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WO2025194049A1 - Cellules immunitaires modifiées et leurs méthodes d'utilisation - Google Patents

Cellules immunitaires modifiées et leurs méthodes d'utilisation

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Publication number
WO2025194049A1
WO2025194049A1 PCT/US2025/019954 US2025019954W WO2025194049A1 WO 2025194049 A1 WO2025194049 A1 WO 2025194049A1 US 2025019954 W US2025019954 W US 2025019954W WO 2025194049 A1 WO2025194049 A1 WO 2025194049A1
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Prior art keywords
acid sequence
seq
amino acid
nucleic acid
cells
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Inventor
Rizwan ROMEE
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Dana Farber Cancer Institute Inc
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Dana Farber Cancer Institute Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4254—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K40/4255—Mesothelin [MSLN]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/39—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by a specific adjuvant, e.g. cytokines or CpG
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2312—Interleukin-12 (IL-12)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the disclosure provided herein is related to engineered immune cells and methods of using the same, such as in treating cancer.
  • Mesothelin and other proteins differentially expressed on cancer cells is differentially expressed in a variety of tumors and cancers.
  • gaps in therapeutic efficacy remain for previous mesothelin-directed therapies and therapies directed to other proteins differentially expressed on cancer cells (see, e.g., Faust et al., Mesothelin: An Immunotherapeutic Target beyond Solid Tumors, Cancers (Basel), 14(6): 1550 (2022)). Therefore, more effective mesothelin-directed therapies are desirable therapies directed to other proteins differentially expressed on cancer cells.
  • engineered cells comprising a natural killer cell (an NK cell), comprising a chimeric antigen receptor (CAR) of the formula:
  • A is a mesothelin antibody means for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain
  • I is interleukin 12 (IL- 12); and
  • C is a collagen binding domain
  • engineered cells comprising a natural killer cell (an NK cell), comprising a chimeric antigen receptor (CAR) of the formula:
  • A is a mesothelin antibody means for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain
  • I is interleukin 12 (IL- 12).
  • engineered cells comprising a natural killer cell (an NK cell), comprising a chimeric antigen receptor (CAR) of the formula:
  • A is an antibody or fragment thereof for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain
  • I is interleukin 12 (IL- 12); and
  • C is a collagen binding domain
  • engineered cells comprising a natural killer cell (an NK cell), comprising a chimeric antigen receptor (CAR) of the formula:
  • A is an antibody or fragment thereof for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain
  • I is interleukin 12 (IL- 12).
  • CARs chimeric antigen receptors
  • A is a mesothelin antibody means for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain
  • I is interleukin 12 (IL- 12); and
  • C is a collagen binding domain.
  • A is a mesothelin antibody means for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain
  • I is interleukin 12 (IL- 12).
  • CARs chimeric antigen receptors
  • A is an antibody or fragment thereof for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain
  • I is interleukin 12 (IL- 12); and
  • C is a collagen binding domain
  • CARs chimeric antigen receptors
  • A is an antibody or fragment thereof for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain
  • I is interleukin 12 (IL- 12).
  • CARs chimeric antigen receptors
  • compositions comprising an engineered cell provided herein of any one of claims 1-30, and a pharmaceutically acceptable carrier.
  • pharmaceutical compositions comprising an engineered cell provided herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
  • kits for treating cancer comprising administering to a subject with cancer: an engineered cell provided herein, or a pharmaceutical composition provided herein.
  • compositions for treating cancer comprising administering to a subject with cancer: an engineered cell provided herein, or a pharmaceutical composition provided herein.
  • compositions for use in treating cancer comprising administering to a subject with cancer: an engineered cell provided herein, or a pharmaceutical composition provided herein.
  • SEQ ID NO: 1 is an amino acid sequence of an LCDR1 embodiment.
  • SEQ ID NO: 2 is an amino acid sequence of an LCDR2 embodiment.
  • SEQ ID NO: 3 is an amino acid sequence of an LCDR3 embodiment.
  • SEQ ID NO: 4 is an amino acid sequence of an LC embodiment.
  • SEQ ID NO: 5 is a nucleic acid sequence of an LC embodiment.
  • SEQ ID NO: 6 is an amino acid sequence of a linker embodiment.
  • SEQ ID NO: 7 is a nucleic acid sequence of a linker embodiment.
  • SEQ ID NO: 8 is an amino acid sequence of an HCDR1 embodiment.
  • SEQ ID NO: 9 is an amino acid sequence of an HCDR2 embodiment.
  • SEQ ID NO: 10 is an amino acid sequence of an HCDR3 embodiment.
  • SEQ ID NO: 11 is an amino acid sequence of an HC embodiment.
  • SEQ ID NO: 12 is a nucleic acid sequence of an HC embodiment.
  • SEQ ID NO: 13 is an amino acid sequence of a CD8 hinge embodiment.
  • SEQ ID NO: 14 is a nucleic acid sequence of a CD8 hinge embodiment.
  • SEQ ID NO: 15 is an amino acid sequence of a CD8 transmembrane domain embodiment.
  • SEQ ID NO: 16 is a nucleic acid sequence of a CD8 transmembrane domain embodiment.
  • SEQ ID NO: 17 is an amino acid sequence of a 4-1BB cytoplasmic domain embodiment.
  • SEQ ID NO: 18 is a nucleic acid sequence of a 4- IBB cytoplasmic domain embodiment.
  • SEQ ID NO: 19 is an amino acid sequence of a CD3 cytoplasmic domain embodiment.
  • SEQ ID NO: 20 is a nucleic acid sequence of a CD3 cytoplasmic domain embodiment.
  • SEQ ID NO: 21 is an amino acid sequence of a P2A embodiment.
  • SEQ ID NO: 22 is a nucleic acid sequence of a P2A embodiment.
  • SEQ ID NO: 23 is an amino acid sequence of an IL-12 signal peptide embodiment.
  • SEQ ID NO: 24 is a nucleic acid sequence of an IL-12 signal peptide embodiment.
  • SEQ ID NO: 25 is an amino acid sequence of an IL-12 P40 subunit embodiment.
  • SEQ ID NO: 26 is a nucleic acid sequence of an IL-12 P40 subunit embodiment.
  • SEQ ID NO: 27 is an amino acid sequence of an elastin linker embodiment.
  • SEQ ID NO: 28 is a nucleic acid sequence of an elastin linker embodiment.
  • SEQ ID NO: 29 is an amino acid sequence of an IL-12 P35 subunit embodiment.
  • SEQ ID NO: 31 is an amino acid sequence of a GS linker embodiment.
  • SEQ ID NO: 32 is a nucleic acid sequence of a GS linker embodiment.
  • SEQ ID NO: 33 is an amino acid sequence of a collagen binding domain embodiment.
  • SEQ ID NO: 34 is a nucleic acid sequence of a collagen binding domain embodiment.
  • SEQ ID NO: 35 is an amino acid sequence of an IL-12 subunit embodiment.
  • SEQ ID NO: 37 is a nucleic acid sequence of vector and pHIV-aMeso-CAR-IL-12-CBD- CBD-A embodiment.
  • SEQ ID NO: 38 is a nucleic acid sequence of a vector and pHIV-aMeso-CAR-CBD-IL-12- MC embodiment.
  • SEQ ID NO: 39 is an amino acid sequence of a GS linker embodiment.
  • FIGS. 3A-3D show an embodiment in which IL12-engineered MSLN-CAR CIML NK cells were characterized.
  • the oxygen consumption rate (OCR; FIGS. 3A, 3C) and extracellular acidification rate (ECAR; FIGS. 3B, 3D) were evaluated in real time.
  • FIGS. 4A-4C show an embodiment in which IL12-engineering of MSLN-CAR CIML NK cells dramatically enhanced the anti-tumor response.
