Genetically engineered B cells and methods of use thereof

Abstract

The present invention relates to genetically engineered B cells, which express and carry a chimeric B cell receptor on their surface, and which further express and secrete antibodies or cytokines. TIFF2025508041000027.tif72169

Description

[Technical field]

[0001] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety, and the disclosures of these publications are incorporated by reference into this application in order to more fully describe the state of the art known to those skilled in the art as of the date of the invention described and claimed herein.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all and any copyright rights whatsoever.

[0003] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 318,317, filed March 09, 2022, the entire contents of which are incorporated herein by reference.

[0004] FIELD OF THEINVENTION The present invention relates to genetically engineered B cells, which express and bear a chimeric B cell receptor (cBCR) on their surface, and which further express and secrete antibodies or cytokines. [Background technology]

[0005] 2. Background of the Invention Over the past decade, immune checkpoint blockade inhibitors (CBIs) and CAR-T cells have revolutionized the way we treat cancer. Although both of these therapies engage the patient's immune system, neither is able to actively mount an anti-tumor immune response. Summary of the Invention

[0006] An embodiment of the present invention is directed to genetically engineered B cells.

[0007] In embodiments, the engineered B cells express and bear a chimeric B cell receptor (cBCR) on their surface, and the engineered B cells further express and secrete antibodies or cytokines.

[0008] In embodiments, the chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

[0009] In an embodiment, the extracellular domain is an antibody or an antibody fragment. In an embodiment, the antibody is a nanobody, bispecific, scFv, or Fab. In an embodiment, the antibody is specific for a tumor-associated antigen. For example, the tumor-associated antigen is selected from the group consisting of CAIX, BCMA, CD138, PD-L1, PD-L2, VEGF, CD70, CD99, CEA, Her-2, GD2, CD171, αFR, PMSA, IL13α, MSLN, TAG-72, and TROP2. For example, the bispecific antibody is specific for PD-1 and CTLA4, PD-1 and TIGIT, TIGIT and CCR4, GITR and TIGIT, or PD-1 and CCR4. In an embodiment, the antibody is an anti-IGHV1-69 antibody. In an embodiment, the antibody is specific for an infectious disease-associated antigen, for example, HA1, HA2, NA, or spike protein. For example, the infectious disease is a viral disease such as influenza, coronavirus, HIV, or tuberculosis.

[0010] In embodiments, expression of the secreted antibody or cytokine is controlled by an inducible response element. For example, the inducible response element is NFAT or NF KIn embodiments, the antibody is a B response element. In embodiments, the antibody is a checkpoint blockade modulator. In embodiments, the antibody is a checkpoint blockade inhibitor. In embodiments, the antibody is specific for CA-9, PD-1, PD-L1, PD-L2, CTLA4, TIGIT, VISTA, CD70, TIM-3, LAG-3, CD40L, CCR4, GITR, or CXCR4. In embodiments, the antibody is specific for HA1, HA2, NA, or spike protein. In embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, CD40-L, or BAFF. In embodiments, the antibody comprises a monoclonal antibody. In embodiments, the antibody comprises a humanized antibody. In embodiments, the antibody comprises a nanobody, scFv, Fab, antibody-cytokine fusion, or bispecific antibody. For example, bispecific antibodies are specific for PD-1 and CTLA4, PD-1 and TIGIT, TIGIT and CCR4, GITR and TIGIT, or PD-1 and CCR4. For example, an antibody-cytokine fusion protein includes anti-PD1-scIL12.

[0011] Aspects of the invention are also directed to a nucleic acid encoding a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a chimeric B cell receptor, the chimeric B cell receptor comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain, and the second polypeptide comprises an antibody or a cytokine.

[0012] In an embodiment, the extracellular domain is an antibody or an antibody fragment. In an embodiment, the antibody is a nanobody, bispecific, scFv, or Fab. In an embodiment, the antibody is specific for a tumor-associated antigen. For example, the tumor-associated antigen is selected from the group consisting of CAIX, BCMA, CD138, PD-L1, PD-L2, VEGF, CD70, CD99, CEA, Her-2, GD2, CD171, αFR, PMSA, IL13α, MSLN, TAG-72, and TROP2. For example, the bispecific antibody is specific for PD-1 and CTLA4, PD-1 and TIGIT, TIGIT and CCR4, GITR and TIGIT, or PD-1 and CCR4. In an embodiment, the antibody is an anti-IGHV1-69 antibody. In an embodiment, the antibody is specific for an infectious disease-associated antigen, for example, HA1, HA2, NA, or spike protein. For example, the infectious disease is a viral disease such as influenza, coronavirus, HIV, or tuberculosis.

[0013] In embodiments, expression of the secreted antibody or cytokine is controlled by an inducible response element. For example, the inducible response element is NFAT or NF K In embodiments, the antibody is specific for CA-9, PD-1, PD-L1, PD-L2, CTLA4, TIGIT, VISTA, CD70, TIM-3, LAG-3, CD40L, CCR4, GITR, or CXCR4. In embodiments, the antibody is specific for HA1, HA2, NA, or spike protein. In embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, CD40-L, or BAFF. In embodiments, the antibody comprises a monoclonal antibody. In embodiments, the antibody comprises a humanized antibody. In embodiments, the antibody comprises a nanobody, scFv, Fab, or bispecific antibody. For example, the bispecific antibody is specific for PD-1 and CTLA4, PD-1 and TIGIT, TIGIT and CCR4, GITR and TIGIT, or PD-1 and CCR4.

[0014] Aspects of the present invention are also directed to vectors comprising the nucleic acid described herein.In embodiments, the vector is a lentivirus vector or an adeno-associated virus (AAV) vector.Lentivirus and AAV are viruses that can be useful as vectors for gene therapy, such as using CRISPR / Cas systems.

[0015] Additionally, aspects of the present disclosure are directed to cells comprising the vectors described herein.

[0016] Aspects of the invention are also directed to a composition comprising a first expression vector and a second expression vector, wherein the genome of the first expression vector comprises a nucleotide sequence encoding a chimeric B cell receptor and the genome of the second expression vector comprises a nucleotide sequence encoding an antibody or a cytokine, In embodiments, the first expression vector and the second expression vector are lentiviral or adeno-associated viral vectors.

[0017] In embodiments, a nucleotide sequence encoding an inducible response element is operably linked to a nucleotide sequence encoding an antibody or cytokine. For example, the inducible response element is NFAT or NF K B response element.

[0018] In embodiments, the chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain. In embodiments, the extracellular domain is an antibody or an antibody fragment. In embodiments, the antibody is a nanobody, scFv, or Fab. In embodiments, the antibody is specific for a tumor associated antigen. For example, the tumor associated antigen is selected from the group consisting of CAIX, BCMA, CD138, PD-L1, PD-L2, VEGF, CD70, CD99, CEA, Her-2, GD2, CD171, αFR, PMSA, IL13α, MSLN, TAG-72, and TROP2. In embodiments, the antibody is an anti-IGHV1-69 antibody. In embodiments, the antibody is specific for an infectious disease associated antigen, such as HA1, HA2, NA, or spike protein. For example, the infectious disease is a viral disease, such as influenza, coronavirus, HIV, or tuberculosis.

[0019] In embodiments, expression of the secreted antibody or cytokine is controlled by an inducible response element. For example, the inducible response element is NFAT or NF KIn embodiments, the antibody is a B response element. In embodiments, the antibody is a checkpoint blockade modulator. In embodiments, the antibody is a checkpoint blockade inhibitor. In embodiments, the antibody is specific for CA-9, PD-1, PD-L1, PD-L2, CTLA4, TIGIT, VISTA, CD70, TIM-3, LAG-3, CD40L, CCR4, GITR, or CXCR4. In embodiments, the antibody is specific for HA1, HA2, NA, or spike protein. In embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, CD40-L, or BAFF. In embodiments, the antibody comprises a monoclonal antibody. In embodiments, the antibody comprises a humanized antibody. In embodiments, the antibody comprises a nanobody, scFv, Fab, antibody-cytokine fusion, or bispecific antibody. For example, bispecific antibodies are specific for PD-1 and CTLA4, PD-1 and TIGIT, TIGIT and CCR4, GITR and TIGIT, or PD-1 and CCR4. For example, an antibody-cytokine fusion includes anti-PD1-scIL12.

[0020] Aspects of the invention are also directed to methods of producing a population of genetically engineered B cells. In embodiments, the methods include isolating a population of B cells from a subject and transducing the population of B cells with a vector described herein, thereby producing a population of genetically engineered B cells. Embodiments may further include activating the population of B cells prior to transduction. Embodiments may further include culturing the population of genetically engineered B cells. Embodiments may further include administering the population of genetically engineered B cells to a subject in need thereof.

[0021] Additionally, aspects of the invention are directed to methods of treating a subject suffering from cancer by administering to the subject a genetically engineered B cell described herein, a nucleic acid described herein, or a composition described herein.

[0022] Additionally, aspects of the invention are directed to methods of preventing cancer in a subject by administering to the subject a genetically engineered B cell described herein, a nucleic acid described herein, or a composition described herein, e.g., the cancer is BCLL, NSCLC, ccRCC, mesothelioma.

[0023] Aspects of the invention are directed to methods of treating a subject suffering from an infectious disease by administering to the subject a genetically engineered B cell described herein, a nucleic acid described herein, or a composition described herein.

[0024] Still further, aspects of the invention are directed to methods of preventing an infectious disease by administering to a subject a genetically engineered B cell described herein, a nucleic acid described herein, or a composition described herein, for example, the infectious disease is a viral disease such as influenza, coronavirus, HIV, or tuberculosis.

[0025] Other objects and advantages of the present invention will become readily apparent from the ensuing description. [Brief description of the drawings]

[0026] [Figure 1] Schematic diagram of CASS B-cell therapy for non-small cell lung cancer. In this example, anti-MSLN is the targeting moiety and anti-TIGIT / anti-PD1 bispecific antibody is the secretory moiety. [Diagram 2] Characterization of exemplary antibodies for CASS B cell development. Panel A) Anti-PD1 mAb is comparable to pembrolizumab in MLR assays. Panel B) BLI assay demonstrates that the antibody blocks the TIGIT / CD155 binding interaction (purple) and shares an epitope with a control Ab (blue). Panel C) MSLN+ cell binding curve (left) and BLI-based kinetic measurements (right) demonstrate that one clone (Glyl-2-H4) binds to a conformational epitope present only on the GPI-linked form of MSLN. [Diagram 3]Figure 2 shows the design of bispecific antibodies (bsAb). Panel A) Schematic of bsAb with different scFvs coloured red or blue. Diagram of the experimental layout of the dual binding assay (Panel B) and the resulting binding cure (Panel C) showing the functionality of both sides of the bsAb. [Figure 4] CASS B cell construct testing and B cell transduction are shown. Panel A) Cells expressing engineered IgG-BCR constructs are able to bind soluble HA. Panel B) Lentivirus can be used to achieve high transduction efficiency using multiple DNA constructs and donors. Panel C) A reporter system has been engineered in Jurkat cells using an NFAT / NFkB inducible response element that shows increasing levels of GFP expression under various stimulation conditions. [Diagram 5] An overview of data on hG6.3 targeting moieties, biological studies, and CASS B-cell design and strategy is shown: (Panel A) IGHV1-69 mAb containing the hG6.3 core binding idiotope against D80, i.e., CDR-H2 residues M53-N58 (green) bound to hG6.3 in the crystal structure (blue); (Panel B) Preferential recognition of non-mutated IGHV1-69 B-CLL patient samples by hG6.3-CAR T cells (G36 is an anti-CAIX control); (Panel C) Schematic of CASS vectors and different CBI payloads and bispecific Ab derivatives; (Panel D) Schematic of CASS B-cell therapy. B cells are harvested by leukapheresis and transduced to express a chimeric BCR targeting cancer cells and encoded to secrete an optimized CBI payload, as determined by ex vivo PDOTS and in vivo humanized mouse studies. [Figure 6] Membrane expression (Panel A) and HA binding (Panel B) in 293T cells are shown. [Figure 7-1] Exemplary combination constructs of embodiments of the present invention are provided. [Figure 7-2] See description of Figure 7-1. [Figure 7-3] See description of Figure 7-1. [Figure 7-4] See description of Figure 7-1. [Figure 8-1] 1 provides exemplary split vector constructs according to embodiments of the present invention. [Figure 8-2] See description of Figure 8-1. [Figure 8-3] See description of Figure 8-1. [Figure 8-4] See description of Figure 8-1. [Figure 8-5] See description of Figure 8-1. [Figure 9-1] Schematic diagram of current immunotherapy. Panel A is adapted from Shifaa M. Abdin et al. J Immunother Cancer 2021;9:e002741. Panel B is adapted from Larson, RC, Maus, MV Nat Rev Cancer 21, 2021. Panel C is adapted from Zhang, C., Hu, Y, Xiao, W. et al. Cell Mol Immunol 2021. [Figure 9-2] See description of Figure 9-1. [Figure 9-3] See description of Figure 9-1. [Figure 10] Diagram of the function of B cells in the immune system adapted from Li Rui et al, Frontiers in Immunology, 2016. [Figure 11] Diagram of B cell signaling pathway adapted from Balaji, S., Ahmed, M., Lorence, E. et al. J Hematol Oncol, 2018. [Figure 12] FIG. 1 shows a diagram of chimeric antibody signaling and secreting (CASS) B cells. [Figure 13] Diagram of first run of B cell transduction adapted from Howell Moffett et al., Science Immunology, 2019. Media: StemMACS HSC Expansion Medium XF+5%FBS+IL4+CD40L-hex+ODN2006. [Figure 14] FIG. 1 shows a diagram of the design of chimeric antibody signaling and secreting (CASS) B cells. [Figure 15-1] A diagram of the construct is shown. [Figure 15-2] See description of Figure 15-1. [Figure 15-3] See description of Figure 15-1. [Figure 15-4] See description of Figure 15-1. [Figure 15-5] See description of Figure 15-1. [Figure 15-6] See description of Figure 15-1. [Figure 16] Representative data from the first trial of B cell transduction is shown. The construct used in this experiment contains an internal BGH domain, which results in premature termination of the construct and inability to integrate via the LTR. Thus, high gene expression is seen immediately after transduction, but within 2 weeks expression has decreased to near zero. This indicates a lack of gene integration rather than expression via transient lentiviral "transfection." [Figure 17] A schematic diagram of the redesign of the CASS B construct is shown. According to the results in Figure 16, the BGH polyA was removed to allow stable genomic integration after transduction. [Figure 18] Representative data of transduction check after BGH removal are shown. B cells were isolated and activated on Nov. 11, 2021; transduced on Nov. 12, 2021; and stained on Nov. 19, 2021. Virus was pseudotyped with VSVG and BGH was removed. B cells were transduced 18-24 hours after activation. However, they are negative 7 days after transduction. [Figure 19] Representative graphs of data showing the duration and manner in which B cell stimulation plays a role in transduction are shown. Adapted from Bovia, Fabrice et al, Gene Therapy 2013. [Figure 20-1]Representative data for optimal envelope proteins for B cell transduction are shown. VSVG: low density lipoprotein receptor (LDL-R); GALV: sodium-dependent phosphate transporters 1 and 2 (GLVR-1 / 2); and BaEV: neutral amino acid (aa) transporters 1 and 2 (ASCT-1 / 2). Panel A is adapted from Caeser, R., Di Re, M., Krupka, JA et al. Nat Commun 2019. Panel B is adapted from Levy C et al, J Thromb Haemost 2016. [Figure 20-2] See description of Figure 20-1. [Figure 21] Schematic and representative data of transfer plasmids optimized for B cell transduction are shown. [Figure 22] Schematic diagram of the two transduction protocols is shown: the Bovia protocol utilizes EL4-B5 feeder cells, whereas the Moffett protocol utilizes soluble CD40L. Moffett:1.HFMoffett,CKHarms,KSFitzpatrick,MRTooley,J.Boonyaratanakornkit,JJTaylor,B cells engineered to express pathogen-specific antibodies protect against infection.Sci.Immunol.4(2019),doi:10.1126 / sciimmunol.aax0644,31101673.Bovia:1.F.Bovia,P.Salmon,T.Matthe s, K. Kvell, THNguyen, C. Werner-Favre, M. Barnet, M. Nagy, F. Leuba, JFArrighi, V. Piguet, D. Trono, RHZubler, Efficient transduction of primary human B lymphocytes and nondividing myeloma B cells with HIV-1-derived lentiviral vectors.Blood.101,1727-1733(2003),12406892. [Figure 23-1] Representative data from a transduction check after 9 days is shown. B cells were isolated from EL4-B5 using CD19 and the virus was pseudotyped using three different envelope proteins (VSVG, BaEV, GALV). It can be seen that for cells activated via the Bovia protocol, high transduction titers are seen in EL4-B5 feeder cells (CD19-) but not in human B cells (CD19+). Following the protocol adopted from Mofett, the high level of GFP expression seen in CD19+ B cells indicates efficient transduction using BaEV pseudotyped virus, but not GALV or VSVG. Retronectin reagent is a recombinant human fibronectin fragment sold by TakaraBio. Without wishing to be bound by theory, it enhances viral transduction through binding to the heparin-binding domain of the viral particle and binding to the cellular integrins VLA-4 and / or VLA-5 via the C domain (https: / / www.takarabio.com / learning-centers / gene-function / t-cell-transduction-and-culture / retronectin-faqs). However, in this assay, the inventors did not see any advantage in using retronectin for transduction. [Figure 23-2] See description of Figure 23-1. [Figure 23-3] See description of Figure 23-1. [Figure 23-4] See description of Figure 23-1. [Figure 24-1]Representative data from day 4 transduction checks are shown. Cells were transduced with BaEV pseudotyped lentivirus. The first column uses the LeGO GFP vector, which constitutively expresses GFP. pHAGE-F10-NFkB is a CASS B cell vector that expresses GFP under the control of the NFkB / NFAT response element. pHAGE-F10 is the extracellular binding domain of CASS B cells only. Importantly, the CASS B cell vector shows both binding of QBend10 (an epitope marker via RQR8) and expression of GFP. [Figure 24-2] See description of Figure 24-1. [Figure 25-1] Representative data of HA binding to F10-CASS B cells are shown. 250k cells were washed with PBS and resuspended in 5μM ZombieViolet dye (in PBS) for 20 minutes at room temperature. Cells were quenched with medium containing FBS and then resuspended in 100ul containing FcX and 4ug / mL biotinylated H1 stem. After 1 hour incubation at 4C, cells were washed and stained with QBend10-FITC and APC-streptavidin. Importantly, nearly all QBend10+ cells also bound HA, indicating that the transduced cells express both a functional chimeric BCR and an epitope tag separated by T2A. [Figure 25-2] See description of Figure 25-1. [Figure 25-3] See description of Figure 25-1. [Figure 26] Representative data of inducible expression of GFP is shown. Transduced B cells were activated for 4 days, and then inducible GFP expression was measured 8 days after transduction. As can be seen here, GFP expression of the NFAT-NFkB construct is not altered by additional BCR stimulation (anti-IgM or HA+Strep) compared to medium alone. The 3x3 NFAT construct does not show any change in GFP expression under any condition. [Figure 27]Schematics of representative payload constructs constructed are shown. ZsG was used for platform optimization, anti-PD(L)l is the anti-cancer therapy, and aSARS is either an irrelevant control for cancer therapy or an anti-SARS therapy for infectious disease therapy. [Figure 28-1] Representative data of CASS B cell secretion of scFv-Fc is shown. B cells were seeded at 1E6 cells / mL in stim medium on May 24, 2022 and harvested on May 28, 2022. High levels of IgG expression were observed in all wells, but antigen-specific scFv-Fc was detected for anti-PD1 P4B3m3. Based on ELISA results with purified protein, this is likely due to the low affinity of mAb 42 (aPDL1) and the incorrect spike target selected for 11A. 11A targets GD03 SARS, but a different strain was used for the ELISA. [Figure 28-2] See description of Figure 28-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description of the Invention Described herein are compositions and methods relating to chimeric antibody signaling and secreting (CASS) B cells as targeted inducible platforms for secreting immune-modulating polypeptides at target sites. For example, CASS B cells can express an engineered tumor-targeting B cell receptor on their surface and, upon engagement, can secrete high levels of dual-targeted bispecific checkpoint blockade modulator antibodies locally at the tumor site. Because B cells also function as professional antigen-presenting cells, CASS B cells can also process and present antigens on MHC class II molecules, further enhancing immune cell recognition of tumors and aiding in neo-antigen spreading. CASS B cells, a key component of immune memory, can simultaneously recruit a broad range of immune cells and reverse tumor-infiltrating lymphocyte exhaustion, providing a robust, lifelong surveillance program that protects against tumor metastasis and recurrence. Embodiments of the CASS B cell platform can be used to prevent and treat cancer and infectious diseases.

