells of claim 4, wherein the IL- 12 polypeptide is an IL-12-Fc fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

7. The population of immune cells of any one of claims 1-6, wherein the one or more immune regulatory agents comprise an CD40L polypeptide, a TNF-a polypeptide, or a combination thereof, and wherein the CD40L polypeptide and / or the TNF-a polypeptide is respectively a CD40L molecule and a TNF-a molecule, or a fusion polypeptide comprising such.

8. The population of immune cells of claim 7, wherein the CD40L polypeptide comprises the amino acid sequence of SEQ ID NO: 24; and / or wherein the TNF-a polypeptide comprises the amino acid sequence of SEQ ID NO: 23.

9. The population of immune cells of claim 7 or claim 8, wherein the CD40L polypeptide is a CD40L-Fc fusion polypeptide, and / or wherein the TNF-a polypeptide is a TNF-a-Fc fusion polypeptide.

10. The population of immune cells of any one of claims 1-9, wherein the one or more immune regulatory agents comprise PD-L1 or OX40L, or a fusion polypeptide comprising PD-L1 or OX40L; optionally wherein the PD-L1 is an extracellular domain of a naturally-occurring PD-L1 molecule (PD-L1 ECD).

11. The population of immune cells of claim 10, wherein the one or more immune regulatory agents comprise a PD-L1 ECD-Fc fusion polypeptide or an OX40L-Fc fusion polypeptide.

12. The population of immune cells of any one of claims 1-11, wherein the engineered B cells further express an immune checkpoint inhibitor.

13. The population of immune cells of claim 12, wherein the immune checkpoint inhibitor is an antibody that binds PD-1 or PD-L1.

14. The population of immune cells of any one of claims 1-13, wherein the plurality of B cells are human B cells, which optionally is derived from one or more healthy human donors.

15. The population of immune cells of any one of claims 1-14, further comprising a plurality of T cells expressing a chimeric antigen receptor (CAR).

16. A method for treating a tumor in a subject, comprising administering to a subject in need thereof an effective amount of the population of immune cells set forth in anyone of claims 1-15, wherein the plurality of engineered B cells in the immune cell population is allogenic to the subject.

17. The method of claim 16, wherein the subject is a human patient having a tumor, which optionally is a solid tumor.

18. The method of claim 16 or claim 17, wherein the immune cells are administered intratumorally to the subject.

19. The method of any one of claims 16-18, wherein the population of immune cells is set forth in any one of claims 1-11, and wherein the method further comprises administering to the subject an immune checkpoint inhibitor, a population of T cells expressing chimeric antigen receptor (CAR), or a combination thereof.

20. The method of claim 19, wherein the method further comprises administering to the subject the immune checkpoint inhibitor, which is an antibody specific to PD-1, or PD- Ll, or a combination thereof.

21. A method for producing a population of engineered B cells, the method comprising: delivering into a population of immune cells comprising B cells one or more nucleic acids encoding one or more immune regulatory agents, which are interleukin- 12 (IL- 12), CD40L, TNF-a, OX40L, 4-1BBL, PD-L1, or a fusion polypeptide comprising such; thereby producing a population of engineered B cells expressing the one or more immune regulatory agents.

22. The method of claim 21 , wherein the population of engineered B cells are human B cells.

23. The method of claim 21 or claim 22, wherein the population of immune cells is from one or more healthy donors, optionally healthy human donors.

24. The method of any one of claims 21-23, wherein the population of immune cells is a population of human peripheral blood mononuclear cells (PBMCs).

25. The method of any one of claims 21-24, wherein the one or more nucleic acids are located on one or more vectors, optionally retroviral vectors, adenoviral vectors, or adeno-associated viral vectors.

26. The method of any one of claims 21-24, wherein the one or more nucleic acids are mRNAs, which optionally are delivered to the B cells by electroporation.

27. The method of any one of claims 21 -26, wherein the one or more immune regulatory agents are set forth in any one of claims 3-9.

28. The method of any one of claims 21-27, wherein the method further comprises delivering to the population of immune cells or the population of engineered B cells one or more nucleic acids encoding an antibody specific to PD-1 or PD-L1.

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