WO2024097877A1 - Anticorps anti-zp4 et récepteurs antigéniques chimériques et leurs méthodes d'utilisation - Google Patents

Anticorps anti-zp4 et récepteurs antigéniques chimériques et leurs méthodes d'utilisation Download PDF

Info

Publication number
WO2024097877A1
WO2024097877A1 PCT/US2023/078519 US2023078519W WO2024097877A1 WO 2024097877 A1 WO2024097877 A1 WO 2024097877A1 US 2023078519 W US2023078519 W US 2023078519W WO 2024097877 A1 WO2024097877 A1 WO 2024097877A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
cdr
domain
antibody
car
Prior art date
Application number
PCT/US2023/078519
Other languages
English (en)
Inventor
Valentina HOYOS
Bing Zhang
Lauren SOMES
Xinpei YI
Jonathan T. LEI
Original Assignee
Baylor College Of Medicine
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Baylor College Of Medicine filed Critical Baylor College Of Medicine
Publication of WO2024097877A1 publication Critical patent/WO2024097877A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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

Definitions

  • the present disclosure relates generally to the fields of immunology, cell biology, molecular biology, and medicine. More particularly, it concerns Zona Pellucida 4 (ZP4) antibodies and related compositions, including at least chimeric antigen receptors and methods of use thereof.
  • ZP4 Zona Pellucida 4
  • CART cells have the potential to cure patients with advanced cancer, as demonstrated by their extraordinary success in hematological malignancies, which resulted in the FDA approval of six CART therapies since 2017. Broadening their application toward solid tumors like breast cancer, however, has proven challenging.
  • One major hurdle is finding a target that is highly expressed by cancer cells but not vital tissues.
  • the present disclosure concerns methods and compositions related to particular antibodies.
  • the antibodies may be utilized in immunotherapies of any kind and for any medical application in which targeting of ZP4 is therapeutic.
  • the present disclosure provides isolated monoclonal antibodies, wherein the antibodies specifically bind to ZP4.
  • Embodiments of the disclosure include an isolated monoclonal antibody, wherein the antibody specifically binds to ZP4 and comprises: (I): (a) a first VH CDR comprising SEQ ID NO: 3; (b) a second VH CDR comprising SEQ ID NO: 4; (c) a third VH CDR comprising SEQ ID NO: 5; (d) a first VL CDR comprising SEQ ID NO: 8; (e) a second VL CDR comprising SEQ ID NO: 9; and (f) a third VL CDR comprising SEQ ID NO: 10; (II): (a) a first VH CDR comprising SEQ ID NO: 13; (b) a second VH CDR comprising SEQ ID NO: 14; (c) a third VH CDR comprising SEQ ID NO: 15; (d) a first VL CDR comprising SEQ ID NO: 18; (e) a second VL CDR comprising SEQ ID NO: 19; and
  • the antibody comprises: (a) a first VH CDR comprising SEQ ID NO: 3; (b) a second VH CDR comprising SEQ ID NO: 4; (c) a third VH CDR comprising SEQ ID NO: 5; (d) a first VL CDR comprising SEQ ID NO: 8; (e) a second VL CDR comprising SEQ ID NO: 9; and (f) a third VL CDR comprising SEQ ID NO: 10.
  • the antibody comprises a VH domain at least about 80% identical to the VH domain of SEQ ID NO: 2 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 7.
  • the antibody comprises a VH domain identical to the VH domain of SEQ ID NO: 2 and a VL domain identical to the VL domain of SEQ ID NO: 7.
  • the antibody comprises: (a) a first VH CDR comprising SEQ ID NO: 13; (b) a second VH CDR comprising SEQ ID NO: 14; (c) a third VH CDR comprising SEQ ID NO: 15; (d) a first VL CDR comprising SEQ ID NO: 18; (e) a second VL CDR comprising SEQ ID NO: 19; and (f) a third VL CDR comprising SEQ ID NO: 20.
  • the antibody comprises a VH domain at least about 80% identical to the VH domain of SEQ ID NO: 12 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 17.
  • the antibody comprises a VH domain identical to the VH domain of SEQ ID NO: 12 and a VL domain identical to the VL domain SEQ ID NO: 17.
  • the antibody comprises: (a) a first VH CDR comprising SEQ ID NO: 23; (b) a second VH CDR comprising SEQ ID NO: 24; (c) a third VH CDR comprising SEQ ID NO: 25; (d) a first VL CDR comprising SEQ ID NO: 28; (e) a second VL CDR comprising SEQ ID NO: 29; and (f) a third VL CDR comprising SEQ ID NO: 30.
  • the antibody comprises a VH domain at least about 80% identical to the VH domain of SEQ ID NO: 22 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 27.
  • the antibody comprises a VH domain identical to the VH domain of SEQ ID NO: 22 and a VL domain identical to the VL domain SEQ ID NO: 27.
  • the antibody comprises: (a) a first VH CDR comprising SEQ ID NO: 33; (b) a second VH CDR comprising SEQ ID NO: 34; (c) a third VH CDR comprising SEQ ID NO: 35; (d) a first VL CDR comprising SEQ ID NO: 38; (e) a second VL CDR comprising SEQ ID NO: 39; and (f) a third VL CDR comprising SEQ ID NO: 40.
  • the antibody comprises a VH domain at least about 80% identical to the VH domain of SEQ ID NO: 32 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 37.
  • the antibody comprises a VH domain identical to the VH domain of SEQ ID NO: 32 and a VL domain identical to the VL domain SEQ ID NO: 37.
