Genetically engineered b cells and methods of use thereof

Abstract

The present invention relates to genetically engineered B cells wherein the B cells express and bear chimeric B cell receptors on their surface, and wherein the genetically engineered B cells further express and secrete antibodies or cytokines.

Description

[0001] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications are hereby incorporated by reference in their entirety into this application in order to more fully describe the state of the art as 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 files and records, but otherwise reserves any and all copyrights whatsoever.

[0003] Related Applications

[0004] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 318,317, filed on March 9, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0005] The present invention relates to genetically engineered B cells, wherein the B cells express and carry on their surface a chimeric B cell receptor (cBCR), and wherein the genetically engineered B cells further express and secrete antibodies or cytokines. Background Art

[0006] Over the past decade, immune checkpoint blockade inhibitors (CBIs) and CAR-T cells have revolutionized the way we treat cancer. While both of these therapies involve the patient's immune system, neither actively initiates an anti-tumor immune response. Summary of the invention

[0007] Aspects of the invention relate to genetically engineered B cells.

[0008] In embodiments, the genetically engineered B cell expresses and carries on its surface a chimeric B cell receptor (cBCR), and wherein the genetically engineered B cell further expresses and secretes an antibody or a cytokine.

[0009] In embodiments, the chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

[0010] In embodiments, the extracellular domain is an antibody or an antibody fragment. In embodiments, the antibody is a nanobody, scFv, bispecific or Fab. In embodiments, the antibody is specific for tumor-associated antigens. For example, the tumor-associated antigen is selected from the group consisting of the following items: 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 embodiments, the antibody is an anti-IGHV 1-69 antibody. In embodiments, the antibody is specific for infectious disease-associated antigens, such as HA1, HA2, NA or spike proteins. For example, infectious diseases are viral diseases, such as influenza, coronavirus, HIV or tuberculosis.

[0011] In an embodiment, the expression of the antibody or the cytokine is controlled by an inducible response element. For example, the inducible response element is NFAT or NF k B response element. In embodiments, the antibody is a checkpoint blocking regulator. In embodiments, the antibody is a checkpoint blocking 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 the following items: 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 a 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. For example, the antibody-cytokine fusion protein comprises anti-PD1-scIL12.

[0012] Aspects of the invention relate to a nucleic acid encoding a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a chimeric B-cell receptor, wherein the chimeric B-cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain, and wherein the second polypeptide comprises an antibody or a cytokine.

[0013] In embodiments, the extracellular domain is an antibody or an antibody fragment. In embodiments, the antibody is a nanobody, scFv, bispecific or Fab. In embodiments, the antibody is specific for tumor-associated antigens. For example, the tumor-associated antigen is selected from the group consisting of the following items: 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 embodiments, the antibody is an anti-IGHV 1-69 antibody. In embodiments, the antibody is specific for infectious disease-associated antigens, such as HA1, HA2, NA or spike proteins. For example, infectious diseases are viral diseases, such as influenza, coronavirus, HIV or tuberculosis.

[0014] In an embodiment, the expression of the antibody or the cytokine is controlled by an inducible response element. For example, the inducible response element is NFAT or NF K B response element. In embodiments, the antibody is a checkpoint blocking regulator. 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 the following items: 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 a 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.

[0015] Aspects of the invention further relate to a vector comprising a nucleic acid as described herein. In embodiments, the vector is a lentiviral vector or an adeno-associated virus (AAV) vector. Lentivirus and AAV are viruses that can be used as gene therapy vectors, such as CRISPR / Cas systems.

[0016] Still further aspects of the present invention relate to a cell comprising a vector as described herein.

[0017] Furthermore, aspects of the invention relate 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 wherein 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 vectors or adeno-associated viral vectors.

[0018] In an embodiment, the nucleotide sequence encoding the inducible response element is operably linked to the nucleotide sequence encoding the antibody or cytokine. For example, the inducible response element is a NFAT response element or a NF K B response element.

[0019] 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 tumor-associated antigens. 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, aFR, PMSA, IL13α, MSLN, TAG-72, and TROP2. In embodiments, the antibody is an anti-IGHV 1-69 antibody. In embodiments, the antibody is specific for infectious disease-associated antigens, such as HA1, HA2, NA, or spike proteins. For example, infectious diseases are viral diseases, such as influenza, coronavirus, HIV, or tuberculosis.

[0020] In an embodiment, the expression of the antibody or the cytokine is controlled by an inducible response element. For example, the inducible response element is NFAT or NF KB response element. In embodiments, the antibody is a checkpoint blocking regulator. In embodiments, the antibody is a checkpoint blocking 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 the following items: 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 a 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. For example, the antibody-cytokine fusion comprises anti-PD1-scIL12.

[0021] Aspects of the present invention also relate to a method for preparing a genetically engineered B cell population. In an embodiment, the method comprises: isolating a B cell population from a subject, and transducing the B cell population with a vector as described herein, thereby producing a genetically engineered B cell population. An embodiment may also include a step of activating the B cell population before transduction. An embodiment may also include a step of culturing the genetically engineered B cell population. An embodiment may also include a step of administering the genetically engineered B cell population to a subject in need.

[0022] Additionally, aspects of the invention relate to methods of treating a subject having cancer by administering to the subject a genetically engineered B cell as described herein, a nucleic acid as described herein, or a composition as described herein.

[0023] Further, aspects of the invention relate to a method of preventing cancer in a subject by administering to the subject a genetically engineered B cell as described herein, a nucleic acid as described herein, or a composition as described herein. For example, the cancer is BCLL, NSCLC, ccRCC, mesothelioma.

[0024] Aspects of the invention relate to methods of treating a subject suffering from an infectious disease by administering to the subject a genetically engineered B cell as described herein, a nucleic acid as described herein, or a composition as described herein.

[0025] Further, aspects of the invention relate to methods for preventing infectious diseases by administering to a subject a genetically engineered B cell as described herein, a nucleic acid as described herein, or a composition as described herein. For example, the infectious disease is a viral disease, such as influenza, coronavirus, HIV, or tuberculosis.

[0026] Other objects and advantages of the present invention will become apparent from the ensuing description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic diagram of CASS B cell therapy for non-small cell lung cancer is shown. In this example, anti-MSLN is the targeting moiety and the anti-TIGIT / anti-PD1 bispecific antibody is the secretion moiety.

[0028] Figure 2 Characterization of exemplary antibodies for CASS B cell development is shown. Panel A) Anti-PD1 mAb is comparable to pembrolizumab in MLR assay. Panel B) BLI assay demonstrates that the antibody blocks TIGIT / CD155 binding interaction (purple) and shares epitopes with control Ab (blue). Panel C) MSLN+ cell binding curves (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.

[0029] Figure 3 The design of bispecific antibodies (bsAbs) is shown. Figure A) Schematic diagram of bsAbs, with different scFvs in red or blue. Schematic diagram of the experimental layout (Figure B) and the resulting binding curves (Figure C) of the dual binding assays demonstrating the functionality of the two sides of the bsAb.

[0030] Figure 4 CASS B cell construct testing and B cell transduction are shown. Panel A) Cells expressing engineered IgG-BCR constructs can bind soluble HA. Panel B) Lentivirus can be used to achieve high transduction efficiencies using multiple DNA constructs and donors. Panel C) A reporter gene system has been engineered in Jurkat cells using NFAT / NFkB inducible response elements that shows increased GFP expression levels under various stimulation conditions

[0031] Figure 5A summary of data on the hG6.3 targeting moiety, biological studies, and CASS B cell design and strategy is shown: (Figure A) hG6.3 core binding idiotype to D80, IGHV1-69 mAb, which contains CDR-H2 residues M53 to N58 (green), bound to Hg 6.3 (blue) in the crystal structure. (Figure B) Preferential recognition of unmutated IGHV1-69B-CLL patient samples by hG6.3-CAR T cells (G36 is an anti-CAIX control); (Figure C) Schematic diagram of CASS vectors and different CBI payloads and bispecific Ab derivatives, (Figure D) Schematic diagram of CASS B cell therapy. B cells were recovered by leukapheresis, transduced to express chimeric BCRs targeting cancer cells, and encoded to secrete optimized CBI payloads, as determined by in vitro PDOTS and in vivo humanized mouse studies.

[0032] Figure 6 Membrane expression (Panel A) and HA binding (Panel B) by 293T cells are shown.

[0033] Figure 7 Exemplary combinatorial constructs of embodiments of the invention are provided.

[0034] Figure 8 Exemplary split vector constructs of embodiments of the invention are provided.

[0035] Fig. 9 A schematic diagram of current immunotherapy is shown. Figure A is adapted from Shifaa M. Abdin et al. J Immunother Cancer 2021;9:e002741. Figure B is adapted from Larson, RC, Maus, MV Nat Rev Cancer 21, 2021. Figure C is adapted from Zhang, C., Hu, Y, Xiao, W. et al. Cell Mol Immunol 2021.

[0036] Fig.10 A diagram showing the function of B cells in the immune system adapted from Li Rui et al., Frontiers in Immunology, 2016 is shown.

[0037] Fig.11 A diagram of the B-cell signaling pathway adapted from Balaji, S., Ahmed, M., Lorence, E. et al., J Hematol Oncol, 2018 is shown.

[0038] Fig.12 A schematic representation of Chimeric Antibody Signaling and Secretion (CASS) B cells is shown.

[0039] Fig.13 Shown is a diagram of the first round of B cell transduction adapted from Howell Moffett et al., Science Immunology, 2019. Culture medium: StemMACS HSC Expansion Medium XF + 5% FBS + IL4 + CD40L-hex + ODN2006.

[0040] Fig.14 A schematic representation of the design of Chimeric Antibody Signaling and Secretion (CASS) B cells is shown.

[0041] Fig.15 A schematic representation of the construct is shown.

[0042] Fig.16 Representative data of the first test of B cell transduction are shown. The construct used in this experiment contained an internal BGH domain, which resulted in premature termination of the construct and failure to incorporate via the LTR. Thus, high gene expression was observed immediately after transduction, however within 2 weeks expression had dropped to near zero. This indicates a lack of gene integration, but rather expression via transient "transfection" of the lentivirus.

[0043] Fig.17 A schematic diagram of the redesigned CASS B construct is shown. Fig.16 In the results presented in Figure 2, the BGH polyA was removed to allow stable genomic integration after transduction.

[0044] Fig.18 Representative data from transduction examinations after BGH removal are shown. Activated B cells were isolated: 11 / 11 / 21; transduced: 11 / 12 / 21; and stained: 11 / 19 / 21. Viruses were pseudotyped with VSVG and BGH was removed. B cells were transduced 18-24 hours after activation. However, they were negative 7 days after transduction.

[0045] Fig.19 Shown are representative graphs of data illustrating the duration and manner in which B cell stimulation exerts its effects on transduction. Adapted from Bovia, Fabrice et al., Gene Therapy 2013.

[0046] Fig. 20Representative 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). Figure A is adapted from Caeser, R., Di Re, M., Krupka, JA, et al., Nat Commun 2019. Figure B is adapted from Levy C, et al., J Thromb Haemost 2016.

[0047] Fig.21 Schematic and representative data of transfer plasmids optimized for B cell transduction are shown.

[0048] Fig. 22 Schematic diagrams of two transduction protocols are shown. The Bovia protocol utilizes EL4-B5 feeder cells, while the Moffett protocol utilizes soluble CD40L. Moffett: 1, HF Moffett, C, K. Harms, K. S Fitzpatrick, MR Tooley, J. Boonyaratanakornkit, J. J Taylor, Engineering B cells to express pathogen-specific antibodies protects against infection. Sci. Immuno 1.4 (2019), doi: 10.1126 / sciimmuno1.aax0644, 31101673. Bovia: 1, F. Bovia, P. Salmon, T. Matthes, K. Kvell, T. Nguyen, C, Werner-Favre, M. Barnet, M. Nagy, F. Leuba, J. F. Arrighi, V. Piguet, D. Trono, R. H. Zubler, Efficient transduction of primary human B lymphocytes and non-dividing myeloma B cells with HIV-1-derived lentiviral vectors. Blood. 101, 1727-1733 (2003), 12406892.

[0049] Fig.23Representative data from transduction examinations after 9 days are shown. CD19 was used to isolate B cells from EL4-B5, and the viruses were pseudotyped using 3 different envelope proteins (VSVG, BaEV, GALV). For cells activated via the Bovia protocol, it can be seen that high transduction titers were observed in EL4-B5 feeder cells (CD19-), but not in human B cells (CD19+). Following the protocol employed by Moffett, high levels of GFP expression were observed in CD 19+ B cells, indicating that efficient transduction was performed using BaEV pseudotyped viruses rather than GALV or VSVG. RetroNectin reagent is a recombinant human fibronectin fragment sold by TakaraBio. Without being bound by theory, it enhances viral transduction by binding of viral particles to the heparin binding domain and to the cellular integrins VLA-4 and / or VLA-5 via the C domain (https: / / www.takarabiocom / leaming-centers / gene-function / t-cell-transduction-and-culture / retronectin-faqs). However, in this assay we did not see a benefit from using RetroNectin for transduction.

[0050] Fig.24 Representative data from day 4 transduction assays are shown. Cells were transduced with BaEV pseudotyped lentivirus. The first column used the LeGO GFP vector that 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 only the extracellular binding domain of the CASS B cells. Importantly, the CASS B cell vector shows binding of QBend10 (epitope marker via RQR8) and expression of GFP.

[0051] Fig.25 Representative data for HA binding to F10-CASS B cells are shown. 250k cells were washed with PBS and resuspended in 5uM Zombie Violet dye (in PBS) for 20 minutes at room temperature. The cells were quenched with FBS-containing culture medium and then resuspended in 100ul of a solution containing FcX and 4ug / ml biotinylated H1 stem. After incubation at 4°C for 1 hour, the cells were washed and stained with QBend10-FITC and APC-streptavidin. Importantly, almost all QBend10+ cells also bind HA, indicating that the transduced cells express functional chimeric BCRs and epitope tags separated by T2A.

[0052] Fig.26The representative data of the inducible expression of GFP are shown. The B cells of transduction are activated for 4 days, and then the inducible GFP expression is measured 8 days after transduction. As seen here, compared with only using culture medium, the GFP expression of NFAT-NFkB construct does not change with additional BCR stimulation (anti-IgM or HA+ streptococcus). 3x3NFAT constructs do not show any change of GFP expression under any conditions.

[0053] Fig. 27 Schematic diagram of representative payload constructs constructed is shown. ZsG is used for platform optimization, anti-PD(L)1 will be an anti-cancer therapy, and aSARS will be an unrelated control in cancer therapy or an anti-SARS therapy in infectious disease therapy.

[0054] Fig.28 Representative data for secretion of scFv-Fcs by CASS B cells are shown. B cells were seeded in stim medium at a concentration of 1E6 cells / ml on 5 / 24 / 22 and harvested on 5 / 28 / 22. High levels of IgG expression were observed in all wells, however, antigen-specific scFv-Fcs against PD1 P4B3m3 were detected. Based on the ELISA results of the purified protein, this may be due to the low affinity of mAb 42 (aPDL1) and the wrong incorporation target selected for 11A. 11A targets GD03SARS, but a different strain was used for the ELISA. DETAILED DESCRIPTION

[0055] Described herein are compositions and methods related to chimeric antibody signaling and secretion (CASS) B cells, which secrete immunomodulatory polypeptides at target sites as a targeted and inducible platform. For example, CASS B cells can express an engineered tumor-targeted B cell receptor on their surface, and once engaged, can locally secrete high levels of dual-targeted bispecific checkpoint blockade regulator antibodies at the tumor site. Since B cells also act as professional antigen-presenting cells, CASS B cells can also process and present antigens on class II molecules, further enhancing immune cell recognition of tumors and helping new antigens spread. As a key component of immune memory, CASS B cells can simultaneously recruit a wide range of immune cells and reverse tumor-infiltrating lymphocyte depletion, providing a robust and lifelong monitoring program to prevent tumor metastasis and recurrence. Embodiments of the CASS B cell platform can be used to prevent and treat cancer and infectious diseases.

[0056] The specific implementation of one or more embodiments is provided herein. However, it should be 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 the basis of the claims and as a representative basis for teaching those skilled in the art to adopt the present invention in any appropriate manner.

[0057] Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents. In the claims and / or specification, when used in conjunction with the term "comprising", the use of the word "a" or "an" may mean "one / an", but is also consistent with "one or more / one or more", "at least one", and "one or more than one / one or more than one".

[0058] Wherever the phrases "for example," "such as," "including," etc. are used herein, unless expressly stated otherwise, they should be understood to be followed by the phrase "and but not limited to." Phrases like "embodiment," "exemplary," etc. should be understood to be non-limiting.

[0059] The term "substantially" allows deviations from the descriptor that do not adversely affect the intended purpose. Even if the term "substantially" is not explicitly recited, the descriptive term should be understood to be modified by the term "substantially".

[0060] The terms "comprising" and "including" and "having" and "involving" (and similar variations, etc.) are used interchangeably and have the same meaning. Specifically, each of these terms is defined to be consistent with the general U.S. patent law definition of "comprising", and thus should be interpreted as open terms meaning "at least the following", and should also be interpreted as not excluding additional features, limitations, aspects, etc. Thus, for example, "a process involving steps a, b, and c" means a process that includes at least steps a, b, and c. Whenever the term "a" or "an" is used, it should be understood to mean "one or more", unless such interpretation does not make sense in the context.

[0061] As used herein, the term "about" may mean approximately, roughly, around, or in the region thereof. When the term "about" is used in conjunction with a numerical range, the term modifies the range by extending the boundaries above and below the numerical values ​​set forth. Typically, the term "about" is used herein to modify numerical values ​​above and below the stated value by a change above or below (higher or lower), such as 20%.

[0062] Chimeric B cell receptor

[0063] Aspects of the invention relate to genetically engineered B cells that are modified to express and carry a chimeric B cell receptor on their surface. In embodiments, the genetically modified B cells may comprise a single chimeric B cell receptor targeting an antigen, or a single chimeric B cell receptor targeting two or more antigens (e.g., a bispecific chimeric B cell receptor, or a multispecific chimeric B cell receptor). In some embodiments, the cell comprises a split chimeric B cell receptor, such as two different scFvs with different costimulatory domains expressed on the surface of the B cell. In addition, some embodiments include fine-tuned chimeric B cell receptors.

[0064] Split chimeric B cell receptors may include two or more chimeric B cell receptors on the surface of a cell, such as a B cell. A chimeric B cell receptor may be specific for two or more antigens. In this embodiment, a first chimeric B cell receptor is specific for a first antigen, and a second chimeric B cell receptor is specific for a second antigen. As described herein, a chimeric B cell receptor may be in any desired orientation. For example, a first chimeric B cell receptor may be specific for a second antigen, and a second chimeric B cell receptor may be specific for a first antigen. A first chimeric B cell receptor and a second chimeric B cell receptor may be expressed by a single nucleic acid construct. In such embodiments, a nucleic acid encoding a cleavable linker may be located between nucleic acids encoding a first chimeric B cell receptor and a second chimeric B cell receptor. In other embodiments, two chimeric B cell receptors may be expressed in the same cell, but from two different nucleic acid constructs.