[0028] Detailed description of one or more embodiments is provided herein. However, it is understood that the present invention can be embodied in various forms. Therefore, the specific details disclosed herein should not be interpreted as limiting, but as a basis for the claims and as a representative basis for teaching a person skilled in the art to use the present invention in any suitable manner.

[0029] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The use of the words "a" or "an" when used in conjunction with the word "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0030] Whenever any of the phrases "for example," "such as," "including," and the like are used herein, unless expressly stated otherwise, it is understood that the phrase "without limitation" is accompanying. Similarly, "an example," "exemplary," and the like are understood to be non-limiting.

[0031] The term "substantially" permits deviations from the descriptors that do not adversely affect the intended purpose. It is understood that the descriptors are modified by the term "substantially" even if the word "substantially" is not expressly recited.

[0032] Terms such as "comprising," "including," "having," and "involving" (and similarly "comprises," "includes," "has," and "involves") are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the general U.S. patent law definition of "comprising," and therefore is to be construed as an open term meaning "at least the following," and not excluding additional features, limitations, aspects, etc. Thus, for example, "a process comprising steps a, b, and c" means that the process includes at least steps a, b, and c. Whenever the terms "a" or "an" are used, they are to be understood as "one or more," unless such an interpretation is insignificant in the context.

[0033] The term "about" as used herein can refer to approximately, roughly, approximately, or within a region thereof. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" is used herein to modify numerical values ​​above and below the set forth value by a variance of up to 20 percent (higher or lower).

[0034] Chimeric B cell receptor Aspects of the present invention are directed to genetically engineered B cells that have been modified to express and carry a chimeric B cell receptor on its surface. In embodiments, the genetically modified B cells can include a single chimeric B cell receptor that targets one antigen, or a single chimeric B cell receptor that targets two or more antigens (e.g., a bispecific chimeric B cell receptor, or a multispecific chimeric B cell receptor). In some embodiments, the cells include a split chimeric B cell receptor, such as two different scFvs expressed on the B cell surface with different co-stimulatory domains. Additionally, some embodiments include fine-tuned chimeric B cell receptors.

[0035] A split chimeric B cell receptor can include two or more chimeric B cell receptors on the surface of a cell, such as a B cell. The chimeric B cell receptor can be specific for two or more antigens. In this example, the first chimeric B cell receptor is specific for a first antigen and the second chimeric B cell receptor is specific for a second antigen. As described herein, the chimeric B cell receptor can be in any orientation desired. For example, the first chimeric B cell receptor can be specific for a second antigen and the second chimeric B cell receptor can be specific for the first antigen. The first and second chimeric B cell receptors can be expressed from a single nucleic acid construct. In such an example, a nucleic acid encoding a cleavable linker can be placed between the nucleic acid encoding the first chimeric B cell receptor and the nucleic acid encoding the second chimeric B cell receptor. In other embodiments, the two chimeric B cell receptors can be expressed from the same cell but from two separate nucleic acid constructs.

[0036] In embodiments, the chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

[0037] Engineered B cell receptors, called chimeric B cell receptors, e.g., B cell receptors containing antibodies or antibody fragments preselected for high affinity to specific disease-associated antigens, are a powerful new approach to disease. Because B cells function as professional antigen-presenting cells, they can process and present antigens on MHC class II molecules, enhance immune cell recognition of tumors, and aid in neoantigen spreading. CASS B cells, a key component of immune memory, simultaneously recruit a wide range of immune cells, providing a robust, lifelong surveillance program that reverses tumor-infiltrating lymphocyte exhaustion and protects against tumor metastasis and recurrence. In certain cases, B cells can include receptors that are chimeric, non-natural, and at least partially engineered by human hands. In certain cases, engineered chimeric B cell receptors have one, two, three, four, or more components, and in some embodiments, one or more components facilitate B cells targeting or binding to one or more antigen-containing cancer cells.

[0038] A chimeric B cell receptor according to the present disclosure comprises at least one transmembrane polypeptide comprising at least one extracellular ligand binding domain and one transmembrane polypeptide comprising at least one intracellular signaling domain, which polypeptides assemble together to form the chimeric B cell receptor.

[0039] The term "extracellular ligand binding domain" or "extracellular domain" as used herein can refer to an oligo or polypeptide capable of binding to a ligand. The domain can interact with a cell surface molecule. For example, the extracellular ligand binding domain can be selected to recognize a ligand that acts as a cell surface marker on a target cell associated with a particular disease state. For example, the disease state can be cancer and the target ligand can be a cancer-associated antigen. In another example, the disease state can be an infectious disease and the target ligand can be an infectious disease-associated antigen. In embodiments, the extracellular ligand binding domain can include an antigen binding domain or antigen recognition domain derived from an antibody against a target antigen. The antigen binding domain or antigen recognition domain can be an antibody fragment. An "antibody fragment" can be a molecule other than an intact antibody that includes a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. For example, embodiments can include chimeric B cell receptors with two scFvs as antigen recognition domains. Embodiments can also include chimeric B cell receptors with one scFv as antigen recognition domains. Additionally, embodiments can include chimeric B cell receptors with bispecific antibodies as antigen recognition domains. For example, bispecific antibodies can be specific for PD-1 and CTLA4, PD-1 and TIGIT, TIGIT and CCR4, GITR and TIGIT, or PD-1 and CCR4. For example, bispecific antibodies can be specific for HA and NA, or influenza HA and coronavirus spike (S), or SARS2. Bispecific or cross-reactive antibodies are known in the art. See, for example, Pilewski, Kelsey A., et al. "Functional HIV-1 / HCV cross-reactive antibodies isolated from a chronically co-infected donor." Cell Reports 42.2 (2023).

[0040] The antigen recognition domain can be directed to any antigen target of interest. In embodiments, the antigen target of interest is on the surface of a cell, such as the surface of a cancer cell (i.e., tumor-associated antigen). The antigen target of interest can also be associated with infectious disease (i.e., infectious disease-associated antigen). Non-limiting examples of antigen targets include TIGIT, PD-1, CAIX, BCMA, CD138, PD-L1, PD-L2, VEGF, CD70, CD99, CEA, Her-2, GD2, CD171, αFR, PMSA, IL13α, MSLN, TAG-72, TROP2, B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CCR4, CTLA-4, GITR, GITRL, ICOS, ICOSL, LAG-3, LIGHT, OX-40, OX40L, TIM3, 4-1BB, VISTA, HEVM, BTLA, and KIR. In embodiments, the antigen target includes CAIX. In embodiments, the antibody targets mesothelin.

[0041] Exemplary antibody compositions (e.g., VH and / or VL sequences or fragments thereof) useful in the design of the chimeric B cell receptors described herein include, but are not limited to, the following: Anti-CAIX antibodies described in PCT / US2006 / 046350 and PCT / US2015 / 067178 Anti-CXCR4 antibodies described in PCT / US20006 / 005691 Anti-CCR4 antibodies described in PCT / US2008 / 088435, PCT / US2013 / 039744, and PCT / US2015 / 054202 - the anti-PD-L1 antibodies described in PCT / US2008 / 088435 and PCT / US2020 / 062815; - the anti-PD-1 antibodies described in PCT / US2020 / 037791 and PCT / US2020 / 037781; Anti-GITR antibodies described in PCT / US2017 / 043504 Anti-claudin 4 antibody described in PCT / US2019 / 022272 - anti-MUC1 antibodies described in PCT / US2020 / 037783, Anti-TIGIT antibodies described in U.S. Provisional Patent Application No. 63 / 242992 Anti-IGHV1-69 antibody described in PCT / US2011 / 038970 Anti-influenza antibodies described in PCT / US2008 / 085876 and PCT / US2016 / 026800 ●Anti-SARS-CoV-2 (Each of these applications is incorporated herein by reference in its entirety).

[0042] The term "antibody" herein is used in the broadest sense and can refer to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts with" can refer to an antibody that reacts with one or more antigenic determinants of a desired antigen and not with other polypeptides. Antibodies of the present disclosure can include, but are not limited to, polyclonal, monoclonal, humanized, fully human, mosaic, bispecific, multispecific, chimeric, dAb (domain antibodies), single chain antibodies, Fab, Fab' and F(ab')2 fragments, scFv, diabodies, minibodies, scFv-Fc fusions, and Fab expression libraries. Unless expressly stated to the contrary, references made herein to "antibody" or "antibodies" include, for example, any (or all) of these molecules so long as they exhibit the desired antigen-binding activity.

[0043] The embodiments described herein may include multispecific antibodies. The term "multispecific antibodies" may refer to antibodies that can specifically bind to different types of epitopes. More specifically, multispecific antibodies are antibodies that have specificity for at least two different types of epitopes, and include antibodies that recognize different antigens as well as antibodies that recognize different epitopes on the same antigen. (For example, if the antigen is a heteroreceptor, the multispecific antibody binds to different domains that make up the heteroreceptor, or if the antigen is monomeric, the multispecific antibody binds to multiple sites on the monomeric antigen). For example, the multispecific antibody may be a pentameric IgM antibody in which each dimer represents an antibody against a different epitope or target protein.

[0044] In embodiments, the antibody comprises a modular tetramer / tetravalent bispecific antibody as described in WO 2018 / 071913, which is incorporated herein by reference in its entirety. For example, a tetravalent bispecific antibody is a dimer of bispecific scFv fragments comprising a first binding site for a first antigen and a second binding site for a second antigen. For example, the bispecific antibody can be specific for PD-1 and CTLA4, PD-1 and TIGIT, TIGIT and CCR4, or PD-1 and CCR4. The two binding sites can be linked together via a linker domain. In embodiments, the scFv fragment is a tandem scFv, and the linker domain is linked to an immunoglobulin hinge region (e.g., IgG 1 , IgG 2 , IgG 3 , or IgG 4 In embodiments, the immunoglobulin hinge region amino acid sequence includes, for example, the amino acid sequence (GGGS) x1-6 , (GGGGS) x1-6 and GSAGSAAGSGEF. In embodiments, the linker domain can be an immunoglobulin Fc domain, e.g., an IgG 1 , IgG 2 , IgG 3 , and IgG4 At least a portion of an Fc domain. At least a portion of an immunoglobulin Fc domain can be a CH2 domain. The Fc domain is an immunoglobulin hinge region (e.g., IgG 1 , IgG 2 , IgG 3 , and IgG 4 The linker domain can be linked to the C-terminus of the amino acid sequence of the hinge region. The linker domain can be linked to the C-terminus of the amino acid sequence of the hinge region of the linker domain. The linker domain can be linked to the C-terminus of the hinge region of the linker domain. x1-6 , (GGGGS) x1-6 , and GSAGSAAGSGEF).

[0045] In an embodiment, the antibody comprises a mosaic antibody. A mosaic antibody is an antibody in which the external amino acid residues of an antibody of one species are rationally replaced or "mosaicized" by the external amino acid residues of an antibody of a second species, so that the antibody of the first species is not immunogenic in the second species, thereby reducing the immunogenicity of the antibody. Since the antigenicity of a protein depends mainly on its surface characteristics, the immunogenicity of an antibody can be reduced by substituting exposed residues that are different from those typically found in antibodies of another mammalian species. This reasonable substitution of the external residues should have little or no effect on the internal domains or inter-domain contacts. Thus, the ligand binding properties should be unaffected, since the changes are limited to the variable region framework residues. This process is called "mosaicism" because only the outer surface or skin of the antibody is altered, while the supporting residues remain unaffected.

[0046] Single-chain Fv ("scFv") polypeptide molecules are covalently linked VH:VL heterodimers that can be expressed from gene fusions containing VH and VL coding genes linked by a peptide-encoding linker. (Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). Numerous methods have been described for identifying chemical structures for converting the naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that will fold into a three-dimensional structure substantially similar to that of an antigen-binding site. See, e.g., U.S. Patent Nos. 5,091,513, 5,132,405, and 4,946,778.

[0047] Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Methods of making bispecific antibodies are known in the art. See, for example, U.S. Patent No. 8,329,178, which is incorporated herein by reference in its entirety.

[0048] Antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from each other in the nature of the heavy chains present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2 and others. Furthermore, in humans, the light chains can be kappa or lambda chains.

[0049] The term "antigen-binding site" or "binding portion" can refer to the portion of an immunoglobulin molecule that is involved in antigen binding. The antigen-binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, called "hypervariable regions", are interposed between more conserved adjacent stretches known as "framework regions" or "FRs". Thus, the term "FR" can refer to the amino acid sequences naturally found between and adjacent to the hypervariable regions of immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity determining regions" or "CDRs".

[0050] In an embodiment, the extracellular ligand-binding domain is a single-chain antibody fragment (scFv) comprising a light chain (VL) and a heavy chain (VH) variable fragment of a target antigen-specific monoclonal antibody joined by a flexible linker. Those skilled in the art will recognize that the embodiment can include different linkers typically known in the art. See, for example, Chen, et al., "Fusion protein linkers: property, design and functionality." Advanced Drug Delivery Reviews 65.10(2013):1357-1369, which is incorporated herein by reference in its entirety. For example, different linkers can be used to fine-tune the dual targeting chimeric B cell receptor construct. The length of the linker can vary depending on the antibodies of the dual targeting chimeric B cell receptor construct, their angle of approach to the target epitope, and the topography of the target on the tumor cell membrane. For example, the flexible linker can include a GGGS1, GGGGS3, GGGGS5, or IgG1 hinge. In some embodiments, the number of G in the linker can be 2, 3, 4, 5, 6, or 7 in combination with any of S1, S2, S3, S4, S5, or S6. For example, the orientation of the scFv relative to the linker can vary. In one nucleic acid construct, the first scFv can be in the first cassette (i.e., before the linker) and the second scFv can be in the second cassette (i.e., after the linker). Alternatively, the first scFv can be in the second cassette and the second scFv can be in the first cassette. Linkers of various lengths and flexibilities can be utilized as described herein. Different orientations of the two scFvs can affect binding.

[0051] Antigen recognition domains useful for constructing chimeric B cell receptors, e.g., scFvs directed to a first antigen and / or a second antigen, can be synthesized, engineered, and / or produced using nucleic acid (e.g., DNA). The DNA encoding the antigen recognition domain can be cloned in frame to DNA encoding the required chimeric B cell receptor elements, such as, but not limited to, the CD8 hinge region, the transmembrane domain, BCR-associated proteins (such as CD79a and CD79b), and the co-stimulatory domain of a molecule of immunological interest, such as, but not limited to, CD19 and CD20.

[0052] As non-limiting examples, binding domains other than scFvs can also be used for predefined targeting of B cells, such as camelid single domain antibody fragments or receptor ligands, antibody binding domains, antibody hypervariable loops, or CDRs.

[0053] In embodiments, the transmembrane domain further comprises a stalk region between the extracellular ligand binding domain and the transmembrane domain. As used herein, the term "stalk region" may refer to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand binding domain. Specifically, the stalk region is used to provide more flexibility and accessibility to the extracellular ligand binding domain. The stalk region may comprise up to 300 amino acids, e.g., 10-100 amino acids, e.g., 25-50 amino acids. The stalk region may be derived from all or a portion of a naturally occurring molecule, such as all or a portion of the extracellular region of CD8, CD4, or CD28, or all or a portion of an antibody constant region (e.g., CH1, CH2, CH3, or both CH2 and CH3 for an IgG antibody, or CH1, CH2, CH3, CH4, or any combination thereof for an IgM antibody). For example, the stalk region may comprise an IgG (CH2-CH3) or a portion thereof. For example, the stalk region may comprise IgG1 (CH2-CH3), IgG2 (CH2-CH3), IgG3 (CH2-CH3), IgG4 (CH2-CH3), or portions thereof. In embodiments, the stalk region does not comprise a constant domain. In embodiments, the stalk region may be a synthetic sequence that corresponds to a naturally occurring stalk sequence, or may be a completely synthetic stalk sequence. In one embodiment, the stalk region is a portion of the human CD8 alpha chain.

[0054] The signaling domain or intracellular signaling domain of the chimeric B cell receptor of the present invention may be involved in intracellular signaling following binding of the extracellular ligand-binding domain to a target, resulting in activation of immune cells and immune responses. In other words, the signaling domain may be involved in the activation of at least one of the normal effector functions of the B cell in which the chimeric B cell receptor is expressed. Thus, the term "signaling domain" refers to, for example, the early activation of Lyn and Syk, and the activation of NFAT and NF. KIt can refer to the portion of a protein that instructs a cell to carry out a specialized function, such as late activation of B. In embodiments, the signaling domain or intracellular signaling domain comprises all or a portion of CD79a and / or CD79b that contain ITAMs for amplifying signaling of the chimeric B cell receptor (BCR).

[0055] In embodiments, the signal transduction domain can comprise two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation, and those that act antigen-independently to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences can comprise signaling motifs known as ITAM immunoreceptor tyrosine-based activation motifs. ITAMs are distinct signaling motifs found in the cytoplasmic tails of various receptors that serve as binding sites for syk / zap70 class tyrosine kinases. Examples of ITAMs that can be used in the present invention can include, by way of non-limiting example, those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d.