  • FIG. 4A Cytotoxicity was assessed (as measured by CCK8 assay) for 0VCAR8, SK0V3, AsPCl, and Capan2 cell lines after 24-hour coculture with untransduced (top down triangle), MSLN-CAR (top up trangle), MSLN-CAR-IL12 (circle), and MSLN-CAR-IL12-A3 (square) CIML NK cells.
  • FIGS. 4A-4C show an embodiment in which IL12-engineering of MSLN-CAR CIML NK cells dramatically enhanced the anti-tumor response.
  • IFNg secretion was measured by ELISA from the untransduced, MSLN-CAR, MSLN-CAR-IL12, and MSLN-CAR- IL12-A3 CIML NK cells after a 24-hour co-culture with the cancer cell lines.
  • FIG. 5 shows an embodiment in which IL12-engineering of MSLN-CAR CIML NK cells dramatically enhanced the anti-tumor response.
  • Apoptotic cell populations were assessed (double positive Apotracker and Live/dead staining gated on EpCAM + cells) after a 24-hour co-culture of 3 different patient centric models, including patient-derived organoids (top) and patient-derived xenograft cells (bottom) with untransduced (tip down triangle), MSLN-CAR (tip up triangle), MSLN-CAR-IL12 (circle), and MSLN-CAR-IL12-A3 (hexagon) CIML NK cells.
  • FIGS. 6A-6B show an embodiment in which a long-term re-challenge experiment assessed NK cell-mediated cytotoxicity against cancer cell lines 0VCAR8 and AsPCl at E:T ratio of 1 : 1. Changes in tumor cell viability across 3 successive challenges over 12 days for untransduced (tip down triangle), MSLN-CAR (tip up triangle), MSLN-CAR-IL12 (circle), and MSLN-CAR-IL12-A3 (hexagon) NK cells.
  • FIGS. 7A-7C show an embodiment in which (FIGS. 7A-7B) expression of key cytolytic mediators was assessed in IL12-engineered NK cells, including CD107a + , IFNg + , Granzyme B (GrB), Perforin (PRE) and TNFa NK cells after 24-hour incubation with cancer cells; 0VCAR8 or AsPCl; and (FIG. 7C) expression was assessed for key NK cell receptors in MSLN-CAR-IL12 and MSLN-CAR-IL12-A3 CIML NK cells compared to MSLN-CAR NK cells post 24-hour co-culture with tumor cells (0VCAR8).
  • FIGS. 7A-7B show an embodiment in which (FIGS. 7A-7B) expression of key cytolytic mediators was assessed in IL12-engineered NK cells, including CD107a + , IFNg + , Granzyme B (GrB), Perforin (PRE) and TNFa NK cells after 24-hour
  • FIGS. 8A-8D show an embodiment in which transactivation was assessed for bystander NK (By-NK) cells by IL12-engineered CAR CIML NK cells.
  • By-NK Bosset NK
  • FIGS. 8A-8B Percentage of CD107a + and IFNg + was assessed among the CTV + By-NK cells co-cultured with MSLN-CAR, MSLN-CAR- IL12, and MSLN-CAR-IL12-A3 followed by their exposure to 0VCAR8 and AsPCl cells.
  • FIGS. 9A-9D show an embodiment in which transactivation was assessed for bystander T (By-T) cells by IL12-engineered CAR CIML NK cells. Percentage of CD107a + and IFNg + among the CTV + By-T cells gated on CD4 + T cells and CD8 + T cells co-cultured with MSLN-CAR, MSLN- CAR-IL12, and MSLN-CAR-IL12-A3 followed by their exposure to 0VCAR8 and AsPCl cells.
  • FIGS. 10A-10B show an embodiment in which transactivation was assessed for bystander T (By-T) cells by IL12-engineered CAR CIML NK cells. Percentage of CD107a + and IFNg + among the CTV + By-T cells gated on CD4 + T cells and CD8 + T cells co-cultured with MSLN-CAR, MSLN- CAR-IL12, and MSLN-CAR-IL12-A3 followed by their exposure to 0VCAR8 and AsPCl cells.
  • FIG. 11 shows an embodiment in which transactivation was assessed for tumor-associated immune cells by IL12-engineered CAR CIML NK cells. Representative histograms are shown for DNAM1, NKp30, NKG2D, CD25 and CD69 in tumor associated NK (TA-NK) cells (gated on CTV CD45 + CD3 CD56 + cells).
  • TA-NK tumor associated NK
  • FIGS. 12A-12B show an embodiment in which transactivation was assessed for tumor- associated immune cells by IL12-engineered CAR CIML NK cells. Representative histograms are shown for key surface receptors CD25, CD69, LAG3, Tim3, CTLA4 and PD1 in tumor associated T (TA-T) cells (gated on CD45 CD3 cells). Data is represented as Mean ⁇ SD.
  • FIGS. 14A-14B show an embodiment in which therapeutic efficacy was assessed for IL12- engineered MSLN-CAR CIML NK cells in an ovarian cancer xenograft model.
  • the engineered cells and pharmaceutical compositions provided herein can be administered to subjects or patients.
  • administration refers to the act of the attending physician or caregiver, prescribing the agent for administration and thereby causing the application of an agent to a subject, through ingestion, infusion, injection, or any other means, whether self-administered or administered by a clinician or other qualified caregiver.
  • a “subject” includes both human patients and veterinary subjects, including human and non-human mammals.
  • the subject or patient has or has a risk of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis.
  • an "antibody” or fragment thereof is an immunoglobulin molecule (or fragment thereof) comprising two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds.
  • the amino terminal portion of each LC and HC includes a variable region of about 100-120 amino acids primarily responsible for antigen recognition via the complementary determining region (CDRs) contained therein.
  • CDRs complementary determining region
  • the CDRs are interspersed with regions that are well-known and generally conserved among and between species (e.g., mouse and human), which are termed framework regions (FRs).
  • FRs framework regions
  • the CDRs are interspersed with FRs.
  • Antibodies disclosed herein have four FRs, termed FR1, FR2, FR3, and FR4.
  • the FRs are human FRs (e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)).
  • the three CDRs of the LC are referred to as “LCDR1, LCDR2, and LCDR3,” and the three CDRs of the HC are referred to as “HCDR1, HCDR2, and HCDR3.”
  • the functional ability of an antibody to bind a particular antigen is largely determined by CDRs. Assignment of amino acids to CDR domains within the LCVR and HCVR regions of the antibodies of the present disclosure is based on the well-known Kabat numbering conventions (Andrew Martin, Protein Sequence and Structure Analysis of Antibody Variable Domains in Antibody Engineering (Roland Kontermann and Stefan Dtibel eds., 2d ed. 2010)).
  • mesothelin antibody means refers to an antibody or fragment thereof that binds mesothelin, bringing the engineered cells and pharmaceutical compositions provided herein in proximity to mesothelin-expressing or mesothelin-overexpressing cells.
  • Antibodies within the scope of mesothelin antibody means are the disclosed antibodies and functional equivalents thereto.
  • Functional equivalent antibodies comprise different specific amino acid residues but bind mesothelin with a KD of less than 10’ 6 molar, such as less than 10‘ 7 molar, less than 10’ 8 molar, less than 10‘ 9 molar, or less than IO' 10 molar.
  • Functional equivalent antibodies having KD of less than 10' 6 molar, such as less than 10’ 7 molar, less than 10' 8 molar, less than 10' 9 molar, or less than IO' 10 molar would differ insubstantially to bind mesothelin, bringing the engineered cells and pharmaceutical compositions provided herein in proximity to mesothelin-expressing or mesothelin-overexpressing cell, and having a therapeutic effect.