  • the antibody comprises: (a) a first VH CDR comprising SEQ ID NO: 43; (b) a second VH CDR comprising SEQ ID NO: 44; (c) a third VH CDR comprising SEQ ID NO: 45; (d) a first VL CDR comprising SEQ ID NO: 48; (e) a second VL CDR comprising SEQ ID NO: 49; and (f) a third VL CDR comprising SEQ ID NO: 50.
  • the antibody comprises a VH domain at least about 80% identical to the VH domain of SEQ ID NO: 42 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 47.
  • the antibody comprises a VH domain identical to the VH domain of SEQ ID NO: 42 and a VL domain identical to the VL domain SEQ ID NO: 47.
  • any antibody encompassed herein may be recombinant.
  • the antibody is an IgG, IgM, IgA or an antigen binding fragment thereof.
  • the antibody may be a Fab', a F(ab')2, a F(ab')3, a monovalent scFv, a bivalent scFv, or a single domain antibody.
  • the antibody may be a human antibody, humanized antibody or de-immunized antibody.
  • the antibody is conjugated to an imaging agent, a chemotherapeutic agent, a toxin, or a radionuclide.
  • the antibody may be part of a fusion protein or part of a chimeric protein.
  • Embodiments of the disclosure include compositions comprising any antibody encompassed herein in a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier there is an isolated polynucleotide molecule comprising a nucleic acid sequence encoding any antibody encompassed herein.
  • Embodiments of the disclosure include recombinant polypeptides comprising an antibody VH domain comprising CDRs 1-3 of the VH domain of Clone 46 (SEQ ID NOs: 3, 4, and 5) and CDRs 1-3 of the VL domain of Clone 46 (SEQ ID NOs: 8, 9, and 10).
  • Embodiments of the disclosure include recombinant polypeptides comprising an antibody VH domain comprising CDRs 1-3 of the VH domain of Clone 108 (SEQ ID NOs: 13, 14, and 15) and CDRs 1-3 of the VL domain of Clone 108 (SEQ ID NOs: 18, 19, and 20).
  • Embodiments of the disclosure include recombinant polypeptides comprising an antibody VH domain comprising CDRs 1-3 of the VH domain of Clone 2 (SEQ ID NOs: 23, 24, and 25) and CDRs 1-3 of the VL domain of Clone 2 (SEQ ID NOs: 28, 29, and 30).
  • Embodiments of the disclosure include recombinant polypeptides comprising an antibody VH domain comprising CDRs 1-3 of the VH domain of Clone 128 (SEQ ID NOs: 33, 34, and 35) and CDRs 1-3 of the VL domain of Clone 128 (SEQ ID NOs: 38, 39, and 40).
  • Embodiments of the disclosure include recombinant polypeptides comprising an antibody VH domain comprising CDRs 1-3 of the VH domain of Clone 164 (SEQ ID NOs: 43, 44, and 45) and CDRs 1-3 of the VL domain of Clone 164 (SEQ ID NOs: 48, 49, and 50).
  • there is an isolated polynucleotide molecule comprising a nucleic acid sequence encoding any polypeptide encompassed herein.
  • Embodiments of the disclosure include host cells comprising one or more polynucleotide molecule(s) encoding any antibody and/or recombinant polypeptide encompassed herein.
  • the host cell is an immune cell, a mammalian cell, a yeast cell, a bacterial cell, a ciliate cell or an insect cell.
  • the cancer is a solid tumor, such as triple negative breast cancer.
  • the antibody is in a pharmaceutically acceptable composition.
  • the antibody may be administered systemically, intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.
  • the method further comprises administering at least a second anticancer therapy to the subject.
  • the second anticancer therapy may be a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, and/or cytokine therapy.
  • the second anticancer therapy may comprise an adoptive T-cell therapy.
  • an engineered Zona Pellucida 4 (ZP4) CAR or TCR having an antigen binding domain comprising: (I): (a) a first VH CDR comprising SEQ ID NO: 3; (b) a second VH CDR comprising SEQ ID NO: 4; (c) a third VH CDR comprising SEQ ID NO: 5; (d) a first VL CDR comprising SEQ ID NO: 8; (e) a second VL CDR comprising SEQ ID NO: 9; and (f) a third VL CDR comprising SEQ ID NO: 10; (II): (a) a first VH CDR comprising SEQ ID NO: 13; (b) a second VH CDR comprising SEQ ID NO: 14; (c) a third VH CDR comprising SEQ ID NO: 15; (d) a first VL CDR comprising SEQ ID NO: 18; (e) a second VL CDR comprising SEQ ID
  • the antigen-binding domain comprises: (a) a first VH CDR comprising SEQ ID NO: 3; (b) a second VH CDR comprising SEQ ID NO: 4; (c) a third VH CDR comprising SEQ ID NO: 5; (d) a first VL CDR comprising SEQ ID NO: 8; (e) a second VL CDR comprising SEQ ID NO: 9; and (f) a third VL CDR comprising SEQ ID NO: 10.
  • the antigen-binding domain may comprise a VH domain at least about 80% identical to the VH domain of SEQ ID NO: 2 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 7.
  • the antigen-binding domain may comprise a VH domain identical to the VH domain of SEQ ID NO: 2 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 7.
  • the antigenbinding domain comprises: (a) a first VH CDR comprising SEQ ID NO: 13; (b) a second VH CDR comprising SEQ ID NO: 14; (c) a third VH CDR comprising SEQ ID NO: 15; (d) a first VL CDR comprising SEQ ID NO: 18; (e) a second VL CDR comprising SEQ ID NO: 19; and (f) a third VL CDR comprising SEQ ID NO: 20.
  • the antigen-binding domain comprises a VH domain at least about 80% identical to the VH domain of SEQ ID NO: 12 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 17.
  • the antigen-binding domain comprises a VH domain identical to the VH domain of SEQ ID NO: 12 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 17.