[0065] In an embodiment, the chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

[0066] Modified B cell receptors, such as B cell receptors containing antibodies or antibody fragments, which are pre-selected by high affinity for specific disease-related antigens, are called chimeric B cell receptors, and are a powerful new method for fighting diseases. Because B cells act as professional antigen-presenting cells, they can process and present antigens on class II molecules, enhance immune cell recognition of tumors, and help new antigens spread. As a key component of immune memory, CASS B cells will simultaneously recruit a wide range of immune cells and reverse tumor infiltrating lymphocyte depletion, providing a robust and lifelong monitoring plan to prevent tumor metastasis and recurrence. In certain cases, B cells may include chimeric, non-natural and at least partially artificially engineered receptors. In certain cases, the engineered chimeric B cell receptor has one, two, three, four or more components, and in some embodiments, one or more components promote B cells to target or bind to one or more antigen-containing cells.

[0067] 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; such that the polypeptides assemble together to form a chimeric B cell receptor.

[0068] As used herein, the term "extracellular ligand binding domain" or "extracellular domain" may refer to an oligomer or polypeptide that can bind 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 is a cell surface marker on a target cell associated with a specific disease state. For example, the disease state may be cancer, and the target ligand may be a cancer-associated antigen. In another embodiment, the disease state may be an infectious disease, and the target ligand may be an infectious disease-associated antigen. In an embodiment, the extracellular ligand binding domain may include an antigen binding domain or an antigen recognition domain that is derived from an antibody against a target antigen. The antigen binding domain or the antigen recognition domain may be an antibody fragment. "Antibody fragments" may be molecules other than intact antibodies that include a portion of an intact antibody that binds to an 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 by antibody fragments. For example, an embodiment may include a chimeric B cell receptor having two scFvs as antigen recognition domains. An embodiment may also include a chimeric B cell receptor having one scFv as an antigen recognition domain. In addition, an embodiment may include a chimeric B cell receptor having a bispecific antibody as an antigen recognition domain. For example, a bispecific antibody may be specific for PD-1 and CTLA4, PD-1 and TIGIT, TIGIT and CCR4, GITR and TIGIT, or PD-1 and CCR4. For example, a bispecific antibody may be specific for HA and NA, or influenza HA and coronavirus spike or SARS2. Bispecific or cross-reactive antibodies are known in the art. For example, see Pilewski, Kelsey A. et al. "Functional HIV-1 / HCV cross-reactive antibodies isolated from a chronically co-infected donor." Cell Reports 42.2 (2023).

[0069] Antigen recognition domains can be directed to any antigen target of interest. In embodiments, the antigen target of interest is on the cell surface, such as the surface of cancer cells (i.e., tumor-associated antigens). The antigen target of interest may also be associated with infectious diseases (i.e., infectious disease-associated antigens). Non-limiting examples of antigen targets include TIGIT, PD-1, CAIX, BCMA, CD 138, PD-L1, PD-L2, VEGF, CD70, CD99, CEA, Her-2, GD2, CD171, aFR, 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 antigenic target comprises CAIX. In embodiments, the antibody targets mesothelin.

[0070] Exemplary antibody compositions (e.g., VH and / or VL sequences or fragments thereof) for designing chimeric B cell receptors as described herein include, but are not limited to:

[0071] Anti-CAIX antibodies described in PCT / US2006 / 046350 and PCT / US2015 / 067178

[0072] Anti-CXCR4 antibodies described in PCT / US20006 / 005691

[0073] Anti-CCR4 antibodies described in PCT / US2008 / 088435, PCT / US2013 / 039744 and PCT / US2015 / 054202

[0074] Anti-PD-L1 antibodies described in PCT / US2008 / 088435 and PCT / US2020 / 062815

[0075] Anti-PD-1 antibodies described in PCT / US2020 / 037791 and PCT / US2020 / 037781

[0076] Anti-GITR antibodies described in PCT / US2017 / 043504

[0077] Anti-clauin-4 antibodies described in PCT / US2019 / 022272

[0078] Anti-MUC 1 antibodies described in PCT / US2020 / 037783

[0079] Anti-TIGIT antibodies described in U.S. Provisional Patent Application 63 / 242,992

[0080] Anti-IGHV 1-69 antibodies described in PCT / US2011 / 038970

[0081] Anti-influenza antibodies described in PCT / US2008 / 085876 and PCT / US2016 / 026800

[0082] Anti-SARS-CoV-2

[0083] (Each of these applications is incorporated herein by reference in its entirety).

[0084] The term "antibody" is used in the broadest sense herein and may refer to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds to (immunoreacts with) an antigen. "Specific binding" or "immunoreacts with" may refer to an antibody that reacts with one or more antigenic determinants of a desired antigen and does not react with other polypeptides. Antibodies of the present disclosure may 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, miniantibodies, scFv-Fc fusions, and Fab expression libraries. Unless otherwise specified, the term "antibody" or "antibodies" referred to herein encompasses, for example, any (or all) of these molecules, as long as they exhibit the desired antigen binding activity.

[0085] Embodiments as described herein may include multispecific antibodies. The term "multispecific antibody" may refer to antibodies that can specifically bind to different types of epitopes. More specifically, multispecific antibodies are antibodies that are specific for at least two different types of epitopes, and in addition to antibodies that recognize different antigens, also include antibodies that recognize different epitopes on the same antigen. (For example, when the antigen is a heterologous receptor, the multispecific antibody binds to different domains that constitute the heterologous receptor: Alternatively, when the antigen is a monomer, 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.

[0086] In an embodiment, 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 a bispecific scFv fragment, comprising a first binding site for a first antigen and a second binding site for a second antigen. For example, a bispecific antibody may be specific for PD-1 and CTLA4, PD-1 and TIGIT, TIGIT and CCR4, or PD-1 and CCR4. The two binding sites may be joined together via a linker domain. In an embodiment, the scFv fragment is a tandem scFv, and the linker domain comprises an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, and IgG4 hinge region) amino acid sequence. In an embodiment, the immunoglobulin hinge region amino acid sequence may be flanked by a flexible linker amino acid sequence, for example, having an amino acid sequence (GGGS) x1-6 、(GGGGS) x1-6 and GSAGSAAGSGEF. In an embodiment, the linker domain comprises at least a portion of an immunoglobulin Fc domain, such as an IgG1, IgG2, IgG3, and IgG4 Fc domain. At least a portion of an immunoglobulin Fc domain may be a CH2 domain. The Fc domain may be connected to the C-terminus of an immunoglobulin hinge region (e.g., an IgG1, IgG2, IgG3, and IgG4 hinge region) amino acid sequence. The linker domain may comprise a flexible linker amino acid sequence (e.g., (GGGS)) at one end or at both ends. x1-6 、(GGGGS) x1-6 and GSAGSAAGSGEF).

[0087] In embodiments, antibody comprises mosaic antibody. Mosaic antibody is such an antibody: the outer amino acid residues of the antibody of one species are reasonably replaced or " mosaicked " by the outer amino acid residues of the antibody of the second species, so that the antibody of the first species is non-immunogenic in the second species, thereby reducing the immunogenicity of the antibody. Since the antigenicity of the protein depends mainly on its surface characteristics, the immunogenicity of the antibody can be reduced by replacing the exposed residues, which are different from those residues found in the antibody of another mammalian species. The rational replacement of this external residue should have almost no effect on the internal domain or inter-domain contact, or have no effect at all. Therefore, since the change is limited to the variable region framework residues, the ligand binding properties should not be affected. This process is called " mosaicking ", because only the outer surface or outer layer of the antibody changes, and the supporting residues remain undisturbed.

[0088] Single-chain Fv ("scFv") polypeptide molecules are covalently linked VH::VL heterodimers that can be expressed by a gene fusion comprising a VH encoding gene and a VL encoding gene connected by a peptide encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). Many methods have been described to identify chemical structures for converting 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 that is substantially similar to that of an antigen binding site. See, e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,946,778.

[0089] Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). Methods for preparing bispecific antibodies are known in the art. See, e.g., U.S. Pat. No. 8,329,178, which is incorporated herein by reference in its entirety.

[0090] Antibody molecules obtained from humans refer to any of the classes of IgG, IgM, IgA, IgE and IgD, which differ from each other by the nature of the heavy chain present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2, etc. Furthermore, in humans, the light chain can be either a kappa chain or a lambda chain.

[0091] The term "antigen binding site" or "binding portion" may refer to the portion of an immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by the amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly different segments within the V regions of the heavy and light chains, called "hypervariable regions," are inserted between more conserved flanking segments called "framework regions" or "FRs." Thus, the term "FR" may refer to the amino acid sequences that naturally occur between and adjacent to the hypervariable regions of an immunoglobulin. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged 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 the bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity determining regions" or "CDRs."

[0092] 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 connected by a flexible linker. The skilled person will recognize that the embodiments may include different linkers generally 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, the use of different linkers will allow people to fine-tune dual-targeted chimeric B cell receptor constructs. The linker length may vary depending on the antibody of the dual-targeted chimeric B cell receptor construct, its angle of approach to the target epitope, and the morphology of the target on the tumor cell membrane. For example, a flexible linker may include a GGGS1, GGGS3, GGGS5, or IgG1 hinge. In some embodiments, the number of Gs in the joint can be a combination of 2, 3, 4, 5, 6 or 7 with S1, S2, S3, S4, S5 or S6. For example, the direction of the scFv relative to the joint can be changed. In a nucleic acid construct, the first scFv can be in the first box (i.e. before the joint), and the second scFv can be in the second box (i.e. after the joint). Alternatively, the first scFv can be in the second box, and the second scFv can be in the first box. As described herein, various lengths and flexible joints can be used. The different orientations of the two scFvs will affect the combination.

[0093] Antigen recognition domains that can be used to construct chimeric B cell receptors, such as scFvs to a first antigen and / or a second antigen, can be synthesized, engineered, and / or produced using nucleic acids (e.g., DNA). The DNA encoding the antigen recognition domain can be cloned in frame to DNA encoding the necessary chimeric B cell receptor elements, such as, but not limited to, the CD8 hinge region, transmembrane domains, BCR-associated proteins (such as CD79a and CD79b), and costimulatory domains of molecules of immunological interest, such as, but not limited to, CD 19 and CD20.

[0094] Binding domains other than scFv may also be used for the predetermined targeting of B cells, such as, by way of non-limiting example, camelid single domain antibody fragments or receptor ligands, antibody binding domains, antibody hypervariable loops or CDRs.

[0095] In one embodiment, the membrane spaning domain is also included in the stem region between the extracellular ligand binding domain and the membrane spaning domain. The term "stem region" as used herein may mean any oligomerization or polypeptide having the function of connecting the membrane spaning domain to the extracellular ligand binding domain. In particular, the stem region is used to provide greater flexibility and accessibility for the extracellular ligand binding domain. The stem region may include up to 300 amino acids, such as 10 to 100 amino acids, or such as 25 to 50 amino acids. The stem region may be derived from all or part of naturally occurring molecules, such as all or part of the extracellular region derived from CD8, CD4 or CD28, or derived from all or part of antibody constant region (such as CH1, CH2, CH3 or both CH2 and CH3 for IgG antibodies, CH1, CH2, CH3, CH4 or any combination thereof for IgM antibodies). For example, the stem region may include IgG (CH2-CH3) or a portion thereof. For example, the stem region may comprise IgG1 (CH2-CH3), IgG2 (CH2-CH3), IgG3 (CH2-CH3), IgG4 (CH2-CH3), or a portion thereof. In an embodiment, the stem region does not comprise a constant domain. In an embodiment, the stem region may be a synthetic sequence corresponding to a naturally occurring stem sequence, or may be a fully synthetic stem sequence. In one embodiment, the stem region is a portion of a human CD8 chain.

[0096] The signal transduction domain or intracellular signal transduction domain of the chimeric B cell receptor of the present invention can be responsible for intracellular signal transduction after the extracellular ligand binding domain binds to the target, resulting in the activation of immune cells and immune responses. In other words, the signal transduction domain can be responsible for the activation of at least one normal function of the B cell expressing the chimeric B cell receptor. Therefore, the term "signaling domain" can refer to a portion of a protein that directs a cell to perform a specialized function, such as, for example, early activation of Lyn and Syk and activation of NFAT and NF K Late activation of B. In embodiments, the signal transduction domain or intracellular signaling domain comprises all or a portion of CD79a and / or CD79b comprising an ITAM that amplifies chimeric B cell receptor (BCR) signaling.

[0097] In an embodiment, the signal transduction domain may include two different types of cytoplasmic signaling sequences, those that initiate antigen-dependent primary activation, and those that provide secondary or costimulatory signals in an antigen-independent manner. The primary cytoplasmic signaling sequence may include a signaling motif, which is an activation motif based on tyrosine of an immune receptor known as ITAM. ITAM is a clear signaling motif found in the cytoplasmic tail of a variety of receptors as a syk / zap70 class tyrosine kinase binding site. Examples of ITAMs that can be used for the present invention may include, as non-limiting examples, those derived from TCR, TCR, TCR, TCR, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, and CD66d.

[0098] Chimeric B cell receptors may contain native transmembrane and / or intracellular domains. In native B cells, engagement of the B cell receptor results in rapid tyrosine phosphorylation and calcium ion polarization of the intracellular region, leading to downstream activation of NFAT and NF-kB. Using NFAT / NF-kB response elements to drive expression of our secretory proteins, we designed an inducible expression system that will be activated by binding of the surface engineered BCR and subsequent downstream signaling pathways.

[0099] In an embodiment, the intracellular signaling domain of the BCR comprises a co-stimulatory signal molecule. In an embodiment, the intracellular signaling domain comprises 2, 3, 4 or more co-stimulatory molecules in series. The co-stimulatory molecule may be a cell surface molecule other than an antigen receptor or its ligand required for an effective immune response.

[0100] "Costimulatory ligand" may 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, for example, ligand binding to the BCR. Costimulatory ligands may 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 molecules (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Costimulatory ligands may also encompass antibodies 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 ligands that specifically bind to CD83.

[0101] "Costimulatory molecules" may refer to cognate binding partners on B cells that specifically bind to costimulatory ligands, thereby mediating costimulatory responses of cells, such as, but not limited to, proliferation, activation, differentiation, etc. Costimulatory molecules may 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 ligands that specifically bind to CD83, etc. See, e.g., Mongini, Patricia KA, and John K. Inman. "Cytokine dependency of human B cell cycleprogression elicited by ligands which coengage BCR and the CD21 / CD 19 / CD81 costimulatory complex." Cellular immunology 207.2 (2001): 127-140.

[0102] In another embodiment, the signal transduction domain is a TNFR-associated factor 2 (TRAF2) binding motif, the intracytoplasmic tail of the costimulatory TNFR family member. The cytoplasmic tail of the 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 the receptor trimerization reaction.

[0103] The distinguishing features of suitable transmembrane polypeptides include the ability to be expressed on the surface of immune cells, particularly B cells, and the ability to interact to direct the cellular response of immune cells to predetermined target cells. Different transmembrane polypeptides of chimeric B cell receptors comprising extracellular ligand binding domains and / or signal transduction domains interact with each other, participate in signal transduction after binding to the target ligand, and induce an immune response. The transmembrane domain can be derived from a natural or synthetic source. The transmembrane domain can be derived from any membrane-bound or transmembrane protein.

[0104] The term "part" as used herein may refer to any subunit of a molecule, i.e., a shorter peptide. Alternatively, a functional variant of the amino acid sequence of a polypeptide may be prepared by mutations in the DNA encoding the polypeptide. Such variants or functional variants include, for example, deletions, insertions or substitutions of residues in the amino acid sequence. Any combination of deletions, insertions and substitutions may also be performed to obtain the final construct, as long as the final construct has the desired activity, especially exhibits specific anti-target cell immune activity. The function of the chimeric B cell receptor of the present invention in a host cell can be detected in an assay suitable for demonstrating the signal transduction potential of the chimeric B cell receptor when it binds to a specific target. Such assays are available to those skilled in the art. For example, the assay allows detection of signal transduction pathways triggered when binding to a target, such as involving measurement of tyrosine phosphorylation of an intracellular domain, or downstream activation of NFAT and NF K B. Analysis of activation.

[0105] In embodiments, polyclonal CASS B cells can be prepared by transducing cells with two lentiviruses (or two adeno-associated viruses) encoding different BCRs and sorting the double-transduced cells. Alternatively, polyclonal CASS B cells can be prepared by generating a BCR library library and using these plasmids to generate a lentivirus (or adeno-associated virus) encoding a BCR pool. In this case, one will obtain a population of CASS B cells expressing different BCRs, some of which may have undergone multiple transduction events and therefore display multiple BCRs on the surface of a single cell.

[0106] cell

[0107] Embodiments of the present disclosure include B cells expressing chimeric B cell receptors. Cells may be of any type, including immune cells that can express chimeric B cell receptors for therapy (i.e., cancer therapy or infectious disease therapy), or cells carrying expression vectors encoding chimeric B cell receptors, such as bacterial cells. As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably. All of these terms also include their progeny, i.e., any and all offspring. For example, due to intentional or unintentional mutations, all progeny do not need to be exactly the same. In the context of expressing heterologous nucleic acid sequences, "host cell" may refer to eukaryotic cells capable of replicating vectors and / or expressing heterologous genes encoded by vectors. Host cells may and have been used as recipients of vectors. Host cells may be "transfected", "converted", or "transduced", which may refer to the process of transferring or introducing exogenous nucleic acids into host cells. Transformed cells include cells of the primary subject and their progeny. As used herein, the terms "engineered" and "recombinant" cells or host cells may refer to cells into which an exogenous nucleic acid sequence, such as, for example, a vector, has been introduced. Thus, recombinant cells are distinguished from natural cells that do not contain recombinantly introduced nucleic acids. In an embodiment of the invention, the host cell is a B cell.

[0108] Some vectors may employ control sequences that allow them to be replicated and / or expressed in both prokaryotic and eukaryotic cells. Those skilled in the art will further appreciate the conditions under which all of the above host cells are incubated to maintain them and allow for vector replication. Also understood and known are techniques and conditions that allow for large-scale production of vectors and production of nucleic acids encoded by the vectors and their cognate polypeptides, proteins or peptides.

[0109] The cells can be autologous, syngeneic, allogeneic, and even in some cases xenogeneic.

[0110] In many cases, one would like to be able to kill the modified B cells, such as when one wishes to terminate treatment, if the cells become neoplastic, if the absence of the cells is of interest in research, or other events. For this purpose, expression of certain gene products can be provided, wherein the modified cells can be killed under controlled conditions, such as inducible suicide genes.

[0111] Armed CASSB cells

[0112] The present invention also includes genetically engineered CASS B cells that are 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 restoration (IR) CASS B cells.

[0113] The term "polypeptide" may encompass the singular "polypeptide" as well as the plural "polypeptides", and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, rather than a specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to one or more chains of two or more amino acids may refer to "polypeptides" herein, and the term "polypeptide" may be used in place of any of these terms or interchangeably with any of these terms. "Polypeptide" may also refer to the product of post-expression modification of a polypeptide (including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage or modification by non-naturally occurring amino acids through known protecting groups). A polypeptide may be derived from a natural biological source or produced by recombinant technology, but need not be translated from a specified nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. With respect to amino acid sequences, those skilled in the art will readily recognize that individual substitutions, deletions or additions to a nucleic acid sequence, peptide sequence, polypeptide sequence 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 results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.

[0114] In embodiments, the polypeptide may be an antibody or fragment thereof, or a cytokine.

[0115] As used herein, "antibody" or "antigen-binding polypeptide" may refer to a polypeptide or polypeptide complex that specifically recognizes an antigen and binds to the antigen. By "specifically binds" or "immunoreacts with" is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides. The antibody may be a complete antibody and any antigen-binding fragment or single chain thereof. For example, an "antibody" may include any protein or peptide containing such a molecule that contains at least a portion of an immunoglobulin molecule having a biological activity of binding to an antigen.