  • mAbs monoclonal antibodies
  • mAbs can be produced, for example, by hybridoma technologies, recombinant technologies, phage display technologies, synthetic technologies (e.g., CDR or specificity-determining residue, SDR, grafting), or combinations of such or other technologies known in the art.
  • mAbs are antibodies derived from a single copy or clone including, for example, any eukaryotic, prokaryotic or phage clone.
  • binding refers to the well-understood interaction between a protein, peptide, or polysaccharide and the cognate binding partner (e.g., an interleukin and an interleukin receptor, such as IL-12 and IL-12 receptor, or a collagen binding domain (CBD) and collagen). Binding can be measured in a variety of ways (see, e.g., The Immunoassay Handbook: Theory and applications of ligand binding, ELISA and related techniques (David Wild ed., 4th ed. 2013)).
  • a particular protein, peptide, or polysaccharide binds to a cognate binding partner and does not bind in a significant amount to other proteins or polysaccharides present in a sample or subject disclosed herein. Binding occurs between the disclosed proteins or polysaccharides (e.g., IL-12 and/or CBD) and a cognate binding partner (e.g., IL- 12 receptor and collagen, respectively). Binding occurs when the interaction has a KD of less than 10‘ 6 molar, such as less than 10' 7 molar, less than 10' 8 molar, less than 10' 9 molar, or less than IO' 10 molar.
  • Chimeric antigen receptors are receptor proteins that have been engineered for expression on an immune cell and to target a specific antigen (“target antigen”) as well as activate an immune cell (e.g., memory-like NK cells).
  • CARs are used in therapies, such as immune cell therapy, including memory-like NK cell therapy.
  • CARs can be engineered into allogeneic immune cells (i.e., immune cells from a donor are engineered) or autologous immune cells (i.e., immune cells from a patient or subject that are re-introduced after engineering).
  • Cytokines are small proteins ( ⁇ 5-25 kDa) involved in cell signaling, typically through binding cognate cytokine receptors at the cell surface.
  • cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors and are produced by various cell types, such as immune cells (e.g., NK cells, macrophages, B lymphocytes, T lymphocytes, and mast cells), endothelial cells, fibroblasts, and stromal cells.
  • cells such as memory-like NK cells, can be engineered to produce or secrete cytokines. Cytokines play a role in immune responses to infection, inflammation, trauma, sepsis, cancer, and reproduction.
  • the cytokines of the present disclosure are interleukins, such as IL- 12.
  • an “effective amount” is a quantity sufficient to achieve a desired effect in a subject. For instance, this can be the amount necessary to prevent, treat, or ameliorate a disease, for example, inhibiting or suppressing cancer, such as solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis.
  • cancer such as solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis.
  • an effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of cancer or a tumor, such as in solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis.
  • Efficacy is first evident in the cellular response, for which a variety of in vitro and cell-based assays are well-known to measure. Kristina V. Kitaeva et al., Cell Culture Based In vitro Test Systems for Anti cancer Drug Screening, 8 Front. Bioeng. Biotechnol. 322(2020)).
  • an effective amount is the amount necessary to significantly inhibit or reduce cancer cell proliferation or migration, invasion, or adhesion.
  • a cellular response manifests as significantly reduced tumor size, reduced or inhibited disease progression, and improvement in survival in a subject or patient. More particularly, an effective amount provides improvement in important cancer endpoints, Overall Survival (OS), Disease-Free Survival (DFS), Objective Response Rate, Complete Response Rate or Progression Free Survival (PFS).
  • OS Overall Survival
  • DFS Disease-Free Survival
  • Objective Response Rate Objective Response Rate
  • Complete Response Rate or Progression Free Survival PFS.
  • Interleukin-12 is a cytokine secreted a variety of lymphocytes and other cell types. IL-12 enhances cytotoxic activity in NK cells and CD8+ cytotoxic T cells, regulates the activation and proliferation of T and natural killer (NK) cells, and is linked to NK cell signal transduction.
  • IL- 12 amino acid and nucleic acid sequences are well-known in the art.
  • IL-12 comprises the amino acid sequences of SEQ ID NOs: 23, 25, 29, and 35; embodiments of nucleic acids that encode IL-12 include SEQ ID NOs: 24, 26, 30, and 36.
  • NK cells Natural Killer Cells
  • Immune-based and CAR-based therapies include NK and CAR-NK cells, such as memory-like NK and memory-like CAR-NK cells.
  • NK cells include memory-like NK cells (e.g., US Patent Pub. 20230149460, incorporated herein by reference in its entirety on March 15, 2024) and memory-like CAR-NK cells (e.g., US Patent Pub. 20230149460, incorporated herein by reference in its entirety on March 14, 2025).
  • parenteral formulations typically comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions or the like as a vehicle.
  • pharmaceutical compositions can additionally include minor amounts of nontoxic auxiliary substances for stability.
  • the carrier may be sterile and/or suspended or otherwise contained in a unit dosage form including one or more measured doses of the composition suitable for administration to a subject of an effective amount of the engineered cells disclosed herein. Medications for use in treatment may also be included in embodiments.
  • the unit dosage form may be in a sealed vial that contains sterile contents or a syringe for injection into a subject, lyophilized for subsequent solubilization and administration, or in a solid or controlled release dosage.
  • a pharmaceutical composition of the present disclosure contains an "effective" or “therapeutically effective” amount, as used interchangeably herein, of an engineered cell of the present disclosure.
  • An effective amount is also one in which any toxic or detrimental effects of the engineered cell of the present disclosure are outweighed by the therapeutically beneficial effects.
  • sequence identity“ is referred to as the similarity between amino acid or nucleic acid sequences, which is expressed as the similarity between the sequences. Sequence identity is frequently measured as percent identity, in which two sequences are considered more similar the higher the percentage. Homologs or variants of a polypeptide or nucleic acid molecule possess a relatively high degree of sequence identity when aligned using standard methods, which are well-known. Ceslovas Venclovas, Methods for Sequence-Structure Alignment in Homology Modeling: Methods and Protocols 55-82 (Andrew Orry and Ruben Abagyan, eds., 2012)).
  • terapéutica in conjunction with engineered cells and pharmaceutical compositions disclosed herein refers to engineered cells suitable for use in human treatment of cancer, such as solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis.
  • Such engineered cells or pharmaceutical compositions express IL-12 and CBD that bind their cognate binding partner (e.g., IL-12 receptor and collagen, respectively) with a KD of less than 10' 6 molar, such as less than 10’ 7 molar, less than 10' 8 molar, less than 10' 9 molar, or less than IO' 10 molar and any toxic or detrimental effects of the engineered cells and pharmaceutical compositions disclosed herein are outweighed by the therapeutic beneficial effects.
  • cognate binding partner e.g., IL-12 receptor and collagen, respectively
  • the engineered cells disclosed herein can be used in therapy.
  • the engineered cells disclosed herein can be used to treat, prevent (such as through prophylactic treatment), or ameliorate a cancer, such as solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis.
  • a cancer such as solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis.
  • preventing refers to inhibiting the full development of a disease, such as cancer, such as solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis.
  • Treating refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number of size of metastases.
  • “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer, such as solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis.
  • a disease such as cancer, such as solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis.
  • a “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing a pathology, such as cancer, such as solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis.
  • cancer such as solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis.
  • a protein is a conservative variant where it contains conservative amino acid substitutions that do not substantially affect or decrease the affinity of a protein.
  • a cytokine that binds its cognate receptor can include at least 1, 2, 5, 10, or 15 conservative substitutions, for example, and bind the cognate receptor.
  • Conservative amino acid substitution tables providing functionally similar amino acids are well-known to one of ordinary skill in the art.
  • the following groups are examples of amino acids that are considered conservative substitutions for one another: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).