[0056] The chimeric B cell receptor can comprise a native transmembrane domain and / or an intracellular domain. In native B cells, engagement of the B cell receptor leads to rapid tyrosine phosphorylation and calcium ion polarization of the intracellular domain, resulting in downstream activation of NFAT and NF-kB. Using NFAT / NF-kB response elements to drive expression of our secreted proteins, we designed an inducible expression system that is activated by binding of the surface engineered BCR and subsequent downstream signaling pathways.

[0057] In an embodiment, the intracellular signaling domain of the BCR of the present invention comprises a costimulatory signal molecule. In an embodiment, the intracellular signaling domain contains two, three, four or more costimulatory molecules in tandem. The costimulatory molecule can be a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient immune response.

[0058] A "costimulatory ligand" can refer to a molecule that specifically binds to a cognate co-stimulatory molecule on a cell, thereby providing a signal that mediates a B cell response, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by binding of the ligand to the BCR. Costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MlCB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, Toll ligand receptor, and in particular binding ligands that specifically bind B7-H3. Costimulatory ligands can also include antibodies that specifically bind to ligands that specifically bind to costimulatory molecules present on B cells, such as, but not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and CD83, among others.

[0059] A "costimulatory molecule" can refer to a cognate binding partner on a B cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation, activation, and differentiation. Costimulatory molecules can include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include CD19, CD21, CD27, CD28, CD8, CD81, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-I (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, TRL7 / 9, and a ligand that specifically binds to CD83, and the like. See, e.g., Mongini, Patricia KA, and John K. Inman, "Cytokine dependency of human B cell cycle progression elicited by ligands which coengage BCR and the CD21 / CD19 / CD81 costimulatory complex." Cellular immunology 207.2 (2001):127-140.

[0060] In another specific embodiment, the signaling domain is a TNFR-associated factor 2 (TRAF2) binding motif that is the intracellular tail of a costimulatory TNFR family member. The cytoplasmic tail of a costimulatory TNFR family member contains a TRAF2 binding motif consisting of a major conserved motif (P / S / A)X(Q / E)E) or a minor motif (PXQXXD), where X is any amino acid. TRAP proteins are recruited to the intracellular tails of many TNFRs in response to receptor trimerization.

[0061] The distinguishing features of suitable transmembrane polypeptides include their ability to be expressed on the surface of immune cells, particularly B cells, and to interact to induce a cellular response of immune cells against predefined target cells. The different transmembrane polypeptides of the chimeric B cell receptor, including the extracellular ligand binding domain and / or the signal transduction domain, interact together to participate in signal transduction after binding to the target ligand and induce an immune response. The transmembrane domain can be derived from either natural or synthetic origin. The transmembrane domain can be derived from any membrane-bound or transmembrane protein.

[0062] The term "part of" as used herein can refer to any subset of a molecule, i.e., a shorter peptide. Alternatively, amino acid sequence functional variants of a polypeptide can be prepared by mutation of the DNA encoding the polypeptide. Such variants or functional variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct has the desired activity, and in particular exhibits specific anti-target cellular immune activity. The functionality of the chimeric B cell receptor of the present invention in a host cell can be detected in an assay suitable for demonstrating the signaling ability of the chimeric B cell receptor upon binding of a specific target. Such assays are available to the skilled artisan. For example, the assays can be used to measure tyrosine phosphorylation of the intracellular domain, or the expression of NFAT and NF-. K These assays allow for the detection of signal transduction pathways that are triggered upon target binding, such as assays involving the measurement of downstream activation of B activation.

[0063] In embodiments, polyclonal CASS B cells can be generated by transducing cells with two lentiviruses (or two adeno-associated viruses) encoding different BCRs and selecting double-transduced cells. Alternatively, polyclonal CASS B cells can be generated by generating a BCR library pool and using these plasmids to generate lentiviruses (or adeno-associated viruses) encoding a pool of BCRs. In this case, a population of CASS B cells expressing different BCRs is obtained, some of which may have undergone multiple transduction events and therefore may display multiple BCRs on the surface of a single cell.

[0064] cell An embodiment of the present disclosure includes a B cell expressing a chimeric B cell receptor. The cell can be of any type, including an immune cell capable of expressing a chimeric B cell receptor for therapy (i.e., cancer therapy or infectious disease therapy), or a cell such as a bacterial cell carrying an expression vector encoding the chimeric B cell receptor. As used herein, the terms "cell," "cell line," and "cell culture" can be used interchangeably. All of these terms include their progeny, which are any subsequent generations. Not all progeny need be identical, e.g., due to deliberate or inadvertent mutation. In the context of expressing a heterologous nucleic acid sequence, a "host cell" can refer to a eukaryotic cell capable of replicating a vector and / or expressing a heterologous gene encoded by the vector. Host cells can be and have been used as recipients of vectors. Host cells can be "transfected," "transformed," or "transduced," which refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A transformed cell includes the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" or host cell can refer to a cell into which an exogenous nucleic acid sequence, such as a vector, has been introduced. Recombinant cells are thus distinguishable from naturally occurring cells that do not contain a recombinantly introduced nucleic acid. In an embodiment of the invention, the host cell is a B cell.

[0065] Some vectors may use control sequences that allow them to be replicated and / or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which all of the above host cells are incubated to maintain them and allow replication of the vectors. Techniques and conditions that allow large-scale production of vectors, and production of nucleic acids encoded by the vectors and their cognate polypeptides, proteins, or peptides, are also understood and known.

[0066] The cells can be autologous, syngeneic, allogeneic, or in some cases, xenogeneic.

[0067] There are many situations in which one would like to be able to kill modified B cells, such as when the cells become neoplastic and one wishes to terminate treatment, in research where one is interested in having the cells exist and then disappear, or for other reasons. To this end, one can provide for the expression of certain gene products that can kill the modified cells under controlled conditions, such as an inducible suicide gene.

[0068] armed CASS B cells The present invention further includes engineered CASS B cells that have been modified to secrete one or more polypeptides. Such CASS B cells may be referred to as factories, CASS B cell factories, armed CASS B cells, or immune recovery (IR) CASS B cells.

[0069] The term "polypeptide" can encompass a single "polypeptide" and multiple "polypeptides" and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to one or more chains of two or more amino acids can refer to "polypeptide" herein, and the term "polypeptide" can be used in place of or interchangeably with any of these terms. "Polypeptide" can also refer to post-expression modified products of a polypeptide, such as, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides can be derived from natural biological sources or produced by recombinant technology and need not necessarily be translated from a specific nucleic acid sequence. Polypeptides can be generated in any manner, including chemical synthesis. With respect to amino acid sequences, one of skill in the art will readily recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, delete, or substitute a single amino acid or a small percentage of amino acids in the encoded sequence are collectively referred to herein as "conservatively modified variants." In some embodiments, the alteration replaces an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

[0070] In embodiments, the polypeptide may be an antibody or fragment thereof, or a cytokine.

[0071] As used herein, an "antibody" or "antigen-binding polypeptide" can refer to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. "Specifically binds" or "immunoreacts" means that the antibody reacts with one or more antigenic determinants of a desired antigen and not with other polypeptides. An antibody can be a whole antibody and any antigen-binding fragment, or single chain thereof. For example, an "antibody" can include any protein or peptide-containing molecule, including at least a portion of an immunoglobulin molecule, that has the biological activity of binding to an antigen.

[0072] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Antibody fragments, regardless of structure, bind to the same antigen recognized by the intact antibody. The term "antibody fragment" can include aptamers (such as spiegelmers), minibodies, and diabodies. The term "antibody fragment" can also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. Antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific (e.g., bispecific), human, humanized or chimeric antibodies, mosaic antibodies, single chain antibodies, epitope-binding fragments such as Fab, Fab' and F(ab')2, Fd, Fvs, single chain Fvs (scFv), single chain antibodies, dAbs (domain antibodies), minibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, nanobodies derived from camelids, and anti-idiotypic (anti-Id) antibodies.

[0073] In an embodiment, the antibody secreted by CASS B cells is a checkpoint blocking antibody. The term "checkpoint blocking antibody" can refer to an antibody that inhibits an immune checkpoint. When stimulated, a key regulator of the immune system suppresses the immune system's response to immune stimuli, such as cancer cells. Checkpoint blocking antibodies can block inhibitory checkpoints and restore immune system function to, such as, cancer cells. Checkpoint blocking antibodies include, but are not limited to, anti-PD-1, anti-PDL-2, and anti-CTLA-4. Other antibodies that regulate the immune system, such as anti-TGFb, and tumor vasculature, such as anti-VEFG, are also viable candidates.

[0074] In embodiments, antibodies secreted by CASS B cells may be specific for HA1, HA2, NA, or spike proteins. Exemplary antibody compositions (e.g., VH and / or VL sequences or fragments thereof) useful in the armed B cells described herein include, but are not limited to, the anti-influenza antibodies described in International Applications PCT / US2008 / 085876 and PCT / US2016 / 026800.

[0075] In embodiments, the antibodies secreted by CASS B cells are specific for TIGIT, CAIX, GITR, PD-L1, PD-L2, PD-1, CCR4, CTLA-4, VISTA, CD70, PD-1, TIM-3, LAG-3, CD40L, or CXCR4. For example, CASS B cell factories can secrete PD-L1 mAbs locally at tumor sites to restore effective anti-cancer immunity and / or reverse T cell exhaustion.

[0076] Exemplary antibody compositions (e.g., VH and / or VL sequences or fragments thereof) useful for engineering armed B cells as described herein include, but are not limited to, the following: Anti-CAIX antibodies described in PCT / US2006 / 046350 and PCT / US2015 / 067178 Anti-CXCR4 antibodies described in PCT / US20006 / 005691 Anti-CCR4 antibodies described in PCT / US2008 / 088435, PCT / US2013 / 039744, and PCT / US2015 / 054202 - the anti-PD-L1 antibodies described in PCT / US2008 / 088435 and PCT / US2020 / 062815; - the anti-PD-1 antibodies described in PCT / US2020 / 037791 and PCT / US2020 / 037781; Anti-GITR antibodies described in PCT / US2017 / 043504 Anti-claudin 4 antibody described in PCT / US2019 / 022272 - anti-MUC1 antibodies described in PCT / US2020 / 037783, -Anti-TIGIT antibodies described in U.S. Provisional Patent Application No. 63 / 242992 Anti-IGHV1-69 antibody described in PCT / US2011 / 038970 Anti-influenza antibodies described in PCT / US2008 / 085876 and PCT / US2016 / 026800 (Each of these applications is incorporated herein by reference in its entirety).

[0077] For exemplary antibodies for treating cancer, see also Yasunaga, Masahiro. Seminars in cancer biology. Vol. 64. Academic Press, 2020.

[0078] In embodiments, the antibody is a bispecific antibody. For example, in embodiments, the antibody comprises a modular tetramer / tetravalent bispecific antibody as described in WO 2018 / 071913, which is incorporated herein by reference in its entirety. For example, a tetravalent bispecific antibody is a dimer of bispecific scFv fragments comprising a first binding site for a first antigen and a second binding site for a second antigen. For example, the bispecific antibody can be specific for PD-1 and CTLA4, PD-1 and TIGIT, TIGIT and CCR4, or PD-1 and CCR4. The two binding sites can be linked together via a linker domain. In embodiments, the scFv fragment is a tandem scFv, and the linker domain is linked to an immunoglobulin hinge region (e.g., IgG 1 , IgG 2 , IgG 3 , and IgG 4 In embodiments, the immunoglobulin hinge region amino acid sequence includes, for example, the linker amino acid sequence (GGGS). x1-6 , (GGGGS) x1-6 and GSAGSAAGSGEF. In embodiments, the linker domain can be an immunoglobulin Fc domain, e.g., an IgG 1 , IgG 2 , IgG 3 , and IgG 4 At least a portion of an Fc domain. At least a portion of an immunoglobulin Fc domain can be a CH2 domain. The Fc domain is an immunoglobulin hinge region (e.g., IgG 1 , IgG 2 , IgG 3 , or IgG 4 The linker domain can be linked to the C-terminus of the amino acid sequence of the hinge region. The linker domain can be linked to the C-terminus of the amino acid sequence of the hinge region of the linker domain. The linker domain can be linked to the C-terminus of the amino acid sequence of the hinge region of the linker domain. x1-6 , (GGGGS) x1-6 , and GSAGSAAGSGEF).

[0079] In embodiments, the cytokine secreted by CASS B cells may be IL-12, IL-15, IL-18, IL-2, IL-7, CD40-L, or BAFF, or may be a cytokine receptor / Fc fusion protein.

[0080] An embodiment of the armed CASS B cell can include a gene expression vector that co-expresses multiple ORFs. The multiple ORFs can be separated by a linker such as an internal ribosome entry site (IRES) or the 2A family of peptides. 2A peptides are short (approximately 18-25 aa) peptides derived from viruses. 2A peptides can be referred to as "self-cleaving" peptides that produce multiple proteins from the same transcript. 2A peptides function by causing the ribosome to skip synthesis of a glycine and proline peptide bond at the C-terminus of the 2A element, causing separation between the end of the 2A sequence and the downstream peptide. As a result, the upstream protein has several extra 2A residues added to its C-terminus, while the downstream protein has an extra proline added to its N-terminus. There are four 2A peptides, P2A, T2A, E2A, and F2A, derived from four different viruses.

[0081] In embodiments, a secretable polypeptide may be expressed from a second expression construct that may be present in the same DNA vector as that encoding the chimeric B cell receptor (e.g., antigen recognition domain). A second expression cassette may be used to encode a secretable polypeptide (i.e., an antibody or cytokine) and may be cloned either before or after the linker (e.g., an IRES or 2A family of peptides).

[0082] In an embodiment, the second expression cassette encoding a secretable polypeptide can include a response element. A "response element" can refer to a part of a gene that must be present for the gene to respond to some hormone or other stimulus. In an embodiment, the response element is an inducible response element. For example, the response element can be an NFAT and / or NF-kB response element.

[0083] In one embodiment, for example, a second expression cassette was inserted after a syn-BCR plasmid with multiple NFAT and / or NF-kB response elements and a minimal IL2 / IL8 promoter upstream of a secreted protein (Ab or other protein). In native B cells, engagement of the BCR results in rapid tyrosine phosphorylation of the IC domain and calcium ion polarization, resulting in downstream activation of NFAT and NF-kB. Using NFAT / NF-kB response elements to drive expression of our secreted protein, we designed an inducible expression system that is activated by antigens expressed on cancer cells. This creates a targeted inducible delivery system that secretes its therapeutic payload only when activated by tumor cells, resulting in localized regions of consistent, high immunomodulatory Ab / protein concentration concentrated around the tumor. This can reverse the suppressive nature of the tumor microenvironment, resulting in improved outcomes and tumor elimination while reducing the on-target / off-tumor side effects often seen with systemic delivery of therapeutic antibodies and cytokines.

[0084] In embodiments, the expression cassette can further comprise a post-transcriptional response element that, when transcribed, creates a tertiary structure that enhances expression. For example, the post-transcriptional response element can be a WPRE.

[0085] Introduction of constructs into B cells The expression vector encoding the chimeric B cell receptor may be introduced as one or more DNA molecules or constructs, in which at least one marker may be present that allows for selection of host cells that contain the construct.

[0086] The constructs can be prepared by conventional methods, and the genes and regulatory regions can be isolated, ligated, cloned into a suitable cloning host, and analyzed by restriction or sequencing or other convenient means, as appropriate. For example, PCR can be used to separate individual fragments containing all or part of the functional unit, and one or more mutations can be introduced using "primer repair", ligation, in vitro mutagenesis, etc., as appropriate. Once the construct is complete and has been demonstrated to have the correct sequence, it can then be introduced into B cells by any convenient means. The constructs may be incorporated and packaged for infection or transduction of cells, such as non-replicating defective viral genomes, such as adenovirus, adeno-associated virus (AAV), Herpes simplex virus (HSV), or retroviral or lentiviral vectors. The constructs can include viral sequences for transfection. Alternatively, the constructs can be introduced by fusion, electroporation, biolistic methods, transfection, lipofection, etc. The host cells can be grown and expanded in culture prior to introducing the construct, and then the construct is introduced and treated appropriately to integrate the construct. The cells are then expanded and screened for the marker present in the construct. Various markers that can be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.

[0087] In some cases, when the construct is integrated into a specific locus, it may have a target site for homologous recombination. For example, an endogenous gene can be knocked out and replaced with the gene encoded by the construct (at the same locus or elsewhere) using materials and methods known in the art for homologous recombination. For homologous recombination, either OMEGA or O-vectors can be used. See, for example, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et al., Nature (1988) 336, 348-352; and Joyner, et al. Nature (1989) 338, 153-156. Furthermore, plasmid-based methods of inducing double-strand breaks have used homologous recombination for genome manipulation. Zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPRs can all direct nucleases to cause specific double-strand breaks.

[0088] The constructs can be introduced as a single DNA molecule encoding at least the CAR and optionally another gene, or as separate DNA molecules carrying one or more genes. Other genes include, for example, genes encoding therapeutic molecules or suicide genes. The constructs can be introduced simultaneously or sequentially, each carrying the same or different markers.

[0089] Vectors containing useful elements such as bacterial or yeast origins of replication, selectable and / or amplifiable markers, promoter / enhancer elements for expression in prokaryotes or eukaryotes, which can be used to prepare construct DNA stocks and perform transfections, are well known in the art and many are commercially available.

[0090] Treatment method Aspects of the present disclosure are directed to methods of preventing or treating a subject suffering from a disease or disorder by administering CASS B cells as described herein. In embodiments, the method comprises administering to a subject suffering from or at risk for a disease or disorder a therapeutically effective amount of CASS B cells as described herein. The therapeutically effective amount may depend on the severity and course of the disease or disorder, previous treatments, the subject's health status, weight, response to drugs, and the judgment of the attending physician.

[0091] "Treatment" and "treating" can refer to the management and care of a subject for the purpose of combating a condition, disease, or disorder, such as cancer or an infectious disease, in any manner in which one or more of the symptoms of the disease or disorder are ameliorated or otherwise beneficially altered. The term can include a full range of treatments for a given condition from which a patient suffers, such as administering an active compound for the purposes of reducing or alleviating symptoms or complications; slowing the progression of the condition, disease, or disorder; curing or eliminating the condition, disease, or disorder; and / or preventing the condition, disease, or disorder, where "preventing" or "prevention" can refer to the management and care of a patient for the purpose of preventing the onset of the condition, disease, or disorder, and can include the administration of an active compound to prevent or reduce the risk of onset of the symptoms or complications.

[0092] An "individual" or "subject" can be a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0093] The CASS B cells according to the present disclosure can be used to prevent or treat a disease or disorder (e.g., cancer or an infectious disease) in a subject in need thereof. In another embodiment, the isolated cells according to the present invention can be used in the manufacture of a medicament for the treatment of cancer or an infectious disease (e.g., a viral infection) in a patient in need thereof.

[0094] An embodiment may depend on a method for treating a patient in need thereof, the method comprising at least one of the steps of: (a) providing a CASS B CLL according to the invention; and (b) administering the cells to the patient.