[0116] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of the structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" may include aptamers (such as spiegelmers), miniantibodies, and diabodies. The term "antibody fragment" may also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. The antibodies, antigen-binding polypeptides, variants or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific (e.g., bispecific), human, humanized or mosaic antibodies, chimeric antibodies, single chain antibodies, epitope binding fragments, such as Fab, Fab′ and F(ab′)2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, dAbs (domain antibodies), miniantibodies, disulfide-linked Fvs (sdFv), fragments comprising a VL domain or a VH domain, fragments produced by a Fab expression library, nanobodies obtained from camels, and anti-idiotypic (anti-Id) antibodies.

[0117] In an embodiment, the antibody secreted by CASS B cells is a checkpoint blocking antibody. The term "checkpoint blocking antibody" may refer to an antibody that inhibits immune checkpoints. When stimulated, key regulators of the immune system inhibit the immune system's response to immune stimuli (such as cancer cells). Checkpoint blocking antibodies can block inhibitory checkpoints and restore immune system function, such as against cancer cells. Checkpoint blocking antibodies include, but are not limited to, anti-PD-1, anti-PDL-2, and anti-CTLA-4. Other antibodies that modulate the immune system, such as anti-TGFb and tumor vasculature, such as anti-VEFG, are also viable candidates.

[0118] In embodiments, antibodies secreted by CASS B cells may be specific for HA1, HA2, NA or spike proteins. As described herein, exemplary antibody compositions (e.g., VH and / or VL sequences or fragments thereof) that can be used for armed B cells include, but are not limited to, anti-influenza antibodies described in PCT / US2008 / 085876 and PCT / US2016 / 026800.

[0119] In embodiments, 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 locally secrete PD-L1 monoclonal antibodies at tumor sites to restore effective anti-cancer immunity and / or reverse T cell exhaustion.

[0120] As described herein, exemplary antibody compositions (e.g., VH and / or VL sequences or fragments thereof) for designing armed B cells include, but are not limited to:

[0121] Anti-CAIX antibodies described in PCT / US2006 / 046350 and PCT / US2015 / 067178

[0122] Anti-CXCR4 antibodies described in PCT / US20006 / 005691

[0123] Anti-CCR4 antibodies described in PCT / US2008 / 088435, PCT / US2013 / 039744 and PCT / US2015 / 054202

[0124] Anti-PD-L1 antibodies described in PCT / US2008 / 088435 and PCT / US2020 / 062815

[0125] Anti-PD-1 antibodies described in PCT / US2020 / 037791 and PCT / US2020 / 037781

[0126] Anti-GITR antibodies described in PCT / US2017 / 043504

[0127] Anti-clauin-4 antibodies described in PCT / US2019 / 022272

[0128] Anti-MUC 1 antibodies described in PCT / US2020 / 037783

[0129] Anti-TIGIT antibodies described in U.S. Provisional Patent Application 63 / 242,992

[0130] Anti-IGHV 1-69 antibodies described in PCT / US2011 / 038970

[0131] Anti-influenza antibodies described in PCT / US2008 / 085876 and PCT / US2016 / 026800

[0132] (Each of these applications is incorporated herein by reference in its entirety).

[0133] See also Yasunaga, Masahiro for exemplary antibodies for treating cancer. Seminars in cancer biology. Vol. 64. Academic Press, 2020,

[0134] In an embodiment, the antibody is a bispecific antibody. For example, in an embodiment, 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 a bispecific scFv fragment, comprising a first binding site for a first antigen and a second binding site for a second antigen. For example, a bispecific antibody may be specific for PD-1 and CTLA4, PD-1 and TIGIT, TIGIT and CCR4, or PD-1 and CCR4. The two binding sites may be joined together via a linker domain. In an embodiment, the scFv fragment is a tandem scFv, and the linker domain comprises an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, and IgG4 hinge region) amino acid sequence. In an embodiment, the immunoglobulin hinge region amino acid sequence may be flanked by a flexible linker amino acid sequence, for example, having an amino acid sequence (GGGS) x1-6 、(GGGGS) x1-6 and GSAGSAAGSGEF. In an embodiment, the linker domain comprises at least a portion of an immunoglobulin Fc domain, such as an IgG1, IgG2, IgG3, and IgG4 Fc domain. At least a portion of an immunoglobulin Fc domain may be a CH2 domain. The Fc domain may be connected to the C-terminus of an immunoglobulin hinge region (e.g., an IgG1, IgG2, IgG3, and IgG4 hinge region) amino acid sequence. The linker domain may comprise a flexible linker amino acid sequence (e.g., (GGGS)) at one end or at both ends. x1-6 、(GGGGS) x1-6 and GSAGSAAGSGEF).

[0135] 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.

[0136] Embodiments of armed CASS B cells may include gene expression vectors that co-express multiple ORFs. Multiple ORFs may be separated by linkers, such as internal ribosome entry sites (IRES) or the 2A peptide family. 2A peptides are short (-18-25aa) peptides derived from viruses. 2A peptides may be referred to as "self-cleaving" peptides that will produce multiple proteins from the same transcript. 2A peptides work by causing the ribosome to skip the synthesis of the glycine and proline peptide bonds at the C-terminus of the 2A element, resulting in separation between the end of the 2A sequence and the downstream peptide. As a result, the C-terminus of the upstream protein will add several additional 2A residues, while the N-terminus of the downstream protein will add an additional proline. There are four 2A peptides, P2A, T2A, E2A, and F2A, which come from four different viruses.

[0137] In embodiments, the secretable polypeptide can be expressed from a second expression construct, which can be in the same DNA vector as the DNA vector encoding the chimeric B cell receptor (e.g., antigen recognition domain). A second expression cassette, which can be used to encode a secretable polypeptide (i.e., an antibody or cytokine), can be cloned before or after a linker (e.g., an IRES or 2A family peptide).

[0138] In embodiments, the second expression cassette encoding the secretable polypeptide may comprise a response element. A "response element" may refer to a portion of a gene that must be present in order for the gene to respond to some hormone or other stimulus. In embodiments, the response element is an inducible response element. For example, the response element may be a NFAT and / or NF-kB response element.

[0139] In one embodiment, for example, a second expression cassette is inserted after a cis BCR plasmid with a minimal IL2 / IL8 promoter upstream of multiple NFAT and / or NF-kB response elements and secreted proteins (Ab or other proteins). In natural B cells, the involvement of BCR leads to rapid tyrosine phosphorylation and calcium ion polarization of the IC domain, leading to downstream activation of NFAT and NF-kB. Using NFAT / NF-kB response elements to drive the expression of our secretory proteins, we designed an inducible expression system that will be activated by antigens expressed on cancer cells. This produces a targeted and inducible delivery system that secretes its therapeutic payload only when activated by tumor cells, resulting in a local area of ​​consistent and high immunomodulatory Ab / protein concentration centered on the tumor. This can reverse the inhibitory nature of the tumor microenvironment, leading to improved outcomes and tumor elimination, while reducing the targeted / non-targeted tumor side effects often seen with systemic delivery of therapeutic antibodies and cytokines.

[0140] In an embodiment, the expression cassette may further comprise a post-transcriptional response element that, when transcribed, produces a tertiary structure that enhances expression. For example, the post-transcriptional response element may be a WPRE.

[0141] Introduction of constructs into B cells

[0142] The expression vector encoding the chimeric B cell receptor may be introduced as one or more DNA molecules or constructs, wherein at least one marker may be present to allow selection of host cells containing the construct.

[0143] Constructs can be prepared in a conventional manner, wherein genes and regulatory regions can be appropriately separated, linked, cloned in an appropriate cloning host, analyzed by restriction enzyme digestion or sequencing or other convenient methods. For example, using PCR, individual fragments including all or part of the functional unit can be isolated, wherein one or more mutations can be introduced using "primer repair", ligation, in vitro mutagenesis, etc., as appropriate. Once the construct is complete and proven to have an appropriate sequence, the construct can be introduced into B cells by any convenient method. The construct can be integrated and packaged into a non-replicating defective viral genome, such as adenovirus, adeno-associated virus (AAV) or herpes simplex virus (HSV) or other viruses, including retroviral vectors or lentiviral vectors, for infection or transduction into cells. The construct may include viral sequences for transfection. Alternatively, the construct can be introduced by fusion, electroporation, gene gun technique, transfection, lipofection, etc. Before the construct is introduced, the host cell can be grown and amplified in a culture medium, followed by appropriate treatment to introduce the construct and integrate the construct. The cells are then amplified and screened by the markers present in the construct. Various markers that may be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, and the like.

[0144] In some cases, there may be a target site for homologous recombination, where the construct is integrated at a specific locus. For example, materials and methods known in the art of homologous recombination can be used to knock out endogenous genes and replace them with genes encoded by the construct (at the same locus or elsewhere). 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. In addition, plasmid-based methods that induce double-strand breaks have used homologous recombination for genome engineering. Zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and CRISPR can all guide nucleases to cause specific double-strand breaks.

[0145] The construct can be introduced as a single DNA molecule encoding at least CAR and optionally another gene, or different DNA molecules with one or more genes. For example, other genes include genes encoding therapeutic molecules or suicide genes. Constructs can be introduced simultaneously or continuously, each of which has the same or different markers.

[0146] 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, etc. Materials useful for preparing construct DNA and for performing transfections are well known in the art, and many are commercially available.

[0147] Treatment

[0148] Aspects of the present disclosure relate to methods for preventing or treating a subject with a disease or disorder by administering CASS B cells as described herein. In embodiments, the method comprises administering a therapeutically effective amount of CASS B cells as described herein to a subject with a disease or disorder or at risk of a disease or disorder. The therapeutically effective amount may depend on the severity and course of the disease or disorder, previous therapy, the subject's health, weight, and response to drugs, and the judgment of the treating physician.

[0149] "Treatment" and "treating" may 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 that ameliorates or otherwise beneficially alters one or more symptoms of the disease or disorder. The term may include a full range of treatments for the specific condition from which the patient suffers, such as the administration of an active compound for the purpose of: alleviating or relieving symptoms or complications; delaying the progression of the condition, disease, or disorder; curing or eliminating the condition, disease, or disorder; and / or preventing the condition, disease, or disorder, wherein "preventing" or "prevention" may refer to the management and care of a patient for the purpose of preventing the development of a condition, disease, or disorder, and includes the administration of an active compound to prevent or reduce the risk of the onset of symptoms or complications.

[0150] An "individual" or "subject" can be a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and 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.

[0151] The CASS B cells according to the present disclosure can be used to prevent or treat a disease or disorder (e.g., cancer or infection) in a subject in need thereof. In another embodiment, the isolated cells according to the present invention can be used to prepare a medicament for treating cancer or infection (such as a viral infection) in a patient in need thereof.

[0152] Embodiments may rely on a method for treating a patient in need thereof, the method comprising at least one of the following steps: (a) providing a CASS B cell according to the invention, and (B) administering the cell to the patient.

[0153] The treatment may be ameliorative, curative or preventative. It may be part of an autologous immunotherapy, or may be part of an allogeneic immunotherapy. Autologous means that the cells, cell lines or cell populations used to treat the patient are derived from the patient or from a human leukocyte antigen (HLA) compatible donor. Allogeneic means that the cells or cell populations used to treat the patient are not from the patient, but from a donor.

[0154] The present invention is suitable for allogeneic immunotherapy because it allows the conversion of B cells obtained from a donor into non-alloreactive cells. This can be done under standard protocols and replicated as many times as needed. The resulting modified B cells can be pooled and administered to one or several patients as an "off-the-shelf" therapeutic product.

[0155] Cells useful in the disclosed methods are described herein. The treatments described herein can be used to treat a patient diagnosed with a disease or disorder.

[0156] Embodiments as described herein can modulate the immune system to treat a subject suffering from a disease or disorder. "Modulate" can refer to upregulating, inducing, stimulating, enhancing and / or alleviating inhibition, as well as inhibiting, attenuating and / or downregulating or inhibiting. In embodiments, the activity of the subject's immune system is modulated.

[0157] The embodiments as described herein may be administered to a subject in combination with one or more therapies for a disease or disorder.

[0158] Methods for treating cancer

[0159] Embodiments include methods for treating subjects with cancer. The term "cancer" may refer to a series of pathological symptoms associated with the onset or progression of malignant tumors and metastasis. The term "tumor" may refer to a new growth of tissue in which the proliferation of cells is uncontrolled and progressive. In embodiments, a tumor may be a malignant tumor in which the primary tumor has the characteristics of invasion or metastasis, or it shows a greater degree of anaplasia than a benign tumor. Therefore, "treatment of cancer" or "treating cancer" may refer to activities that prevent, alleviate or improve any major phenomenon (onset, progression, metastasis) or secondary symptoms associated with the disease.

[0160] An emerging mechanism associated with tumor progression is the immune checkpoint pathway, which involves cellular interactions that, under normal conditions, prevent T cell overactivation, allowing T cells to function in a self-limited manner. As an evasion mechanism, many tumors are able to stimulate the expression of immune checkpoint molecules, resulting in an anergic phenotype of T cells that cannot suppress tumor development. For example, emerging clinical data highlight the importance of a pair of inhibitory ligands and receptors as immune checkpoints: programmed death ligand 1 (PD-L1; B7-H1 and CD274) and programmed death receptor-1 (PD-1; CD279) that prevent cytotoxic T lymphocytes from killing cancer cells. The PD 1 receptor is expressed by many cell types, such as T cells, B cells, natural killer (NK) cells, and host tissues. Tumors and antigen presenting cells (APCs) expressing PD-L 1 can block T cell receptor (TCR) signaling of cytotoxic T lymphocytes by binding to the receptor PD-1, reducing cytokine production and T cell proliferation. PD-L1 overexpression can be found in many tumor types and can also mediate immunosuppressive functions through interactions with other proteins, including CD80 (B7.1), blocking its ability to activate T cells by binding to CD28.

[0161] Genetically engineering human B cells to express tumor-directed chimeric B-cell receptors can generate antitumor effector cells that can bypass tumor immune escape mechanisms caused by abnormal protein-antigen processing and presentation. In addition, these transgenic receptors can target tumor-associated antigens of nonprotein origin.

[0162] For example, aspects of the present disclosure relate to methods of killing cancer cells, such as renal cancer cells.

[0163] Aspects of the present disclosure also relate to methods of halting or slowing cancer progression or promoting cancer regression in a subject.

[0164] Still further, aspects of the present disclosure relate to methods of reducing cell proliferation of cancer cells in a subject.

[0165] "Cancer" and "cancerous" may refer to or describe, for example, the physiological condition in mammals characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, chronic lymphocytic leukemia, non-small cell lung cancer, clear cell renal 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 cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoma, various types of head and neck cancer. For example, the cancer is renal cell carcinoma, such as ccRCC.

[0166] The subject may have cancer, such as a liquid cancer (ie, a blood cancer) and / or a solid cancer (ie, a tumor). The cancer may be benign or malignant, and may be a cancer affected by the immune system.

[0167] Treatable cancers include tumors that are not vascularized or substantially not vascularized, and vascularized tumors. Cancers may include non-solid tumors (such as blood tumors, such as leukemias and lymphomas) or may include solid tumors. The types of cancers treated with the CAR of the present invention include, but are not limited to, cancers, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, cancers, and melanomas. Adult tumors / cancers and childhood tumors / cancers are also included.

[0168] In cancer, normal cell-cell interactions in tissues are disrupted, and the tumor microenvironment evolves to adapt to the growing tumor. The tumor microenvironment (TME) can refer to the cellular environment in which the tumor exists, including components such as surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and extracellular matrix (ECM). The tumor microenvironment is complex and is greatly influenced by the immune system.

[0169] The present invention provides CASS B cell therapy for treating or preventing cancer. CASS B cells secrete mono-, bi- or tri-specific mini-antibodies, antibodies or mini-antibody / antibody fusion proteins or cytokines at tumor sites, which can provide additional benefits by changing (i.e., regulating) the immunosuppressive tumor microenvironment. For example, the microenvironment surrounding cancer cells and / or tumors can be modulated to reduce microenvironment-dependent immunosuppression, thereby modulating (or allowing) the immune system to kill tumor cells.

[0170] In embodiments of the present invention, the method of the present invention for clinical aspects is combined with other drugs such as anticancer agents that effectively treat hyperproliferative diseases. "Anticancerous" agents can have a negative impact on the cancer of a subject, for example, by killing cancer cells, inducing apoptosis of cancer cells, reducing the growth rate of cancer cells, reducing the incidence or quantity of metastasis, reducing tumor size, inhibiting tumor growth, reducing the blood supply of tumors or cancer cells, promoting the immune response for cancer cells or tumors, preventing or inhibiting the development of cancer, or extending the life span of cancer subjects. For example, these other compositions will be provided with a combined amount that effectively kills or inhibits cell proliferation. The process may include contacting cancer cells with expression constructs and medicaments or multiple factors simultaneously. This can be achieved by contacting cells with a single composition or pharmacological preparation comprising two medicaments, or by contacting cells with two different compositions or preparations simultaneously, wherein one composition comprises an expression construct, and the other comprises a second medicament.

[0171] Tumor cell resistance to chemotherapeutic and radiotherapeutic 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 similarly be used in combination with chemotherapeutic, radiotherapeutic or immunotherapeutic interventions and pro-apoptotic or cell cycle modulators.

[0172] Alternatively, the therapy of the present invention can be spaced a few minutes to a few weeks before or after other medicaments are treated. In another medicament and the present invention are respectively applied to individual embodiments, it is generally ensured that there will not be a long time interval between each administration, so that the medicine and the therapy of the present invention can still produce a favorable combined effect on cells. In such cases, cells can be contacted with two modalities within about 12-24 hours from each other (for example, within about 6-12 hours from each other). However, in some cases, it may be desirable to significantly extend the treatment time, wherein the corresponding administration is separated by several days (2, 3, 4, 5, 6 or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8 days).

[0173] The treatment cycle will be repeated as necessary. For example, various standard therapies and surgical interventions can be used in combination with the cell therapy of the present invention.

[0174] Cancer therapy also includes a variety of combination therapies based on chemotherapy and radiotherapy. Combination chemotherapy includes, but is not limited to, for example, paclitaxel, hexamethylmelamine, docetaxel, Herceptin, methotrexate, Noanto, Norade, cisplatin (CDDP), carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, dactinomycin D, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, raloxifene, estrogen receptor binding agents, paclitaxel, gemcitabine, navelbine, transferase-protein transferase inhibitors, anti-platinum, 5-fluorouracil, vincristine, vinblastine and methotrexate, or any analog or derivative variant of the foregoing, and also and their combinations. In a specific embodiment, individual chemotherapy is used in combination with the present invention, for example, before, during and / or after the application of the present invention.

[0175] Other factors that cause DNA damage and have been widely used include the so-called gamma rays, X-rays and / or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also useful, such as microwaves and UV radiation. Most likely, all of these factors produce extensive damage to DNA, DNA precursors, replication and repair of DNA, and assembly and maintenance of chromosomes. The dosage range of X-rays ranges from a dosage of 50 roentgens to 200 roentgens per day over an extended period of time (3 to 4 weeks) to a single dose of 2000 roentgens to 6000 roentgens. The dosage range of radioisotopes varies greatly, depending on the half-life of the isotope, the intensity and type of radiation, and the absorption of tumor cells.

[0176] As applied to cells, the terms "contacting" and "exposing" are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to or placed in direct juxtaposition with a target cell. To achieve cell killing or stasis, the two agents are delivered to the cell in a combined amount effective to kill the cell or prevent it from dividing.