  • the engineered cells comprise a natural killer cell (an NK cell), comprising a chimeric antigen receptor (CAR) of the formula:
  • A is a mesothelin antibody means for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain
  • I is interleukin 12 (IL- 12); and C is a collagen binding domain.
  • the engineered cells comprise a natural killer cell (an NK cell), comprising a chimeric antigen receptor (CAR) of the formula:
  • A is a mesothelin antibody means for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain; and I is interleukin 12 (IL- 12).
  • the engineered cells comprise a natural killer cell (an NK cell), comprising a chimeric antigen receptor (CAR) of the formula:
  • a - B - I - C (Formula III), wherein: A is an antibody or fragment thereof for binding a target antigen;
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain
  • I is interleukin 12 (IL- 12); and
  • C is a collagen binding domain
  • the engineered cells comprise a natural killer cell (an NK cell), comprising a chimeric antigen receptor (CAR) of the formula:
  • A is an antibody or fragment thereof for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain
  • the immune cell is selected from the group consisting of T cells, natural killer (NK) cells, macrophages, dendritic cells, hematopoietic stem cells (HSC), induced pluripotent stem cells, cord blood stem cells, and/or derivatives thereof.
  • the immune cell is a T cell.
  • the immune cell is selected from the list consisting of: a cytotoxic lymphocyte, T cell, cytotoxic T cell (CD8 + T cell), T helper cell (CD4 + T cell), a T cell and/or yd T cell, Thl7 T-cell, NK T (NKT) cell, and regulatory T (Treg) cell.
  • nucleic acids encode a chimeric antigen receptor (CAR) of the formula:
  • A is a mesothelin antibody means for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain
  • I is interleukin 12 (IL- 12); and C is a collagen binding domain.
  • the nucleic acids encode a chimeric antigen receptor (CAR) of the formula:
  • A is a mesothelin antibody means for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain; and I is interleukin 12 (IL-12).
  • IL-12 interleukin 12
  • the nucleic acids encode a chimeric antigen receptor (CAR) of the formula:
  • A is an antibody or fragment thereof for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain
  • I is interleukin 12 (IL- 12); and C is a collagen binding domain.
  • the nucleic acids encode a chimeric antigen receptor (CAR) of the formula: A B I (Formula IV), wherein:
  • A is an antibody or fragment thereof for binding a target antigen
  • B comprises a hinge region, a transmembrane domain, and a cytoplasmic domain
  • I is interleukin 12 (IL- 12).
  • mesothelin antibody means are contemplated in Formulas I and II, and various antibodies or fragments thereof are contemplated in Formulas III and IV.
  • the CAR of Formulas I-FV are specific for mesothelin, a glycosylphosphatidylinositol-anchored cell surface protein present in a variety of cancers.
  • the mesothelin antibody means or antibodies or fragments thereof comprise a mesothelin-binding antibody fragment.
  • the mesothelin antibody means or antibodies or fragments thereof comprise a mesothelin-binding scFv.
  • a mesothelin-binding scFvs comprise a light chain variable domain (LCVR) is linked to a heavy chain variable domain fragment (HCVR) that binds mesothelin.
  • the linkage comprises a GGGGS (SEQ ID NO: 39) linker or other linker disclosed herein, such as a linker comprising GGGGS GGGGS GGGGS (SEQ ID NO: 6).
  • A comprises the complementarity determining regions (CDRs) LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is SEQ ID NO: 1, the amino acid sequence of LCDR2 is SEQ ID NO:2, the amino acid sequence of LCDR3 is SEQ ID NOG.
  • A comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 8, the amino acid sequence of HCDR2 is SEQ ID NO:9, the amino acid sequence of HCDR3 is SEQ ID NO: 10.
  • A comprises the complementarity determining regions (CDRs) LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of LCDR1 is SEQ ID NO: 1, the amino acid sequence of LCDR2 is SEQ ID NO:2, the amino acid sequence of LCDR3 is SEQ ID NOG, the amino acid sequence of HCDR1 is SEQ ID NO: 8, the amino acid sequence of HCDR2 is SEQ ID NOV, and the amino acid sequence of HCDR3 is SEQ ID NO: 10.
  • CDRs complementarity determining regions
  • A comprises a light chain variable region (LCVR), wherein the amino acid sequence of the LCVR is SEQ ID NO: 4, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 5.
  • LCVR light chain variable region
  • HCVR heavy chain variable region
  • amino acid sequence of the HCVR is SEQ ID NO: 11
  • A comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the amino acid sequence of the LCVR is SEQ ID NO: 4, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 5; the amino acid sequence of the HCVR is SEQ ID NO: 11, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 12.
  • the antibodies and fragments thereof of formulas III and IV are directed to other differentially expressed proteins, peptides, and protein domains on tumor or cancer cells, such as anti-CD19 antibodies and fragments thereof and anti-CD17 antibodies and fragments thereof.
  • hinge regions also known as spacers
  • a person or ordinary skill in the art understands that hinge regions or spacers in a chimeric antigen receptor (CAR) typically sit between a target-binding region (e.g., A of Formulas I-IV, a mesothelin antibody means or antibody or fragment thereof, such as svFc) and the outer membrane of a cell in which the hinge region or spacer is expressed (e.g., a memory-like NK cell).
  • Hinge regions or spacers typically vary in size and can be derived from a variety of extant proteins, such as extracellular proteins or protein domains (e.g., US Patent No.
  • the hinge region comprises a hinge domain derived from CD3( ⁇ , CD4, CD8a, CD28, IgGl, IgG2, or IgG4.
  • the hinge region comprises a hinge domain derived from CD28.
  • the hinge region comprises the amino acid sequence of SEQ ID NO: 13.
  • the hinge region comprises the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 14.
  • transmembrane domains are contemplated for Formulas I-IV.
  • a person of ordinary skill in the art understands that transmembrane domains in a chimeric antigen receptor (CAR) typically sit between a hinge region or spacer and an intracellular domain.
  • Transmembrane domains can vary in size and are often derived from extant transmembrane proteins or protein domains (e.g., US Patent No. 10,597,456, incorporated herein by reference in its entirely on March 14, 2025).
  • the transmembrane domain of Formulas I-IV comprises a transmembrane domain derived from CD3 , CD4, CD8a, CD28, or CD137.
  • the transmembrane domain of Formulas I-IV comprises a transmembrane domain derived from CD28.
  • the transmembrane domain of Formulas I-IV comprises the amino acid sequence of SEQ ID NO: 15.
  • the transmembrane domain of Formulas I-IV comprises the amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 16.
  • Various the cytoplasmic domains are contemplated for R3 of Formulas I and II.
  • cytoplasmic domains in a chimeric antigen receptor are typically linked to a transmembrane domain and are within the intracellular compartment of a cell in which the cytoplasmic domain is expressed (e.g., memory -like NK cell).
  • Cytoplasmic domains typically vary in size and and include a signaling domain and/or a costimulatory domain can be derived from a variety of extant proteins, such as intracellular signaling, and/or costimulatory proteins or protein domains (e.g., US Patent No. 10,597,456, incorporated herein by reference in its entirely on March 14, 2025).
  • the cytoplasmic domain of Formulas I-IV comprises a signaling domain and a costimulatory domain.
  • the cytoplasmic domain of Formulas I-IV is of the formula:
  • R2 comprises a costimulatory domain.
  • the cytoplasmic domain of Formulas I-IV comprises R2 - Rl.
  • the cytoplasmic domain of Formulas I-IV comprises Rl - R2.
  • Rl comprises a signaling domain derived from CD3( ⁇ , CD27, CD28, CD40, KIR2DS2, MyD88, or 0X40.
  • Rl comprises a costimulatory domain derived from CD3( ⁇ .
  • Rl comprises a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 19.
  • Rl comprises a costimulatory domain comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 20.