  • the antigen-binding domain comprises: (a) a first VH CDR comprising SEQ ID NO: 23; (b) a second VH CDR comprising SEQ ID NO: 24; (c) a third VH CDR comprising SEQ ID NO: 25; (d) a first VL CDR comprising SEQ ID NO: 28; (e) a second VL CDR comprising SEQ ID NO: 29; and (f) a third VL CDR comprising SEQ ID NO: 30.
  • the antigen-binding domain comprises a VH domain at least about 80% identical to the VH domain of SEQ ID NO: 22 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 27. In specific embodiments, the antigen-binding domain comprises a VH domain identical to the VH domain of SEQ ID NO: 22 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 27.
  • the antigen-binding domain comprises: (a) a first VH CDR comprising SEQ ID NO: 33; (b) a second VH CDR comprising SEQ ID NO: 34; (c) a third VH CDR comprising SEQ ID NO: 35; (d) a first VL CDR comprising SEQ ID NO: 38; (e) a second VL CDR comprising SEQ ID NO: 39; and (f) a third VL CDR comprising SEQ ID NO: 40.
  • the antigen-binding domain comprises a VH domain at least about 80% identical to the VH domain of SEQ ID NO: 32 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 37. In specific embodiments, the antigen-binding domain comprises a VH domain identical to the VH domain of SEQ ID NO: 32 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 37.
  • the antigen-binding domain comprises: (a) a first VH CDR comprising SEQ ID NO: 43; (b) a second VH CDR comprising SEQ ID NO: 44; (c) a third VH CDR comprising SEQ ID NO: 45; (d) a first VL CDR comprising SEQ ID NO: 48; (e) a second VL CDR comprising SEQ ID NO: 49; and (f) a third VL CDR comprising SEQ ID NO: 50.
  • the antigen-binding domain comprises a VH domain at least about 80% identical to the VH domain of SEQ ID NO: 42 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 47. In specific embodiments, the antigen-binding domain comprises a VH domain identical to the VH domain of SEQ ID NO: 42 and a VL domain at least about 80% identical to the VL domain of SEQ ID NO: 47.
  • the CAR comprises one or more signaling domains selected from the group consisting of CD3 ⁇ , CD28, OX40/CD134, 4-1BB/CD137, ICOS, CD27 and a combination thereof.
  • the CAR or TCR may be encoded by a viral vector, such as a lentiviral vector, adenoviral vector, adeno-associated viral vector, or retroviral vector.
  • the CAR may comprise a hinge and/or a transmembrane domain.
  • the CAR or TCR, or cells expressing same may further comprise a transduction marker and/or safety switch, and the transduction marker and/or safety switch may be linked on a vector to the CAR by a cleavage peptide, such as 2A peptide, or IRES.
  • the CAR may further comprise a second antigen binding domain, such as one that targets a cancer antigen other than the antigen targeted by the other antigen binding domain.
  • Expression vectors encoding the CAR or TCR are encompassed herein.
  • a host cell engineered to express a ZP4 CAR and/or a ZP4 TCR.
  • the cell may be engineered to express any CAR or TCR encompassed herein.
  • the cell may be an immune cell, such as a T cell, including a primary human T cell or TIL and including a CD4+ T cell or CD8+ T cell.
  • the primary human T cell may or may not be obtained from a healthy donor.
  • the immune cell can be autologous or allogeneic with respect to a recipient individual.
  • the cell is engineered using a CRISPR or transposase system.
  • Embodiments of the disclosure include pharmaceutical compositions comprising ZP4 targeted T cells and a pharmaceutical carrier, wherein the ZP4 targeted T cells are engineered to express a CAR or TCR of the disclosure.
  • Embodiments of the disclosure include compositions comprising an effective amount of ZP4 targeted T cells for the treatment of cancer in a subject, wherein the ZP4 targeted T cells are engineered to express a CAR or TCR of the disclosure.
  • Embodiments of the disclosure include use of a composition comprising an effective amount of ZP4 targeted T cells for the treatment of cancer in a subject, wherein the ZP4 targeted T cells are engineered to express a CAR or TCR of the disclosure.
  • Embodiments of methods for treating cancer in a subject comprise administering an effective amount of ZP4 targeted T cells to the subject, wherein the ZP4 targeted T cells are engineered to express a CAR or TCR of the disclosure.
  • the cancer may or may not be a solid tumor.
  • the cancer may be triple negative breast cancer.
  • the ZP4 targeted T cells are administered intravenously, intradermally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, or locally.
  • any method further comprises administering at least a second anti cancer therapy to the subject, such as a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, immunotherapy, and/or cytokine therapy.
  • the cancer is a ZP4-expressing cancer.
  • FIGS. 1A-1D Identification of ZP4 as a target for TNBC 1
  • A ZP4 mRNA expression in normal human tissues in the GTEx study.
  • IB An MS/MS spectrum supporting the identification of one out of the seven identified ZP4 specific peptides in CPTAC samples.
  • 1C ZP4 mRNA expression in breast tumors in the METABRIC cohort.
  • ID ZP4 IHC staining of normal tissues demonstrates positivity restricted to ZP surrounding oozytes.
  • FIGS. 2A-2B ZP4 expression on BCM PDX models.
  • 2A ZP4 mRNA expressed in 33/103 (32%) of TNBC PDX models and ZP4 protein detected in 4/103 (4%) of TNBC PDX models.
  • 2B IHC confirms expression of ZP4 in 2 of the PDX models, BCM-9161 and BCM-0046.
  • FIGS. 3A-3D ZP4 CAR engineering.
  • 3A Schema of an example of a ZP4 CAR retroviral vector.
  • 3B Supernatant from hybridoma clones detected ZP4 expression on ZP4.RFP+ 293 T cells measured by flow cytometry. 293 T transduced with empty RFP vector used as negative control.