[0177] Immunotherapy relies on the use of immune effector cells and molecules to target and destroy cancer cells.Immuno effectors can be, for example, antibodies that are specific to certain markers on the surface of tumor cells.The antibody itself can serve as the effector of therapy, or it can recruit other cells to actually achieve cell killing.Antibodies can also be combined with drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, cholera toxin, pertussis toxin, etc.) and are used only as targeting agents.Alternatively, the effector can be a lymphocyte carrying a surface molecule that interacts directly or indirectly with a tumor cell target.Various effector cells include cytotoxic T cells and NK cells.

[0178] Therefore, immunotherapy in addition to the therapy of the present invention described herein can be used as part of a combination therapy with the cell therapy of the present invention. Methods of combination therapy are discussed herein. For example, tumor cells must carry some markers that are easy to target, i.e., not present on most other cells. There are many tumor markers, and any of these markers are suitable for targeting in the context of the present invention. Common tumor markers include PD-1, PD-L 1, 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.

[0179] In yet another embodiment, the secondary treatment is gene therapy, wherein a therapeutic polynucleotide is administered before, after, or simultaneously with the clinical embodiments of the invention.A variety of expression products are encompassed within the present invention, including cell proliferation inducers, cell proliferation inhibitors, or programmed cell death regulators.

[0180] Approximately 60% of cancer patients will undergo some type of surgery, including preventive, diagnostic or staging, curative, and palliative surgery. Curative surgery is a cancer treatment that can be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies.

[0181] Therapeutic surgery includes excision, in which 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 also include laser surgery, cryosurgery, electrosurgery, and delusion control surgery (Mohs surgery). For example, the present invention can be used to remove surface cancer, precancerous lesions, or an occasional amount of normal tissue.

[0182] After removing part or all of the cancer cells, tissues or tumors, a cavity is formed in the body. Treatment can be completed by perfusion, direct injection or local application of the area and other anticancer therapies. Such treatments 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 also be different doses.

[0183] In some embodiments, other agents may 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 connections, cell growth inhibitors and differentiation agents, cell adhesion inhibitors, or agents that increase the sensitivity of hyperproliferative cells to apoptosis inducing agents. Immunomodulators include tumor necrosis factor; interferon α, β and γ; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1β, MCP-1, RANTES and other chemokines. In some embodiments, the 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 hyperproliferative cells. Increasing intercellular signaling by increasing the number of GAP connections will increase the anti-hyperproliferative effect on adjacent hyperproliferative cell populations. In other embodiments, cell inhibitors or differentiation agents may 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 antibody c225, may be used in combination with the present invention to enhance the efficacy of treatment.

[0184] According to one embodiment of the invention, the treatment may be administered to a patient receiving immunosuppressive therapy. The invention uses cells or cell populations that are resistant to such immunosuppressants due to inactivation of a gene encoding at least one immunosuppressant receptor. In this regard, immunosuppressive therapy should facilitate selection and expansion of T cells according to the invention in a patient.

[0185] In other embodiments, the cell compositions as described herein can be administered to a patient in combination with (e.g., before, at the same time, or after) bone marrow transplantation, T cell ablation therapy using chemotherapeutic 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 ablation therapy, such as agents that react with CD20, e.g., rituximab (Rituxan). For example, in one embodiment, a subject may receive standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In certain embodiments, after transplantation, the subject receives an infusion of the expanded immune cells of the present invention. In another embodiment, the expanded cells are administered before or after surgery. The modified cells obtained by any of the methods described herein can be used in a specific aspect of the present invention for treating host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD) in a patient in need thereof; therefore, within the scope of the present invention is a method for treating host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD) in a patient in need thereof, comprising treating the patient by administering to the patient an effective amount of modified cells comprising inactivated TCRα and / or TCRβ genes.

[0186] Methods of treating infectious diseases

[0187] Aspects of the invention relate to methods of treating or preventing an infectious disease in a subject by administering to the subject a composition as described herein. The term "infectious disease" may 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. "Anti-infectious disease" treatment refers to treatment that prevents, ameliorates, or eradicates an infectious disease and / or its causative agent.

[0188] 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 virus, viral meningitis, herpes, anthrax, Lyme disease, and Escherichia coli, etc. See, e.g., Pelegrin, Mireia, Mar Naranjo-Gomez, and Marc Piechaczyk. "Antiviral monoclonal antibodies: more than just simple neutralizers?" 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.

[0189] Cell administration

[0190] The present disclosure is particularly suitable for allogeneic immunotherapy because it allows B cells obtained from a donor to be converted into non-alloreactive cells. This can be done under standard protocols and replicated as many times as needed. The resulting modified B cells can be pooled and administered to one or several patients as an "off-the-shelf" therapeutic product.

[0191] Depending on the nature of the cells, the cells can be introduced into a host organism, such as a mammal, in a variety of ways. In a specific embodiment, the cells can be introduced at the tumor site, although in an alternative embodiment, the cells are directed to cancer or modified to target cancer. The number of cells used will depend on many situations, the purpose of introduction, the life span of the cells, the scheme to be used, such as the number of administrations, the ability of cell proliferation, the stability of the recombinant construct, etc. The cells can be applied in the form of a dispersion, for example, injected into or near the site of interest. The cells can be in a physiologically acceptable culture medium.

[0192] In some embodiments, cells are encapsulated to inhibit immune recognition and placed at the tumor site.

[0193] Cells can be administered as needed. Various regimens can be used depending on the desired response, mode of administration, life span of the cells, and number of cells present. The amount administered will depend at least in part on the above factors.

[0194] The administration of cells or cell colonies according to the present invention can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. Compositions as described herein can be administered to patients by subcutaneous, intradermal, intratumoral, intrasegmental, intramedullary, intramuscular, intravenous or intralymphatic injection or intraperitoneal administration. In one embodiment, the cell composition of the present invention is administered by intravenous injection.

[0195] The administration of cells or cell colonies can be composed of administering 104-109 cells / kg body weight, for example, 105-106 cells / kg body weight, including the number of cells of all integer values ​​within these ranges. Cells or cell colonies can be administered in one or more dosages. In another embodiment, the cells of the effective amount are administered in a single dose. In another embodiment, the cells of the effective amount are administered in more than one dosage over a period of time. The administration timing is determined by the attending physician and depends on the clinical condition of the patient. Cells or cell colonies can be obtained from any source, such as a blood bank or a donor. Although individual needs are different, it is within the technical scope of the art to determine the effective amount of a given cell type for a specific disease or condition. Effective amount refers to the amount providing a therapeutic or preventive benefit. The administered dosage will depend on the age, health and weight of the recipient, the type of simultaneous treatment (if any), the frequency of treatment and the nature of the desired effect.

[0196] It should be understood that the system is subject to many variables, such as cellular response to the ligand, expression efficacy and appropriate secretion levels, activity of the expression product, specific needs of the patient (which may vary over time and circumstances), rate of loss of cell activity due to loss of expression activity by the cell or individual cells, etc. Therefore, for each individual patient, even if there are universal cells that can be administered to the entire population, each patient's individual appropriate dosage must be monitored, and such practice of monitoring patients is routine in the art.

[0197] Nucleic acid-based expression systems

[0198] The chimeric B cell receptors of the present disclosure can be expressed from expression vectors. Recombinant techniques for generating such expression vectors are well known in the art.

[0199] As described herein, a DNA construct may also be referred to as a "DNA vector" and may be cloned into a vector for transduction and production of CASS B cells that secrete polypeptides and / or fragments thereof. For example, a DNA construct may be cloned into a lentiviral vector for production of a lentivirus that will be used to transduce and produce chimeric antigen receptor T cells that secrete mono-, bi- or tri-specific immunomodulatory antibodies / minibodies and / or antibody fusion proteins at a tumor site. For example, a DNA construct may be cloned into an adeno-associated viral vector for production of an adeno-associated virus that will be used to transduce and produce chimeric antigen receptor T cells that secrete mono-, bi- or tri-specific immunomodulatory antibodies / minibodies and / or antibody fusion proteins at a tumor site.

[0200] In embodiments, the DNA construct may comprise a nucleic acid encoding one or more polypeptides, such as a chimeric B cell receptor and / or a secreted polypeptide.

[0201] Carrier

[0202] The term "vector" may refer to a carrier nucleic acid molecule into which a nucleic acid sequence may be inserted for introduction into a cell where it can replicate. A nucleic acid sequence may be "exogenous," meaning that it is exogenous to the cell into which the vector is introduced, or that the sequence is homologous to a sequence in the cell, but that the sequence is not normally present at a position in the host cell nucleic acid. Vectors include plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One skilled in the art is able to construct vectors by standard recombinant techniques (see, e.g., Maniatis et al., 1988 and Ausubel et al., 1994, both of which are incorporated herein by reference in their entirety).

[0203] 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 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. An expression vector may contain a variety of "control sequences," which refer to nucleic acid sequences necessary for transcribing and translating an operably linked coding sequence in a specific host cell. In addition to control sequences for controlling transcription and translation, vectors and expression vectors may also contain nucleic acid sequences with other functions, as described below.

[0204] Promoters and enhancers

[0205] A "promoter" is a control sequence, which is a region of a nucleic acid sequence that controls the initiation and rate of transcription. It may contain genetic elements to which regulatory proteins and molecules may 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 a promoter is in the correct functional position and / or orientation relative to a nucleic acid sequence to control transcription initiation and / or expression of that sequence.

[0206] The promoter contains a sequence for locating the start site of RNA synthesis. The most famous example in this regard is the TATA box, but in some promoters lacking a TATA box, such as the promoter of the mammalian terminal deoxynucleotidyl transferase gene and the promoter of the SV40 late gene, discrete elements covering the start site itself help to fix the start site. Additional promoter elements regulate the frequency of transcription initiation. These are located in the region 30-110 BP upstream of the start site, although many promoters are also shown to contain functional elements downstream of the start site. In order to place the coding sequence under the control of the promoter, people place the 5' end of the transcription start site of the transcription reading frame "downstream" (i.e., 3') of the selected promoter. "Upstream" promoters stimulate the transcription of DNA and promote the expression of coding RNA.

[0207] The spacing between promoter elements is usually flexible so that promoter function is retained when elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can activate transcription either collaboratively or independently. A promoter may or may not be used with an "enhancer," which refers to a cis-acting regulatory sequence that participates in the activation of transcription of a nucleic acid sequence.

[0208] The promoter may be a promoter naturally associated with a nucleic acid sequence, which may be obtained by isolating a 5' non-coding sequence upstream of a coding segment and / or exon. Such promoters may be referred to as "endogenous". Similarly, an enhancer may be an enhancer naturally associated with a nucleic acid sequence, located downstream or upstream of the sequence. Alternatively, certain advantages may be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not usually associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not usually 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 any other virus or prokaryotic or eukaryotic cell, as well as promoters or enhancers that are not "naturally present", i.e., different elements containing different transcriptional regulatory regions, and / or mutations that alter expression. For example, the most commonly used promoters in recombinant DNA construction include beta-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to synthesizing nucleic acid sequences for generating promoters and enhancers, sequences can be generated using recombinant cloning and / or nucleic acid amplification techniques, including PCR.TM, in conjunction with the compositions disclosed herein (see U.S. Pat. Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference). In addition, control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, etc., can also be used.

[0209] Naturally, it is important to use promoters and / or enhancers that effectively direct the expression of the DNA fragment in the organelle, cell type, tissue, organ or organism selected for expression. Those skilled in the art of molecular biology generally know the use of promoters, enhancers and cell type combinations in protein expression (see, e.g., Sambrook et al., 1989, incorporated herein by reference). The promoter employed may be constitutive, tissue-specific, inducible, and / or may be used to direct high-level expression of the introduced DNA fragment under suitable conditions, such as to facilitate large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous.

[0210] In addition, any promoter / enhancer combination can also be used to drive expression. The use of T3, T7 or SP6 cytoplasmic expression systems is another embodiment. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters, whether as part of a delivery complex or as an additional gene expression construct, if the appropriate bacterial polymerase is provided.

[0211] The identities of tissue-specific promoters or elements, as well as assays to characterize their activity, are well known to those skilled in the art.

[0212] Effective translation of the coding sequence may also require specific start signals. These signals include the ATG start codon or adjacent sequences. It may be necessary to provide exogenous translation control signals, including the ATG start codon. Those of ordinary skill in the art will easily determine this and provide the necessary signals.

[0213] In certain embodiments of the present disclosure, internal ribosome entry site (IRES) elements are used to generate multigenic or polycistronic messages, and these may be used in the present invention.

[0214] A vector may include a multiple cloning site (MCS), which is a region of nucleic acid containing multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant techniques to digest the vector. "Restriction enzyme digestion" refers to the catalytic cleavage of a nucleic acid molecule with an enzyme that acts only at specific locations within the nucleic acid molecule. Many of these restriction endonucleases are commercially available. The use of these enzymes is widely understood by those skilled in the art. Typically, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to allow exogenous sequences to be attached to the vector. "Ligation" refers to the process of forming a phosphodiester bond between two nucleic acid fragments, which may or may not be contiguous to each other. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombinant technology.

[0215] Splice sites, termination signals, origins of replication and selectable markers may also be employed.

[0216] Plasmid vector

[0217] In certain embodiments, plasmid vectors can be used to transform host cells. Plasmid vectors containing replicons and control sequences are used together with these hosts, and the replicons and control sequences are derived from species compatible with the host cells. The vector usually carries a replication site, and a marker sequence that can provide phenotypic selection in the transformed cell. In a non-limiting example, Escherichia coli is usually transformed with a derivative of pBR322, which is a plasmid derived from an Escherichia coli species. pBR322 contains ampicillin and tetracycline resistance genes, and therefore provides a simple method for identifying transformed cells. pBR plasmids, or other microbial plasmids or phages must also contain, or be modified to contain, for example, a promoter that can be used by microorganisms to express their own proteins.

[0218] In addition, phage vectors containing replicons and control sequences compatible with the host microorganisms can be used as transformation vectors associated with these hosts. For example, phage lambda GEM.TM.11 can be used to prepare recombinant phage vectors that can be used to transform host cells, such as, for example, E. coli LE392.

[0219] Other useful plasmid vectors include pIN vectors (Inouye et al., 1985); and pGEX vectors for generating soluble fusion proteins of glutathione S transferase (GST) for subsequent purification and separation or cleavage. Other suitable fusion proteins are fusion proteins with β-galactosidase, ubiquitin, etc.

[0220] Bacterial host cells, such as E. coli, containing the expression vector are grown in a number of suitable culture media, such as LB. As will be appreciated by those skilled in the art, expression of recombinant proteins in certain vectors can be induced by contacting the host cells with agents specific to certain promoters, such as by adding IPTG to the culture medium or by switching the incubation to a higher temperature. After further culturing the bacteria for a period of time, such as between 2 and 24 hours, the cells are collected by centrifugation and washed to remove residual culture medium.

[0221] Viral vectors

[0222] Some viruses infect cells or enter cells via receptor-mediated endocytosis, and the ability to integrate into the host cell genome and stably and effectively express viral genes makes it an attractive candidate for transferring exogenous nucleic acids into cells (e.g., mammalian cells). The components of the present invention may be viral vectors encoding one or more CARs of the present invention. Non-limiting examples of viral vectors that can be used to deliver nucleic acids of the present invention are described below.

[0223] Adenoviral vector

[0224] Specific nucleic acid delivery methods include the use of adenoviral expression vectors. Although adenoviral vectors are known to have a low ability to integrate into genomic DNA, this feature is offset by the high efficiency of gene transfer provided by these vectors. "Adenoviral expression vectors" are meant to include those constructs containing adenoviral sequences sufficient to (a) support packaging of the construct, and (b) ultimately express tissue or cell specific constructs that have been cloned therein. Knowing the genetic structure of adenovirus (a 36 kb linear double-stranded DNA virus), large segments of adenoviral DNA can be replaced with exogenous sequences up to 7 kb (Grunhaus and Horwitz, 1992).

[0225] AAV vectors

[0226] Adenovirus-assisted transfection can be used to introduce nucleic acids into cells. It is reported that transfection efficiency is increased in cell systems using adenovirus coupling systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). Adeno-associated virus (AAV) is an attractive vector system for cells of the present invention because it has a high integration frequency and it can infect non-dividing cells, making it useful for gene delivery to mammalian cells, such as in tissue culture (Muzyczka, 1992) or in vivo. AAV has a wide range of infectious hosts (Tratschin et al., 1984; Lebkowski et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details on the generation and use of rAAV vectors are described in U.S. Patents 5,139,941 and 4,797,368, each of which is incorporated herein by reference.

[0227] Retroviral vectors

[0228] Retroviruses are useful as delivery vectors because of their ability to integrate their genes into the host genome, transfer large amounts of foreign genetic material, infect a broad spectrum of species and cell types, and be packaged in specialized cell lines (Miller, 1992).

[0229] To construct a retroviral vector, a nucleic acid (e.g., a nucleic acid encoding a sequence of interest) is inserted into the position of certain viral sequences in the viral genome to produce a replication-defective virus. To produce virions, a packaging cell line containing gag, pol, and env genes but without LTR and packaging components was constructed (Mann et al., 1983). When a recombinant plasmid containing cDNA is introduced into a specific cell line together with the retroviral LTR and packaging sequence (e.g., by calcium phosphate precipitation), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles and then secreted into the culture medium (Nicolas and Rubenstein, 1988: Temin, 1986; Mann et al., 1983). The culture medium containing the recombinant retrovirus is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).

[0230] Lentiviruses are complex retroviruses that contain, in addition to the common retroviral genes gag, pol, and env, other genes with regulatory or structural functions. Lentivirus vectors are well known in the art (see, e.g., Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U.S. Patents 6,013,516 and 5,994,136). Some examples of lentiviruses include human immunodeficiency virus: HIV-1, HIV-2, and simian immunodeficiency virus: SIV. Lentivirus vectors were generated by multiple attenuation of HIV virulence genes, e.g., genes env, vif, vpr, vpu, and nef were deleted, making the vector biologically safe.

[0231] Recombinant lentiviral vectors can infect non-dividing cells and can be used for in vivo and ex vivo gene transfer and expression of nucleic acid sequences. For example, recombinant lentiviruses can infect non-dividing cells, where suitable host cells are transfected with two or more vectors carrying packaging functions, i.e., gag, pol and env, as well as rev and tat, as described in U.S. Pat. No. 5,994,136, which is incorporated herein by reference. One can target the recombinant virus by linking the envelope protein to antibodies or specific ligands to target receptors of specific cell types. For example, by inserting a sequence of interest (including a regulatory region) into a viral vector together with another gene encoding a ligand for a receptor on a specific target cell, the vector is now target-specific.

[0232] Other viral vectors

[0233] Other viral vectors can be used as vaccine constructs of 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 provide several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

[0234] Delivery using modified viruses

[0235] The nucleic acid to be delivered can be contained in an infectious virus that has been engineered to express a specific binding ligand. Thus, the viral particle will specifically bind to the cognate receptor of the target cell and deliver the contents to the cell. A new approach designed for specific targeting of retroviral vectors has been developed based on chemical modification of retroviruses by chemical addition of lactose residues on the viral envelope. This modification allows specific infection of hepatocytes via the salivary glycoprotein receptor.

[0236] Another method for targeting recombinant retroviruses was designed, in which biotinylated antibodies to retroviral envelope proteins and specific cell receptors were used. Antibodies were coupled via the biotin component using streptavidin (Roux et al., 1989). Antibodies to major histocompatibility complex class I and class II antigens were used, which demonstrated the infection of a variety of human cells with these surface antigens in vitro with ecotropic viruses (Roux et al., 1989).