[0095] The treatment may be ameliorative, curative or preventative. It may be part of an autoimmunotherapy or part of an allogeneic immunotherapy treatment. Autologous means that the cell, cell line or population of cells used to treat the patient is derived from the patient or from a human leukocyte antigen (HLA)-matched donor. Allogeneic means that the cell or population of cells used to treat the patient is derived from a donor, rather than from the patient.

[0096] The present invention is suitable for allogeneic immunotherapy insofar as it allows the transformation of B cells obtained from a donor into non-allo-reactive cells. This can be carried out under standard protocols and can be replicated as many times as necessary. The resulting modified B cells can be pooled and administered to one or several patients, making them available as an "off the shelf" therapeutic product.

[0097] Cells that can be used in the disclosed methods are described herein. The methods can be used to treat a patient diagnosed with a disease or disorder.

[0098] The embodiments described herein can modulate the immune system to treat a subject suffering from a disease or disorder. "Modulating" can refer to upregulation, induction, stimulation, enhancement, and / or release of inhibition, as well as inhibition, attenuation, and / or downregulation or suppression. In embodiments, the activity of the immune system of a subject is modulated.

[0099] The embodiments described herein can be administered to a subject in combination with one or more therapies for a disease or disorder.

[0100] How to Treat Cancer The embodiments include a method of treating a subject suffering from cancer. The term "cancer" can refer to the range of pathological symptoms associated with the initiation or progression of malignant tumors, as well as metastasis. The term "tumor" can refer to new growth of tissue in which cell proliferation is uncontrolled and progressive. In embodiments, a tumor can be a malignant tumor in which the primary tumor has invasive or metastatic properties, or exhibits a greater degree of anaplasia than a benign tumor. Thus, "treatment of cancer" or "treating cancer" can refer to activities that prevent, alleviate, or ameliorate either the primary phenomena (initiation, progression, metastasis) or secondary symptoms associated with the disease.

[0101] An emerging mechanism related to tumor progression is the immune checkpoint pathway, which involves cellular interactions that prevent excessive activation of T cells under normal conditions and allow T cell function in a self-limiting manner. As an evasion mechanism, many tumors can stimulate the expression of immune checkpoint molecules, resulting in an anergic phenotype of T cells that cannot suppress tumor progression. For example, emerging clinical data highlight the importance of one inhibitory ligand and receptor pair as an immune checkpoint: programmed death-ligand 1 (PD-L1; B7-H1 and CD274) and programmed cell death receptor 1 (PD-1; CD279) in preventing the killing of cancer cells by cytotoxic T lymphocytes. The PD1 receptor is expressed by many cell types, such as T cells, B cells, natural killer cells (NK), and host tissues. Tumors and antigen-presenting cells (APCs) expressing PD-L1 can block T cell receptor (TCR) signaling of cytotoxic T lymphocytes via binding to the receptor PD-1, reducing cytokine production and T cell proliferation. PD-L1 overexpression can be found in many tumor types and may mediate immunosuppressive functions through its interactions with other proteins, including CD80 (B7.1), and block its ability to activate T cells through binding to CD28.

[0102] Genetic engineering of human B cells to express tumor-specific chimeric B cell receptors can generate antitumor effector cells that circumvent tumor immune evasion mechanisms through abnormalities in protein-antigen processing and presentation. Moreover, these transgenic receptors are directed against tumor-associated antigens that are not protein-derived.

[0103] For example, aspects of the disclosure are directed to methods of killing cancer cells, such as renal cancer cells.

[0104] Aspects of the present disclosure are further directed to methods of halting or reducing the progression of, or promoting the regression of, cancer in a subject.

[0105] Still further, aspects of the present disclosure are directed to a method of reducing cell proliferation of cancer cells in a subject.

[0106] "Cancer" and "cancerous" can refer to or describe, for example, a physiological condition in a mammal characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, chronic lymphocytic leukemia, non-small cell lung cancer, renal clear cell carcinoma, mesothelioma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer. For example, the cancer is renal cell carcinoma, such as ccRCC.

[0107] The subject may be afflicted with cancer, such as a liquid cancer (i.e., a blood cancer) and / or a solid cancer (i.e., a tumor). The cancer may be benign or malignant, and may be influenced by the immune system.

[0108] Cancers that can be treated include tumors that are not vascularized or are not yet substantially vascularized, as well as vascularized tumors.Cancers can include non-solid tumors (e.g., hematological tumors, such as leukemias and lymphomas) or can include solid tumors.Types of cancers that can be treated with the CAR of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas.Adult tumors / cancers and pediatric tumors / cancers are also included.

[0109] In cancer, normal cell-cell interactions in tissues are disrupted and a tumor microenvironment evolves to accommodate the growing tumor. The tumor microenvironment (TME) can refer to the cellular environment in which a tumor resides, including components such as surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM). The tumor microenvironment is complex and is strongly influenced by the immune system.

[0110] The present invention provides CASS B cell therapy for the treatment or prevention of cancer. Secretion of monospecific, bispecific, or trispecific minibodies, antibodies, or minibody / antibody fusion proteins or cytokines by CASS-B cells at the tumor site may provide additional benefits by altering (i.e., modulating) the immunosuppressive tumor microenvironment. For example, the microenvironment surrounding the cancer cells and / or tumor can be modulated such that microenvironment-dependent immunosuppression is reduced to modulate (or enable) the immune system to kill tumor cells.

[0111] In an embodiment of the invention, the method of the invention for clinical aspects is combined with other agents effective in treating hyperproliferative diseases, such as anti-cancer agents. An "anti-cancer" agent can adversely affect cancer in a subject, for example, by killing cancer cells, including apoptosis in the cancer cells, reducing the rate of proliferation of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing blood supply to the tumor or cancer cells, promoting an immune response to the cancer cells or tumor, preventing or inhibiting the progression of cancer, or increasing the life span of a subject with cancer. For example, these other compositions will be provided in a combined amount effective to kill or inhibit proliferation of the cells. This process can include contacting the cancer cells with the expression construct and the agent or multiple factors simultaneously. This can be accomplished by contacting the cells with a single composition or pharmacological formulation that includes both agents, or by contacting the cells with two different compositions or formulations at the same time, one composition that includes the expression construct and another that includes the second agent.

[0112] Tumor cell resistance to chemotherapy and radiotherapy agents is a major problem in clinical oncology. One goal of current cancer research is to find ways to improve the efficacy of chemotherapy and radiotherapy by combining them with other therapies. In one embodiment, cell therapy can be used in conjunction with chemotherapy, radiotherapy, or immunotherapy interventions, as well as proapoptotic or cell cycle regulating agents.

[0113] Alternatively, the therapy of the present invention may precede or follow the treatment of the other agent by intervals ranging from minutes to weeks. In embodiments in which the other agent and the present invention are applied separately, it is generally ensured that no significant period of time expires between the time of each delivery, so that the agent and the treatment of the present invention can still exert their advantageously combined effect on the cells. In such cases, the cells may be contacted with both modalities within about 12-24 hours of each other (e.g., within about 6-12 hours of each other). In some situations, it may be desirable to significantly extend the treatment period, where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between the respective administrations.

[0114] Treatment cycles would be repeated as necessary. For example, various standard therapies, as well as surgical interventions, may be applied in combination with the cell therapy of the present invention.

[0115] Cancer therapy also includes a variety of combination therapies using both chemical and radiation-based treatments. Combination chemotherapy includes, but is not limited to, for example, Abraxane, altretamine, docetaxel, herceptin, methotrexate, novantrone, zoladex, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabine, navelbine, farnesyl protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate, or variants of any of the foregoing analogs or derivatives, and combinations thereof. In certain embodiments, chemotherapy for an individual is employed in combination with the present invention, eg, before, during, and / or after administration of the present invention.

[0116] Other agents that cause DNA damage and have been used widely include what are commonly known as gamma radiation, X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging agents, such as microwave and ultraviolet radiation, are also useful. All of these agents most likely cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens for prolonged periods (3-4 weeks) to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

[0117] As used herein, the terms "contact" and "exposure" as applied to a cell are used to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to or directly juxtaposed with a target cell. To achieve cell killing or stasis, both agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

[0118] Immunotherapy relies on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector can be, for example, an antibody specific for some marker on the surface of the tumor cell. The antibody alone can function as the effector of therapy or can recruit other cells to actually kill the cell. The antibody can also be conjugated to a drug or toxin (chemotherapeutic agent, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and simply function as a targeting agent. Alternatively, the effector can be a lymphocyte carrying a surface molecule that interacts directly or indirectly with the tumor cell target. Various effector cells include cytotoxic T cells and NK cells.

[0119] Thus, immunotherapies other than the therapies of the present invention described herein can be used as part of a combination therapy in combination with the cell therapy. The combination therapy approach is described herein. For example, tumor cells must have some markers that are amenable to targeting, i.e., not present on the majority of other cells. Many tumor markers exist, any of which may be suitable for targeting in the context of the present invention. Common tumor markers include PD-1, PD-L1, CTLA4, carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B, and p155.

[0120] In yet another embodiment, the secondary treatment is gene therapy in which a therapeutic polynucleotide is administered before, after, or simultaneously with the clinical embodiments of the invention.A variety of expression products are encompassed by the invention, including inducers of cell proliferation, inhibitors of cell proliferation, or regulators of programmed cell death.

[0121] Approximately 60% of cancer patients will undergo some type of surgery, including preventative, diagnostic, staging, curative and palliative surgery. Curative surgery is a cancer treatment that may be used in combination with other therapies, such as the treatment of the present invention, chemotherapy, radiation therapy, hormonal therapy, gene therapy, immunotherapy and / or alternative therapies.

[0122] Curative surgery includes resection, where all or part of the cancerous tissue is physically removed, excised, and / or destroyed. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs surgery). For example, the present invention may be used in conjunction with the removal of superficial cancers, precancers, or incidental amounts of normal tissue.

[0123] When all cancerous cells, tissues, or parts of tumors are removed, a cavity may be formed in the body. Treatment can be achieved by perfusion, direct injection, or local application to the site with additional anti-cancer therapy. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments can be of various dosages as well.

[0124] In some embodiments, other agents can be used in combination with the present invention to improve the therapeutic efficacy of the treatment. These additional agents include immunomodulators, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, cell adhesion inhibitors, or agents that enhance the sensitivity of hyperproliferative cells to apoptosis inducers. Immunomodulators include tumor necrosis factor, interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs, or MIP-1, MIP-1β, MCP-1, RANTES, and other chemokines. In some embodiments, upregulation of cell surface receptors or their ligands, such as Fas / Fas ligand, DR4, or DR5 / TRAIL, will enhance the apoptosis-inducing ability of the present invention by establishing an autocrine or paracrine effect on the hyperproliferative cells. Increasing intercellular signaling by increasing the number of GAP junctions enhances the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors can also be used to improve the efficacy of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. In some embodiments, other agents that increase the sensitivity of hyperproliferative cells to apoptosis, such as the antibody c225, can be used in combination with the present invention to improve the efficacy of the treatment.

[0125] According to one embodiment of the present invention, the treatment can be administered to a patient undergoing immunosuppressive treatment. The invention uses a cell or a population of cells that have been made tolerant to at least one immunosuppressant drug due to the inactivation of a gene encoding a receptor for the immunosuppressant. In this aspect, the immunosuppressive treatment should aid in the selection and expansion of T cells according to the invention in the patient.

[0126] In further embodiments, the cell compositions described herein can be administered to a patient in combination with (e.g., before, simultaneously with, or after) bone marrow transplantation, T cell ablative therapy using any of the following: chemotherapy agents, such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies, such as OKT3 or CAM PATH. In another embodiment, the cell compositions of the present invention are administered after B-cell ablative therapy, such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, the subject can receive standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, after transplant, the subject receives an infusion of the expanded immune cells of the present invention. In further embodiments, the expanded cells are administered before or after surgery. The modified cells obtained by any one of the methods described herein can be used in certain aspects of the present invention to treat patients in need thereof for host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD). Thus, within the scope of the present invention is a method of treating a patient in need thereof against host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), comprising treating the patient by administering to the patient an effective amount of modified cells comprising an inactivated TCR alpha and / or TCR beta gene.

[0127] How to Treat an Infection Aspects of the present invention are directed to methods of treating or preventing an infectious disease in a subject by administering to the subject a composition described herein. The term "infectious disease" can refer to an organism (e.g., a virus, fungus, or bacteria) that is harmful to its host. In some embodiments, the agent is harmful to the human host. An "anti-infectious disease" treatment refers to a treatment that prevents, ameliorates, or eradicates an infectious disease and / or its disease-causing agents.

[0128] Examples of infectious diseases include, but are not limited to, HIV, West Nile virus, Hepatitis A, B, C, smallpox, tuberculosis, vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV), human papillomavirus (HPV), SARS, influenza, coronavirus, Ebola, viral meningitis, herpes, anthrax, Lyme disease, and E. coli. See, e.g., Pelegrin, Mireia, Mar Naranjo-Gomez, and Marc Piechaczyk, "Antiviral monoclonal antibodies: can they be more than simple neutralizing agents?," Trends in microbiology 23.10 (2015): 653-665; Salazar, Georgina, et al., "Antibody therapies for the prevention and treatment of viral infections," npj Vaccines 2.1 (2017): 1-12.

[0129] Cell administration The present disclosure is particularly suitable for allogeneic immunotherapy inasmuch as it allows the transformation of B cells obtained from a donor into non-allo-reactive cells. This can be performed under standard protocols and can be replicated as many times as necessary. The resulting modified B cells can be pooled and administered to one or several patients, making them available as an "off the shelf" therapeutic product.

[0130] Depending on the nature of the cells, the cells can be introduced into a host organism, e.g., a mammal, in a wide variety of ways. In certain embodiments, the cells can be introduced into the site of a tumor, while in alternative embodiments, the cells home to the cancer or are modified to home to the cancer. The number of cells used depends on various circumstances, the purpose of the introduction, the lifespan of the cells, the protocol used, e.g., number of administrations, ability of the cells to grow, stability of the recombinant construct, etc. The cells can be applied, for example, as a dispersion injected at or near the site of interest. The cells can be in a physiologically acceptable medium.

[0131] In some embodiments, the cells are encapsulated to inhibit immune recognition and are located at the site of the tumor.

[0132] The cells can be administered as desired. A variety of protocols can be used depending on the desired response, the method of administration, the longevity of the cells, and the number of cells present. The number of administrations will depend, at least in part, on the factors listed above.

[0133] Administration of the cells or populations of cells according to the invention can be by any convenient method, including aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscular, intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the invention are administered by intravenous injection.

[0134] Administration of the cells or population of cells can consist of administration of 104-109 cells per kg of body weight, for example, 105-106 cells / kg of body weight, including all integer values ​​of cell numbers within these ranges. The cells or population of cells can be administered in one or more doses. In another embodiment, the effective amount of cells is administered as a single dose. In another embodiment, the effective amount of cells is administered as two or more doses over a period of time. The timing of administration is within the discretion of the attending physician and depends on the clinical condition of the patient. The cells or population of cells can be obtained from any source, such as a blood bank or a donor. While individual needs vary, determining the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of the art. An effective amount means an amount that provides a therapeutic or prophylactic benefit. The dosage administered will depend on the age, health and weight of the recipient, the type of concurrent treatment, if any, the frequency of treatment, and the nature of the desired effect.

[0135] It will be appreciated that the system is subject to many variables that may change over time and circumstances, such as cellular response to ligand, expression efficiency and, where appropriate, secretion levels, activity of the expressed product, the specific needs of the patient, loss of cells or the rate of loss of cellular activity as a result of expression activity of individual cells, etc. Thus, with regard to individual patients, even if there are universal cells that can be administered to the entire population, each patient will be monitored for appropriate individual dosing, and such practices of monitoring patients are routine in the art.

[0136] Nucleic Acid-Based Expression Systems The chimeric B cell receptor of the present disclosure can be expressed from an expression vector. Recombinant techniques for making such expression vectors are well known in the art.

[0137] The DNA constructs, which may also be referred to as "DNA vectors" described herein, can be cloned into vectors used to transduce and produce CASS B cells that secrete the polypeptides and / or fragments thereof. For example, the DNA constructs can be cloned into lentiviral vectors for the production of lentiviruses used to transduce and produce chimeric antigen receptor T cells that secrete monospecific, bispecific, or trispecific immunomodulatory antibodies / minibodies and / or antibody fusion proteins at tumor sites. For example, the DNA constructs can be cloned into adeno-associated viral vectors for the production of adeno-associated viruses used to transduce and produce chimeric antigen receptor T cells that secrete monospecific, bispecific, or trispecific immunomodulatory antibodies / minibodies and / or antibody fusion proteins at tumor sites.

[0138] In embodiments, the DNA construct may include nucleic acid encoding one or more polypeptides, such as a chimeric B cell receptor and / or a secreted polypeptide.

[0139] vector The term "vector" can refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. The nucleic acid sequence can be "exogenous", meaning that it is foreign to the cell into which it is introduced, or that the sequence is homologous to a sequence in the cell, but is in a location within the host cell nucleic acid where the sequence is not normally found. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), artificial chromosomes (YACs, etc.). Those skilled in the art will be well equipped to construct vectors using standard recombinant techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both of which are incorporated herein by reference).

[0140] The term "expression vector" refers to any type of genetic construct that contains a nucleic acid encoding an RNA that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors can contain nucleic acid sequences that also perform other functions, as described below.

[0141] Promoters and Enhancers A "promoter" is a control sequence, which is a region of a nucleic acid sequence at which the initiation and rate of transcription are controlled. It can contain genetic elements to which regulatory proteins and molecules bind, such as RNA polymerase and other transcription factors, to initiate specific transcription of a nucleic acid sequence. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence to control transcription initiation and / or expression of that sequence.

[0142] Promoters contain sequences that function to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, for example the promoters of the mammalian terminal deoxynucleotidyl transferase gene and the promoters of the SV40 late genes, separate elements that cover the start site themselves help to anchor the start location. Additional promoter elements regulate the frequency of transcription initiation. These are located in the region 30-110 bp upstream of the start site, but some promoters have been shown to contain functional elements downstream of the start site as well. To place a coding sequence "under the control" of a promoter, the 5' end of the transcription start site of the transcriptional reading frame is placed "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates transcription of the DNA, promoting expression of the encoded RNA.

[0143] In many cases, the spacing between promoter elements is flexible, so that promoter function is maintained even if elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased by up to 50 bp before activity begins to decline. Depending on the promoter, individual elements may function either cooperatively or independently to activate transcription. Promoters may or may not be used in combination with "enhancers," which refer to cis-acting regulatory sequences involved in the transcriptional activation of a nucleic acid sequence.