  • 3C Flow showing SUM159 transduced with ZP4.
  • 3D Cytotoxicity assay demonstrating ZP4 CART killing of ZP4+ TNBC cell line.
  • FIG. 4 mAB clone 46 used to stain ovary tissue, showing detection of ZP4 on Zona pellucida surrounding oocytes.
  • FIG. 5 mAB clone 46 used to stain normal organ tissues, showing no detection of ZP4.
  • FIG. 6 mAB clone 46 used to stain normal organ tissues, showing no detection of ZP4.
  • FIG. 7 mAB clone 46 used to stain normal organ tissues, showing no detection of ZP4.
  • FIG. 8 mAB clone 46 used to stain normal organ tissues, showing some possible ZP4 detection on testes tissue.
  • FIGS. 9A-E illustrates an exemplary structure of CAR constructions.
  • FIG. 9B contains graphs showing ZP4 CAR expression on the surface of T-cells.
  • FIG. 9C illustrates all three CAR cells are predominantly CD8+.
  • FIG. 9D shows the CAR cells have a predominantly effector memory (T em ) phenotype.
  • FIG. 9E illustrates the calculation of percentage of specific lysis in each clone (clones 108, 128, and 164).
  • FIGS. 10A-G illustrates an exemplary cycle for re-seeding, stimulating and harvesting T-cells.
  • FIG. 10B is a collection of representative flow diagrams displaying the results of the 72-hour co-cultures form weeks 1, 3, and 4.
  • FIG. 10C is a graph of T-cell expansion quantified by as the number of cells per well.
  • FIG. 10D is a graph of T cell activation after 72- hour coculture with target cells as measured via 41BB.
  • FIG. 10E is percent T cells that remained in each co-culture after they were quantified.
  • FIG. 10F is percent residual tumor cells that remained in each co-culture after they were quantified.
  • FIG. 10G is a collection of graphs measuring the polyfunctionality of the ZP4 CAR T cells as measured using Isoplexis.
  • FIGS. 11 A-B show photographs of CAR T cell persistence as monitored using the Lumina In Vivo Imaging System (IVIS).
  • FIG. 1 IB is a graph of in vivo persistence quantified as average radiance.
  • FIGS. 12A-E are views of photographs of tumor burden as monitored using IVIS.
  • FIG. 12B is a graph of tumor burden quantified as average radiance.
  • FIG. 12C is a graph of tumorvolume at days post tumor injection.
  • FIG. 12D is a graph of the probability of survival.
  • FIG. 12E is a collection of photographs and graphs illustrating clone 128 and clone 108 CAR T cells reduced overall metastatic burden, especially in the lungs and liver.
  • essentially free in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
  • “a” or “an” may mean one or more.
  • the words “a” or “an” may mean one or more than one.
  • Treating” or treatment of a disease or condition refers to executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, “treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition, “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.
  • therapeutic benefit refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.
  • treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.
  • Subject and “patient” refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.
  • phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate.
  • the preparation of a pharmaceutical composition comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure.
  • animal (e.g., human) administration it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.
  • “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art.
  • aqueous solvents e.g.
  • an antibody or a fragment thereof that binds to at least a portion of ZP4 and inhibits ZP4 activity of any kind, including at least signaling are contemplated.
  • the term “antibody” is intended to refer broadly to any immunologic binding agent, such as IgG, IgM, IgA, IgD, IgE, and genetically modified IgG as well as polypeptides comprising antibody CDR domains that retain antigen binding activity.
  • the antibody may be selected from the group consisting of a chimeric antibody, an affinity matured antibody, a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, or an antigen-binding antibody fragment or a natural or synthetic ligand.
  • the anti- ZP4 antibody is a monoclonal antibody or a humanized antibody.
  • polyclonal or monoclonal antibodies, antibody fragments, and binding domains and CDRs may be created that are specific to ZP4, one or more of its respective epitopes, or conjugates of any of the foregoing, whether such antigens or epitopes are isolated from natural sources or are synthetic derivatives or variants of the natural compounds.
  • antibody fragments suitable for the present embodiments include, without limitation: (i) the Fab fragment, consisting of VL, VH, CL, and CHI domains; (ii) the “Fd” fragment consisting of the VH and CHI domains; (iii) the “Fv” fragment consisting of the VL and VH domains of a single antibody; (iv) the “dAb” fragment, which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules (“scFv”), wherein a VH domain and a VL domain are linked by a peptide linker that allows the two domains to associate to form a binding domain; (viii) bi-specific single chain Fv dimers (see U.S.
  • Fv, scFv, or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains.
  • Minibodies comprising a scFv joined to a CH3 domain may also be made.
  • Antibody-like binding peptidomimetics are also contemplated in embodiments. Liu et al. (2003) describe “antibody like binding peptidomimetics” (ABiPs), which are peptides that act as pared-down antibodies and have certain advantages of longer serum half-life as well as less cumbersome synthesis methods.
  • ABSiPs antibody like binding peptidomimetics
  • Animals may be inoculated with an antigen, such as a ZP4 extracellular domain (ECD) protein, in order to produce antibodies specific for ZP4.
  • an antigen is bound or conjugated to another molecule to enhance the immune response.
  • a conjugate is any peptide, polypeptide, protein, or non-proteinaceous substance bound to an antigen that is used to elicit an immune response in an animal.
  • Antibodies produced in an animal in response to antigen inoculation comprise a variety of non-identical molecules (polyclonal antibodies) made from a variety of individual antibody producing B lymphocytes.
  • a polyclonal antibody is a mixed population of antibody species, each of which may recognize a different epitope on the same antigen.