[0237] Vector delivery and cell transformation

[0238] Suitable methods for nucleic acid delivery for transfection or transformation of cells are known to those of ordinary skill in the art. These methods include, but are not limited to, direct delivery of DNA, such as by ex vivo transfection, injection, etc. Cells can be stably or transiently transformed by applying techniques known in the art.

[0239] In vitro transformation

[0240] Methods for transfecting eukaryotic cells and tissues taken out from an organism in an ex vivo environment are well known to those skilled in the art. Therefore, nucleic acid of the present invention can be used to remove and transfect cells or tissues in vitro. In some embodiments, the transplanted cells or tissues can be placed in an organism. In other embodiments, the nucleic acid is expressed in transplanted cells.

[0241] Pharmaceutical composition

[0242] The present invention provides a therapeutic composition or "pharmaceutical composition" or "formulation" comprising a CASS B cell as described herein and a pharmaceutically acceptable carrier. The present invention also provides a therapeutic composition comprising a nucleic acid as described herein and a pharmaceutically acceptable carrier.

[0243] As used herein, "pharmaceutical composition" or "pharmaceutical formulation" may refer to a composition or pharmaceutical composition suitable for administration to a subject (such as a mammal, especially a human), and refers to a combination of one or more active agents (e.g., genetically engineered cells) or ingredients with a pharmaceutically acceptable carrier or excipient, such that the composition is suitable for in vitro, in vivo or ex vivo diagnostic, therapeutic or preventive use. According to the present invention, the pharmaceutical composition may be sterile and free of contaminants that can cause undesirable reactions in the subject (e.g., the compounds in the pharmaceutical composition are pharmaceutical grade). The pharmaceutical composition may be designed for administration to a subject or patient in need thereof via a variety of different routes of administration, including oral, intravenous, oral, rectal, parenteral, intraperitoneal, intradermal, intratubular, intramuscular, subcutaneous, inhalation, and the like.

[0244] In an embodiment, the pharmaceutical composition may include components that ensure the viability of CASS B cells therein. In particular, the cells can be provided in the form of a pharmaceutical composition that includes an isotonic excipient for human administration prepared under sufficiently sterile conditions. Regarding the general principles of pharmaceutical preparations, the reader can refer to Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, edited by G. Morstyn & W. Sheridan, Cambridge University Press, 1996, which is incorporated herein by reference in its entirety. The selection of the cell excipient and any accompanying ingredients of the composition will be adjusted according to the device used for administration. For example, the composition may include a suitable buffer system to a suitable pH, such as a pH close to neutral (e.g., a phosphate or carbonate buffer system), and may include sufficient salt to ensure isotonic conditions for the cells, i.e., to prevent osmotic stress. For example, a suitable solution for these purposes may be phosphate buffered saline (PBS) known in the art. In addition, the composition may include a carrier protein, such as albumin, which can increase the viability of the cells. To ensure exclusion of non-human animal material, the albumin may be of human origin (eg, isolated from human material or recombinantly produced). Suitable concentrations of albumin are generally known.

[0245] Therefore, the pharmaceutical composition according to the present invention and used according to the present invention may include a pharmaceutically acceptable excipient, carrier, buffer, preservative, stabilizer, antioxidant or other substances well 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 substance will depend on the route of administration. The composition may include one or more cell protection molecules. Such substances can make cells independent of their environment.

[0246] "Pharmaceutically acceptable excipient", "pharmaceutically acceptable diluent", "pharmaceutically acceptable carrier" or "pharmaceutically acceptable adjuvant" may refer to excipients, diluents, carriers and / or adjuvants that can be used to prepare pharmaceutical compositions that are generally safe, non-toxic and have no adverse effects biologically or otherwise, and include acceptable excipients, diluents, carriers and adjuvants for veterinary use and / or human pharmaceutical use. Pharmaceutically acceptable excipients, diluents, carriers and / or adjuvants used in the specification and claims include one or more such excipients, diluents, carriers and adjuvants.

[0247] The present invention also encompasses methods for producing the pharmaceutical composition by mixing cells of the present invention and / or nucleic acid with one or more other components as described above. Liquid pharmaceutical compositions typically include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oils or synthetic oils. Physiological saline solutions, tissue or cell culture media, glucose or other sugar solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

[0248] The composition can be in the form of an acceptable aqueous solution for parenteral administration, which does not contain pyrogens and has suitable pH, isotonicity and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonic carriers such as sodium chloride, Ringer's injection or lactated Ringer's injection. The composition can be prepared using biological fluids such as artificial cerebrospinal fluid. In other aspects, the present invention relates to a device, which is arranged to include surgical instruments for applying compositions at tissue dysfunction or injury sites, and also includes pharmaceutical compositions as defined above, wherein the device is suitable for applying pharmaceutical compositions at tissue dysfunction or injury sites. For example, suitable surgical instruments can inject liquid compositions comprising genetically engineered cells as described herein at neural dysfunction or injury sites. Cells can be implanted in patients by any technology known in the art, including the technology described in the following: Freed et al. 1997. Cell Transplant 6:201-202; Kordower et al., 1995. N Engl J Med 332:1118-1124; 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 these documents are incorporated herein by reference.

[0249] Method for preparing genetically engineered B cell populations

[0250] Embodiments of the present invention also relate to methods for preparing genetically engineered B cell populations.For example, such methods include isolating a B cell population from a subject, and transducing the B cell population with a vector as described herein, thereby producing a genetically engineered B cell population.

[0251] The cells, such as B cells, into which the polynucleotide is to be introduced can be obtained from a source such as the subject itself, a donor subject, or a cell bank. For example, the cells can be obtained from the subject, just as B cells can be used for autologous transplantation.

[0252] In embodiments, the polynucleotide may be introduced into the cell by transduction, such as by phage or viral transfer; transformation, such as by extracellular uptake of naked DNA or microinjection.

[0253] Positive and negative controls can be used as needed. For example, a positive control for transduction efficiency can be an empty plasmid lentiviral stock carrying mCherry, eGFP or other fluorescent molecule tags such as YFP, BFP or RFP.

[0254] In embodiments, the method further comprises the step of activating the B cell population prior to transduction.

[0255] In embodiments, the method may further include the step of culturing a genetically engineered B cell population. Cultivating cells may refer to a process in which cells are maintained under conditions suitable for maintenance and / or growth, wherein conditions may refer to, for example, the temperature at which the cells are maintained, nutrient availability, atmospheric CO2 content, and cell density. Cells may be cultured in vivo or in vitro. Suitable culture conditions for maintaining, proliferating, expanding, and differentiating different types of cells are known to the skilled person. See, for example, Moffett, HF et al. (2019). B cells are engineered to express pathogen-specific antibodies to protect against infection. Science Immunology, 4(35).

[0256] Kit of the present invention

[0257] Any composition described herein may be included in a kit. In a non-limiting example, one or more cells for cell therapy and / or reagents for generating one or more cells for cell therapy may be included in a kit, the reagents carrying a recombinant expression vector. The kit components are provided in a suitable container.

[0258] Some components of the test kit may be packaged in an aqueous medium or in a lyophilized form. The container means of the test kit may include at least one vial, test tube, flask, bottle, syringe or other container means, in which the components may be placed and appropriately aliquoted. When there is more than one component in the test kit, the test kit may also include a second, third or other additional container, in which the additional components may be placed separately. However, various combinations of components may be included in vials. The test kit of the present invention may also include a device for enclosing and containing components in commercial sales. Such containers may include injection or blow molded plastic containers, in which the desired vials are retained.

[0259] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, of which sterile aqueous solutions are particularly useful. In some cases, the container device 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 even applied to and / or mixed with other components of the kit.

[0260] However, the components of the kit may be provided as dry powders. When the reagents and / or components are provided as dry powders, the powders may be reconstituted by adding a suitable solvent. For example, the solvent may also be provided in another container means. The kit may also include a second container means for containing a sterile, pharmaceutically acceptable buffer and / or other diluent.

[0261] In a specific embodiment of the invention, cells for cell therapy are provided in a kit, and in some cases, the cells are substantially the only component of the kit. The kit may include reagents and materials for preparing the desired cells. In a specific embodiment, the reagents and materials include primers, nucleotides, suitable buffers or buffering agents, salts, etc. for amplifying the desired sequence, and in some cases, the reagents include vectors and / or DNA encoding chimeric B cell receptors as described herein and / or their regulatory elements.

[0262] In certain embodiments, one or more devices are present in the kit that are suitable for extracting one or more samples from an individual. The device may be a syringe, a scalpel, etc.

[0263] 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, hormone therapy and / or immunotherapy. The kit can be customized for a particular cancer in an individual and include a corresponding second cancer therapy for that individual.

[0264] Example

[0265] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes for preparing and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples for illustrative purposes only, because similar results can be obtained using alternative methods.

[0266] Example 1

[0267] Engineered chimeric antibody signaling and secreting (CASS) B cells as a targeting and inducible platform at tumor sites Secretory immunomodulatory proteins

[0268] Membrane Ig constructs (synthetic BCRs) were designed by linking the scFv to a membrane-tethered IgG or IgM hinge-CH2-CH3 with native transmembrane and intracellular domains. A secondary cassette was inserted behind the syn-BCR plasmid with multiple NFAT and / or NF-k response elements and a minimal IL2 / IL8 promoter upstream of the secreted protein (Ab or other protein, such as cytokine).

[0269] In native B cells, engagement of the BCR results in rapid tyrosine phosphorylation of the intracellular domain and calcium ion polarization, leading to downstream activation of NFAT and NF-kB. Using NFAT / NF-kB response elements to drive expression of our secretory proteins, we designed an inducible expression system that will be activated by antigens expressed on cancer cells. This creates a targeted and inducible delivery system that secretes its therapeutic payload only when activated by tumor cells, resulting in localized areas of consistent and high immunomodulatory Ab / protein concentrations centered on the tumor. This can reverse the suppressive nature of the tumor microenvironment, leading to improved outcomes and tumor elimination, while reducing the targeted / non-targeted tumor side effects often seen with systemic delivery of therapeutic antibodies and cytokines.

[0270] Non-limiting examples include anti-CAIX CASS B cells that will secrete anti-PD 1 / anti-CTLA4 bispecific antibodies, and anti-mesothelin CASS B cells that will secrete anti-PD1 / anti-TIGIT bispecific antibodies. Non-limiting examples of other secreted antibodies include anti-CCR4, anti-PDL 1, 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.

[0271] Figure 1 This is a schematic diagram of a CASS B cell. This schematic uses separate plasmids for the syn-BCR and secreted Ab, however this can also be combined into one plasmid.

[0272] Current treatments include CAR T cells and systemic Ab delivery. CAR T cell therapy is associated with cytotoxicity. Unlike CAR-T cells, the embodiments herein use B cells, which do not cause direct cytotoxicity to tumors as seen with CAR T cells. Instead, the genetically 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 will allow the rest of the immune system to destroy the tumor and provide lifelong protection against tumor regrowth and metastasis. The addition of an inducible system is also a key component because it will reduce the serum concentration of our Ab and reduce tumor / target side effects.

[0273] Therefore, the embodiments described herein can be used to treat solid tumors, especially solid tumors with a very strong immunosuppressive tumor microenvironment. The embodiments described herein can also be used to prevent and / or treat other indications, including infectious diseases.

[0274] Example 2

[0275] Engineering chimeric antibody signaling and secreting (CASS) B cells for cancer therapy

[0276] summary

[0277] Immune checkpoint blockade inhibitors (CBI) and CAR T cells have revolutionized the way we treat cancer. Although both therapies involve the patient's immune system, they cannot actively initiate anti-tumor immune responses, and their scope of use and efficacy are significantly limited. To address this problem, this article describes chimeric antibody secretion and signaling (CASS) B cells, which express engineered tumor-targeted B cell receptors and, once engaged, will locally secrete high levels of dual-targeted bispecific checkpoint blockade regulator antibodies (e.g., dual-targeted bispecific checkpoint inhibitor antibodies) at the tumor site. Since B cells also act as professional antigen-presenting cells, they can process and present antigens on class II molecules, further enhancing immune cell recognition of tumors and helping new antigens spread. As a key component of immune memory, CASS B cells will simultaneously recruit a wide range of immune cells and reverse tumor-infiltrating lymphocyte depletion, providing a robust and lifelong monitoring plan to prevent tumor metastasis and recurrence. Non-small cell lung cancer (NSCLC) was selected as a model to develop anti-MSLN-directed CASS B cells that secrete immunomodulatory anti-PD1 / TIGIT bispecific antibodies (bsAbs). Objective 1 will focus on the development of the CASS B cell platform, by the end of this phase we will identify three antibodies and optimize the signaling domains comprising the CASS B cells. In Objective 2, in vitro characterization and efficacy testing will be performed, providing a clear understanding of the link between CASS B cell activation and bsAb secretion, while providing key analysis of CASS B cell efficacy compared to CAR T cells at the functional and molecular level. Objective 3 will perform in vivo experiments in humanized mice using cell line-derived and patient-derived NSCLC models. Multiparameter flow cytometry, single-cell RNA sequencing, and immunohistochemistry will provide a detailed assessment of the molecular and mechanistic efficacy of the immunomodulatory bsAbs and the CASS B cell platform as a whole.

[0278] Research objectives

[0279] Without being bound by theory, we will develop a new type of combined cellular immunotherapy, namely chimeric antibody secretion and signaling (CASS) B cells. These B cells will express engineered tumor-associated antigens (TAAs) targeting B cell receptors (BCRs), and once engaged, will secrete high levels of checkpoint blockade regulators (e.g., dual-targeted bispecific checkpoint inhibitor antibodies) bispecific antibodies (bsAbs) locally at the tumor site. This will allow reversal of the immunosuppressive tumor microenvironment (TME) and restoration of the patient's natural innate and adaptive immunity to eliminate cancer cells. In addition, unlike T cells, B cells act as professional antigen presenting (APC) cells and should enhance tumor cell recognition and help new antigens spread, resulting in reversal of tumor infiltrating lymphocytes (TIL) exhaustion and induction of a broader and more robust anti-tumor immune response. For proof of principle studies, we propose to design a CASS B cell that secretes anti-PD 1 / TIGIT bsAbs that targets mesothelin (MSLN) for the treatment of NSCLC1-3.

[0280] The first goal focuses on the construction and optimization of engineered anti-MSLN IgG-BCR, anti-PD 1 / TIGIT bsAbs that will be delivered to tumor sites, and inducible response elements (REs) that drive bsAb expression. Our laboratory has characterized panels of anti-MSLN, anti-PD1, and anti-TIGIT antibodies, and functional assays will identify lead candidates. Concurrent efforts will focus on the development of inducible response elements and optimization of B cell transduction conditions.

[0281] The second goal is to evaluate the function of anti-MSLN CASS B cells in vitro. Activation assays will be used to quantify bsAb and cytokine secretion levels. Patient-derived organotypic tumor spheres (PDOTS) will be used to evaluate CASS B cell efficacy and compare molecules / mechanisms with CAR T cells via cytokine profiles, IHC, and single-cell RNA sequencing (scRNAseq). Moreover, the implementation scheme can use mesothelioma tumors for PDOTs.

[0282] The ultimate goal will be to generate cell line-derived (CDX) and patient-derived xenograft (PDX) models of NSCLC using HLA-matched humanized mice to test CASS B cell efficacy. The models will be paired with various analytical techniques (IHC, flow cytometry, scRNAseq) to further explore the impact of CASS B cell therapy on the surrounding TME.

[0283] background

[0284] Checkpoint blockade inhibitor (CBI) monoclonal antibodies (mAbs) and adoptive cell therapy have had a transformative impact on cancer treatment, shifting the focus from simply killing tumor cells to activating the patient's natural anti-tumor immunity and reversing the immunosuppressive tumor microenvironment (TME). Although these represent the most promising anti-cancer therapies to date, only a small percentage of patients experience complete or durable responses, and many patients experience varying degrees of severity. 4-7 To address this issue, combined CBI therapies such as anti-PD(L)1 / anti-TIGIT have been tested and are currently under investigation in clinical trials. 8-11 Another approach is to develop armored or immune-restoring CAR T cells that are engineered to secrete immunomodulatory payloads directly at the tumor site, thereby improving efficacy while reducing systemic delivery. 12，13 On-target / off-tumor side effects seen in

[0285] In addition to T cells, various immune cells can be used to generate new CARs, including natural killer cells (NK-CAR) and macrophages (CAR-M), all of which have in common that they provide direct anti-tumor activity. 14，15 B cells are an important component of humoral immunity and the antibodies they produce were the basis for the initial development of immunotherapy. However, they do not possess intrinsic cytotoxic capacity and have been largely excluded from these advances.

[0286] The Chimeric Antibody Secretion and Signaling (CASS) B cell platform will bring B cell research into the 21st century by developing a unique B cell-based cell therapy that does not rely on direct cytotoxicity, but instead exploits 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, leading to the recruitment of CD4+ T cells and allowing for enhanced tumor cell recognition and neoantigen spread. While the inducible targeted delivery of CBIs will reduce irAEs, the ability to act as professional APCs makes the CASS B cell platform unique among cell therapy platforms as CASS B cells have the ability to initiate robust anti-tumor responses. This impact has been demonstrated in the demonstration of MHC class II neoantigens in the innate anti-tumor response. 16，17 This is further exemplified by the work of the United Nations in which the United Nations plays a key role.

[0287] Although this example focuses on MSLN+ targeted CASS B cells secreting anti-PD 1 / TIGIT bsAbs against NSCLC, the modular design of CASS B cells allows for easy targeting of other TAAs, and the secreted payload can be tuned to target relevant immune axes, allowing CASS B cells to be adapted to multiple cancers. Because B cells are long-lived and are an important component of immune memory, CASS B cells continuously deliver therapeutic payloads to tumors and provide a lifelong immune surveillance system against metastasis and recurrence after the primary tumor has been eradicated.

[0288] B cell engineering is a recent achievement that has focused primarily on creating B cells that secrete neutralizing, anti-pathogen antibodies against RS and HIV21-23. Unlike the cancer treatments proposed here, the B cells described in these works 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, which is a requirement for combating infectious diseases but not for targeting immune markers. The sum of these works indicates that it is possible to manipulate B cells to express selected engineered antibodies in an inducible manner and that upon activation, these engineered B cells differentiate not only into Ab-secreting cells but also into memory B cells that provide long-term protection24.

[0289] Non-limiting examples of research plans

[0290] Objective 1: Engineering and optimization of CASS B cell constructs - Appropriate scFvs (anti-MSLN, PD1, TIGIT) will be selected for the development of CASS B cells, followed by optimization of signaling domains and inducible response elements25-28. Various lentiviral envelope proteins and culture conditions will also be utilized 29-32 Optimize protocols for efficient transduction and exponential expansion of primary B cells.

[0291] Non-limiting examples of experimental design and procedures

[0292] Antibody discovery, engineering and optimization: We have previously established a 27 billion member human phage library for the isolation of many therapeutic antibodies33-37. Anti-PD1 ( Figure 2 Figure A), TIGIT ( Figure 2 Figure B) and MSLN ( Figure 2 The group in panel C) has been used for further engineering and optimization.

[0293] Bispecific antibody design and engineering: Since PD1 / TIGIT is expressed on immune cells, immune depletion is not required 38，39The current design utilizes a tandem scFv construct previously developed and characterized in the Marasco lab, however other designs will also be considered ( Figure 3 Figures A to C).