[0144] A promoter may be one that is naturally associated with a nucleic acid sequence, as may be obtained by isolating 5 prime' non-coding sequences located upstream of a coding segment and / or exon. Such a promoter may be referred to as "endogenous". Similarly, an enhancer may be one that is naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from other viruses, or from prokaryotic or eukaryotic cells, and promoters or enhancers that are "non-naturally occurring", i.e., that contain different elements of different transcriptional regulatory regions, and / or mutations that alter expression. For example, promoters most commonly used in recombinant DNA construction include the lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR™, in conjunction with the compositions disclosed herein (see U.S. Patent Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference.) Additionally, control sequences that direct transcription and / or expression of sequences in non-nuclear organelles, such as mitochondria, chloroplasts, etc., may be used as well.

[0145] Of course, it will be important to use a promoter and / or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in the art of molecular biology are generally aware of the use of promoter, enhancer, and cell type combinations for protein expression (see, for example, Sambrook et al. 1989, incorporated herein by reference). The promoter used may be constitutive, tissue-specific, inducible, and / or useful under appropriate conditions to direct high-level expression of the introduced DNA segment, which is advantageous for large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0146] Additionally, any combination of promoters / enhancers can be used to drive expression. Use of the T3, T7, or SP6 cytoplasmic expression systems is another embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided as part of the delivery complex or as an additional gene expression construct.

[0147] The identity of tissue-specific promoters or elements, as well as assays to characterize their activity, are well known to those of skill in the art.

[0148] Specific initiation signals may also be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would be able to readily determine this and provide the necessary signals.

[0149] In certain embodiments of the present disclosure, the use of internal ribosome entry site (IRES) elements is used to create multigenic, or polycistronic, messages, which can be used in the present invention.

[0150] A vector can contain a multiple cloning site (MCS), a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used to digest the vector in conjunction with standard recombinant techniques. "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at a specific location in the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is widely understood by those of skill in the art. Restriction enzymes that cut within the MCS are often used to linearize or fragment a vector so that an exogenous sequence can be ligated into the vector. "Ligation" refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be adjacent to each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.

[0151] Splice sites, termination signals, origins of replication, and selectable markers may also be used.

[0152] Plasmid vectors In certain embodiments, plasmid vectors can be used to transform host cells. Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in connection with these hosts. The vectors usually carry a replication site, as well as marking sequences that can provide phenotypic selection in transformed cells. In a non-limiting example, E. coli is often transformed using derivatives of pBR322, a plasmid derived from E. coli species. pBR322 contains genes for ampicillin and tetracycline resistance, allowing easy identification of transformed cells. The pBR plasmid, or other microbial plasmids or phages, must also contain, or be modified to contain, a promoter that the microorganism can use for expression of its own proteins, for example.

[0153] Additionally, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, phage lambda GEM.TM.11 can be utilized in generating recombinant phage vectors that can be used to transform host cells such as E. coli LE392.

[0154] Further useful plasmid vectors include the pIN vectors (Inouye et al., 1985); and the pGEX vectors for use in generating soluble fusion proteins with glutathione S-transferase (GST) for subsequent purification and isolation or cleavage. Other suitable fusion proteins are those with galactosidase, ubiquitin, etc.

[0155] Bacterial host cells, e.g., E. coli, containing the expression vector are grown in any of a number of suitable media, e.g., LB. As will be appreciated by those skilled in the art, expression of the recombinant protein in a particular vector can be induced by contacting the host cells with an agent specific for the particular promoter, e.g., by adding IPTG to the medium, or by switching the incubation to a higher temperature. The bacteria are further cultured, e.g., for 2-24 hours, after which the cells are harvested by centrifugation and washed to remove residual medium.

[0156] Viral Vectors The ability of certain viruses to infect cells via receptor-mediated endocytosis, enter cells, integrate into the genome of host cells, and stably and efficiently express viral genes makes them attractive candidates for the introduction of foreign nucleic acid into cells (e.g., mammalian cells). A component of the present invention can be a viral vector encoding one or more CARs of the present invention. Non-limiting examples of viral vectors that can be used to deliver the nucleic acid of the present invention are described below.

[0157] Adenovirus Vectors A particular method for delivery of nucleic acids involves the use of adenoviral expression vectors. Adenoviral vectors are known to have a low capacity for integration into genomic DNA, but this feature is offset by the high gene transfer efficiency afforded by these vectors. "Adenoviral expression vector" is meant to include constructs that contain sufficient adenoviral sequences (a) to support packaging of the construct, and (b) to ultimately express the tissue- or cell-specific construct cloned therein. The genetic makeup or knowledge that adenovirus is a 36 kb, linear, double-stranded DNA virus allows for the replacement of large pieces of adenoviral DNA with foreign sequences up to 7 kb in size (Grunhaus and Horwitz, 1992).

[0158] AAV vectors Adenovirus assisted transfection can be used to introduce nucleic acid into cells. Increased transfection efficiency has been reported in cell lines using the adenovirus coupled system (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated virus (AAV) is an attractive vector system for use in the cells of the invention because of its high frequency of integration and its ability to infect non-dividing cells, and is therefore useful for gene delivery to mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broad host range for infectivity (Tratschin et al., 1984; Laughlin et al, 1986; Lebkowski et al, 1988; McLaughlin et al, 1988). Details regarding the generation and use of rAAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368, each of which is incorporated herein by reference.

[0159] Retroviral Vectors Retroviruses are useful as delivery vectors due to their ability to integrate their genes into the host genome, to introduce large amounts of foreign genetic material, to infect a wide range of species and cell types, and to be packaged in specialized cell lines (Miller, 1992).

[0160] To construct a retroviral vector, a nucleic acid (e.g., encoding a sequence of interest) is inserted into the viral genome in place of a certain viral sequence to produce a virus that is replication-defective. To produce virions, a packaging cell line is constructed that contains the gag, pol, and env genes, but does not contain the LTR and packaging components (Mann et al., 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences, is introduced into a special cell line (e.g., by calcium phosphate precipitation), the packaging sequences allow the RNA transcripts of the recombinant plasmid to be packaged into viral particles that are then secreted into the culture medium (Nicolas and Rubenstein, 1988: Temin, 1986; Mann et al., 1983). The medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors can infect a variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).

[0161] Lentiviruses are complex retroviruses that contain the common retroviral genes gag, pol, and env, plus other genes with regulatory or structural functions. Lentiviral vectors are well known in the art (see, e.g., Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; and U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency viruses: HIV-1, HIV-2, and simian immunodeficiency virus: SIV. Lentiviral vectors are generated by multiple attenuation of HIV pathogenicity genes, e.g., genes env, vif, vpr, vpu, and nef are deleted, making them biologically safe vectors.

[0162] Recombinant lentivirus vectors can infect non-dividing cells and can be used for gene transfer and expression of nucleic acid sequences both in vivo and ex vivo. For example, recombinant lentiviruses can infect non-dividing cells in which suitable host cells are transfected with two or more vectors carrying packaging functions, namely gag, pol, and env, as well as rev and tat, as described in U.S. Patent No. 5,994,136, which is incorporated herein by reference. To target receptors on specific cell types, recombinant viruses can be targeted by binding of envelope proteins with antibodies or specific ligands. For example, by inserting a sequence of interest (including regulatory regions) into a viral vector together with another gene that codes for a ligand for a receptor on a specific target cell, the vector is now target-specific.

[0163] Other viral vectors Other viral vectors can be used as vaccine constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), Sindbis virus, cytomegalovirus, and herpes simplex virus can be used. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

[0164] Delivery using modified viruses The nucleic acid to be delivered may be housed within an infectious virus engineered to express a specific binding ligand. Thus, the viral particle specifically binds to the cognate receptor of the target cell and delivers its contents to the cell. A new approach designed to enable specific targeting of retroviral vectors was developed based on the chemical modification of retroviruses by chemical addition of lactose residues to the viral envelope. This modification allows specific infection of hepatocytes via sialoglycoprotein receptors.

[0165] Another approach to targeting recombinant retroviruses was designed, using biotinylated antibodies against retroviral envelope proteins and specific cellular receptors. The antibodies were conjugated via the biotin moiety by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated in vitro infection of various human cells bearing these surface antigens by ecotropic viruses (Roux et al., 1989).

[0166] Vector delivery and cell transformation Suitable methods for nucleic acid delivery for transfection or transformation of cells are known to those skilled in the art. Such methods include, but are not limited to, direct delivery of DNA by ex vivo transfection, injection, etc. Cells can be stably or transiently transformed by application of techniques known in the art.

[0167] Ex vivo transformation Methods for transfecting eukaryotic cells and tissues ex vivo that have been removed from a living organism are known to those skilled in the art. Thus, the nucleic acids of the present invention can be used to remove cells or tissues and transfect them ex vivo. In some aspects, the transplanted cells or tissues can be placed into an organism. In other embodiments, the nucleic acid is expressed in the transplanted cells.

[0168] Pharmaceutical Compositions The invention provides therapeutic compositions, or "pharmaceutical compositions" or "formulations", comprising the CASS B cells described herein and a pharma- ceutically acceptable carrier. The invention also provides therapeutic compositions comprising a nucleic acid as described herein and a pharma- ceutically acceptable carrier.

[0169] The phrase "pharmaceutical composition" or "pharmaceutical formulation" can refer to a composition or pharmaceutical composition suitable for administration to a subject, such as a mammal, particularly a human, and can refer to a combination of ingredients, including an active agent (e.g., genetically engineered cells) or a pharma- ceutical acceptable carrier or excipient, making the composition suitable for diagnostic, therapeutic, or prophylactic use in vitro, in vivo, or ex vivo. According to the present invention, a "pharmaceutical composition" can be sterile and free of contaminants that may induce an undesirable response in a subject (e.g., the compounds in the pharmaceutical composition are pharmaceutical grade). The pharmaceutical composition can be designed to be administered to a subject or patient in need thereof via many different routes of administration, including oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, inhalation, and the like.

[0170] In embodiments, the pharmaceutical composition may contain ingredients that ensure the viability of the CASS B cells therein. In particular, the cells may be provided in the form of a pharmaceutical composition that includes an isotonic excipient prepared under sterile conditions sufficient for administration to humans. For general principles in pharmaceutical formulations, the reader is referred to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996, which is incorporated herein in its entirety. The choice of cell excipient and any accompanying elements of the composition is adapted according to the device used for administration. For example, the composition may include a suitable buffer system (e.g., a phosphate or carbonate buffer system) to a suitable pH, e.g., near neutral pH, and may include sufficient salt to ensure isotonic conditions for the cells, i.e., to prevent osmotic stress. For example, a solution suitable for these purposes may be phosphate buffered saline (PBS), as known in the art. Additionally, the composition may include a carrier protein, e.g., albumin, that may enhance the viability of the cells. To ensure the exclusion of non-human animal materials, the albumin may be of human origin (e.g., isolated from human material or recombinantly produced). Appropriate concentrations of albumin are generally known.

[0171] Thus, pharmaceutical compositions according to the present invention and for use according to the present invention may contain pharma- ceutically acceptable excipients, carriers, buffers, preservatives, stabilizers, antioxidants, or other materials known to those skilled in the art. Such materials should be non-toxic and should not interfere with the activity of cells or nucleic acids. The exact nature of the carrier or other materials depends on the route of administration. The composition may contain one or more cytoprotective molecules. Such materials may make cells independent of their environment.

[0172] A "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," or "pharmaceutically acceptable adjuvant" can refer to an excipient, diluent, carrier, and / or adjuvant that is generally safe, non-toxic, and not biologically or otherwise undesirable and is useful in preparing a pharmaceutical composition, and includes excipients, diluents, carriers, and adjuvants that are acceptable for use in veterinary and / or human medicine. As used herein and in the claims, "pharmaceutically acceptable excipient, diluent, carrier, and / or adjuvant" includes one or more such excipients, diluents, carriers, and adjuvants.

[0173] The invention also encompasses methods of producing the pharmaceutical compositions by mixing the cells and / or nucleic acids of the invention with one or more additional ingredients as described above. Liquid pharmaceutical compositions generally include a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Saline, tissue or cell culture medium, dextrose or other saccharide solutions or glycols (e.g., ethylene glycol, propylene glycol, or polyethylene glycol) may be included.

[0174] The composition may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are well able to prepare suitable solutions using isotonic vehicles such as, for example, sodium chloride, Ringer's injection, or lactated Ringer's injection. The composition may be prepared using a biological fluid such as artificial cerebrospinal fluid. In a further aspect, the present invention relates to a device comprising a surgical instrument for administering the composition to a site of tissue dysfunction or pathology, further comprising a pharmaceutical composition as defined above, the device being adapted to administer the pharmaceutical composition to a site of tissue dysfunction or pathology. For example, a suitable surgical instrument may be capable of injecting a liquid composition comprising the genetically engineered cells described herein to a site of neurological dysfunction or pathology. The cells can be transplanted into the patient by any technique known in the art, including those described in Freed et al. 1997. Cell Transplant 6:201-202; Kordower et al. 1995. N Engl J Med 332:1 118-1 124; Freed et al. 1992. N Engl J Med 327:1549-1555; Tateishi-Yuyama,Eriko,et al. The Lancet 360.9331(2002):427-435; THOMA,CHRISTINE,et al. Nature medicine 3.3(1997); Kondziolka,D,et al. Neurology 55.4(2000):565-569, the entire disclosures of each of which are incorporated herein by reference.

[0175] Methods for producing genetically engineered populations of B cells

[0013] Embodiments of the present invention are also directed to methods of making a population of genetically engineered B cells. For example, such methods include isolating a population of B cells from a subject and transducing the population of B cells with a vector as described herein, thereby producing a population of genetically engineered B cells.

[0176] The cells, such as B cells, into which the polynucleotides are introduced can be obtained from the subject themselves, a donor subject, or a source such as a cell bank. For example, cells can be harvested from the subject, as in the case of B cells that may be used for autologous transplantation.

[0177] In embodiments, the polynucleotide may be introduced into the cell by transduction, such as transfer by a bacteriophage or virus, transformation, such as uptake of naked DNA from outside the cell, or by microinjection.

[0178] Positive and negative controls can be used as needed. For example, a positive control for transduction efficiency can be an empty plasmid lentivirus stock with mCherry, eGFP, or other fluorescent molecule tags such as YFP, BFP, or RFP.

[0179] In embodiments, the method further comprises activating the population of B cells prior to transduction.

[0180] In embodiments, the method can further comprise culturing the population of genetically engineered B cells. Culturing cells can refer to the process of maintaining cells under conditions suitable for maintenance and / or proliferation, conditions including, for example, the temperature at which the cells are maintained, nutrient availability, atmospheric CO2, and the like. 2 The term may refer to cell content, cell density, etc. Cells can be cultured in vivo or in vitro. Appropriate culture conditions for maintaining, growing, expanding, and differentiating different types of cells are known to those skilled in the art. See, for example, Moffett, HF et al (2019). B cells engineered to express pathogen-specific antibodies protect against infection. Science Immunology, 4(35).

[0181] Kits of the Invention Any of the compositions described herein may be included in a kit. In a non-limiting example, the kit may include one or more cells for use in cell therapy and / or reagents for generating one or more cells for use in cell therapy that contain a recombinant expression vector. The components of the kit are provided in suitable container means.

[0182] Some components of the kit may be packaged in either aqueous media or lyophilized form. The container means of the kit may include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed and suitably dispensed. If there is more than one component in the kit, the kit may also contain a second, third, or other additional container into which the additional components may be placed separately. However, various combinations of components may be included in the vial. The kit of the present invention may also include a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0183] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly useful. In some cases, the container means itself may be a syringe, pipette, and / or other similar device from which the formulation may be applied to an infected area of ​​the body, injected into an animal, and / or applied to and / or mixed with the other components of the kit.

[0184] However, the components of the kit may also be provided as a dry powder. When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. For example, the solvent can also be provided in another container means. The kit may also comprise a second container means for containing a sterile pharma- ceutically acceptable buffer and / or other diluent.

[0185] In certain embodiments of the invention, the cells used for cell therapy are provided in a kit, and in some cases, the cells are essentially the only component of the kit. The kit may include reagents and materials for producing the desired cells. In certain embodiments, the reagents and materials include primers for amplifying the desired sequence, nucleotides, suitable buffers or buffer reagents, salts, etc., and in some cases, the reagents include vectors and / or DNA encoding the chimeric B cell receptor described herein and / or regulatory elements therefor.

[0186] In certain embodiments, there are one or more devices in the kit suitable for extracting one or more samples from an individual. The device may be a syringe, a scalpel, etc.

[0187] In some cases of the invention, in addition to the cell therapy embodiment, the kit also includes a second cancer therapy, such as, for example, chemotherapy, hormonal therapy, and / or immunotherapy. The kit can be tailored to the individual's particular cancer and can include the individual's respective second cancer therapy. EXAMPLES

[0188] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes of making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, and are for illustrative purposes only, since alternative methods can be used to obtain similar results.

[0189] Example 1 Engineering chimeric antibody signaling and secretion (CASS) B cells as a targeted inducible platform to secrete immunomodulatory proteins at tumor sites Membrane Ig constructs (synthetic BCRs) were designed by linking scFvs to membrane-tethered IgG or IgM hinge-CH2-CH3 with native transmembrane and intracellular domains. A secondary cassette was inserted upstream of a secreted protein (Ab or other protein, e.g., cytokine) after a syn-BCR plasmid with multiple NFAT and / or NF-kB response elements and a minimal IL2 / IL8 promoter.

[0190] In natural B cells, engagement of the BCR results in rapid tyrosine phosphorylation and calcium ion polarization of the intracellular domain, leading to downstream activation of NFAT and NF-κB. Using NFAT / NF-κB response elements to drive expression of our secreted proteins, we engineered an inducible expression system that is activated by antigens expressed on cancer cells. This creates a targeted inducible delivery system that secretes its therapeutic payload only when activated by tumor cells, resulting in localized regions of consistent high immunomodulatory Ab / protein concentration concentrated around the tumor. This can reverse the suppressive nature of the tumor microenvironment, resulting in improved outcomes and tumor elimination while reducing the on-target / off-tumor side effects often seen with systemic delivery of therapeutic antibodies and cytokines.

[0191] Non-limiting examples include anti-CAIX CASS B cells secreting anti-PD1 / anti-CTLA4 bispecific antibodies, and anti-mesothelin CASS B cells secreting anti-PD1 / anti-TIGIT bispecific antibodies. Other non-limiting examples of secreting antibodies include anti-CCR4, anti-PDL1, anti-VEGF, anti-CAIX, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA4, anti-TIGIT, anti-VISTA, anti-CD70, anti-TIM-3, anti-LAG-3, anti-CD40L, anti-CCR4, anti-GITR, or anti-CXCR4.

[0192] Figure 1 shows the CASS B cell schematic, which uses separate plasmids for syn-BCR and secreted Ab, but which can also be combined into one plasmid.

[0193] Current therapeutic methods include CAR T cells and systemic Ab delivery. CAR T cell therapy is associated with cytotoxicity. The embodiments herein use B cells that, unlike CAR-T cells, do not result in direct tumor cytotoxicity as seen with CAR T cells. Rather, the engineered B cells described herein focus on reversing the suppressive nature of the tumor microenvironment by secreting high levels of Ab / cytokines locally around the tumor. This allows the remaining immune system to destroy the tumor and also provides lifelong protection against regrowth and metastasis. The addition of an inducible system is also an important component as it reduces the serum concentration of our Abs, thereby reducing off-tumor / on-target side effects.