  • a monoclonal antibody is a single species of antibody wherein every antibody molecule recognizes the same epitope because all antibody producing cells are derived from a single B-lymphocyte cell line.
  • the methods for generating monoclonal antibodies generally begin along the same lines as those for preparing polyclonal antibodies.
  • rodents such as mice and rats are used in generating monoclonal antibodies.
  • rabbit, sheep, or frog cells are used in generating monoclonal antibodies.
  • the use of rats is well known and may provide certain advantages.
  • Mice e.g., BALB/c mice
  • BALB/c mice are routinely used and generally give a high percentage of stable fusions.
  • Hybridoma technology involves the fusion of a single B lymphocyte from a mouse previously immunized with a ZP4 antigen with an immortal myeloma cell (usually mouse myeloma).
  • This technology provides a method to propagate a single antibody-producing cell for an indefinite number of generations, such that unlimited quantities of structurally identical antibodies having the same antigen or epitope specificity (monoclonal antibodies) may be produced.
  • ZP4 cells can be isolated.
  • Human ZP4 is overexpressed on the surface of murine L cells (an immortalized fibroblast cell line). These cells can then be sorted to enrich the ZP4-high population and used to immunize mice and generate antibody clones. This method ensures that the antibodies generated exclusively recognize extracellular epitopes.
  • the lymph nodes from the mice are removed and used to make hybridomas.
  • Supernatant from each hybridoma clone can be screened for specificity to ZP4.
  • RNA from the hybridomas that produce selected ZP4 mAb clones can then be used to obtain the sequences of the mAb variable regions and construct CARs.
  • Plasma B cells (CD45 + CD5'CD19 + ) may be isolated from freshly prepared rabbit peripheral blood mononuclear cells of immunized rabbits and further selected for ZP4 binding cells. After enrichment of antibody producing B cells, total RNA may be isolated and cDNA synthesized. DNA sequences of antibody variable regions from both heavy chains and light chains may be amplified, constructed into a phage display Fab expression vector, and transformed into A. coli. ZP4 specific binding Fab may be selected out through multiple rounds enrichment panning and sequenced.
  • Selected C ZP4 D79b binding hits may be expressed as full-length IgG in rabbit and rabbit/human chimeric forms using a mammalian expression vector system in human embryonic kidney (HEK293) cells (Invitrogen) and purified using a protein G resin with a fast protein liquid chromatography (FPLC) separation unit.
  • HEK293 human embryonic kidney
  • FPLC fast protein liquid chromatography
  • the antibody is a chimeric antibody, for example, an antibody comprising antigen binding sequences from a non-human donor grafted to a heterologous nonhuman, human, or humanized sequence (e.g., framework and/or constant domain sequences).
  • a heterologous nonhuman, human, or humanized sequence e.g., framework and/or constant domain sequences.
  • Methods have been developed to replace light and heavy chain constant domains of the monoclonal antibody with analogous domains of human origin, leaving the variable regions of the foreign antibody intact.
  • “fully human” monoclonal antibodies are produced in mice transgenic for human immunoglobulin genes.
  • Methods have also been developed to convert variable domains of monoclonal antibodies to more human form by recombinantly constructing antibody variable domains having both rodent, for example, mouse, and human amino acid sequences.
  • “humanized” monoclonal antibodies only the hypervariable CDR is derived from mouse monoclonal antibodies, and the framework and constant regions are derived from human amino acid sequences (see U.S. Pat. Nos. 5,091,513 and 6,881,557). It is thought that replacing amino acid sequences in the antibody that are characteristic of rodents with amino acid sequences found in the corresponding position of human antibodies will reduce the likelihood of adverse immune reaction during therapeutic use.
  • a hybridoma or other cell producing an antibody may also be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced by the hybridoma.
  • Antibodies may be produced from any animal source, including birds and mammals.
  • the antibodies are ovine, murine (e.g., mouse and rat), rabbit, goat, guinea pig, camel, horse, or chicken.
  • newer technology permits the development of and screening for human antibodies from human combinatorial antibody libraries.
  • bacteriophage antibody expression technology allows specific antibodies to be produced in the absence of animal immunization, as described in U.S. Pat. No. 6,946,546, which is incorporated herein by reference.
  • antibodies to ZP4 will have the ability to neutralize or counteract the effects of ZP4 regardless of the animal species, monoclonal cell line, or other source of the antibody.
  • Certain animal species may be less preferable for generating therapeutic antibodies because they may be more likely to cause allergic response due to activation of the complement system through the “Fc” portion of the antibody.
  • whole antibodies may be enzymatically digested into “Fc” (complement binding) fragment, and into antibody fragments having the binding domain or CDR. Removal of the Fc portion reduces the likelihood that the antigen antibody fragment will elicit an undesirable immunological response, and thus, antibodies without Fc may be preferential for prophylactic or therapeutic treatments.
  • antibodies may also be constructed so as to be chimeric or partially or fully human, so as to reduce or eliminate the adverse immunological consequences resulting from administering to an animal an antibody that has been produced in, or has sequences from, other species.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • substitutions may be nonconservative such that a function or activity of the polypeptide is affected.
  • Non-conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.
  • Proteins may be recombinant, or synthesized in vitro.
  • a nonrecombinant or recombinant protein may be isolated from bacteria. It is also contemplated that a bacteria containing such a variant may be implemented in compositions and methods. Consequently, a protein need not be isolated.
  • compositions there is between about 0.001 mg and about 10 mg of total polypeptide, peptide, and/or protein per ml.
  • concentration of protein in a composition can be about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml or more (or any range derivable therein).
  • about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% may be an antibody that binds ZP4 .
  • An antibody or preferably an immunological portion of an antibody can be chemically conjugated to, or expressed as, a fusion protein with other proteins.