[0294] B cell isolation, expansion, and transduction: B cells will be isolated and various expansion media formulations will be tested 23，40，41 For transduction, B cells will be activated, transduced, and sorted 72 hours after transduction.

[0295] Generation of IgG-BCR constructs: Our engineered IgG-BCRs were constructed using scFv fused to a membrane-bound IgG1 hinge Fc that is expressed at high levels and binds to target antigens ( Figure 4 Figure A) 42 Transduction experiments using primary B cells demonstrated high titer transduction of multiple donors and DNA constructs ( Figure 4 Figure B). We have previously developed an inducible T cell activation assay using the NFAT / NFkB RE ( Figure 4 Panel C). For some experiments, a fluorescent protein will be used instead of a secreted bsAb.

[0296] The monoclonal antibody engineering techniques routinely employed in our laboratory will allow us to develop panels of previously discovered monoclonal antibodies to find lead candidates for each target. Without being bound by theory, we were able to achieve high transduction efficiencies and exponential expansion of transduced cells in in vivo experiments.

[0297] Milestones: The first milestone will be the identification of lead antibodies for each target. The second milestone will be the construction of vectors and successful transduction / expansion of CASS B cells.

[0298] Objective 2: In vitro testing and efficacy of CASSB cells - In-depth in vitro characterization will be performed to determine activation thresholds and quantify bsAb secretion levels. Final in vitro assays will be performed using patient-derived organotypic tumor spheroids (PDOTS), allowing us to observe CASSB cell homing and perform detailed analysis of bsAb payloads.

[0299] Experimental design and procedures

[0300] CASS B cell activation assay: Supernatants from activated CASS B cells will be screened via ELISA to measure bsAb and cytokine concentrations.

[0301] NSCLC PDOT Generation and Evaluation: Generation of NSCLC PDOTs will be performed in the laboratory of Dr. David Barbie following the protocols outlined by Jenkins et al. and Aref et al. 43，44. In addition to testing bsAb and CASS B cell efficacy, a comparative experiment will be performed to determine the therapeutic differences between CASS B cell and CAR T cell therapy. Immune profiling via IHC, scRNAseq, and cytokine profiling will be performed. Due to the limited availability of NSCLC tumor tissue, a backup plan has been designed to use mesothelioma tumors from PDOT generation. Mesothelioma has a highly suppressive TME and immune infiltrating cells show high levels of exhaustion markers, making it an ideal alternative for in vitro assays. 45-48 .

[0302] Without being bound by theory, we can provide a deep understanding of the activation potential of CASS B cells and identify the best anti-MSLN scFv for selective targeting of MSLN+ tumors. PDOT will provide a comprehensive data set of CASSB cell efficacy via cytokine secretion and transcriptional profiles, and without being bound by theory, anti-MSLN CASS B cells will traffic to tumors and secreted bsAbs will reverse immune cell suppression. Additionally, and without being bound by theory, we can see that CASS B cells have greater epitope spreading and bystander immune cell activation compared to CAR T cell therapy.

[0303] Milestones: The first milestone in Aim 2 may be the generation of CASS B cell activation curves and quantification of bsAb secretion. The next milestone will be the establishment of PDOTS and efficacy testing of the bsAb system as a CASS B cell payload. The final milestone will be the comparison of CASS B cell and CAR T cell therapies via IHC and scRNAseq and the generation of immune profiles for each therapy.

[0304] 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 in final testing will improve tumor integrity and phenotypic characterization while accurately mimicking the in vivo tumor TME.

[0305] Experimental design and procedures

[0306] NSCLC CDX and PDX models established in humanized mice: NSCLC cell lines and PDX models will be screened for PDL1 and CD155 expression levels prior to fluorescent treatment 49 Human immune system reconstitution will proceed as reported by us and others 50-52 To generate growth curves, NSCLC cell lines / PDX tumors were transplanted into the flanks of humanized mice at different concentrations. 53 .

[0307] Evaluate the efficacy of CASS B cells 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 multiparametric FACS, IHC / ISH, and scRNASeq following established protocols commonly used in our laboratory. 54-63 .

[0308] We have extensive experience in generating humanized mice and CDX / PDX models and will develop successful NSCLC models. These models will demonstrate that CASS B cells accumulate around tumors and secrete high levels of bsAbs, reducing the immunosuppressive nature of the TME and recruiting additional anti-tumor immune cells. IHC / ISH and scRNAseq will provide molecular evidence of bsAb efficacy, CASS B cell homing, and APC pathway activation in CASS B cells and CD4 T cells, while 5′ scRNA-seq of TCR / BCRs will be used to directly monitor epitope spreading in TILs.

[0309] Statistical considerations: With 5 animals per group for single pairwise comparisons between conditions of interest and different outcome measures, we have a power of 0.93 to detect differences in means equal to 2.5 SD at the 0.05 level using a two-sample two-tailed t-test. Additional statistical analyses will be provided by consultation with the Dana-Farber Biostatistics Center.

[0310] Milestones: The first milestone for Aim 3 will be the generation of CDX models and PDX models for NSCLC. Subsequent milestones will be the initiation and completion of planned animal studies to test bsAbs in CDX models and CASSB cells in CDX and PDX models. Due to the large amount of data generated by these experiments, the third milestone will be the completion of data analysis for each animal study.

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[0336] 25. Uchibori, R. et al., Functional Analysis of an Inducible Promoter Drivenby Activation Signals from a Chimeric Antigen Receptor. Mol. Ther.-Oncolytics 12, 16-25 (2019).

[0337] 26. Zhang, L et al., Improving adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment. Mol. Ther. 19, 751-759 (2011).

[0338] 27. Hooijberg, E., Bakker, AQ, Ruizendaal, JJ & Spits, H. NFAT-controlled expression of GFP permits visualization and isolation of antigen-stimulated primary human T cells. Blood 96, 459-466 (2000).

[0339] 28. Lin, T. et al., Establishment of NF-κB sensing and interleukin-4 secreting mesenchymal stromal cells as an "on-demand" drug delivery system to modulate inflammation. Cytotherapy 19, 1025-1034 (2017).

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[0341] 30. Tomas, H.A. et al., Improved GaLV-TR Glycoproteins to Pseudotype Lentiviral Vectors: Impact of Viral Protease Activity in the Production of LV Pseudotypes. Mol. Ther.-Methods Clin. Dev. 15, 1-8 (2019).

[0342] 31. Bernadin, O. et al., Baboon envelope LVs efficiently transduced human adult, fetal, and progenitor T cells and corrected SCID-X1 T-cell deficiency. Blood Adv. 3, 461-475 (2019).

[0343] 32. Winiarska, M. et al., Selection of an optimal promoter for gene transfer in normal B cells. Mol. Med. Rep. 16, 3041-3048 (2017).

[0344] 33. Xu, C. et al., Unique biological properties of catalytic domain-directed human anti-CAIX antibodies discovered through phage-display technology. PLoS One 5, (2010).

[0345] 34. Sui, J. et al., Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat. Struct.

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[0348] 36. Chang, D.-K. et al., Humanization of an anti-CCR4 antibody that kills cutaneous T-cell lymphoma cells and abrogates Suppression by T-regulatory cells. Mol. Cancer Ther. 11, 2451 - 2461 (2012).

[0349] 37. Chang, D.K. et al., Humanized mouse G6 anti-idiotypic monoclonal antibody has therapeutic potential against IGHV1-69 germline gene-based B-CLL. MAbs 8, 787 - 798 (2016).

[0350] 38. Marin-Acevedo, J.A. et al., Next generation of immune checkpoint therapy in cancer: New developments and challenges. J. Hematol. Oncol. 11, 1-20 (2018).

[0351] 39. Saunders, K.O. Conceptual approaches to modulating antibody effector functions and circulation half-life. Front. Immunol. 10, 1-20 (2019).

[0352] 40. Su, K.-Y., Watanabe, A., Yeh, C.-H., Kelsoe, G. & Kuraoka, M. Efficient Culture of Human Naive and Memory B Cells for Use as APCs. J. Immunol. 197, 4163-4176 (2016).

[0353] 41. Kuraoka, M. et al., Complex Antigens Drive Permissive Clonal Selection in Germinal Centers. Immunity 44, 542-552 (2016).

[0354] 42. Wan, Z. et al., The activation of IgM- or isotype-switched IgG- and IgE-BCR exhibits distinct mechanical force sensitivity and threshold. Elife 4, 1-24 (2015).

[0355] 43. Aref, A.R. et al., 3D microfluidic: Ex vivo culture of organotypic tumor spheroids to model immune checkpoint blockade. Lab Chip 18, 3129-3143 (2018).

[0356] 44. Jenkins, R.W. et al., Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids. Cancer Discov. 176, 139-148 (2018).

[0357] 45. Lv, J. & Li, P. Mesothelin as a biomarker for targeted therapy. Biomark Res. 7, (2019).

[0358] 46. Marcq, E. et al., Abundant expression of TIM-3, LAG-3, PD-1 and PD-L1 as immunotherapy checkpoint targets in effusions of mesothelioma patients. Oncotarget 8, 89722-89735 (2017).

[0359] 47. Klampatsa, A. et al., Phenotypic and functional analysis of malignant mesothelioma tumor-infiltrating lymphocytes. Oncoimmunology 8, 1-12 (2019).

[0360] 48. Alay, A. et al., Integrative transcriptome analysis of malignant pleural mesothelioma reveals a clinically relevant immune-based classification. J. Immunother. Cancer 9, (2021).

[0361] 49. Liu, J.F. et al., Establishment of patient-derived tumor xenograft models of epithelial ovarian cancer for preclinical evaluation of novel therapeutics. Clin. Cancer Res. 23, 1263-1273 (2017).

[0362] 50. Biswas, S. et al., Humoral immune responses in humanized BLT mice immunized with West Nile Virus and HIV-1 envelope proteins are largely mediated via human CD5+ B cells. Immunol. 134, 419 - 433 (2011).

[0363] 51. Chang, H. et al., Human B-cell ontogeny in humanized NOD / SCIDγc(null) mice generates a diverse yet auto / poly- and HIV-1-reactive antibody repertoire. Genes Immun. 13, 399 - 410 (2012).

[0364] 52. Billerbeck, E. et al., Development of human CD4+FoxP3+ regulatory T cells in human stem cell factor-, granulocyte-macrophage colony-stimulating factor-, and interleukin-3-expressing NOD-SCID IL2Rγnull humanized mice. Blood 117, 3076 - 3086 (2011).

[0365] 53. Lin, S. et al., Establishment of peripheral blood mononuclear cell-derived humanized lung cancer mouse models for studying efficacy of PD-L1 / PD-1 targeted immunotherapy. MAbs 10, 1301 - 1311 (2018).

[0366] 54. Stoeckius, M. et al., Cell Hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics. Genome Biol. 19, 1 - 12 (2018).

[0367] 55. Tian, L. et al., scPipe: A flexible R / Bioconductor preprocessing pipeline for single - cell RNA - sequencing data. PLoS Comput. Biol. 14, 1 - 15 (2018).

[0368] 56. Amezquita, R.A. et al., Orchestrating single - cell analysis with Bioconductor. Nat. Methods 17, 137 - 145 (2020).

[0369] 57. Hao, Y. et al., Integrated analysis of multimodal single - cell data. Cell 184, 3573 - 3587.e29 (2021).

[0370] 58. Lytal, N., Ran, D. & An, L Normalization Methods on Single - Cell RNA - seq Data: An Empirical Survey. Front. Genet. 11, 1 - 14 (2020).

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[0372] 60. Haghverdi, L, Lun, ATL, Morgan, MD & Marioni, JCBatch effects insingle-cell RNA-sequencing data are corrected by matching mutual nearest neighbors. Nat. Biotechnol. 36, 421-427 (2018).

[0373] 61. Chen, Y. et al. edgeR: differential analysis of sequence read countdata User’s Guide. RPackag. 1-121 (2020).

[0374] 62. Subramanian, A. et al., Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545-15550 (2005).

[0375] 63. DeTomaso, D. et al., Functional interpretation of single cell similarity maps. Nat. Commun. 10, (2019).

[0376] Example 3

[0377] Exploring the B-CLL TME in vitro and in vivo using CASSB cells and patient-derived tumor cells

[0378] Chimeric Antibody Signaling and Secretion (CASS) B cells will be used to study the CLL B tumor microenvironment (TME) in vitro and in vivo in tumor-bearing mice. CASS B cells inducibly secrete checkpoint blockade modulator Abs (e.g., checkpoint inhibitor Abs) at tumor sites. Unlike CAR T and NK cells, CASS B cells are antigen presenting cells (APCs) and will promote neoantigen recognition and spread upon reversal of T cell exhaustion. Studies will continue in tumor spheroids and tumor-bearing mice.

[0379] Over the past decade, immune checkpoint blockade inhibitor (CBI) antibodies and CAR T cells have revolutionized the way we treat cancer. While both therapies involve the patient's immune system, neither can actively initiate an anti-tumor immune response, and there are significant limitations to their scope of use and efficacy. To address this issue, we will develop chimeric antibody secreting and signaling (CASS) B cells that express an engineered tumor-targeting B cell receptor (BCR) and, once engaged, will secrete high levels of CBI locally at the tumor site. Because B cells also act as professional antigen-presenting cells, they can process and present antigens on class II molecules, further enhancing immune cell recognition of tumors and helping new antigens spread. As a key component of immune memory, CASS B cells will simultaneously recruit a wide range of immune cells and reverse tumor-infiltrating lymphocyte exhaustion, providing a robust and lifelong monitoring program to prevent tumor metastasis and recurrence.

[0380] B CLL was chosen as a model to develop anti-IGHV 1-69 directed CASS B cells that secrete immunomodulatory CBIs. The discovery that the IGHV1-69 encoded BCR expressed on B-CLL cells is always an unmutated VH fragment provides an opportunity for anti-idiotypic CASS B cell therapy. We have humanized the G6 (hG6.3) mAb, completed co-crystallography studies of (G6-id+) hG6.3 with its BCR target, and demonstrated that hG6.3 binds to germline-encoded residues in the VH complementarity determining region (CDR-H2). We have also isolated several potent CBI- and bi-specific Abs that will be studied. In Aim 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 will characterize the immune profile of these tumors using scRNASeq, immune receptor profiling, CITESeq, and multiplexed IHC to investigate the tumor microenvironment (TME) of IGHV1-69+ and IGHV1-69-B CLL. In Aim 2, we will treat PDOTs with CASS B cells secreting different CBIs, followed by molecular analysis to determine the effects of different CBIs on TME immune signaling. These ex vivo studies will lead to 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 the functional and molecular levels. In Aim 3, we will treat humanized mice bearing B-CLL tumors with CASS B cells, which showed the greatest restoration of anti-tumor immunity in Aim 2. The scRNASeq and other interrogations described above will provide a detailed assessment of the molecular and mechanistic efficacy of the CBI and CASS B cell platform as a whole. At the end of this phase, we will gain a deep understanding of the B CLL TME and CASS B cell interventions at the molecular level that can best restore anti-B CLL immunity.

[0381] Checkpoint blockade inhibitor (CBI) monoclonal antibodies (mAbs) and adoptive cell therapy have had a transformative impact on cancer therapy, shifting the focus from simply killing tumor cells to activating the patient's natural anti-tumor immunity and reversing the immunosuppressive tumor microenvironment (TME). Although these represent the most promising anti-cancer therapies to date, only a small percentage of patients experience complete or durable responses, and many patients experience immune-related adverse events (irAEs) of varying severity. Another approach is to develop armored or immune-restored CAR T cells that are engineered to secrete immunomodulatory payloads directly at the tumor site, thereby improving efficacy while reducing the on-target / off-tumor side effects that occur with systemic delivery. In addition to T cells, various immune cells have been used to generate new CARs, including natural killer cells (NK-CARs) and macrophages (CAR-Ms), and what all these immune cell therapies have in common is that they provide direct anti-tumor killing activity. B cells are an important component of humoral immunity, and the antibodies they produce are the basis for the initial development of immunotherapy. However, they do not have intrinsic cytotoxic capacity and have therefore been largely excluded from these advances.

[0382] The Chimeric Antibody Signaling and Secretion (CASS) B cell platform is a novel and high-risk program designed to advance B cell research to promote its participation in 21st century therapies. We will develop CASS B cell therapies to study and treat aggressive, therapy-resistant IGHVl-69-derived B CLL. CASS B cells are a unique B cell-based cell therapy that does not rely on direct cytotoxicity, but instead exploits 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, leading to the recruitment of CD4+ T cells and allowing enhanced tumor cell recognition and neoantigen spread. While the inducible targeted delivery of CBIs will reduce irAEs, the ability to act as professional antigen presenting cells (APCs) makes the CASS B cell platform unique among cell therapy platforms because CASS B cells have the ability to initiate robust anti-tumor responses. MHC class II neoantigens play a role in innate anti-tumor responses. We will perform ex vivo and in vivo studies in humanized mice bearing B-CLL patient leukemia cells treated with CASS B cells secreting the different CBI payloads we have discovered.

[0383] Example 4

[0384] The role of B cells in our immune system is to recognize foreign invaders from microorganisms to cancer cells and eliminate them by producing antibodies (Abs) that bind and remove threats. B cells achieve this by expressing membrane-bound Abs (B cell receptors BCRs) that bind to tumor antigens, causing B cells to transform from cells expressing BCRs to cells that secrete Abs. After BCR involvement, rapid tyrosine phosphorylation and calcium ion polarization mediated by two major tyrosine kinases Lyn and Syn. These biochemical events lead to the activation of downstream signaling pathways, leading to further downstream activation of NF-kB and NFAT signals, and ultimately leading to B cell expansion and robust mAb secretion. Without being bound by theory, we will design chimeric antibody signaling and secretion (CASS) B cells that will use these signaling pathways to induce clonal anti-tumor CASS B cell expansion and secretion of anti-PDL1 antibodies at tumor sites, which will help restore anti-tumor immunity.

[0385] Without being bound by theory, human B cells can be engineered to express human B cell receptors (aBCRs) against tumor-associated antigens (TAAs), which would allow them to migrate to solid tumors where they would be activated upon binding of the aBCR to the TAAs. Furthermore, once at the tumor site, CASS B cells would be further engineered such that upon activation they would secrete anti-PDL1 antibodies that act as checkpoint blockade inhibitors (CBIs) to reverse the immunosuppressive tumor microenvironment (TME).

[0386] In Aim 1, we will engineer B cells to express an IgG-type membrane-bound BCR against the TAA carbonic anhydrase IX (CAIX) expressed on the surface of clear cell renal cell carcinoma (ccRCC). For proof-of-principle studies, a second Ab signaling plasmid containing an NFAT or NF-kB response element will be used to drive secretion of an anti-PDL1 Ab. Upon localization to the tumor site and BCR binding to CAIX, anti-PDL1 antibody secretion will be induced by NFAT or NF-kB activation. This activation should result in high concentrations of anti-PDL1 antibodies at the tumor site. In Aim 2, we will use our orthotopic ccRCC mouse model to determine whether anti-CAIX CASSB cells can migrate to the tumor and be activated by ccRCC to secrete anti-PDL1 antibodies. We will also use this model to test the safety of this therapy, as CAIX is also expressed, albeit at lower levels and with a different cytoplasmic distribution compared to ccRCC. We have engineered the anti-CAIX targeting moiety to preferentially recognize high-density CAIX on tumor cells, but not healthy bile duct cells.