[0194] Thus, the embodiments described herein can be used to treat solid tumors, especially those with a highly immunosuppressive tumor microenvironment. The embodiments described herein can also be used to prevent and / or treat other indications, including infectious diseases.

[0195] Example 2 Engineered chimeric antibody signaling and secreting (CASS) B cells to achieve cancer cures summary Immune checkpoint blockade inhibitors (CBIs) and CAR T cells have revolutionized the way we treat cancer. Although both of these therapies engage the patient's immune system, neither is able to actively mount an anti-tumor immune response, presenting significant limitations in the scope of their use and efficacy. To address this, chimeric antibody-secreting and signaling (CASS) B cells are described herein that express an engineered tumor-targeting B cell receptor and upon engagement secrete high levels of dual-targeted bispecific checkpoint blockade modulator antibodies (e.g., dual-targeted bispecific checkpoint inhibitor antibodies) locally at the tumor site. As B cells also function as professional antigen-presenting cells, they can process and present antigens on MHC class II molecules, further enhancing immune cell recognition of tumors and aiding in neo-antigen spread. CASS B cells, a key component of immune memory, provide a robust, lifelong surveillance program that simultaneously recruits a broad range of immune cells, reverses tumor-infiltrating lymphocyte exhaustion, and protects against tumor metastasis and recurrence. Non-small cell lung cancer (NSCLC) was selected as a model to develop anti-MSLN-directed CASS B cells secreting immunomodulatory anti-PD1 / TIGIT bispecific antibodies (bsAbs). Objective 1 focused on the development of the CASS B cell platform, and at the end of this stage, we identified three antibodies and optimized the signaling domains that comprise CASS B cells. Objective 2 involved in vitro characterization and efficacy testing, providing a clear understanding of the link between CASS B cell activation and bsAb secretion, while providing a critical analysis of CASS B cell efficacy compared to CAR T cells at both functional and molecular levels. Objective 3 performed in vivo experiments using cell line-derived and patient-derived NSCLC models in humanized mice. Multiparameter flow cytometry, single-cell RNA sequencing, and immunohistochemistry provided a detailed assessment of the molecular and mechanistic efficacy of the immunomodulatory bsAbs and the entire CASS B cell platform.

[0196] Research purpose Without wishing to be bound by theory, the inventors develop a new type of combination cellular immunotherapy, chimeric antibody-secreting and signaling (CASS) B cells. These B cells express engineered tumor-associated antigens (TAA) that target the B cell receptor (BCR) and, upon engagement, secrete high levels of checkpoint blockade modulators (e.g., dual-targeted bispecific checkpoint inhibitor antibodies) bispecific antibodies (bsAbs) locally to the tumor site. This allows for the reversal of the immunosuppressive tumor microenvironment (TME) and the restoration of the patient's natural innate and adaptive immunity to eliminate cancerous cells. In addition, unlike T cells, B cells should act as professional antigen-presenting (APC) cells, enhancing tumor cell recognition and assisting neoantigen spreading, thereby both reversing tumor-infiltrating lymphocyte (TIL) exhaustion and inducing a broader and stronger antitumor immune response. For proof-of-principle studies, we propose to engineer anti-PD1 / TIGIT bsAb-secreting CASS B cells that target mesothelin (MSLN) for the treatment of NSCLC. 1~3 .

[0197] The first objective will focus on the construction and optimization of an engineered anti-MSLN IgG-BCR, an anti-PD1 / TIGIT bsAb delivered to the tumor site, and an inducible response element (RE) to drive bsAb expression. A panel of anti-MSLN, anti-PD1, and anti-TIGIT antibodies will be identified by our lab, and functional assays will identify lead candidates. Concurrent efforts will focus on the development of the inducible response element and optimization of B cell transduction conditions.

[0198] The secondary objective is functional evaluation of anti-MSLN CASS B cells in vitro. Activation assays will be used to quantify bsAb and cytokine secretion levels. Patient-derived organotypic tumor spheroids (PDOTS) will be used to evaluate CASS B cell potency and perform molecular / mechanistic comparisons with CAR T cells via cytokine profiling, IHC, and single-cell RNA sequencing (scRNAseq). Also, embodiments can use mesothelioma tumors for PDOT.

[0199] The ultimate goal is to utilize HLA-matched humanized mice to generate cell line-derived (CDX) and patient-derived xenograft (PDX) models of NSCLC to test CASS B cell efficacy. The models will be combined with various analytical techniques (IHC, flow cytometry, scRNAseq) to further investigate the effect of CASS B cell therapy on the surrounding TME.

[0200] background Checkpoint blockade inhibitor (CBI) monoclonal antibodies (mAbs) and adoptive cell therapy have had a transformative effect on cancer therapy by shifting the focus from simply killing tumor cells to activating the patient's natural antitumor immunity and reversing the immunosuppressive tumor microenvironment (TME). While these therapies represent some of the most promising anticancer treatments to date, only a small proportion of patients experience a complete or durable response, and many experience immune-related adverse events (irAEs) of varying severity. 4~7 To combat this, combination CBI therapies such as anti-PD(L)1 / anti-TIGIT are being tested and have demonstrated significant promise in clinical trials. 8~11 An alternative approach is the development of armored or immune-restorative CAR T cells that are engineered to secrete immune-modulating payloads directly to the tumor site, improving efficacy while reducing the on-target / off-tumor side effects seen with systemic delivery. 12、13 .

[0201] In addition to T cells, a variety of immune cells can be utilized to generate new CARs, including natural killer cells (NK-CARs) and macrophages (CAR-M), and one consistent feature of all of these cells is that they provide direct antitumor activity. 14、15 B cells, a key component of humoral immunity, produce antibodies and the field of immunotherapy was initially based on them, but they have no inherent cytotoxic capacity and therefore have been largely excluded from these advances.

[0202] The chimeric antibody secreting and signaling (CASS) B cell platform will bring B cell research into the 21st century by developing unique B cell-based cell therapies that do not rely on direct cytotoxicity but instead exploit two intrinsic capabilities of B cells: their ability to secrete high levels of CBI antibodies to reverse the immunosuppressive TME, and their ability to process and present antigens on MHC class II molecules, resulting in the recruitment of CD4+ T cells and enabling enhanced tumor cell recognition and neo-antigen spreading. While the inducible and targeted delivery of CBI reduces irAEs, the ability to function as professional APCs makes the CASS B cell platform unique in the cell therapy arena, as CASS B cells have the ability to mount potent anti-tumor responses. This impact has been further illustrated in studies that have demonstrated that MHC class II neo-antigens play a critical function in innate anti-tumor responses. 16、17 .

[0203] While this example focuses on MSLN+ targeted CASS B cells secreting anti-PD1 / TIGIT bsAbs for NSCLC, the modular design of CASS B cells allows for easy targeting of other TAAs and the secreted payload can be tailored to target relevant immunological axes, allowing adaptation of CASS B cells to a wide variety of cancers. Because B cells are long-lived and an important part of immune memory, CASS B cells can continuously deliver therapeutic payloads to tumors and provide a lifelong immune surveillance system against metastasis and recurrence once the primary tumor has been eradicated.

[0204] B cell engineering is a more recent achievement and has been primarily focused on generating B cells that secrete neutralizing anti-pathogenic antibodies against RSV and HIV. 21~23 Unlike the cancer therapeutics proposed herein, the B cells described in these studies focus on the systemic production of antibodies to neutralize viral infections and utilize CRISPR / Cas9 to insert recombinant mAbs into the Ig loci of B cells. This has the added benefit of allowing the antibodies to continue to undergo affinity maturation, a requirement for fighting infections but not for targeting immune markers. Taken together, these studies show that B cells can be engineered to express selected engineered antibodies in an inducible manner, and that upon activation, these engineered B cells not only differentiate into Ab-secreting cells, but also into memory B cells that provide long-term protection. 24 .

[0205] Non-limiting examples of research plans Objective 1: Engineering and optimization of CASS B cell constructs - Select appropriate scFvs (anti-MSLN, PD1, TIGIT) for CASS B cell development, followed by optimization of signaling domains and inducible response elements. 25~28 Protocols for highly efficient transduction and exponential expansion of primary B cells have also been optimized utilizing various lentiviral envelope proteins and culture conditions. 29~32 .

[0206] Non-limiting examples of experimental designs and procedures Antibody discovery, engineering, and optimization: The inventors have previously constructed a 27 billion member human phage library that has been used to isolate several therapeutic antibodies. 33~37 The anti-PD1 (Figure 2 Panel A), TIGIT (Figure 2 Panel B), and MSLN (Figure 2 Panel C) sets have been identified for further engineering and optimization.

[0207] Designing and engineering bispecific antibodies: PD1 / TIGIT is expressed on immune cells, so immune depletion is undesirable 38、39 The current design utilizes tandem scFv constructs previously developed and characterized in the Marasco laboratory, although other designs will be considered (Figure 3 panels A-C).

[0208] B cell isolation, expansion, and transduction: Isolate B cells and test various expansion media formulations 23、40、41 For transduction, B cells are activated, transduced, and selected 72 hours after transduction.

[0209] Generation of IgG-BCR constructs: Our engineered IgG-BCR is constructed using an scFv fused to a membrane-bound IgG1 hinge-Fc that is expressed at high levels and binds the target antigen (Figure 4 panel A). 42 Transduction experiments using primary B cells demonstrate high titer transduction for multiple donors and DNA constructs (Figure 4 Panel B). We have previously utilized the NFAT / NFkB RE to develop an inducible T cell activation assay (Figure 4 Panel C). In certain experiments, a fluorescent protein is used in place of the secreted bsAb.

[0210] The mAb engineering techniques routinely used by our laboratory allow us to develop a panel of previously discovered mAbs to find lead candidates for each target. Without wishing to be bound by theory, we are able to achieve high transduction efficiency and exponential expansion of transduced cells for in vivo experiments.

[0211] Milestones: The first milestone is the identification of lead antibodies for each target. The second milestone is the successful construction of vectors and transduction / expansion of CASS B cells.

[0212] Aim 2: In Vitro Testing and Efficacy of CASS B Cells - Perform detailed in vitro characterization to identify activation thresholds and quantitate bsAb secretion levels. The final in vitro assay will be performed using patient-derived organotypic tumor spheroids (PDOTS), allowing us to observe CASS B cell homing and perform detailed analysis of the bsAb payload.

[0213] Experimental design and procedures CASS B cell activation assay: Supernatants from activated CASS B cells are screened by ELISA to measure bsAb and cytokine concentrations.

[0214] NSCLC PDOT generation and evaluation: The generation of NSCLC PDOT was performed according to the method described by Jenkins et al. and Aref et al. 43,44 The study will be performed in the laboratory of Dr. David Barbie, following the protocol outlined in. In addition to testing the efficacy of the bsAb and CASS B cells, comparative experiments will be performed to identify therapeutic differences between CASS B cell and CAR T cell treatments. Immune profiling by IHC, scRNAseq, and cytokine profiling will be performed. Due to the limited availability of NSCLC tumor tissue, a backup plan has been devised to use mesothelioma tumors for PDOT generation. Mesothelioma has a highly suppressive TME and immune infiltrating cells display high levels of exhaustion markers, making this an ideal alternative to in vitro assays. 45~48 .

[0215] Without wishing to be bound by theory, the inventors can provide a deeper understanding of CASS B cell activation ability and identify optimal anti-MSLN scFv for selective targeting of MSLN+ tumors. PDOT provides a comprehensive data set of CASS B cell efficacy through cytokine secretion and transcriptional profiles, and without wishing to be bound by theory, anti-MSLN CASS B cells are transported to tumors and secreted bsAb reverses immune cell suppression. Furthermore, without wishing to be bound by theory, the inventors can see greater epitope spreading and bystander immune cell activation by CASS B cells compared to CAR T cell therapy.

[0216] Milestones: The first milestone in objective 2 will be the generation of CASS B cell activation curves and quantification of bsAb secretion. The next milestones will be the establishment of PDOTS and efficacy testing of bsAb systemically and as a CASS B cell payload. The final milestone will be the comparison of CASS B cell and CAR T cell therapies and the generation of immune profiles for each treatment via IHC and scRNAseq.

[0217] Objective 3: In vivo efficacy using HLA-matched humanized NSCLC mouse models - In vivo experiments will be performed on cell line-derived xenograft (CDX) models in humanized mice. The use of validated patient-derived xenograft (PDX) models from publicly available repositories for final testing will improve tumor integrity and phenotypic characterization while accurately mimicking the TME of in vivo tumors.

[0218] Experimental design and procedures Establishment of NSCLC CDX and PDX models in humanized mice: NSCLC cell lines and PDX models are screened for PDL1 and CD155 expression levels prior to luciferization 49 Human immune system reconstitution is performed as described by the present inventors and others. 50~52To generate growth curves, various concentrations of NSCLC cell lines / PDX tumors are implanted into the hind flanks of humanized mice. 53 .

[0219] Assessment of CASS B cell efficacy in humanized NSCLC mouse models: We will use CDX and PDX models to test the efficacy of bsAbs and CASS B cells in vivo. Samples will be analyzed by multi-parameter FACS, IHC / ISH, and scRNASeq following established protocols routinely used in our laboratory. 54~63 .

[0220] We have considerable experience in generating humanized mice and CDX / PDX models and successfully develop NSCLC models. These models demonstrate that CASS B cells cluster around tumors and secrete high levels of bsAb, reducing the immunosuppressive nature of the TME and recruiting additional anti-tumor immune cells. IHC / ISH and scRNAseq provide molecular evidence of bsAb efficacy, CASS B cell homing, and activation of the APC pathway in both CASS B cells and CD4 T cells, while 5'scRNAseq of TCR / BCR is used to directly monitor epitope spreading among TILs.

[0221] Statistical considerations: With 5 animals per group for individual pairwise comparisons between conditions and different outcome measures of interest, we have a power of 0.93 to detect differences in means equal to 2.5 SD using a two-sample two-tailed t-test at the 0.05 level. Additional statistical analyses will be provided in consultation with the Dana-Farber Biostatistics Core.

[0222] Milestones: The first milestone in Objective 3 is the generation of CDX and PDX models for NSCLC. Subsequent milestones will be the initiation and completion of planned animal studies testing bsAb in CDX models and CASS B cells in CDX and PDX models. Due to the wealth of data generated by these studies, the third milestone will be the completion of data analysis for each animal study.

[0223] References cited in this Example. TIFF2025508041000002.tif209147TIFF2025508041000003.tif209147TIFF2025508041000004.tif200146TIFF20255080410 00005.tif200147TIFF2025508041000006.tif218146TIFF2025508041000007.tif218146TIFF2025508041000008.tif146147

[0224] Example 3 Using CASS B cells and patient-derived tumor cells in vitro and in vivo to explore the B-CLL TME Chimeric antibody signaling and secreting (CASS) B cells are used to study the B CLL tumor microenvironment (TME) ex vivo in tumor-bearing mice. CASS B cells inducibly secrete checkpoint blockade modulator Abs (e.g., checkpoint inhibitor Abs) at the tumor site. Unlike CAR T cells and NK cells, CASS B cells are antigen-presenting cells (APCs) and promote neo-antigen recognition and spreading upon reversal of T cell exhaustion. Testing in tumor spheroids and tumor-bearing mice continues.

[0225] Over the past decade, immune checkpoint blockade inhibitor (CBI) antibodies and CAR T cells have revolutionized the way we treat cancer. Although both of these therapies engage the patient's immune system, neither is able to actively mount an antitumor immune response, presenting significant limitations in the scope of their use and efficacy. To address this, we developed chimeric antibody-secreting and signaling (CASS) B cells that express an engineered tumor-targeting B cell receptor (BCR) and, upon engagement, secrete high levels of CBI locally at the tumor site. As B cells also function as professional antigen-presenting cells, they can process and present antigens on MHC class II molecules, further enhancing immune cell recognition of tumors and aiding in neo-antigen spread. CASS B cells, a key component of immune memory, simultaneously recruit a broad range of immune cells, providing a robust, lifelong surveillance program that reverses tumor-infiltrating lymphocyte exhaustion and protects against tumor metastasis and recurrence.

[0226] B-CLL was chosen as a model to develop anti-IGHV1-69 directed CASS B cells secreting immunomodulatory CBIs. The finding that IGHV1-69 encoded BCR expressed on B-CLL cells is always a non-mutated VH segment provides an opportunity for anti-idiotypic CASS B cell therapy. We humanized G6 (hG6.3) mAb and completed co-crystallographic studies of (G6-id+)hG6.3 with its BCR target, demonstrating that hG6.3 binds to germline encoded residues in the VH complementarity determining region (CDR-H2). We also isolated several potent CBIs and bispecific Abs to be studied. In Objective 1, we will continue to develop B-CLL humanized mouse models and patient-derived organotypic spheroids (PDOTs) containing tumor and immune cells from these mice. We characterize the immune profiles of these tumors using scRNASeq, immune receptor profiling, CITESeq, and multiplex IHC to examine the tumor microenvironment (TME) for both IGHV1-69+ and IGHV1-69-B CLL. In objective 2, we treat PDOT with CASS B cells secreting different CBIs, then perform molecular profiling to determine the effect of different CBIs on TME immune signatures. These ex vivo studies provide a clear understanding of the link between CASS B cell activation and CBI secretion, while providing a critical analysis of CASS B cell efficacy compared to CAR T cells at both functional and molecular levels. In objective 3, we treat humanized mice bearing B-CLL tumors with CASS B cells that show the greatest recovery of anti-tumor immunity from objective 2. The above scRNASeq and other studies provide a detailed evaluation of the molecular and mechanistic efficacy of the CBI and CASS B cell platform as a whole. At the end of this stage, we may have molecular insight into the B CLL TME and CASS B cell intervention that can optimally restore anti-B CLL immunity.

[0227] Checkpoint blockade inhibitor (CBI) monoclonal antibodies (mAbs) and adoptive cell therapy have had a transformative effect on cancer therapy, shifting the focus from simply killing tumor cells to activating the patient's natural antitumor immunity to reverse the immunosuppressive tumor microenvironment (TME). While these therapies represent some of the most promising anticancer treatments to date, only a small proportion of patients experience a complete or durable response, and many experience immune-related adverse events (irAEs) of varying severity. An alternative approach is the development of armored or immune-restorative CAR T cells that are engineered to secrete immune-modulating payloads directly to the tumor site, improving efficacy while lowering the on-target / off-tumor side effects seen with systemic delivery. In addition to T cells, a variety of immune cells, including natural killer cells (NK-CARs) and macrophages (CAR-Ms), have been utilized to create novel CARs, and a common thread in all of these immune cell therapies is that they provide direct antitumor killing activity. B cells, a key component of humoral immunity, produce antibodies, and the field of immunotherapy was initially developed around them. However, they do not have inherent cytotoxic capabilities and therefore have been largely excluded from these advances.