  • a fusion protein with other proteins.
  • all such fused proteins are included in the definition of antibodies or an immunological portion of an antibody.
  • Embodiments provide antibodies and antibody-like molecules against ZP4, polypeptides and peptides that are linked to at least one agent to form an antibody conjugate or payload.
  • it is conventional to link or covalently bind or complex at least one desired molecule or moiety.
  • a molecule or moiety may be, but is not limited to, at least one effector or reporter molecule.
  • Effector molecules comprise molecules having a desired activity, e.g., cytotoxic activity.
  • Non-limiting examples of effector molecules that have been attached to antibodies include toxins, therapeutic enzymes, antibiotics, radio-labeled nucleotides and the like.
  • reporter molecule is defined as any moiety that may be detected using an assay.
  • reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands, such as biotin.
  • a metal chelate complex employing, for example, an organic chelating agent such as a diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachl oro-3 -6-diphenylglycouril-3 attached to the antibody.
  • DTP A diethylenetriaminepentaacetic acid anhydride
  • ethylenetriaminetetraacetic acid N-chloro-p-toluenesulfonamide
  • tetrachl oro-3 -6-diphenylglycouril-3 attached to the antibody.
  • Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate.
  • Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate.
  • Certain embodiments of the present disclosure concern obtaining and administering cells to a subject as an immunotherapy to target cancer cells.
  • the cells may deliver antibody compositions encompassed herein but themselves may or may not be immune cells.
  • the cells are immune cells. Examples of cells include T cells (including 0.0 T cells or y8 T cells), Natural Killer (NK) cells, invariant NKT (iNKT) cells, B cells, macrophages, stem cells of any kind (including MSCs or induced pluripotent stem cells), or dendritic cells.
  • T cells including 0.0 T cells or y8 T cells
  • NK Natural Killer
  • iNKT invariant NKT
  • B cells macrophages
  • stem cells of any kind including MSCs or induced pluripotent stem cells
  • dendritic cells dendritic cells.
  • TILs tumor-infiltrating lymphocytes
  • APCs artificial antigen-presenting cells
  • beads coated with T cell ligands and activating antibodies or cells isolated by virtue of capturing target cell membrane
  • allogeneic cells naturally expressing anti-host tumor T cell receptor (TCR)
  • non-tumor-specific autologous or allogeneic cells genetically reprogrammed or "redirected" to express tumor-reactive TCR or chimeric TCR molecules displaying antibody-like tumor recognition capacity known as "T-bodies”.
  • the T cells are derived from the blood, bone marrow, lymph, or lymphoid organs.
  • the cells are human cells.
  • the cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen.
  • the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4 + cells, CD8 + cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
  • the cells may be allogeneic and/or autologous.
  • the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs).
  • the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, as described herein, and re-introducing them into the same patient, before or after cryopreservation.
  • T cells e.g., CD4 ⁇ and/or CD8 + T cells
  • TN naive T
  • TEFF effector T cells
  • memory T cells and sub-types thereof such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T (TrEMRA)cells, tumor-infdtrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells
  • helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
  • one or more of the T cell populations is enriched for or depleted of cells that are positive for a specific marker, such as surface markers, or that are negative for a specific marker.
  • a specific marker such as surface markers
  • such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (e.g., memory cells).
  • the cells are enriched for (i.e., positively selected for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and/or CD62L and/or depleted of (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA.
  • cells are enriched for or depleted of cells positive or expressing high surface levels of CD122, CD95, CD25, CD27, and/or IL7-Ra (CD 127).
  • CD8 + T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L.
  • T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14.
  • a CD4 + or CD8 + selection step is used to separate CD4 + helper and CD8 + cytotoxic T cells.
  • Such CD4 + and CD8 + populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
  • CD8 + cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation.
  • enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations.
  • combining TCM- enriched CD8 + T cells and CD4 + T cells further enhances efficacy.
  • the T cells are autologous T cells.
  • tumor samples are obtained from patients and a single cell suspension is obtained.
  • the single cell suspension can be obtained in any suitable manner, e.g., mechanically (disaggregating the tumor using, e.g., a gentleMACSTM Dissociator, Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase).
  • Single-cell suspensions of tumor enzymatic digests are cultured in interleukin-2 (IL-2).
  • the cells are cultured until confluence (e.g., about 2> ⁇ 10 6 lymphocytes), e.g., from about 5 to about 21 days, preferably from about 10 to about 14 days.
  • the cells may be cultured from 5 days, 5.5 days, or 5.8 days to 21 days, 21.5 days, or 21.8 days, such as from 10 days, 10.5 days, or 10.8 days to 14 days, 14.5 days, or 14.8 days.
  • the cultured T cells can be pooled and rapidly expanded. Rapid expansion provides an increase in the number of antigen-specific T-cells of at least about 50-fold (e.g., 50-, 60-, 70-, 80-, 90-, or 100-fold, or greater) over a period of about 10 to about 14 days, preferably about 14 days. More preferably, rapid expansion provides an increase of at least about 200-fold (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, or greater) over a period of about 10 to about 14 days, preferably about 14 days.
  • rapid expansion provides an increase in the number of antigen-specific T-cells of at least about 50-fold (e.g., 50-, 60-, 70-, 80-, 90-, or 100-fold, or greater) over a period of about 10 to about 14 days, preferably about 14 days. More preferably, rapid expansion provides an increase of at least about 200-fold (e.g., 200-, 300-, 400
  • T cells can be rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL- 15), with IL-2 being preferred.
  • the non-specific T-cell receptor stimulus can include around 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, N.J.).
  • T cells can be rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell) of the cancer, which can be optionally expressed from a vector, such as an human leukocyte antigen A2 (HLA-A2) binding peptide, in the presence of a T-cell growth factor, such as 300 lU/ml IL-2 or IL- 15, with IL-2 being preferred.