[0387] Non-limiting example of study design: For Objective 1, we will construct an engineered anti-CAIX BCR and transfer it into B cells using lentiviral transduction, with expression driven by the internal spleen focus forming virus (SFFV) promoter. Lentiviral particles will be pseudotyped with gibbon ape leukemia virus (GALV) or engineered baboon envelope glycoprotein to facilitate transduction. In addition, we will design a reporter plasmid to allow induction of NFAT or NF-kB to drive expression of GFP after BCR engagement, and the leader promoter will be used to drive secretion of anti-PDL monoclonal antibody. B cells will be transduced with both anti-CAIX BCR and NFAT / NF-kB inducible GFP lentiviral vectors, and soluble CAIX-Fc will be used to crosslink the BCR. GFP expression will then be measured to determine the best response element, and then anti-PDL 1 mAb will be used instead of GFP for secretion.

[0388] For Aim 2, CASS B cells will be constructed by transduction with tumor-specific anti-CAIX BCR and inducible anti-PDL1mAb secreting lentiviral vectors and tested by mixing CASS B cells with CAIX+PDL 1+ or PDL 1-SKRC-59ccRCC cells. In addition to measuring soluble anti-PDL 1 secretion and binding to ccRCC cells, B cell activation will measure activation markers by FACS. In vivo experiments in humanized PBL NSG-SGM3 mice bearing SKRC-59 tumors will be used to evaluate the efficacy and persistence of CASS B cells. Efficacy will be tested by measuring secreted Abs and increased B cell populations around tumor sites and changes in tumor size. To measure persistence, CASS B cells will be detected in the blood and TME. Finally, scRNAseq will be used to analyze the effects of anti-PDL1mAb on regulating the TME.

[0389] Without being bound by theory, there will be localized areas of high anti-PDL1 antibody concentration centered on the tumor, resulting in improved anti-tumor outcome and tumor elimination compared to systemic delivery.

[0390] CASS B cells are a new concept in cell therapy that will lead to the restoration of local anti-tumor immunity in the TME by blocking the immunosuppressive PD-1 / PD(L)-1 axis. It is also a combination immunotherapy that facilitates single administration and the cost of a single cell infusion.

[0391] Example 5

[0392] Engineering chimeric antibody signaling and secreting (CASS) B cells for cancer therapy

[0393] Innovation: Cancer cells are life forms that have learned to commandeer our immune system for their own growth advantage. They do this by subverting our cellular DNA repair mechanisms, leading to the upregulation of growth factors and their receptors, which in turn leads to uncontrolled tumor growth. This molecular hijacking also results in the surface expression and secretion of molecules involved in immune checkpoint blockade (ICB). Immunotherapy through anti-PD 1 / PDL 1 blockade represents an important advance in the field of cancer and is a first-line 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 treatments is well documented, they do not typically result in a cure for cancer.

[0394] Cell therapy is a method of using the immune system to kill cancer cells. This is largely achieved through T cell receptor (TCR) and chimeric antigen receptor (CAR) T cells, which are able to home in on and target cancer cells. While its success in treating solid tumors is increasing, it still has limitations because cancer cells can also co-opt these T cells and render them ineffective.

[0395] This article describes a new cell therapy, called chimeric antibody signaling and secretion (CASS) B cells, which will use the B cells of the humoral immune system to do what they are best at, that is, secrete high levels of antibodies. These CASS B cells will be engineered to recognize tumor-associated antigens (TAA) by engineered B cell receptors (BCRs), which will activate CASS B cells and induce the production of high levels of immunomodulatory bispecific antibodies (BsAb) at the tumor site when TAA participates. This will allow the reversal of the immunosuppressive tumor microenvironment, just as when immune checkpoint blocking monoclonal antibodies (mAbs) are delivered systemically. However, the interesting feature here is that BsAb secretion will only be conditionally expressed when CASS B cells are combined with TAAs, and most of them are located at the tumor site, although some low levels of peripheral leakage may still occur. Without being bound by theory, this is a step towards cell therapy, in which locally secreted monoclonal antibodies are committed to changing the tumor microenvironment and restoring local anti-tumor immunity.

[0396] Rationale: Monoclonal antibody (mAb) drugs that directly kill cancer cells, act as immune checkpoint blockade regulators (e.g., inhibitors), or destroy tumor vasculature are one of the most promising anticancer therapies being developed. However, the idea of ​​engineering human B cells to find cancer cells and secrete these monoclonal antibodies at tumor sites in vivo is novel, untested, but can provide a powerful new method for treating both primary and metastatic tumors. It will also provide a lifelong anti-tumor immune monitoring system to prevent cancer recurrence and achieve "cure". The main role of B cells in our immune system is to identify foreign invaders and remove them by producing antibodies that can bind to and remove the threat of microorganisms or cancer cells. It achieves this by expressing membrane-bound antibodies, which act as B cell receptors (BCRs) that bind tumor antigens, causing B cells to transform from BCR-expressing cells to antibody-secreting cells. After BCR involvement, rapid tyrosine phosphorylation and calcium ion polarization mediated by two major tyrosine kinases Lyn and Syn. These biochemical events lead to the activation of downstream signaling pathways, resulting in further downstream activation of NF-kB and NFAT signaling, and ultimately in B cell expansion and robust mAb secretion (5). We will design chimeric antibody signaling and secretion (CASS) B cells that will exploit these signaling pathways to induce clonal CASS B cell expansion and secretion of immunomodulatory BsAbs at tumor sites.

[0397] Without being bound by theory, the development of the CASS B cell platform may provide therapeutic benefits for a variety of 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 impact on the treatment of NSCLC in particular, becoming a first-line therapy for many patients. However, this therapy has limitations in efficacy, durability, and scope of use. To address these challenges, current trials are focused on anti-PD 1 / anti-TIGIT combination therapy, and clinical trial data recently released by Merck and Roche indicate that this combination therapy shows considerable promise (6-7). We will use NSCLC as a model for the use of anti-PD1 / anti-TIGIT bispecific antibodies.

[0398] Objective: To target CASS B cells to NSCLC tumor sites, an engineered, membrane-bound, single-chain IgG BCR against mesothelin (MSLN) will be used. The plasmid will also contain a second cassette driven by an aNFAT or NF-kB response element that drives the secretion of the anti-TIGIT / anti-PD1 BsAb. Following localization to the tumor site and BCR activation, expression of the B sAb will be induced by the natural signaling pathway of BCR binding by MSLN on NSCLC. Since this will be an inducible expression system, there will be local areas of high antibody concentration at the tumor site, significantly reducing the impact on the target and outside the tumor.

[0399] Methods: CASS B cells will be designed in two parts. Without being bound by theory, we will transfer the engineered BCR into B cells. To effectively transduce B cells, lentiviral particles will be pseudotyped with gibbon ape leukemia virus (GALV) or engineered baboon envelope glycoprotein, and in the transfer vector, expression of the BCR will be driven by the spleen focus forming virus (SFFV) or human elongation factor-1α (EF1α) promoter (8). The second step will be to design NFAT and / or NF-kB inducible expression cassettes for BsAb secretion (9). To decide which response element to use, plasmids will be designed to express GFP. Raji cells and primary B cells will be transduced with the engineered anti-MSLN BCR and NFAT / NF-kB inducible GFP lentiviral vector, and soluble biotinylated MSLN will be added to the medium with streptavidin to crosslink the BCR. GFP expression will then be measured to determine the optimal response element. Preliminary work to construct an engineered membrane-bound IgG (memlgG) was performed using anti-influenza antibodies. Figure 1 Our memlgG construct was shown to be expressed at high levels and to be functionally active as it was able to bind soluble HA.

[0400] Next, CASS B cells will be constructed by lentiviral transduction with a vector encoding a tumor-specific anti-MSLN BCR and an inducible BsAb replacing GFP. Anti-TIGIT / PD 1 BsAb secreted by CASS B cells will be quantified, first by inducing expression using soluble biotinylated MSLN + streptavidin, followed by co-incubation with MSLN + A549 NSCLC. Secondary experiments will test in vitro inhibition of exhaustion by co-culturing CASSB cells with CD3 + T cells and A549 cells expressing various combinations of PDL1 and CD 155 (ligands for TIGIT). In addition to measuring soluble antibody concentrations, cell activation / exhaustion will be measured by FACS staining. These tumor cells will also be stained for CASS B cell shedding of anti-MSLN binding. Next, in vivo experiments will be performed in humanized PBL NSG-SGM3 mice bearing A549 tumors to evaluate the efficacy and persistence of CASS B cells. Efficacy will be measured by local BsAb secretion by immunohistochemical staining, increase in B cell populations around tumor sites, and changes in tumor size. We will also measure BsAb leakage to the periphery by examining serum secreted BsAb concentrations, and to measure persistence, CASS B cells will be detected in peripheral blood and tumor microenvironment (TME). Finally, scRNAseq will be used to analyze the role of BsAb in regulating the TME. Ideally, there will be local areas of high BsAb concentrations around the tumor, leading to improved outcomes and tumor elimination compared to systemic delivery.

[0401] Impact: The goal of developing new cancer therapies should be to achieve a "cure." By understanding the immune elements that are common or "public" to all individuals, host anti-tumor immunity can be restored at the tumor site. Once this is done, we must develop internal immune surveillance systems that will always be there to prevent cancer recurrence. We will develop new immune surveillance systems using B cells of the immune system, which have never been developed for this purpose. We will determine the feasibility of engineering chimeric antibody signaling and secretion (CASS) B cells to target NSCLC expressing mesothelin (MSLN). These anti-MSLN CASS B cells will return to the tumor site, where they will secrete high levels of bispecific anti-TIGIT / anti-PDL1 antibodies that will act as dual checkpoint blockade inhibitors. This will lead to dynamic changes in the tumor microenvironment, which will help reverse T cell exhaustion and restore anti-tumor immunity. An important and practical point is that this combination cellular immunotherapy will be administered once and will have a cost to the payer for the patient's lifetime.

[0402] References cited in this example

[0403] 1.Rolfo C, Caglevic C, Santarpia M, Araujo A, Giovannetti E, Gallardo CD, Pauwels P, Mahave M. Immunotherapy in NSCLC: A Promising and Revolutionary Weapon. Adv Exp Med Biol. 2017;995:97-125.doi:10.1007 / 978-3-319-531564_5.PMID:28321814 Comment.

[0404] 2. M.-O. Grimm, K. Leucht, V. Grunwald, S. Foller, New First Line Treatment Options of Clear Cell Renal Cell Cancer Patients with PD-1 or PD-L1 Immune-Checkpoint Inhibitor-Based Combination Therapies. J. Clin. Med. 9, 565 (2020).

[0405] 3.X. Wu et al., Application of PD-1 Blockade in Cancer Immunotherapy. Comput. Struct. Biotechnol. J. 17, 661-674 (2019).

[0406] 4. Ryeong Lee B, Sehyun C, Moon J, Joon Kim M, Lee H, Wan Ko H, Chul Cho B, Sup Shim H, Hwang D, Ryun Kim H, and Ha S-J. Combination of PD-L1 and PVR determines sensitivity to PD-1 blockade. JCI insite 2020;5(14):e128633. https: / / doi.org / 10.1172 / jci.insight.128633.

[0407] 5. Tolar P, Hanna J, Krueger PD, Pierce SK. The constant region of the membrane immunoglobulin mediates B cell-receptor clustering and signaling in response to membrane antigens. Immunity. 2009;30(1):44-55. Epub 2009 / 01 / 13. doi:10.1016 / j.immuni.2008.11.007. PubMed PMID: 19135393; PMCID: PMC2656684.45.

[0408] 6. Bendell JC, Bedard P, Bang YJ et al.: Phase la / Ib dose-escalation study of the anti-TIGIT antibody tiragolumab as a single agent and in combination with atezolizumab in patients with advanced solid tumors. 2020 AACR Virtual Annual Meeting II. Abstract CT302.

[0409] 7.M.-J.Ahn et al., 1400P Vibostolimab, an anti-TIGIT antibody, as monotherapy and in combination with pembrolizumab in anti-PD-1 / PD-L1-refractory NSCLC. Ann. Oncol. 31, S887 (2020).

[0410] 8.Levy C, Fusil F, Amirache F, Costa C, Girard-Gagnepain A, Negre D, Bernadin O, Garaulet G, Rodriguez A, Nair N, Vandendriessche T, Chuah M, Cosset FL, Verhoeyen E. Baboon envelope pseudotyped lentiviral vectors efficiently transduce human B cells and allow active factor IX B cell secretion in vivo in NOD / SCIDgammac(- / -) mice. J Thromb Haemost. 2016;14(I2):2478-92. Epub 2016 / 09 / 30. doi:10.1111 / jth.13520. PubMed PMID: 27685947.

[0411] 9.Uchibori R, Teruya T, Ido H, Ohmine K, Sehara Y, Urabe M, Mizukami H, Mineno J, Ozawa K. Functional Analysis of an Inducible Promoter Driven by Activation Signals from a Chimeric Antigen Receptor. Mol Ther Oncolytics. 2019;12:16-25. Epub 2019 / 01 / 22. doi:10.1016 / j.omto.2018.11.003. PubMed PMID: 30662937; PMCID: PMC6325072.

[0412] Example 6

[0413] CASSB cell culture and transduction protocol

[0414] Day 0

[0415] 1. Isolate B cells (from stem cells) from fresh or frozen PBMCs using any of the following methods:

[0416] 19054 Easy Sep TM Human B Cell Enrichment Kit

[0417] 19554Easy Sep TM Human Pan-B Cell Enrichment Kit (including plasma cells)

[0418] 2. Activate B cells prior to transduction, such as between -18-24 hours and 3-5 days

[0419] a. Usually cultured in 24-well plates, 1 ml of culture medium, 0.5-1E6 cells / ml

[0420] Activation medium 1 (for Milteym 130-106-196 protocol, but with 1ug / ml ODN 2006 added): Milteyni stem ACS HSC medium + 5% FBS + IL4 + polymerized CD40L + ODN2006

[0421] Activation medium 2 (based on Moffett et al. DOI 10.1126 / sciimmuno1.aax 0644): IMDM + 10% FBS + CD40L-Fc (3.735ug / ml) + ODN2006 (1ug / ml) + IL2 (50ng / ml) + IL10 (50ng / ml) + IL15 (10ng / ml)

[0422] Initially used activation medium 1, but later changed to activation medium 2

[0423] Day 1

[0424] 1. Thaw the virus

[0425] 2. Add DEAE to each well at a final concentration of 10ug / ml

[0426] 3. Add virus and pipette to mix

[0427] 4. Centrifuge the virus at 1200 x g for 55 minutes at 37°C

[0428] 5. Transfer the plate to an incubator and let it sit overnight. There is no need to resuspend the cells.

[0429] Day 2

[0430] 1. Collect the cells in a 15 ml tube, wash twice with PBS, and then resuspend in fresh activation medium.

[0431] a. Ideally, cells can be grown at a concentration of 1E6 cells / ml in ~1 ml, but resuspend accordingly depending on cell number and viability

[0432] b. Maintaining cells at 0.75-1E6 cells / ml is important for rapid expansion at this stage 2. Cultivate cells for 48 hours to allow expression of transduced genes and expansion of cells before sorting

[0433] Day 4

[0434] 1. In the morning, use fresh or frozen irradiated 3T3-msCD40L feeder cells (HIV Reagent #12535)

[0435] a. Cells γ-irradiated at 78 Gy

[0436] 2. 8.75E5 cells / cm 2 Place in DMEM + 10% FBS and allow cells to attach before sorting (> 4 hours)

[0437] 3. Collect cells, wash with PBS, and stain with Zombie Violet (a live / dead exclusion dye from Biolegend)

[0438] a. According to the manufacturer's protocol, 1:1000 diluted dye, 100ul diluted dye / 1E6 cells

[0439] 4. Block Fc receptors with human TruStain FcX (Biolegend 422302)

[0440] 5. Stain the cells with appropriate markers for sorting

[0441] a. QBend10-PE, CD19-APC, Zombie Purple use BFP channel

[0442] 6. Aspirate the medium from the 6-well plate with adherent 3T3-msCD40L feeder cells and replace with expansion medium (IMDM + 10% FBS + 5ug / ml human insulin + 50ug / ml transferrin + 50ng / ml Il2 + 10ng / ml Il15 + 20ng / ml IL21)

[0443] 7. Sorting cells on Sony MA900 directly into 6-well plates with feeder cells

[0444] 8. If the cells are cultured for more than 14 days, add culture medium appropriately. If the culture exceeds 7 days, split the cultured feeder cells into fresh plates on the 6th or 7th day.

[0445] Example 7

[0446] Exemplary constructs include:

[0447] (1)EF1alpha-F105leader-F10-memIgG1-T2A-RQR8-3x3NFAT-inIL2pro-ZsG,

[0448] (2)EF1alpha-F105leader-F10-memIgG1-T2A-RQR8-5xNFAT-inIL2pro-ZsG,

[0449] (3) EF1alpha-F105leader-F10-memIgG1-T2A-RQR8-NFAT-NFkB-ZsG, or

[0450] (4) Any combination thereof.

[0451] EFlalpha Promoter:

[0452]

[0453] F105 leader sequence:

[0454]

[0455] F10 scFv:

[0456]

[0457] Fc domain (CH2 in bold, CH3 in underline)

[0458]

[0459] Transmembrane and intracellular domains:

[0460]

[0461] T2A:

[0462]

[0463] RQR8:

[0464]

[0465] ZsG:

[0466]

[0467] NFAT binds to a 9 bp element with the consensus sequence (A / T)GGAAA(A / N)(A / T / C)N, where N represents any base.

[0468] 3X NFAT RE:

[0469]

[0470] 5X NFAT RE:

[0471]

[0472] ILS NFAT RE:

[0473]

[0474] minIL2 promoter:

[0475]

[0476] minIL8 promoter:

[0477]

[0478] WPRE:

[0479]

[0480] Example 8

[0481] Engineering chimeric antibody signaling and secreting (CASS) B cells for cancer therapy

[0482] summary

[0483] B cells are an integral part of host immunity, and although other immune cells including T cells, NK cells, and bone marrow cells have been engineered to have chimeric antigen receptors (CARs), B cells have been overlooked. The role of B cells in our immune system is to recognize foreign invaders via membrane-bound antibodies (B cell receptors, BCRs) and eliminate them by producing antibodies that bind and remove threats. We can develop a chimeric antibody signaling and secretion (CASS) B cell platform by engineering both target binding domains and secretory antibodies to develop effective anticancer therapeutics. The target binding domain will be designed to recognize tumor-associated antigens, and when bound, it will lead to the secretion of bispecific checkpoint blockade inhibition (CBI) antibodies to reverse the exhaustion of immune cells that have reached the tumor site. The second main 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 CASSB cells will provide a lifelong anti-tumor surveillance network to protect patients from metastasis and recurrence after the initial tumor is eliminated.

[0484] While the CASS B cell platform is applicable to many cancers, we will use non-small cell lung cancer (NSCLC) as an experimental model. NSCLC was chosen because it expresses the tumor-associated antigen mesothelin (MSLN) at high levels and because of the effectiveness of anti-PD 1 and anti-TIGIT antibody combination therapies seen in clinical trials. Objective 1 will focus on engineering CASS B cells, first by constructing a synthetic B cell receptor (BCR) that recognizes MSLN. These cells will be further engineered to express bispecific antibodies targeting PD1 and TIGIT, which are controlled by a switch that is activated only upon binding to MSLN at the tumor site. Objective 2 will use tissue culture experiments to demonstrate that our engineering is successful and show the ability of the bispecific antibodies to restore anti-cancer immunity. Experiments in Objective 3 will be performed using mice with a human immune system to demonstrate the effectiveness of the CASSB cell platform in vivo and allow for detailed molecular characterization of anti-tumor immune responses. Without being bound by theory, the development of the CASS B cell platform supports translational research in the development of new therapeutic platforms that can impact a wide range of clinical indications and play a decisive role in improving healthcare and saving lives.