  • R2 comprises a costimulatory domain derived from of CD3y, CD35, CD3s, CD3i CD27, CD40, CD28, CD72, CD80, CD86, CLEC-1, 4-1BB, TYROBP (DAP12), Dectin-1, FcaRI, FcyRI, FcyRII, FcyRIII, FceRI, IL-2RB, ICOS, KIR2DS2, MyD88, 0X40, or ZAP70.
  • R2 comprises a costimulatory domain derived from 4-1BB.
  • R2 comprises a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 17.
  • R2 comprises a costimulatory domain comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 18.
  • Various collagen binding domains are contemplated for Formulas I and III.
  • the collagen binding domains contemplated herein are linked to the cytoplasmic domain and are within the intracellular compartment of a cell (e.g., memory-like NK cell).
  • Collagen binding domains typically vary in size, ranging from about 7 to 224 amino acids, and can be derived from a variety of extant proteins, such as proteins or protein domains (e.g., US Patent Pub. No. 20200331984, incorporated by reference herein in its entirely on March 14, 2025).
  • the collagen binding domain of Formulas I and III comprises a collagen binding domain derived from von Willebrand Factor (vWF or vWBF), fibronectin protein (FN), Clostridium histolyticum protein ColH, or collagenase-derived peptide (aCBD).
  • the collagen binding domain of Formulas I and III comprises a collagen binding domain derived from vWF.
  • the collagen binding domain of Formulas I and III comprises a collagen binding domain comprising the amino acid sequence of SEQ ID NO: 33.
  • the collagen binding domain of Formulas I and III comprises a collagen binding domain comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 34.
  • IL-12 amino acid sequences such as amino acid sequences comprising various IL- 12 subunits and IL-12-derived peptides, are contemplated for Formulas I -IV.
  • the IL-12 amino acid sequences contemplated herein are linked to the cytoplasmic domain and are within the intracellular compartment of a cell (e.g., memory-like NK cell).
  • IL-12 amino acid sequences such as amino acid sequences comprising various IL-12 subunits and IL-12-derived peptides typically vary in size (e.g., SEQ ID NOs: 23, 25, 29, and 35).
  • the IL-12 of Formulas I-IV comprises an IL-12 protein, protein domain, or peptide derived from human IL-12.
  • the IL-12 of Formulas I-IV comprises an IL-12 protein, protein domain, or peptide comprising the amino acid sequence of SEQ ID NOs: 23, 25, 29, and 35.
  • the IL-12 of Formulas I-IV comprises an IL-12 protein, protein domain, or peptide comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NOs: 24, 26, 30, and 36.
  • Formulas I -IV can include one or more IL-12.
  • the IL-12 of Formulas I-IV comprises SEQ ID NOs: 23, 25, and 29.
  • the IL-12 of Formulas I-IV comprises SEQ ID NO: 35 and SEQ ID NO: 25.
  • peptide linkers are contemplated for linking the various elements of Formulas I-V.
  • a person of ordinary skill in the art understands that peptide linkers are typically about one to 100 amino acids in length (e.g., 1, 2, 3, 4, 5, 10, 12, 1-10, 1-12, or 1-20, or more amino acids) containing various types of amino acids (e.g., US Patent No. 11,041,023, incorporated by reference herein in its entirety on March 14, 2025).
  • the amino acids include a combination of one or more glycine(s) and/or serine(s).
  • a peptide linker of Formulas I-V comprises the amino acid sequence comprising GGGGS (SEQ ID NO: 39).
  • a peptide linker of Formulas I-V consists of the amino acid sequence GGGGS (SEQ ID NO: 39).
  • a peptide linker of Formulas I-V comprises or consists of 2, 3, 4, 5, 6, or 7 consecutive repeats of the amino acid sequence GGGGS (SEQ ID NO: 39).
  • a peptide linker of Formulas I-V comprises the amino acid sequence of SEQ ID NO: 6.
  • a peptide linker of Formulas I-V comprises an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 7.
  • a peptide linker of Formulas I-V comprises the amino acid sequence of SEQ ID NO: 31.
  • a peptide linker of Formulas I-V comprises an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 32.
  • a peptide linker of Formulas I-V is a linker derived from elastin.
  • a peptide linker of Formulas I-V comprises the amino acid sequence of SEQ ID NO: 27.
  • a collagen binding domain of Formulas I-V comprises a costimulatory domain comprising an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 28.
  • a peptide linker of Formulas I-V is a cleavable linker.
  • a peptide linker of Formulas I-V is a linker derived from P2A peptide (e.g., US Patent No. 11820819, incorporated herein by reference in its entirety on March 14, 2025).
  • a peptide linker of Formulas I-V comprises the amino acid sequence of SEQ ID NO: 21.
  • a peptide linker of Formulas I-V comprises an amino acid sequence encoded by the nucleic acid sequence of SEQ ID NO: 22.
  • a vector is transduced into an immune cell ex vivo, such as a viral or retroviral vector.
  • an immune cell ex vivo such as a viral or retroviral vector.
  • a lentiviral vector is transduced ex vivo into a memory -like NK cell (see, e.g., Zhang et al., Engineering CAR-T cells, Biomark Res. 5:22 (2017); US Patent Pub. No. 20230149460, incorporated herein by reference in its entirety on March 14, 2025).
  • engineered cells and pharmaceutical compositions provided herein are formulated for administration by any suitable route, such as intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local, systemic, otic, inhalational, buccal (e.g., sublingual), and transdermal and can include additional agents that are biologically active, can facilitate or enhance delivery, or can control release. Other biologically active agents can also be administered sequentially, intermittently or simultaneously, such as in the same composition. Controlled release formulations and devices are contemplated, such as by pump. Additional means of formulation, administration, storage, preparation, manufacturing are contemplated (e.g., US Patent Pub. No. 20230149460, incorporated herein by reference in its entirety on March 14, 2025).
  • engineered cells or pharmaceutical compositions provided herein can be formulated into suitable pharmaceutical preparations, such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, patches, or sustained release formulations.
  • engineered cells or pharmaceutical compositions provided herein are formulated in a dried or liquid form.
  • engineered cells or pharmaceutical compositions provided herein are formulated in a liquid form, for example, as a suspension for injection (direct administration) or frozen suspension that is thawed prior to use, dried soluble form, and emulsion.
  • injection or infusion administration is contemplated, for example, subcutaneous, intramuscular, intratumoral, intravenous, or intradermal administration is contemplated.
  • excipients such as water, saline, dextrose, or glycerol
  • carriers such as a diluents, adjuvants, anti-adherents, binders, coatings, fdlers, flavors, colors, lubricants, glidants, preservatives, detergents, sorbents or combinations thereof, are contemplated.
  • aqueous vehicles e.g., aqueous vehicles, non-aqueous vehicles, isotonic agents, buffers, antioxidants, local anesthetics, suspending agents, dispersing agents, emulsifying agents, sequestering agents, chelating agents, or combinations thereof are contemplated herein as excipients and carriers.
  • a therapeutically effective amount of engineered cells or pharmaceutical compositions provided herein are formulated, for example, as single-unit or multi-unit dosage formulations. Additional formulations and modes of administration are contemplated (e.g., US Patent Pub. No. 20230149460, incorporated herein by reference in its entirety on March 14, 2025).
  • SEQ ID NO: 38 vector embodiment (pHIV-aMeso-CAR-CBD-IL-12-MC) gtcgacggatcgggagatctcccgatccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgt gtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttag gcgttttgcgctgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagtttcata gcccatatatgg
  • engineered cells and pharmaceutical compositions comprising the engineered cells disclosed herein and one or more acceptable carriers, diluents, or excipients.
  • methods of treating cancer comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer, such as solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis) a pharmaceutical composition provided herein.