  • HLA-A2 human leukocyte antigen A2
  • T-cell growth factor such as 300 lU/ml IL-2 or IL- 15, with IL-2 being preferred.
  • the in vz'Zro-induced T-cells are rapidly expanded by re-stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2- expressing antigen-presenting cells.
  • the T-cells can be re-stimulated with irradiated, autologous lymphocytes or with
  • the autologous T-cells can be modified to express a T-cell growth factor that promotes the growth and activation of the autologous T-cells.
  • Suitable T-cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, and IL-12.
  • IL interleukin
  • Suitable methods of modification are known in the art. See, for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3 rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001 ; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.
  • modified autologous T-cells express the T-cell growth factor at high levels.
  • T-cell growth factor coding sequences such as that of IL- 12, are readily available in the art, as are promoters, the operable linkage of which to a T-cell growth factor coding sequence promote high- level expression.
  • the cell can be genetically engineered to express engineered antigen receptors such as engineered TCRs or chimeric antigen receptors (CARs).
  • engineered antigen receptors such as engineered TCRs or chimeric antigen receptors (CARs).
  • the autologous T-cells are modified to express a T cell receptor (TCR) having antigenic specificity for a cancer antigen, such as ZP4.
  • TCRs include, for example, those with antigenic specificity for a melanoma antigen, e.g., gplOO or MART-1. Suitable methods of modification are known in the art. See, for instance, Sambrook and Ausubel, supra.
  • the T cells may be transduced to express a TCR having antigenic specificity for a cancer antigen using transduction techniques described in Heemskerk et al. Hum Gene Ther. 19:496-510 (2008) and Johnson et al. Blood 114:535-46 (2009).
  • the T cells comprise one or more nucleic acids introduced via genetic engineering that encode one or more antigen receptors, and genetically engineered products of such nucleic acids.
  • the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived.
  • the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature e.g., chimeric).
  • the CAR contains an extracellular antigen-recognition domain that specifically binds to ZP4.
  • the antigen is a protein expressed on the surface of cells.
  • the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.
  • MHC major histocompatibility complex
  • Exemplary antigen receptors including CARs and recombinant TCRs, as well as methods for engineering and introducing the receptors into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, W02013/071154, W02013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S.
  • the genetically engineered antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 Al.
  • the CAR comprises: a) an intracellular signaling domain, b) a transmembrane domain, and c) an extracellular domain comprising an antigen binding region.
  • the engineered antigen receptors include CARs, including activating or stimulatory CARs, costimulatory CARs (see WO2014/055668), and/or inhibitory CARs (iCARs, see Fedorov et al., 2013).
  • the CARs generally include an extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s). Such molecules typically mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone.
  • nucleic acids including nucleic acids encoding an antigen-specific CAR polypeptide, including a CAR that has been humanized to reduce immunogenicity (hCAR), comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising one or more signaling motifs.
  • the CAR may recognize an epitope comprising the shared space between one or more antigens.
  • the binding region can comprise complementary determining regions of a monoclonal antibody, variable regions of a monoclonal antibody, and/or antigen binding fragments thereof.
  • that specificity is derived from a peptide (e.g., cytokine) that binds to a receptor.
  • the human CAR nucleic acids may be human genes used to enhance cellular immunotherapy for human patients.
  • the disclosure includes a full-length CAR cDNA or coding region.
  • the antigen binding regions or domain can comprise a fragment of the VH and VL chains of a single-chain variable fragment (scFv) derived from a particular human monoclonal antibody, such as those described in U.S. Patent 7,109,304, incorporated herein by reference.
  • the fragment can also be any number of different antigen binding domains of a human antigen-specific antibody.
  • the fragment is an antigen-specific scFv encoded by a sequence that is optimized for human codon usage for expression in human cells.
  • the arrangement could be multimeric, such as a diabody or multimers.
  • the multimers are most likely formed by cross pairing of the variable portion of the light and heavy chains into a diabody.
  • the hinge portion of the construct can have multiple alternatives from being totally deleted, to having the first cysteine maintained, to a proline rather than a serine substitution, to being truncated up to the first cysteine.
  • the Fc portion can be deleted. Any protein that is stable and/or dimerizes can serve this purpose.
  • One could use just one of the Fc domains, e.g., either the CH2 or CH3 domain from human immunoglobulin.
  • One could also use just the hinge portion of an immunoglobulin.
  • the CAR nucleic acid comprises a sequence encoding other costimulatory receptors, such as a transmembrane domain and a modified CD28 intracellular signaling domain.
  • costimulatory receptors include, but are not limited to one or more of CD28, CD27, OX-40 (CD134), and 4-1BB (CD137).
  • CAR is constructed with a specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker, and/or an antigen intended to induce a dampening response, such as an antigen expressed on a normal or non-diseased cell type.
  • a particular antigen or marker or ligand
  • the CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and/or antibody molecules.
  • the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).
  • an antibody molecule such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb).
  • the sequence of the open reading frame encoding the chimeric receptor can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g., via PCR), or combinations thereof. Depending upon the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof as it is found that introns stabilize the mRNA. Also, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA.
  • the chimeric construct can be introduced into immune cells as naked DNA or in a suitable vector.
  • Methods of stably transfecting cells by electroporation using naked DNA are known in the art. See, e.g., U.S. Patent No. 6,410,319.
  • naked DNA generally refers to the DNA encoding a chimeric receptor contained in a plasmid expression vector in proper orientation for expression.
  • the RNA can be transfected through lipid nanoparticles.