[0485] Scientific Abstract

[0486] Immune checkpoint blockade inhibitors (CBIs) and CAR T cells have revolutionized the way we treat cancer. While both of these therapies involve the patient's immune system, with CBI therapy suppressing immunosuppressive signals generated by the tumor microenvironment and activated CAR T cells binding to bystander cells via the release of stimulatory and pro-inflammatory cytokines, neither therapy can actively engage and initiate an anti-tumor immune response. To address this issue, we can develop chimeric antibody secreting and signaling (CASS) B cells that express engineered tumor-associated antigens targeting the BCR and, once bound, will locally secrete high levels of bispecific antibodies (bsAbs) at the tumor site, acting as CBIs. Since B cells are also professional antigen-presenting cells (APCs), they will process and present additional tumor antigens on class II molecules, further enhancing tumor cell recognition and helping new antigens spread. As a component of immune memory, CASS B cells will provide a lifelong surveillance program to prevent tumor metastasis and recurrence. By recruiting a variety of immune cells to the tumor site and reversing the immunosuppressive tumor microenvironment to enable these cells to function, CASS B cell therapy will provide a robust and durable anti-cancer therapy.

[0487] Mesothelin (MSLN) is a tumor-associated antigen, and due to its limited expression in healthy tissues, many biotherapies targeting this marker have been designed, including CAR T and recombinant antibodies. Non-small cell lung cancer (NSCLC) has been indicated to upregulate MSLN expression, and data from clinical trials have demonstrated the effectiveness of anti-PD1 / anti-TIGIT antibody combination therapy. Based on this, NSCLC was selected as a model to develop and test the efficacy of anti-MSLN directed CASS B cells that secrete immunomodulatory anti-PD1 / TIGIT bsAb. Purpose 1 will be dedicated to the development of CASS B cell platforms and BCR engineering to identify mesothelin (MSLN). Additional engineering will be performed to develop an inducible system using the natural BCR signaling pathway so that the involvement of MSLN at the tumor site leads to high levels of bsAb secretion. In Purpose 2, the in vitro efficacy of CASS B cells will be tested, and the activation threshold and the secretion level produced will be characterized to fine-tune CASS B cell signaling and secretion. Objective 3 will conduct in vivo experiments in mice reconstituted with the human immune system using aNSCLC cell line models and patient-derived xenograft models. Multiparameter flow cytometry (FACS), single-cell RNA sequencing, and immunohistochemistry will provide a detailed assessment of the molecular and mechanistic efficacy of the immunomodulatory bsAbs and the CASS B cell platform as a whole.

[0488] Without being bound by theory, the development of the CASS B cell platform supports innovative translational research in the development of new therapeutic platforms that can impact a wide range of clinical indications, and while this example focuses on NSCLC, this research project will allow our team to further refine this novel translational approach and expand it beyond cancer treatment. The development of the CASS B cell platform has potential therapeutic benefits not only in cancer treatment, but also in many other clinical indications, including autoimmune / rheumatic and cardiovascular diseases and neurological diseases, where monoclonal antibody treatments play a decisive role in improving health care and saving lives.

[0489] Non-limiting unique and innovative aspects

[0490] Since the FDA approved the first biologic (Humulin) in 1982, followed by the first monoclonal antibody therapy (muromonab-CD3) in 1986, we have seen an explosion of new biologics. After the initial success of anti-PD1 and CTLA4 therapies, the race has been on how to develop antibodies against the next generation of checkpoint molecules. As researchers began to identify new biomarkers, it became clear that it would be difficult to find receptors comparable to PD 1 and CTLA4, and that these alone would not be sufficient for most patients. As a result, the field turned to combination therapies, combining anti-PD(L)1 therapies with a wide range of other compounds, including standard and experimental treatments. These antibody-based therapies rely on restoring the anti-tumor activity of the patient's immune system, but different cancers require multiple checkpoint blockade pathways to evade our immune system, and not all cancers respond to the same immunotherapies. The next breakthrough was the development of CAR T-cell therapy, in which a patient's T cells are removed and engineered to recognize tumor-associated antigens, allowing for robust targeting and cytotoxic activity. Although CAR T activity indirectly activates other immune cells via the release of cytokines and chemokines, CAR T cells produce significant cytotoxic effects. In addition, because CAR T cells are "living drugs", they will expand and become part of the patient's immune system, providing long-term protection that monoclonal antibody therapy cannot achieve.

[0491] Chimeric antibody secretion and signaling (CASS) B cells are a new form of cell therapy that uses engineered B cells to locally secrete high levels of checkpoint blockade modulator antibodies (e.g., checkpoint inhibitor antibodies) at tumor sites. One of the challenges of systemic delivery of CBI antibody therapies is the wide distribution of targets and the role these receptors play in immune homeostasis, potentially leading to immune-related adverse events (irAEs) of varying severity, including colitis, dermatitis, myocarditis, encephalitis, or peripheral neuropathy. To overcome this challenge, many groups are exploring targeted delivery of CBI therapies, in which CAR T cells are engineered to secrete therapeutic payloads at tumor sites, increasing local concentrations relative to serum concentrations. The unique aspect here is that by utilizing the natural BCR signaling pathway, engineered IgG-BCRs are designed to selectively induce high levels of CBI bsAb expression only when activated by the target antigen, creating pockets of high bsAb concentrations and reversing immunosuppression around the tumor. The use of bsAbs is also an innovative solution to improve the efficacy of single antibody and combination antibody therapies, because potential synergistic activity can be obtained by tethering the binding domains together. Like CAR T cells, CASS B cells will act as an active drug and become a permanent part of the patient's immune system, providing lifelong immune surveillance and preventing metastasis and relapse.

[0492] While CASS B cells do not provide the direct cytotoxic effects seen with CAR T cells, upon stimulation, B cells process and present antigens on class II molecules, recruit CD4+ T cells, and allow for enhanced tumor cell recognition and neoantigen spread. The antigen presentation capabilities of the CASS B cell platform make it unique among cell therapy platforms. Like antibody-based therapies, CASS B cells require activation of the patient's immune system, however, as professional antigen-presenting cells, CASS B cells have the ability to initiate robust anti-tumor responses by engaging a broad range of immune cells. Another important role of B cells in the immune system is that memory B cells are an important complement to immune memory and are able to rapidly produce large amounts of antibodies upon restimulation. This provides a method for lifelong monitoring of tumor metastasis and recurrence.

[0493] The CASS B cell platform could represent a new avenue of research based on the Marasco lab’s expertise in antibody engineering and CAR T cell development. Our work provides extensive experience in the discovery and design of therapeutic quality antibodies against a variety of targets, including viral and tumor-associated antigens, as well as many immune checkpoint markers7-11. Additionally, we have used these antibodies to develop a range of bispecific and multispecific antibody constructs, including knobs-in-hole constructs, IgG fusions, and tandem single-chain antibodies. The development of CAR T cells and our CAR T cell factory provides an opportunity for the lab’s first foray into cell therapy and the targeted delivery of immunomodulatory antibodies to tumor sites. 2 These CAR T projects have provided the laboratory with valuable experience and pipeline development for the evaluation and analysis of biologic therapies both in vitro and in vivo. The skills and techniques gained from these prior lines of research will provide a foundation for our laboratory to explore new avenues of research in CASS B cell development.

[0494] Since the development of the first generation of CAR T cells in the early 1990s, significant improvements in efficacy, durability, and safety have led to the generation of second and third generation CAR T cells in use today. Other immune cells have been used to create new CARs, including natural killer cells (NK-CARs) and macrophages (CAR-Ms), all of which have in common that they provide direct anti-tumor activity12,13. Although B cells are a key component of humoral immunity and produce the antibodies upon which initial developments in the field of immunotherapy were based, they do not possess intrinsic cytotoxic capacity of their own and have been largely excluded from these advances. The CASS B cell platform aims to bring B cell research into the 21st century by developing a unique B cell-based cellular therapy that does not rely on direct cytotoxicity but instead exploits the antibody secretion function of B cells to reverse the immunosuppressive environment, combined with antigen presentation function to activate and recruit additional immune effector cells to eliminate tumors.

[0495] While this example focuses on the use of CASS B cells to treat NSCLC, the CASS B cells described in this proposal are therapeutically applicable to a variety of MSLN+ cancers with immunosuppressive properties, including ovarian and pancreatic cancer. Due to their modular design, CASS B cells can be easily directed to other tumor-associated antigens, and the secreted payload can be regulated to target relevant immune axes, allowing CASS B cells to adapt to a variety of cancers. Because B cells are long-lived and are an important component 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 is eradicated. The long duration and inducible nature of the CASS B cell platform can be easily applied to other diseases treated with biologic therapies, such as cardiovascular and autoimmune diseases and neurological disorders, which often require long-term disease maintenance and rapid administration of therapeutic agents to treat acute symptoms. Therefore, the development of the CASS B cell platform will not only benefit cancer research, but also a wide variety of clinical indications.

[0496] Example 9

[0497] Chimeric Antibody Signaling and Secretion (CASS) B Cells

[0498] -1. Constitutive expression of α-tumor antigen-synthesizing BCR

[0499] -2. Inducible expression of immunomodulatory payloads

[0500] - Checkpoint blockade therapy (α-PD(L)1, α-PD1 / TIGIT)

[0501] -T cell engagers (α-CD3xMSLN, α-CD3xCD28xMSLN)

[0502] -Cytokine fusion (α-PD1-scIL12)

[0503] B cell basics:

[0504] B cell isolation: Stemcell EasySep TM Human Pan B Cell Enrichment Kit (19554)

[0505] Stimulation medium: IMDM + 10% H1FBS + CD40L-Fc (3.735ug / ml) + ODN2006 (lug / ml) + IL2 (50ng / ml) + IL10 (50ng / ml) + IL15 (10ng / ml)

[0506] Long-term expansion medium: IMDM + 10% fetal bovine serum + human insulin (5ug / ml) + transferrin (50ug / ml) + IL2 (50ng / ml) + IL21 (20ng / ml) + IL15 (10ng / ml)

[0507] Transduction examination after BGH removal

[0508] Isolation and activation of B cells: 11 / 11 / 21

[0509] Transduction: 11 / 12 / 21

[0510] Dyeing: 11 / 19 / 21

[0511] The virus was pseudotyped with VSVG and BGH was removed. B cells were transduced 18-24 hours after activation. However, they were negative 7 days after transduction.

[0512] Transduction testing with our vectors

[0513] Carrier:

[0514] LeGO-hEuMAR-GFP

[0515] pHAGE-F 10-RQR8

[0516] pHAGE-F 10-RQR8NFAT / NFkB

[0517] B cell stimulation (4 days):

[0518] Culture medium: IMDM + 10% HI FBS + CD40L-Fc (3.735ug / ml) + ODN2006 (1ug / ml) + IL2 (50ng / ml) + IL10 (50ng / ml) + IL15 (10ng / ml)

[0519] B cell transduction: using GALV and BaEV envelopes, without retronection

[0520] Equivalent

[0521] 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 described herein. Such equivalents are considered to be within the scope of the present invention and are covered by the appended claims.

Claims

1. A genetically engineered B cell, wherein the genetically engineered B cell expresses and carries a chimeric B cell receptor on its surface, and wherein the genetically engineered B cell further expresses and secretes antibodies or cytokines.

2. The genetically engineered B cell of claim 1, wherein the chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

3. The genetically engineered B cell of claim 1, wherein the extracellular domain is an antibody or an antibody fragment.

4. The genetically engineered B cell of claim 3, wherein the antibody is a nanobody, a scFv, a bispecific antibody or a Fab.

5. The genetically engineered B cell of claim 3, wherein the antibody is specific for a tumor-associated antigen.

6. The genetically engineered B cell of claim 5, wherein the tumor-associated antigen is selected from the group consisting of CAIX, BCMA, CD 138, PD-L1, PD-L2, VEGF, CD70, CD99, CEA, Her-2, GD2, CD171, αFR, PMSA, IL13α, MSLN, TAG-72, and TROP2.

7. The genetically engineered B cell of claim 3, wherein the antibody is an anti-IGHV 1-69 antibody.

8. The genetically engineered B cell of claim 3, wherein the antibody is specific for an antigen associated with an infectious disease.

9. The genetically engineered B cell of claim 8, wherein the infectious disease is a viral disease.

10. The genetically engineered B cell of claim 8, wherein the infectious disease comprises influenza, coronavirus, HIV or tuberculosis.

11. The genetically engineered B cell of claim 8, wherein the infectious disease-associated antigen comprises HA1, HA2, NA or spike protein.

12. The genetically engineered B cell of claim 1, wherein expression of the antibody or the cytokine is controlled by an inducible response element.

13. The genetically engineered B cell of claim 12, wherein the inducible response element is NFAT or NF K B response element.

14. The genetically engineered B cell of claim 1, wherein the antibody is a checkpoint blockade modulator.

15. The genetically engineered B cell of claim 14, wherein the antibody is a checkpoint blockade inhibitor.

16. The genetically engineered B cell of claim 1, wherein 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.

17. The genetically engineered B cell of claim 1, wherein the cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, CD40-L or BAFF.

18. The genetically engineered B cell of claim 1, wherein the antibody is specific for HA1, HA2, NA or spike protein.

19. The genetically engineered B cell of claim 1, wherein the antibody comprises a monoclonal antibody.

20. The genetically engineered B cell of claim 1, wherein the antibody comprises a nanobody, a scFv, a Fab, an antibody-cytokine fusion protein, or a bispecific antibody.

21. The genetically engineered cell of claim 1, wherein the antibody comprises a humanized antibody.

22. The genetically engineered B cell of claim 20, wherein the antibody-cytokine fusion protein comprises anti-PD1-scIL12.

23. The genetically engineered B cell of claim 20, wherein 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.

24. A nucleic acid encoding a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a chimeric B cell receptor, wherein the chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain, and wherein the second polypeptide comprises an antibody or a cytokine.

25. The nucleic acid of claim 24, wherein the extracellular domain is an antibody.

26. The nucleic acid of claim 25, wherein the antibody is a nanobody, a scFv, a bispecific or a Fab.

27. The nucleic acid of claim 25, wherein the antibody is specific for a tumor-associated antigen.

28. The nucleic acid of claim 27, wherein 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.

29. The nucleic acid of claim 25, wherein the antibody is an anti-IGHV 1-69 antibody.

30. The nucleic acid of claim 25, wherein the antibody is specific for an antigen associated with an infectious disease.

31. The nucleic acid of claim 30, wherein the infectious disease is a viral disease.

32. The nucleic acid of claim 30, wherein the infectious disease comprises influenza, coronavirus, HIV, or tuberculosis.

33. The nucleic acid of claim 30, wherein the infectious disease-associated antigen comprises HA1, HA2, NA or spike protein.

34. The nucleic acid of claim 24, wherein the antibody comprises a monoclonal antibody.

35. The nucleic acid of claim 24, wherein the antibody comprises a nanobody, a scFv, an antibody-cytokine fusion, or a bispecific antibody.

36. The nucleic acid of claim 24, wherein the antibody comprises a humanized antibody.

37. The nucleic acid of claim 24, wherein 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.

38. The nucleic acid of claim 35, wherein the antibody-cytokine fusion comprises anti-PD1-scIL12.

39. The nucleic acid of claim 35, wherein 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.

40. The nucleic acid of claim 24, wherein the cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, CD40-L, or BAFF.

41. A vector comprising the nucleic acid according to any one of claims 24 to 40.

42. The vector of claim 41, wherein the vector is a lentiviral vector or an adeno-associated viral vector.

43. A cell comprising a vector according to claim 41 or claim 42.

44. 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 wherein the genome of the second expression vector comprises a nucleotide sequence encoding an antibody or a cytokine.

45. The composition of claim 44, wherein the nucleotide sequence encoding the inducible response element is operably linked to the nucleotide sequence encoding the antibody or cytokine.

46. ​​The composition of claim 45, wherein the inducible response element is NFAT or NF K B response element.

47. The composition of claim 44, wherein the chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain.

48. The composition of claim 47, wherein the extracellular domain is an antibody.

49. The composition of claim 48, wherein the antibody is a nanobody, a scFv, a bispecific or a Fab.

50. The composition of claim 48, wherein the antibody is specific for a tumor-associated antigen.

51. The composition of claim 50, wherein 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, aFR, PMSA, IL13α, MSLN, TAG-72, and TROP2.

52. The composition of claim 48, wherein the antibody is an anti-IGHV 1-69 antibody.

53. The composition of claim 48, wherein the antibody is specific for an antigen associated with an infectious disease.

54. The composition of claim 53, wherein the infectious disease is a viral disease.

55. The composition of claim 53, wherein the infectious disease comprises influenza, coronavirus, HIV, or tuberculosis.

56. The composition of claim 53, wherein the infectious disease-associated antigen comprises HA1, HA2, NA or spike protein.

57. The composition of claim 44, wherein the antibody is a checkpoint blockade modulator.

58. The composition of claim 57, wherein the checkpoint blockade modulator comprises a checkpoint blockade inhibitor.

59. The composition of claim 44, wherein 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.

60. The composition of claim 44, wherein the cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, CD40-L, or BAFF.

61. The composition of claim 44, wherein the antibody comprises a monoclonal antibody.

62. The composition of claim 44, wherein the antibody comprises a nanobody, a scFv, a Fab, an antibody-cytokine fusion, or a bispecific antibody.

63. The composition of claim 43, wherein the antibody comprises a humanized antibody.

64. The composition of claim 62, wherein the antibody-cytokine fusion comprises anti-PD1-scIL12.

65. The composition of claim 62, wherein 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.

66. The composition of claim 44, wherein the first expression vector and the second expression vector are lentiviral vectors.

67. A method for preparing a genetically engineered B cell population, the method comprising: isolating a population of B cells from the subject, and The B cell population is transduced with a vector according to claim 41 or claim 42, thereby generating a genetically engineered B cell population.

68. The method of claim 67, further comprising the step of activating the B cell population prior to transduction.

69. The method of claim 67, further comprising the step of culturing the genetically engineered B cell population.

70. The method of claim 67, further comprising the step of administering the genetically engineered B cell population to a subject in need thereof.

71. A method of treating a subject having cancer, the method comprising administering to the subject a genetically engineered B cell according to claims 1 to 23, a nucleic acid according to claims 24 to 39, or a composition according to claims 44 to 66.

72. A method of preventing cancer in a subject, the method comprising administering to the subject a genetically engineered B cell according to claims 1 to 23, a nucleic acid according to claims 24 to 39, or a composition according to claims 44 to 66.

73. The method of claim 66 or claim 67, wherein the cancer is BCLL, NSCLC, ccRCC, mesothelioma.

74. A method of treating a subject having an infectious disease, the method comprising administering to the subject a genetically engineered B cell according to claims 1 to 23, a nucleic acid according to claims 24 to 39, or a composition according to claims 44 to 66.

75. A method of preventing an infectious disease, the method comprising administering to a subject a genetically engineered B cell according to claims 1 to 23, a nucleic acid according to claims 24 to 39, or a composition according to claims 44 to 66.

76. The method of claim 74 or claim 75, wherein the infectious disease is a viral disease.

77. The method of claim 74 or claim 75, wherein the infectious disease comprises influenza, coronavirus, HIV or tuberculosis.

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