  • a patient or subject in need thereof e.g., a patient or subject with or at risk of cancer, such as solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis
  • a patient or subject in need thereof e.g., a patient or subject with or at risk of cancer, such as solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis
  • kits for treating cancer selected from the list consisting of: solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis) an effective amount of a pharmaceutical composition comprising an engineered cell provided herein.
  • a patient or subject in need thereof e.g., a patient or subject with or at risk of cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mesothelin,
  • the provided herein are methods of treating cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer selected from the list consisting of: solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis) an effective amount of an engineered cell provided herein.
  • a patient or subject in need thereof e.g., a patient or subject with or at risk of cancer selected from the list consisting of: solid cancers, collagen containing tumors, cancers overexpressing mesothelin,
  • compositions provided herein for use in treating cancer comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis) a pharmaceutical composition provided herein.
  • a patient or subject in need thereof e.g., a patient or subject with or at risk of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis
  • engineered cells provided herein for use in treating cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis) an effective amount of an engineered cell provided herein.
  • a patient or subject in need thereof e.g., a patient or subject with or at risk of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis
  • compositions provided herein for use in treating cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis) an effective amount of a pharmaceutical composition comprising an engineered cell provided herein.
  • a patient or subject in need thereof e.g., a patient or subject with or at risk of cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mes
  • compositions provided herein for use in treating cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis) an effective amount of an engineered cell provided herein.
  • a patient or subject in need thereof e.g., a patient or subject with or at risk of cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, he
  • a pharmaceutical composition provided herein for treating cancer comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis) a pharmaceutical composition provided herein.
  • a patient or subject in need thereof e.g., a patient or subject with or at risk of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis
  • an engineered cell provided herein for treating cancer comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis) an effective amount of an engineered cell provided herein.
  • a patient or subject in need thereof e.g., a patient or subject with or at risk of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis
  • a pharmaceutical composition provided herein for treating cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis) an effective amount of a pharmaceutical composition comprising an engineered cell provided herein.
  • a patient or subject in need thereof e.g., a patient or subject with or at risk of cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mes
  • an engineered cell provided herein for treating cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mesothelin, hematological malignancies, lung adenocarcinomas, ovarian carcinomas, acute myeloid leukemia, pancreatic cancer, and myelofibrosis) an effective amount of an engineered cell provided herein.
  • a patient or subject in need thereof e.g., a patient or subject with or at risk of cancer selected from the list consisting of solid cancers, collagen containing tumors, cancers overexpressing mesothelin,
  • tumor cells were targeted with an MSLN-CAR along with the incorporation of a secreted IL12 fused with a collagen binding domain (A3) for trapping the CAR in a collagen- rich TME in order to enhance activity while limiting systemic IL 12 exposure and side effects.
  • A3 collagen binding domain
  • NK and T cells were extracted from the same donor leukapheresis collars. NK cells were differentiated into CIML and transduced. T cells were activated using CD3/CD28 beads (TransAct, Miltenyi Biotec®) for 3 days and then transduced. Both NK and T cells were transduced with the following constructs: MSLN- CAR and MSLN-CAR-IL12 and then cultured for 10-12 days.
  • the same donor untransduced and transduced cells were co-cultured with and without tumor target cells (OVCAR8) at E:T ratio of 1 : 1 (250,000 cells in a 24-well plate) for 24 hours, and then the supernatant was collected and stored at - 80 °C. At a later time point, this supernatant was thawed and then evaluated for cytokine/chemokines using a 48-plex panel by Eve Technologies.
  • the cytokine/chemokine are reported as pg/mL, ⁇ OOR values are reported as 0, and the values >OOR were extrapolated from the calibration curve, per manufacturer directions.
  • a MSLN-CAR gene construct was designed with the following components: signal peptide (CD8), MSLN scFv derived from the YP218 antibody, transmembrane (CD8), followed by intracellular signaling domains, 4- 1BB and CD3z.
  • the CAR construct was linked to eGFP via P2A self-cleaving peptide that allowed for assessment of the transduction efficiency.
  • IL12 or IL12-A3 (IL12-A3) were cloned by replacing GFP in a MSLN-CAR construct.
  • the IL12 sequence consisted of p35 followed by p40 with an elastin linker.
  • Collagen binding A3 domain from human vWF factor was linked to the to the N- terminus of p35 subunit using a flexible linker (G4S)e.
  • G4S flexible linker
  • HA tag on the CAR protein was used to determine the transduction efficiency.
  • Lentiviral supernatants were produced using a Baboon lentiviral (BaLV) system and titrated using Jurkat cells.
  • NK MACS media Miltenyi Biotec® supplemented with 5% human serum and 1% Pen/Strep (complete NK MACS media) was added to the transduced cells in the presence of IL2 (500 U/mL). The cells were washed after 3 days and cultured in NK MACS in the presence of low dose IL15 (1 ng/mL).
  • IL12-engineered CAR NK cells Characterization of IL12-engineered CAR NK cells.
  • the transduced cells were evaluated for transduction efficiency by gating on HA-tag + cells and by intracellular staining for IL12 using flow cytometry.
  • the supernatant from the engineered NK cells was collected and evaluated for secreted IL12 using ELISA (Human IL12 p70, Abeam® AB213791).
  • NK MACS media + 1 ng/mL IL 15 After 40 minutes incubation, devices were hydrated with culture media (NK MACS media + 1 ng/mL IL 15). After 72-96 hours, microfluidic chips were stained with IL12 antibody incubated at room temperature for 30 minutes, washed twice with PBS and imaged. Image capture and analysis were performed using a Nikon® Eclipse 80i fluorescence microscope equipped with Z-stack (Prior®), motorized stage (ProScan®) and ZYLA5.5 sCMOS camera (Andor®) and NIS-Elements® AR software package.
  • IL12-engineered CAR-NK cells were cultured for 8-10 days in NK MACS media supplemented with 1 ng/mL of IL-15. The cells were then stained using our flow panels to evaluate the following activating and inhibitory receptors on NK cells: CD56, CD16, NKp30, NKp44, DNAM1, KLRG1, NKG2D, NKG2A, Siglec7, CD39, CD69, NKG2C, CD2, TRAIL, CD158 (KIR2DL1/S1/S3/S5), FasL, TIGIT and Tim3 distributed across 4 panels using flow cytometry.
  • IL12-engineered CAR-NK cells were cultured for 6 days. The cells were then collected, washed once with PBS, and dry pellets were frozen in -80 °C. Bulk RNA sequencing analysis was performed by MedGenome Inc. (California, USA) and analyzed using Partek® Flow® Genomic analysis software.
  • IL12-engneered NK cells Metabolic analysis of IL12-engneered NK cells.
  • untransduced, MSLN- CAR, MSLN-CAR-IL12 and MSLN-CAR-IL12-A3 cells were rested in media and then plated at a density of 300,000 cells per well in Agilent® Seahorse® XFe96 cell culture microplate.
  • the Mito stress test and Glycolysis test was performed using the Agilent® Seahorse® XF T cell metabolic profding kit and XF Glycolysis stress test kit following the manufacturer’s instructions.
  • the data was analyzed using the Agilent® wave software.
  • CAR construct design, transduction and characterization of IL12-engineered MSLN-CAR CIML NK cells CAR expression was assessed by HA-tag staining. Quantification of IL12 secretion from untransduced, MSLN-CAR, MSLN-CAR-IL12, and MSLN-CAR-IL12-A3 CIML NK cells was assessed using ELISA. Collagen binding activity of IL12-A3 was assessed using a microfluidic device containing a 3-Dimensinal collagen gel and transduced CIML NK cells loaded into the side-channel. After 96-hour incubation, the devices were washed and stained for IL12 cytokine.
  • the data is represented as mean fluorescence intensity (MFI) measured over the area of the device.