[0228] The Chimeric Antibody Signaling and Secretion (CASS) B-cell platform is a novel and high-risk project as it seeks to advance B-cell research and facilitate its entry into 21st century therapy. We will develop CASS B-cell therapy to test and treat aggressive types of therapy-resistant IGHV1-69 derived B-cells. CASS B CLL is a unique B-cell-based cellular therapy that does not rely on direct cytotoxicity but utilizes two intrinsic capabilities of B cells: the ability to secrete high levels of CBI antibodies to reverse the immunosuppressive TME, and the ability to process and present antigens on MHC class II molecules, resulting in the recruitment of CD4+ T cells and allowing enhanced tumor cell recognition and neo-antigen spreading. While the inducible and targeted delivery of CBI reduces irAEs, the ability to function as professional antigen-presenting cells (APCs) makes the CASS B-cell platform unique in the cellular therapy arena, as CASS B cells have the ability to mount a potent anti-tumor response. MHC class II neo-antigens played a role in the innate anti-tumor response. The inventors will perform both ex vivo and in vivo studies on humanized mice bearing B-CLL patient leukemia cells treated with CASS B cells secreting the different CBI payloads that the inventors have discovered.

[0229] Example 4 The role of B cells in the immune system is to recognize foreign invaders, from microbes to cancer cells, and eliminate them by the production of antibodies (Abs) that bind and eliminate the threat. B cells achieve this by expressing membrane-bound Abs (B cell receptor BCR) that bind to tumor antigens, switching B cells from BCR-expressing cells to Ab-secreting cells. After BCR engagement, there is rapid tyrosine phosphorylation mediated by two major tyrosine kinases, Lyn and Syn, as well as calcium ion polarization. These biochemical events lead to activation of downstream signaling pathways, leading to further downstream activation of NF-κB and NFAT signaling, ultimately resulting in B cell expansion and potent mAb secretion. Without wishing to be bound by theory, we engineered chimeric antibody signaling-secreting (CASS) B cells that utilize these signaling pathways to induce clonal anti-tumor CASS B cell expansion and secretion of anti-PDL1 Abs at tumor sites that aid in the restoration of anti-tumor immunity.

[0230] Without wishing to be bound by theory, human B cells can be engineered to express artificial B cell receptors (aBCRs) against tumor-associated antigens (TAA), which allows the human B cells to migrate to solid tumors where they become activated following aBCR binding to the TAA. Additionally, once at the tumor site, CASS B cells can be further engineered to secrete anti-PDL1 Abs that, upon activation, act as checkpoint blockade inhibitors (CBIs) to reverse the immunosuppressive tumor microenvironment (TME).

[0231] In objective 1, we engineer B cells to express an IgG-type membrane-bound BCR against the TAA carbonic anhydrase IX (CAIX) expressed on the surface of renal clear cell carcinoma (ccRCC). For proof-of-principle studies, we use a second Ab signaling plasmid containing an NFAT or NF-kB response element to drive the secretion of anti-PDL1 Ab. Upon localization at the tumor site and binding of the BCR to CAIX, anti-PDL1 Ab secretion is induced via NFAT or NF-kB activation. This activation should result in high anti-PDL1 Ab concentrations at the tumor site. In objective 2, we use an orthotopic ccRCC mouse model to determine whether anti-CAIX CASS B cells can migrate to tumors and be activated by ccRCC to secrete anti-PDL1 Ab. Because CAIX is also expressed at lower levels and with a different cytoplasmic distribution compared to ccRCC, we also use this model to test the safety of this therapy. We engineered an anti-CAIX targeting moiety to preferentially recognize high density CAIX on tumor cells but not on healthy cholangiocytes.

[0232] Non-limiting examples of study design: For objective 1, we construct and transfer engineered anti-CAIX BCR into B cells using lentiviral transduction with expression driven from an internal spleen focus forming virus (SFFV) promoter. Lentiviral particles are pseudotyped with gibbon ape leukemia virus (GALV) or engineered baboon envelope glycoproteins to facilitate transduction. Separately, we design reporter plasmids that allow NFAT or NF-kB induction after BCR engagement to drive expression of GFP, and use the lead promoter to drive anti-PDL mAb secretion. B cells are transduced with both anti-CAIX BCR and NFAT / NF-kB inducible GFP lentiviral vectors, and soluble CAIX-Fc is used to crosslink the BCR. GFP expression is then measured to determine the optimal response element, and GFP is then replaced with anti-PDL1 mAb for secretion.

[0233] For objective 2, CASS B cells are constructed by transduction with tumor-specific anti-CAIX BCR and inducible anti-PDL1 mAb secreting lentiviral vectors and tested by mixing CASS B cells with CAIX+PDL1+ or PDL1-SKRC-59 ccRCC cells. In addition to measuring soluble anti-PDL1 secretion and binding to ccRCC cells, B cell activation is measured by FACS for activation markers. In vivo experiments in humanized PBL NSG-SGM3 mice bearing SKRC-59 tumors are used to evaluate efficacy and persistence of CASS B cells. Efficacy is tested by measuring secreted Ab and increased B cell population around the tumor site, as well as changes in tumor size. To measure persistence, CASS B cells are detected in both blood and TME. Finally, scRNAseq is used to analyze the effect of anti-PDL1 mAb on the regulation of TME.

[0234] Without wishing to be bound by theory, compared to systemic delivery, there is a local region of high anti-PDL1 antibody concentration centered on the tumor, resulting in improved anti-tumor outcomes and tumor elimination.

[0235] CASS B cells are a new concept in cell therapy that results in the restoration of local antitumor immunity in the TME by blocking the immunosuppressive PD-1 / PD(L)-1 axis. It is also a combination immunotherapy due to the ease of single administration and cost of single cell infusion.

[0236] Example 5 Engineering chimeric antibody signaling and secreting (CASS) B cells to achieve cancer cures Innovation: Cancer cells are a lifestyle that has learned to outwit the immune system for the advantage of their self-proliferation. They do this through impairment of cellular DNA repair mechanisms that result in upregulation of growth factors and their receptors resulting in uncontrolled tumor growth. This molecular hijacking also results in the surface expression and secretion of molecules involved in immune checkpoint blockade (ICB). Immunotherapy with anti-PD1 / PDL1 blockade represents a significant advance in the cancer field and is a frontline or standard treatment option for a variety of cancers, including non-small cell lung cancer, melanoma, colorectal cancer, and renal cell carcinoma (1-3). However, while the success of the treatment is well documented, it very often does not result in a cure for cancer.

[0237] Cell therapy is a method of harnessing the immune system to kill cancer cells. This is primarily achieved by T cell receptor (TCR) and chimeric antigen receptor (CAR) T cells that can home to and target cancer cells. Their success in treating solid tumors is improving, but remains limited by the fact that cancer cells can also dominate these T cells and disable them.

[0238] Described herein is a new cell therapy called chimeric antibody signaling and secreting (CASS) B cells that harnesses the B cells of the humoral immune system to do what they do best: secrete high levels of antibodies. These CASS B cells are engineered to recognize tumor-associated antigens (TAA) via engineered B cell receptors (BCRs), which upon engagement of the TAA activate the CASS B cells and induce the production of high levels of immunomodulatory bispecific antibodies (BsAbs) at the tumor site. This allows for the reversal of the immunosuppressive tumor microenvironment, as is the case when immune checkpoint blockade monoclonal antibodies (mAbs) are delivered systemically. However, the interesting feature here is that the BsAb secretion is conditionally expressed only upon CASS B cell engagement with the TAA, and is largely localized to the tumor site, although some low-level leakage to the periphery may still occur. Without wishing to be bound by theory, this is a step towards cell therapy where locally secreted monoclonal antibodies are dedicated to altering the tumor microenvironment and restoring local anti-tumor immunity.

[0239] Rationale: Monoclonal antibody (mAb) drugs that either directly kill cancer cells, act as immune checkpoint blockade modulators (e.g., inhibitors), or disrupt tumor vasculature are among the most promising anti-cancer therapeutics in development. However, the idea of ​​engineering human B cells to seek out cancer cells and secrete these mAbs in vivo at the tumor site is novel and untested, but may provide a powerful new way to treat both primary and metastatic tumors. It also provides a lifelong anti-tumor immune surveillance system to prevent cancer recurrence and achieve a "cure (CURES)". The primary role of B cells in the immune system is to recognize foreign invaders and eliminate them by the production of antibodies that bind to microbes or cancer cells and eliminate the threat. This is accomplished by expressing membrane-bound antibodies that act as B cell receptors (BCRs) that bind tumor antigens, switching B cells from BCR-expressing to antibody-secreting cells. Following BCR engagement, there is rapid tyrosine phosphorylation mediated by two major tyrosine kinases, Lyn and Syn, as well as calcium ion polarization. These biochemical events lead to the activation of downstream signaling pathways, leading to further downstream activation of NF-κB and NFAT signaling, ultimately resulting in B cell expansion and potent mAb secretion. (5) We engineer chimeric antibody signaling-secreting (CASS) B cells that exploit these signaling pathways to induce clonal CASS B cell expansion and secretion of immunomodulatory BsAbs at tumor sites.

[0240] Without wishing to be bound by theory, the development of the CASS B cell platform may provide therapeutic benefit to numerous clinical indications that are sensitive to checkpoint blockade modulators (e.g., inhibitors) or have an immunosuppressive microenvironment. Anti-PD1 / PDL1 therapy has had a transformative effect, particularly for the treatment of NSCLC, and has become the state-of-the-art therapy for many patients. However, this therapy has limitations in efficacy, durability, and scope of use. To combat these challenges, current trials are focusing on anti-PD1 / anti-TIGIT combination therapy, and Merck and Roche have recently published clinical trial data showing that this combination shows considerable promise (6-7). We use NSCLC as a model utilizing anti-PD1 / anti-TIGIT bispecific antibodies.

[0241] Objective: To use an engineered membrane-bound single-chain IgG BCR against mesothelin (MSLN) to target CASS B cells to NSCLC tumor sites. The plasmid also contains a second cassette driven by an aNFAT or NF-kB response element that drives secretion of an anti-TIGIT / anti-PD1 BsAb. Upon localization to the tumor site and BCR activation, BsAb expression is induced by native signaling pathways upon BCR engagement of MSLN on the NSCLC. Because this is an inducible expression system, there are localized regions of high antibody concentration at the tumor site, significantly reducing on-target, off-tumor effects.

[0242] Methods: CASS B cells are engineered in two parts. Without wishing to be bound by theory, we are able to transfer engineered BCRs into B cells. To efficiently transduce B cells, lentiviral particles are pseudotyped with gibbon leukemia virus (GALV) or engineered baboon envelope glycoproteins, and in the transfer vector, BCR expression is driven from the spleen focus forming virus (SFFV) or human elongation factor-1 alpha (EFlα) promoter (8). The second step is to design NFAT and / or NF-kB inducible expression cassettes for BsAb secretion (9). To determine which response element to use, a plasmid is engineered to express GFP. Raji cells and primary B cells are transduced with engineered anti-MSLN BCR and NFAT / NF-kB inducible GFP lentiviral vectors, and soluble biotinylated MSLN is added to the culture medium along with streptavidin to crosslink the BCR. GFP expression is then measured to determine the optimal response element. Preliminary work was carried out to construct an engineered membrane-bound IgG (memIgG) using anti-influenza antibodies. Figure 1 shows that our memIgG construct is expressed at high levels and is functionally active, as it can bind soluble HA.

[0243] Next, CASS B cells are constructed by lentiviral transduction with a vector encoding an inducible BsAb that replaces the tumor-specific anti-MSLN BCR and GFP. CASS B cell secretion of anti-TIGIT / PD1 BsAb is quantified by first inducing expression using soluble biotinylated MSLN+streptavidin, followed by co-incubation with MSLN+A549 NSCLC cells. Secondary experiments test for in vitro inhibition of exhaustion by co-culturing CASS B cells with CD3+T cells and A549 cells expressing various combinations of PDL1 and CD155 (ligand for TIGIT). In addition to measuring soluble antibody concentrations, cell activation / exhaustion is measured by FACS staining. These tumor cells are also stained for binding of CASS B cell-shedding anti-MSLN. Next, in vivo experiments are performed in humanized PBL NSG-SGM3 mice bearing A549 tumors to evaluate efficacy and persistence of CASS B cells. Efficacy is measured by local BsAb secretion by immunohistochemical staining, increased B cell population around the tumor site, and changes in tumor size. We also measure BsAb leakage to the periphery by testing serum secreted BsAb concentration to measure persistence. CASS B cells are detected in both peripheral blood and the tumor microenvironment (TME). Finally, scRNAseq is used to analyze the effect of BsAb in modulating the TME. Ideally, there will be a localized region of high BsAb concentration centered around the tumor periphery, resulting in improved outcome and tumor elimination, compared to systemic delivery. Impact: The goal of developing new cancer therapies should be aimed at achieving a "cure". Restoration of host anti-tumor immunity at the tumor site can be achieved by understanding immune elements that are common or "exposed" to all individuals. Once this is done, we must develop an internal immune surveillance system that will be permanently present to prevent cancer from recurring. We develop a new immune surveillance system using B cells of the immune system that have never been developed for this purpose. We determine the feasibility of engineering chimeric antibody signaling and secreting (CASS) B cells to target mesothelin (MSLN)-expressing NSCLC cells. These anti-MSLN CASS B cells home to the tumor site where they secrete high levels of bispecific anti-TIGIT / anti-PDL1 antibodies that act as dual checkpoint blockade inhibitors. This results in a dynamic shift in the tumor microenvironment that helps reverse T cell exhaustion and restore anti-tumor immunity. An important and practical point is that this combination cellular immunotherapy is one-time administration and will cost payers over the patient's lifetime.

[0244] References cited in this example TIFF2025508041000009.tif58146TIFF2025508041000010.tif244147

[0245] Example 6 CASS B cell culture and transduction protocol Day 0 1. Isolate B cells from fresh or frozen PBMCs using one of the following (from Stemcell): 19054 Easy Sep™ Human B Cell Enrichment Kit 19554 Easy Sep™ Human Pan-B Cell Enrichment Kit (including plasma cells) 2. Activate B cells approximately 18-24 hours and 3-5 days prior to transduction. a. Typically, in a 24-well plate, culture in 1 mL of medium containing 0.5-1E6 cells / mL Activation medium 1 (Milteyni 130-106-196 protocol was used, but with the addition of 1ug / mL ODN2006): Milteyni StemMACS HSC medium + 5% FBS + IL4 + multimerized CD40L + ODN2006 Activation medium 2 (based on Moffett et al DOI 10.1126 / sciimmunol.aax0644): IMDM + 10% FBS + CD40L-Fc (3.735ug / mL) + ODN2006 (1ug / mL) + IL2 (50ng / mL) + IL10 (50ng / mL) + IL15 (10ng / mL) Initially, Activation Medium 1 was used, but has since been changed to Activation Medium 2. Day 1 1. Thawing the virus 2. Add DEAE to each well to a final concentration of 10 μg / mL. 3. Add virus and mix with a pipette. 4. Centrifuge the virus at 1200 x g for 55 minutes at 37C. 5. Transfer the plate to an incubator and leave it overnight; there is no need to resuspend the cells. Day 2 1. Cells were collected in a 15mL tube and washed twice with PBS before being resuspended in fresh Activation Medium. a. Ideally about 1 mL, 1E6 cells / mL, but resuspend accordingly based on cell number and viability. b. Maintaining cells at 0.75-1E6 cells / mL is important for rapid expansion during this stage. 2. The cells are incubated for 48 hours to allow expression of the transduced genes and expansion of the cells before selection. Day 4 1. In the morning, use fresh or frozen irradiated 3T3-msCD40L feeder cells (HIV reagent #12535). a. Irradiate the cells with γ-78 Gy. 2. 8.75E5 cells / cm in DMEM+10%FBS 2 and allow cells to attach before selection (>4 hours). 3. Cells are harvested, washed with PBS and stained with Zombie Violet (live / dead exclusion dye from Biolegend). a. Follow manufacturer's protocol: 1:1000 dilution of dye, 100 μl diluted dye / 1E6 cells. 4. Block Fc receptors with human TruStain FcX (Biolegend 422302). 5. Stain cells with appropriate markers for selection. a.QBend10-PE, CD19-APC, and Zombie Violet use the BFP channel. 6. Aspirate medium from 6-well plates containing adherent 3T3-msCD40L feeder cells and replace with Expansion Medium (IMDM + 10% FBS + 5 μg / mL influenza virus + 50 μg / mL transferrin + 50 ng / mL IL2 + 10 ng / mL IL15 + 20 ng / mL IL21). 7. Sort cells on a Sony MA900 directly into 6-well plates containing feeder cells. 8. Culture the cells for 14 days or more, adding medium as needed. If culturing for more than 7 days, split onto fresh plates containing irradiated feeder cells on the 6th or 7th day.

[0246] Example 7 Exemplary constructs include: (1) EF1 alpha-F 105 leader-F10-memIgG 1-T2A-RQR 8-3x3NFAT-minIL2pro-ZsG (2) EF1 alpha-F105 leader-F10-memIgG1-T2A-RQR8-5xNFAT-minIL2pro-ZsG (3) EF1 alpha-F105 leader-F10-memIgG1-T2A-RQR8-NFAT-NFkB-ZsG, or (4) Any combination thereof.

[0247] EFl alpha promoter: TIFF2025508041000011.tif84135TIFF2025508041000012.tif9135

[0248] F105 leader sequence: TIFF2025508041000013.tif5128

[0249] F10 scFv: TIFF2025508041000014.tif23135

[0250] Fc domain (CH2 in bold, CH3 underlined) TIFF2025508041000015.tif19135

[0251] Transmembrane and intracellular domains: TIFF2025508041000016.tif5130

[0252] T2A: TIFF2025508041000017.tif5128

[0253] RQR8: TIFF2025508041000018.tif13135

[0254] ZsG: TIFF2025508041000019.tif22135

[0255] NFAT is a consensus sequence Binding to a 9 bp element having TIFF2025508041000020.tif6128, where N represents any base.

[0256] 3X NFAT RE: TIFF2025508041000021.tif22128

[0257] 5X NFAT RE: TIFF2025508041000022.tif5128

[0258] IL8 NFAT RE: TIFF2025508041000023.tif5128

[0259] minIL2 promoter: TIFF2025508041000024.tif14134

[0260] minIL8 promoter: TIFF2025508041000025.tif5128

[0261] WPRE: TIFF2025508041000026.tif49135

[0262] Example 8 Engineering chimeric antibody signaling and secreting (CASS) B cells to achieve cancer cures summary B cells are components of host immunity, and while other immune cells, including T cells, NK cells, and myeloid cells, have been engineered with chimeric antigen receptors (CARs), B cells have been neglected. The role of B cells in our immune system is to recognize foreign invaders through membrane-bound antibodies (B cell receptors, BCRs) and eliminate them by producing antibodies that bind and eliminate the threat. We can develop a chimeric antibody signaling and secretion (CASS) B cell platform by engineering both target binding domains and secreted antibodies to develop potent anti-cancer therapeutics. The target binding domains are designed to recognize tumor-associated antigens, and upon binding, lead to the secretion of bispecific checkpoint blockade inhibitory (CBI) antibodies to reverse the exhaustion of immune cells that have reached the tumor site. The second major function of B cells is to present foreign protein fragments to T cells, leading to their activation and further recruitment of various anti-tumor immune cells. Finally, B cells play a role in immune memory, and CASS B cells provide a lifelong anti-tumor surveillance network that protects patients from metastasis and recurrence after the initial tumor has been eliminated.

[0263] Although the CASS B cell platform is applicable to several cancers, we use non-small cell lung cancer (NSCLC) as an experimental model. NSCLC was chosen because it can express high levels of the tumor-associated antigen, mesothelin (MSLN), and because of the efficacy of combination therapy with anti-PD1 and anti-TIGIT antibodies seen in clinical trials. Aim 1 focuses on engineering CASS B cells by first constructing a synthetic B cell receptor (BCR) that recognizes MSLN. The cells are further engineered to express a bispecific antibody targeting PD1 and TIGIT under the control of a switch that is activated only upon MSLN binding at the tumor site. The second aim is to use tissue culture experiments to corroborate the success of our engineering and to demonstrate the ability of the bispecific antibody to restore anti-cancer immunity. Experiments in aim 3 are performed using mice with a human immune system to demonstrate the efficacy of the CASS B cell platform in vivo and to allow detailed molecular characterization of the anti-tumor immune response. Without wishing to be bound by theory, the development of the CASS B cell platform will support translational research for the development of new therapeutic platforms that can impact a wide range of clinical indications and have played a critical role in improving healthcare and saving lives.

[0264] scientific summary Immune checkpoint blockade inhibitors (CBI) and CAR T cells have revolutionized the way we treat cancer. Both of these therapies engage the patient's immune system, with CBI therapy inhibiting immunosuppressive signals generated by the tumor microenvironment, and activated CAR T cells engaging bystander cells through the release of stimulatory and proinflammatory cytokines, but neither is able to actively participate in and initiate antitumor immune responses. To address this, we can develop chimeric antibody-secreting and signaling (CASS) B cells that express engineered tumor-associated antigens that target the BCR and, upon engagement, secrete high levels of bispecific antibodies (bsAbs) locally at the tumor site that act as CBIs. As B cells are also professional antigen-presenting cells (APCs), they process and present additional tumor antigens on MHC class II molecules, further enhancing tumor cell recognition and aiding neoantigen spreading. As components of immune memory, CASS B cells provide a lifelong surveillance program that protects against tumor metastasis and recurrence. Driven by the recruitment of a broad range of immune cells to the tumor site and the reversal of the immunosuppressive tumor microenvironment that allows these cells to function, CASS B cell therapy provides a robust and durable anti-cancer therapy.

[0265] Mesothelin (MSLN) is a tumor-associated antigen due to its limited expression on healthy tissues, and numerous biologic therapies have been devised to target this marker, including CAR T and recombinant antibodies. Non-small cell lung cancer (NSCLC) has been shown to upregulate MSLN expression, and data from clinical trials have demonstrated the efficacy of anti-PD1 / anti-TIGIT antibody combination therapy. Based on this, we selected NSCLC as a model to develop and test the efficacy of anti-MSLN-directed CASS B cells secreting immunomodulatory anti-PD1 / TIGIT bsAbs. Aim 1 focuses on the development of a CASS B cell platform and the engineering of the BCR to recognize mesothelin (MSLN). Additional engineering will be performed to use the native BCR signaling pathway to develop an inducible system such that MSLN engagement at the tumor site results in high levels of bsAb secretion. Aim 2 will test the in vitro efficacy of CASS B cells and perform characterization of activation thresholds and resulting secretion levels to allow fine-tuning of CASS B cell signaling and secretion. In objective 3, we will perform in vivo experiments using both aNSCLC cell line models and patient-derived xenograft models in mice reconstituted with the human immune system. Multiparameter flow cytometry (FACS), single-cell RNA sequencing, and immunohistochemistry will provide a detailed assessment of the molecular and mechanistic efficacy of the immunomodulatory bsAb and the entire CASS B cell platform.

[0266] Without wishing to be bound by theory, the development of the CASS B-cell platform supports innovative and translational research for the development of new therapeutic platforms that can impact a wide range of clinical indications. Although this example focuses on NSCLC, this research program will enable our team to further refine this new translational approach and extend it beyond cancer treatment. The development of the CASS B-cell platform has potential therapeutic benefit not only in cancer treatment, but also in numerous other clinical indications, including autoimmune / rheumatic and cardiovascular diseases and neurological disorders, where mAb therapy has played a crucial role in improving health care and saving lives.

[0267] Non-limiting unique and innovative aspects The FDA approved the first biologic (Humulin) in 1982, followed by the first monoclonal antibody therapy (Muromonab-CD3) in 1986, and in the years since then, we have seen a proliferation of new biologics. Since the initial success of anti-PD1 and CTLA4 therapy, the race has been on to see how to develop antibodies against the next generation of checkpoint molecules. As researchers began to identify new biomarkers, it became clear that it was difficult to find receptors comparable to PD1 and CTLA4, and that these alone would not be sufficient for most patients. Thus, the field turned to combination therapies that combine anti-PD(L)1 therapy with a wide range of other compounds, both standard of care and experimental. These antibody-based therapies rely on restoring the antitumor activity of the patient's immune system, but different cancers command diverse checkpoint blockade pathways to evade the immune system, and not all cancers respond to the same immunotherapies. The next breakthrough came with the development of CAR T cell therapy, where patient T cells are ablated and engineered to recognize tumor-associated antigens, allowing robust targeting and cytotoxic activity. CAR T activity indirectly activates other immune cells via cytokine and chemokine release, but the significant cytotoxic effect comes from the CAR T cells. Furthermore, because CAR T cells are "living drugs," they expand and become part of the patient's immune system, providing long-term protection not achieved with monoclonal antibody therapy.

[0268] Chimeric antibody-secreting and signaling (CASS) B cells are a new form of cell therapy that uses engineered B cells to secrete high levels of checkpoint blockade modulator antibodies (e.g., checkpoint inhibitor antibodies). One of the challenges with systemic delivery of CBI antibody therapy is the broad target distribution and the role these receptors play in immune homeostasis, potentially resulting in immune-related adverse events (irAEs) of various severity, including colitis, dermatitis, myocarditis, encephalitis, or peripheral neuropathy. To overcome this challenge, targeted delivery of CBI therapy has been explored by several groups, in which CAR T cells are engineered to secrete therapeutic payloads at the tumor site, increasing local concentrations relative to serum concentrations. The unique aspect here is that by utilizing the native BCR signaling pathway, engineered IgG-BCRs are designed to selectively induce high levels of CBI bsAb expression only upon activation by the target antigen, generating pockets of high bsAb concentration and reversal of immune suppression around the tumor. The use of bsAbs is also an innovative solution to improve the efficacy seen with single and combination antibody therapies, since potential synergistic activity can be obtained by tethering the binding domains together. Similar to CAR T cells, CASS B cells function as living drugs and become a permanent part of the patient's immune system, providing lifelong immune surveillance and protection from metastasis and recurrence.

[0269] Although CASS B cells do not provide the direct cytotoxic effects seen by CAR T cells, upon stimulation, the B cells process and present antigens on MHC class II molecules, recruiting CD4+ T cells and allowing enhanced tumor cell recognition and neo-antigen spreading. The antigen presentation capabilities of the CASS B cell platform make it unique in the cell therapy arena. Similar to antibody-based therapies, CASS B cells require activation of the patient's immune system, but as professional antigen-presenting cells, CASS B cells have the ability to mount a potent anti-tumor response by engaging a broad set of immune cells. Another important role of B cells in the immune system is that memory B cells are a critical complement to immune memory and can rapidly produce large amounts of antibodies upon restimulation. This provides a means for lifelong surveillance of tumor metastasis and recurrence.

[0270] Building on the Marasco Lab's expertise in antibody engineering and CAR T cell development, the CASS B cell platform could be a new research pathway. Our research has provided extensive experience in discovering and engineering therapeutic-quality antibodies against a diverse list of targets, including viral and tumor-associated antigens as well as multiple immune checkpoint markers. 7~11 Furthermore, we have used these antibodies to develop a series of bispecific and multispecific antibody constructs using knob-in-hole, IgG fusions, and tandem scFvs. The development of CAR T cells and our CAR T cell factories has provided our lab's first perspective into cell therapy and the targeted delivery of immune-modulating antibodies to tumor sites. 2 These CAR T programs have provided the laboratory with valuable experience and pipeline development for in vitro and in vivo evaluation and analysis of biologic therapies. The skills and techniques gained from these past lines of work provide the foundation that will enable our laboratory to pursue new avenues of investigation into CASS B cell development.

[0271] Since the first generation of CAR T cells were developed in the early 1990s, significant improvements in efficacy, durability, and safety have led to the creation of the second and third generation CAR T cells used today. Other immune cells, including natural killer cells (NK-CAR) and macrophages (CAR-M), have been harnessed to generate new CARs, and one consistent feature of all of these cells is that they provide direct antitumor activity. 12、13 Although B cells are a key component of humoral immunity, producing antibodies, and the field of immunotherapy was initially developed around them, they have no inherent cytotoxicity capabilities and have therefore been largely excluded from these advances. The CASS B cell platform seeks to bring B cell research into the 21st century by developing unique B cell-based cellular therapies that do not rely on direct cytotoxicity but instead utilize the antibody secretion function of B cells to reverse the immunosuppressive environment, combined with their antigen-presenting function to activate and recruit additional immune effector cells to eliminate tumors.

[0272] Although this example focuses on the use of CASS B cells for the treatment of NSCLC, the CASS B cells described in this proposal are therapeutically applicable to a number of MSLN+ cancers with immunosuppressive properties, including ovarian and pancreatic cancers. Due to their modular design, CASS B cells can be easily directed to other tumor-associated antigens and the secreted payload can be tailored to target relevant immune axes, allowing the adaptation of CASS B cells to a wide variety of cancers. Because B cells are long-lived and an important part of immune memory, CASS B cells continuously deliver therapeutic payloads to tumor sites and provide a lifelong immune surveillance system against metastasis and recurrence after the primary tumor has been eradicated. The long-term persistence and inducibility nature of the CASS B cell platform can be easily adapted to other diseases treated with biological therapies (e.g., cardiovascular and autoimmune diseases as well as neurological disorders), which typically require long-term disease maintenance and rapid administration of therapeutic agents to treat acute symptoms. Thus, the development of the CASS B cell platform can benefit not only cancer research but also a diverse range of clinical indications.

[0273] Example 9 Chimeric antibody signaling and secreting (CASS) B cells -1. Constitutive expression of α tumor antigen-synthesizing BCR -2. Inducible expression of immune-modulating payloads -Checkpoint blockade therapy (α-PD(L)1, α-PD1 / TIGIT) -T cell engagers (α-CD3xMSLN, α-CD3xCD28xMSLN) -Cytokine fusion (α-PD1-scIL12) B Cell Basics: B Cell Isolation: Stemcell EasySep™ Human Pan-B Cell Enrichment Kit (19554) Stimulation medium: IMDM + 10% HI FBS + CD40L-Fc (3.735 ug / mL) + ODN2006 (1 ug / mL) + IL2 (50 ng / mL) + IL10 (50 ng / mL) + IL15 (10 ng / mL) Long-term expansion medium: IMDM + 10% FBS + heparin (5ug / mL) + transferrin (50ug / mL) + IL2 (50ng / mL) + IL21 (20ng / mL) + IL15 (10ng / mL) Transduction check after BGH removal B cells will be isolated and activated on November 11, 2021. The trait will be introduced on November 12, 2021. It will be dyed on November 19, 2021. The virus was pseudotyped with VSVG and BGH was removed. B cells were transduced 18–24 h after activation. However, they were negative 7 days after transduction. Transduction test using our vector vector: LeGO-hEuMAR-GFP pHAGE-F10-RQR8 pHAGE-F10-RQR8 NFAT / NFkB B cell stimulation (4 days): Culture medium: IMDM + 10% HI FBS + CD40L-Fc (3.735 ug / mL) + ODN2006 (1 ug / mL) + IL2 (50 ng / mL) + IL10 (50 ng / mL) + IL15 (10 ng / mL) B cell transduction: using GALV and BaEV envelopes without retronectin.

[0274] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific substances and procedures specifically described herein, which equivalents are considered to be within the scope of this invention and are encompassed by the following claims.

Claims

1. A genetically modified B cell that expresses and carries a chimeric B cell receptor on its surface and further expresses and secretes an antibody, wherein the antibody is specific to CA-9, PD-1, PD-L1, PD-L2, CTLA4, TIGIT, VISTA, CD70, TIM-3, LAG-3, CD40L, CCR4, GITR, or CXCR4.

2. (a) The chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain, or (b) The extracellular domain is optionally an antibody or antibody fragment. (i) The antibody is a nanobody, scFv, bispecific antibody, or Fab, (ii) The antibody is specific to a tumor-associated antigen, optionally the tumor-associated antigen is selected from the group consisting of CAIX, BCMA, CD138, PD-L1, PD-L2, VEGF, CD70, CD99, CEA, Her-2, GD2, CD171, αFR, PMSA, IL13α, MSLN, TAG-72, and TROP2, or (iii) The antibody is an anti-IGHV1-69 antibody, or (iv) The antibody is specific to an infection-related antigen, optionally the infection is a viral disease, or optionally the infection includes influenza, coronavirus, HIV, or tuberculosis, or optionally the infection-related antigen includes HA1, HA2, NA, or spike protein, (c) The expression of the antibody is controlled by an inductive response element, optionally the inductive response element being NFAT or NF Ｋ B is a response element, or (d) The antibody is a checkpoint blocking modulator, optionally the antibody is a checkpoint blocking inhibitor, or (e) The antibody is specific to HA1, HA2, NA, or the spike protein, or (f) The antibody includes a monoclonal antibody, or (g) The antibody comprises a nanobody, scFv, Fab, antibody-cytokine fusion protein, or a bispecific antibody, (h) The antibody may include a humanized antibody, the antibody-cytokine fusion protein may include anti-PD1-scIL12, or the bispecific antibody may be specific to PD-1 and CTLA4, PD-1 and TIGIT, TIGIT and CCR4, GITR and TIGIT, or PD-1 and CCR4. Genetically modified B cells according to claim 1.

3. A nucleic acid encoding a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a chimeric B cell receptor, the chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain, and the second polypeptide comprises an antibody, the antibody being specific to CA-9, PD-1, PD-L1, PD-L2, CTLA4, TIGIT, VISTA, CD70, TIM-3, LAG-3, CD40L, CCR4, GITR, or CXCR4.

4. (a) The extracellular domain is optionally an antibody, (i) The antibody is a nanobody, scFv, bispecific antibody, or Fab, (ii) The antibody is specific to a tumor-associated antigen, optionally the tumor-associated antigen is selected from the group consisting of CAIX, BCMA, CD138, PD-L1, PD-L2, VEGF, CD70, CD99, CEA, Her-2, GD2, CD171, αFR, PMSA, IL13α, MSLN, TAG-72, and TROP2, or (iii) The antibody is an anti-IGHV1-69 antibody, or (iv) The antibody is specific to an infection-related antigen, optionally the infection is a viral disease, or optionally the infection includes influenza, coronavirus, HIV, or tuberculosis, or optionally the infection-related antigen includes HA1, HA2, NA, or spike protein, (b) The antibody comprises a monoclonal antibody, or (c) The antibody comprises a nanobody, scFv, antibody-cytokine fusion, or bispecific antibody, optionally the antibody-cytokine fusion comprises anti-PD1-scIL12, or optionally the bispecific antibody is specific to PD-1 and CTLA4, PD-1 and TIGIT, TIGIT and CCR4, GITR and TIGIT, or PD-1 and CCR4. (d) The antibody includes a humanized antibody, The nucleic acid according to claim 3.

5. A vector comprising the nucleic acid described in claim 4, wherein the vector is optionally a lentiviral vector or an adeno-associated virus vector.

6. A cell comprising the vector according to claim 5.

7. A composition comprising a first expression vector and a second expression vector, wherein the first expression vector comprises a nucleotide sequence encoding a chimeric B cell receptor, and the second expression vector comprises a nucleotide sequence encoding an antibody, wherein the antibody is specific to CA-9, PD-1, PD-L1, PD-L2, CTLA4, TIGIT, VISTA, CD70, TIM-3, LAG-3, CD40L, CCR4, GITR, or CXCR4.

8. (a) a nucleotide sequence encoding an inducible response element is operably linked to a nucleotide sequence encoding an antibody, optionally the inducible response element is NFAT or NF Ｋ B is a response element, or (b) The chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain, optionally the extracellular domain being an antibody, (i) The antibody is a nanobody, scFv, bispecific antibody, or Fab, (ii) The antibody is specific to a tumor-associated antigen, optionally the tumor-associated antigen is selected from the group consisting of CAIX, BCMA, CD138, PD-L1, PD-L2, VEGF, CD70, CD99, CEA, Her-2, GD2, CD171, αFR, PMSA, IL13α, MSLN, TAG-72, and TROP2, or (iii) The antibody is an anti-IGHV1-69 antibody, or (iv) The antibody is specific to an infection-related antigen, optionally the infection is a viral disease, or optionally the infection includes influenza, coronavirus, HIV, or tuberculosis, or optionally the infection-related antigen includes HA1, HA2, NA, or spike protein, (c) The antibody is a checkpoint block modulator, optionally the checkpoint block modulator includes a checkpoint block inhibitor, or (d) The antibody includes a monoclonal antibody, or (e) The antibody comprises a nanobody, scFv, Fab, antibody-cytokine fusion, or bispecific antibody, optionally the antibody-cytokine fusion comprises anti-PD1-scIL12, or optionally the bispecific antibody is specific to PD-1 and CTLA4, PD-1 and TIGIT, TIGIT and CCR4, GITR and TIGIT, or PD-1 and CCR4. (f) The antibody includes a humanized antibody, or (g) The first expression vector and the second expression vector are lentiviral vectors. The composition according to claim 7.

9. A method for creating a population of genetically modified B cells, Prepared from a population of B cells in a sample isolated from the target, Transduction of the vector described in claim 5 into the aforementioned population of B cells is performed to produce a population of genetically modified B cells. The method, including the method described above.

10. (a) A step of activating the population of B cells before transduction, or (b) A step of culturing the population of genetically modified B cells, The method according to claim 9, further comprising:

11. A pharmaceutical composition for use in the treatment or prevention of cancer or infection in a subject, comprising genetically modified B cells according to claim 1 or 2, nucleic acids according to claim 3 or 4, or a first expression vector and a second expression vector as defined in claim 7 or 8.

12. The pharmaceutical composition for use according to claim 11, wherein the cancer is BCLL, NSCLC, ccRCC, or mesothelioma, or the infection is a viral disease, optionally comprising influenza, coronavirus, HIV, or tuberculosis.

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