  • MFI mean fluorescence intensity
  • Expression of key activation, inhibitory and cytotoxic/killer ligands in untransduced, MSLN-CAR, MSLN-CAR-IL12, and MSLN-CAR-IL12-A3 CIML NK cells was measured by flow cytometry and represented as percentage positive cells in the heat map.
  • the data are represented as mean ⁇ SD.
  • IL12-engineered MSLN-CAR CIML NK cells Characterization of IL12-engineered MSLN-CAR CIML NK cells (FIGS 3A-3D),
  • OCR oxygen consumption rate
  • ECAR extracellular acidification rate
  • IL12-engineering of MSLN-CAR CIML NK cells dramatically enhances their anti-tumor responses (FIGS. 4A-4C)
  • cis-activation of the CAR CIML NK cells by secreted IL12 or IL12-A3 molecules was used. Cytotoxicity (as measured by CCK8 assay) of 0VCAR8, SKOV3, AsPCl, and Capan2 cell lines after 24-hour co-culture with untransduced, MSLN-CAR, MSLN-CAR-IL12, and MSLN-CAR-IL12-A3 CIML NK cells.
  • IFNg secretion as measured by ELISA from the untransduced, MSLN-CAR, MSLN-CAR-IL12, and MSLN-CAR-IL12-A3 CIML NK cells after 24-hour co-culture with the cancer cell lines.
  • the data are is represented as mean ⁇ SD.
  • EOC cells (0VCAR8, SK0V3) and PDAC cells (AsPCl, Capan2) were plated in a 96 flat bottom plate (10,000 cells per well) and allowed to adhere overnight.
  • Untransduced (UNT), MSLN-CAR, MSLN-CAR-IL12, or MSLN-CAR-IL12-A3 NK cells were added to the cancer cells at E:T ratios of 1 :2, 1 : 1, and 2: 1 and co-cultured for 24 hours in the presence of IL15 (1 ng/mL).
  • the PDX cells from all the models exhibited 3D-spheroidal architecture, and hence, the spheroid count was used to set up the co-culture experiments using 500 spheroids per condition.
  • Untransduced, MSLN-CAR, or MSLN-CAR-IL12-A3 CIML NK cells were added at E:T ratio of 5: 1 and 2: 1.
  • the cells were dissociated with TrypLE dissociation solution (Gibco®) for 8-10 min and then washed once with FACS buffer. The single cells were stained for EpCAM, Apotracker green (Biolegend®), and Zombie NIR for 25 minutes at room temperature.
  • IL12-engineering of MSLN-CAR CIML NK cells dramatically enhances their anti -turn or responses (FIG. 5)
  • apoptotic cell population double positive Apotracker and live/dead staining gated on EpCAM + cells
  • apoptotic cell population after 24-hour co-culture of 3 different patient centric models including patient-derived organoids (top panel) and patient-derived xenograft cells (bottom panel) with untransduced, MSLN-CAR, MSLN-CAR-IL12, and MSLN-CAR-IL12-A3 CIML NK cells.
  • the data are represented as mean ⁇ SD and mean ⁇ SEM.
  • OVCAR8-mcLuc or AsPCl-mcLuc were plated in a 96 flat bottom plate (10,000 cells per well) and allowed to adhere overnight.
  • Untransduced (UNT), MSLN-CAR, MSLN-CAR-IL12, or MSLN-CAR-IL12-A3 NK cells were added to the cancer cells at E:T ratios of 1 :2 and 1 :1.
  • the viability of cancer cells was measured using mCherry every 3 hours. Cancer cells (10,000 cells per well) were rechallenged every 3 days for a total of 3 rechallenges across 12 days in the presence of IL15 (1 ng/mL).
  • CD 107a degranulation
  • cytolytic mediators including IFNg, Granzyme B (GrB), Perforin (PRF) and TNFa NK cells after 24-hour incubation with cancer cells; 0VCAR8 or AsPC 1 a E:T ratio of 2: 1.
  • GolgiPlug® and GolgiStop® were added after 1 hour of co-culture and then further incubated for 18 hours.
  • CD56, CD3, and CD107a surface proteins (CD56, CD3, and CD107a) (antibody details in Table SI) as well as live/dead dye (Invitrogen®).
  • the cells were washed once with staining buffer before fixation (BD Cytofix/perm). Post fixation, cells were washed twice with Perm wash (IX), and intracellular staining performed (IFNg, GrB, PRF, and TNFa for 30 minutes on ice, then washed once with FACs buffer and data acquired using BD LSR Fortessa® Cell Analyzer. Data was reported as percentage of CD107a, IFNg, GrB, PRF, and TNFa positive NK cells calculated by gating on CD3 CD56 + NK cells.
  • IL12-engineered NK cells including CD107a + , IFNg + , Granzyme B (GrB), Perforin (PRF) and TNFa NK cells after 24-hour incubation with cancer cells; OVCAR8 or AsPCl.
  • the data are represented as mean + SD from four independent NK donors.
  • naive NK cells 25,000 cells were co-cultured with MSLN-CAR or IL12-engineered MSLN-CAR NK cells (25,000 cells) for 48 hours.
  • the naive NK cells were pre-stained with CTV (CellTraceViolet) to differentiate them from the IL12-engineered CARNK cells.
  • the CAR-NK plus naive NK cells were then cultured with the cancer cells (OVCAR8 or AsPCl, 25,000 cells per well).
  • GolgiPlug® and GolgiStop® (Greeneldin A and Monensin, BD biosciences®) were added after 1 hour of co-culture and then further incubated for 5 hours.
  • the cells were then stained using the same method as described above to assess degranulation (CD 107a) and IFNg production.
  • the data are reported as percentage of CD 107a and IFNg positive NK cells calculated by gating on CTV + CD3 CD56 + NK cells.
  • the T cells were first transduced with an MSLN-CAR as T cells need a tumor specific CAR or a TCR for their optimal activation by the target cells.
  • the CAR T cells were also pre-stained with CTV to differentiate them from the IL12- engineered CAR NK cells.
  • CD3 + MSLN-CAR T cells (25,000 cells) were co-cultured with IL12- engineered NK cells (25,000 cells) for 96 hours.
  • the CAR-T cells plus engineered CAR-NK cells were then exposed to the cancer cells (OVCAR8 or AsPCl, 25,000 cells per well) for additional 24 hours.
  • the cells were then stained using the same method as described above to assess degranulation (CD 107a) and IFNg production by the T cells.
  • the data are reported as percentage of CD 107a and IFNg positive T cells calculated by gating on CTV + CD3 + T cells.
  • the CTV + bystander NK or T cells in the trans-activation experiment was used to evaluate their proliferation. Briefly, the CTV-labeled Bystander NK or MSLN-CAR T cells were co-cultured with IL 12- engineered CAR NK cells for 48 and 96 hours, respectively. The proliferation was evaluated by the cell divisions. The intensity of the CTV signal and percentage of CTV-negative T cells were also quantified using FlowJo® and the proliferative index was defined as the total number of divisions divided by the number of cells that went into divisions.
  • Transactivation of the bystander NK (By-NK) cells by IL12-engineered CAR CIML NK cells was used.
  • co-culture of IL12-engineered MSLN-CAR CIML NK cells with the same donor (bystander) NK cells (CTV + ) for 48 hours followed by their exposure to the cancer cells for 6 hours was used.
  • Percentage of CD107a + and IFNg + among the CTV + By-NK cells co-cultured with MSLN-CAR, MSLN-CAR-IL12, and MSLN-CAR-IL12-A3 followed by their exposure to OVCAR8 and AsPCl cells was determined.
  • Transactivation of the bystander patient derived immune cells upon co-culture with the engineered CIML NK cells (FIG. 11).
  • the fluid was processed by centrifugation at 1500 rpm, and the cell pellets were filtered through 100 pm filter.
  • the cells were washed once with PBS and then RBC lysis was performed.
  • the cells were washed again and frozen at -80° C for future use.
  • the ascites cells were screened from each patient to determine the percentage of immune cells including NK and T cells.
  • ascites cells (100,000) were co-cultured with IL12-engineered CAR-CIML NK cells (100,000 cells per well and pre-stained with CTV to differentiate from the ascites cells) for 48 hours in a 96-well plate.
  • the co-cultured cells were then exposed to tumor cells (OVCAR8, 7000 cells per well) for additional 48 hours. After a total of 96 hours, the cells were collected, stained for CD45, CD3, CD56, DNAM1, NKp30, CD25 and CD69 for 30 minutes on ice, fixed using 1% PF A, washed with FACs buffer, and acquired using BD LSR Fortessa® Cell Analyzer.
  • the data is represented as mean Fluorescence intensity (MFI) and percentage positive cells for DNAM1, NKp30, CD25 and CD69 gated on the hCD45 + , CTV, CD3', and CD56 + ascites cells, and the CAR-NK cells were excluded by pre-staining with CTV.
  • MFI Fluorescence intensity
  • ascites cells (100,000) were incubated with CD3/CD28 beads (1 uL per well, TransAct, Miltenyi®) and low IL2 (20 U/mL) for 48 hours. The ascites cells were then co-cultured with IL12-engineered CAR-CIML NK cells (100,000 cells per well) for additional 96 hours. The cells were then collected, stained for CD45, CD3, CD56, CD25, CD69, LAG3, Tim3, CTLA4 and PD1 for 30 mins on ice, fixed using 1% PF A, washed with FACs buffer, and acquired using BD LSR Fortessa® Cell Analyzer (antibody details in Table SI).
  • the data is represented as mean fluorescence intensity (MFI) and percentage positive cells for CD25, CD69, LAG3, Tim3, CTLA4 and PD1 gated on the hCD45 + , and CD3 + ascites cells, and the CAR-NK cells were excluded by pre-staining with CTV.
  • MFI mean fluorescence intensity
  • NK and T cells tumor-associated immune cells derived from the ascitic fluid of patients with advanced ovarian cancer upon co-culture with IL12-engineered MSLN-CAR CIML NK cells was used.
  • Representative histograms are shown of DNAM1, NKp30, NKG2D, CD25 and CD69 in tumor associated NK (TA-NK) cells (gated on CTV CD45 CD3' CD56 + cells).
  • Representative histograms are shown of key surface receptors CD25, CD69, LAG3, Tim3, CTLA4 and PD1 in tumor associated T (TA-T) cells (gated on CD45 + CD3 + cells). The data are represented as mean ⁇ SD.
  • Transactivation of the bystander patient derived immune cells upon co-culture with the engineered CIML NK cells (FIGS. 12A-12B).
  • fluid was processed by centrifugation at 1500 rpm, and the cell pellets were filtered through 100 um filter.
  • the cells were washed once with PBS and then RBC lysis was performed.
  • the cells were washed again and frozen at -80° C for future use.
  • the ascites cells were screened from each patient to determine the percentage of immune cells including NK and T cells.
  • ascites cells (100,000) were co-cultured with IL12-engineered CAR-CIML NK cells (100,000 cells per well, pre-stained with CTV to differentiate from the ascites cells) for 48 hours in a 96-well plate.
  • the co-cultured cells were then exposed to tumor cells (OVCAR8, 7000 cells per well) for additional 48 hours. After a total of 96 hours, the cells were collected, stained for CD45, CD3, CD56, DNAM1, NKp30, CD25 and CD69 for 30 minutes on ice, fixed using 1% PF A, washed with FACs buffer and acquired using BD LSR Fortessa® Cell Analyzer.
  • the data is represented as mean fluorescence intensity (MFI) and percentage positive cells for DNAM1, NKp30, CD25 and CD69 gated on the hCD45 + , CTV, CD3', and CD56 + ascites cells, and the CAR-NK cells were excluded by pre-staining with CTV.
  • MFI mean fluorescence intensity
  • ascites cells (100,000) were incubated with CD3/CD28 beads (1 uL per well, TransAct, Miltenyi®) and low ZL2 (20 U/mL) for 48 hours. The ascites cells were then co-cultured with IL12-engineered CAR-CIML NK cells (100,000 cells per well) for additional 96 hours. The cells were then collected, stained for CD45, CD3, CD56, CD25, CD69, LAG3, Tim3, CTLA4 and PD1 for 30 mins on ice, fixed using 1% PF A, washed with FACs buffer, and acquired using BD LSR Fortessa® Cell Analyzer.
  • NK and T cells tumor-associated immune cells derived from the ascitic fluid of patients with advanced ovarian cancer upon co-culture with IL12-engineered MSLN-CAR CIML NK cells was used.
  • Representative histograms of DNAM1, NKp30, NKG2D, CD25 and CD69 in tumor associated NK (TA-NK) cells (gated on CTV CD45 + CD3 CD56 + cells).
  • Representative histograms of key surface receptors CD25, CD69, LAG3, Tim3, CTLA4 and PD1 in tumor associated T (TA-T) cells (gated on CD45 + CD3 + cells). The data are represented as mean ⁇ SD.
  • IL12-engineered MSLN-CAR CIML NK cells in an ovarian cancer xenograft model (FIGS. 13A-13B).
  • a tumor model and NK cell therapy were used; Tg-IL15 NSG mice received 0VCAR8-mcLuc (5xl0 ? , IP) followed by PBS (control), MSLN-CAR- IL12, MSLN-CAR-IL12-A3 CIML NK cells (IxlO 6 , IP) on day 15 after tumor cell injection.
  • the arrow indicates the day of NK cell injection.
  • the data are represented as mean ⁇ SD or mean ⁇ SEM.
  • the mice in the treatment group received a single injection of 1 million NK cells intraperitoneally (i.p.).
  • the NK cells were cultured ex vivo in NK MACS media supplemented with low dose IL15 (1 ng/mL) for 8-10 days after transduction prior to being injected into the mice.
  • the tumor progression was monitored with weekly BLI imaging. Mice were monitored for survival and euthanized upon disease progression, over-distended abdomen (due to the growth of solid tumors and/or ascites), 15% loss in body weight, or poor body condition (BSC).
  • BSC body condition

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Abstract

L'invention concerne des cellules immunitaires modifiées et leurs méthodes d'utilisation. Dans des modes de réalisation, des cellules immunitaires modifiées se lient à la mésothéline ou à une autre protéine exprimée de manière différentielle dans le cancer. Dans des modes de réalisation, les méthodes comprennent celles qui consistent à traiter le cancer.
PCT/US2025/019954 2024-03-15 2025-03-14 Cellules immunitaires modifiées et leurs méthodes d'utilisation Pending WO2025194049A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114907487A (zh) * 2021-11-26 2022-08-16 郑州大学 新型嵌合抗原受体以及包含其的免疫细胞
WO2022178367A2 (fr) * 2021-02-19 2022-08-25 University Of Southern California Récepteurs d'antigènes synthétiques à chaîne unique et à chaînes multiples pour diverses cellules immunitaires
US20230174623A1 (en) * 2018-09-28 2023-06-08 Massachusetts Institute Of Technology Collagen-localized immunomodulatory molecules and methods thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230174623A1 (en) * 2018-09-28 2023-06-08 Massachusetts Institute Of Technology Collagen-localized immunomodulatory molecules and methods thereof
WO2022178367A2 (fr) * 2021-02-19 2022-08-25 University Of Southern California Récepteurs d'antigènes synthétiques à chaîne unique et à chaînes multiples pour diverses cellules immunitaires
CN114907487A (zh) * 2021-11-26 2022-08-16 郑州大学 新型嵌合抗原受体以及包含其的免疫细胞

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