  • a viral vector e.g., a retroviral vector, adenoviral vector, adeno- associated viral vector, or lentiviral vector
  • Suitable vectors for use in accordance with the method of the present disclosure are non-replicating in the immune cells.
  • a large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.
  • the antigen-specific binding, or recognition component is linked to one or more transmembrane and intracellular signaling domains.
  • the CAR includes a transmembrane domain fused to the extracellular domain of the CAR.
  • the transmembrane domain that naturally is associated with one of the domains in the CAR is used.
  • the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • the transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, and CD3 delta. Alternatively, the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • TCR T Cell Receptor
  • the genetically engineered antigen receptors include recombinant TCRs and/or TCRs cloned from naturally occurring T cells.
  • a "T cell receptor” or “TCR” refers to a molecule that contains variable oc and 0 chains (also known as TCRa and TCR0, respectively) or variable y and 8 chains (also known as TCRy and TCRS, respectively) and that is capable of specifically binding to an antigen peptide bound to a MHC receptor.
  • the TCR is in the u.0 form.
  • TCRs that exist in 0.0 and yd forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions.
  • a TCR can be found on the surface of a cell or in soluble form.
  • a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • a TCR also can contain a constant domain, a transmembrane domain and/or a short cytoplasmic tail (see, e.g., Janeway et al, 1997).
  • each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end.
  • a TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction.
  • the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including TCRs in the aP form or y8 form.
  • TCR includes any TCR or functional fragment, such as an antigen-binding portion of a TCR that binds to a specific antigenic peptide bound in an MHC molecule, i.e. MHC-peptide complex.
  • An "antigen-binding portion" or antigenbinding fragment" of a TCR which can be used interchangeably, refers to a molecule that contains a portion of the structural domains of a TCR, but that binds the antigen (e.g. MHC-peptide complex) to which the full TCR binds.
  • an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable P chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex, such as generally where each chain contains three complementarity determining regions.
  • variable domains of the TCR chains associate to form loops, or complementarity determining regions (CDRs) analogous to immunoglobulins, which confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule and determine peptide specificity.
  • CDRs complementarity determining regions
  • the CDRs are separated by framework regions (FRs) (see, e.g., lores et al., 1990; Chothia et al., 1988; Lefranc et al., 2003).
  • CDR3 is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the beta chain interacts with the C-terminal part of the peptide.
  • CDR2 is thought to recognize the MHC molecule.
  • the variable region of the P-chain can contain a further hypervariability (HV4) region.
  • the TCR chains contain a constant domain.
  • the extracellular portion of TCR chains e.g., a-chain, P-chain
  • the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains, and two membrane-distal variable domains containing CDRs.
  • the constant domain of the TCR domain contains short connecting sequences in which a cysteine residue forms a disulfide bond, making a link between the two chains.
  • a TCR may have an additional cysteine residue in each of the a and P chains such that the TCR contains two disulfide bonds in the constant domains.
  • the TCR chains can contain a transmembrane domain.
  • the transmembrane domain is positively charged.
  • the TCR chains contains a cytoplasmic tail.
  • the structure allows the TCR to associate with other molecules like CD3.
  • a TCR containing constant domains with a transmembrane region can anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex.
  • CD3 is a multi-protein complex that can possess three distinct chains (y, 8, and s) in mammals and the i ⁇ -chain.
  • the complex can contain a CD3y chain, a CD38 chain, two CD3s chains, and a homodimer of CD3( ⁇ chains.
  • the CD3y, CD38, and CD3s chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain.
  • the transmembrane regions of the CD3y, CD38, and CD3s chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains.
  • the intracellular tails of the CD3y, CD38, and CD3s chains each contain a single conserved motif known as an immunoreceptor tyrosine -based activation motif or ITAM, whereas each CD3 chain has three.
  • ITAMs are involved in the signaling capacity of the TCR complex.
  • These accessory molecules have negatively charged transmembrane regions and play a role in propagating the signal from the TCR into the cell.
  • the TCR may be a heterodimer of two chains a and P (or optionally y and 8) or it may be a single chain TCR construct.
  • the TCR is a heterodimer containing two separate chains (a and P chains or y and 8 chains) that are linked, such as by a disulfide bond or disulfide bonds.
  • a TCR for a target antigen e.g., a cancer antigen
  • nucleic acid encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences.
  • the TCR is obtained from a biological source, such as from cells such as from a T cell (e.g. cytotoxic T cell), T cell hybridomas or other publicly available source.
  • the T cells can be obtained from in vivo isolated cells.
  • a high-affinity T cell clone can be isolated from a patient, and the TCR isolated.
  • the T cells can be a cultured T cell hybridoma or clone.
  • the TCR clone for a target antigen has been generated in transgenic mice engineered with human immune system genes (c. ., the human leukocyte antigen system, or HLA).
  • phage display is used to isolate TCRs against a target antigen.
  • the TCR or antigen-binding portion thereof can be synthetically generated from knowledge of the sequence of the TCR.
  • Vectors include but are not limited to, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g. derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV etc), lentiviral vectors (e.g.
  • adenoviral vectors including replication competent, replication deficient and gutless forms thereof, adeno-associated viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, polio virus vectors, vesicular stomatitis virus vectors, maraba virus vectors and group B adenovirus enadenotucirev vectors.
  • Viral vectors encoding an antigen receptor may be provided in certain aspects of the present disclosure.
  • non-essential genes are typically replaced with a gene or coding sequence for a heterologous (or non-native) protein.
  • a viral vector is a kind of expression construct that utilizes viral sequences to introduce nucleic acid and possibly proteins into a cell. The ability of certain viruses to infect cells or enter cells via receptor mediated- endocytosis, and to integrate into host cell genomes and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells ( .g., mammalian cells).
  • Non-limiting examples of virus vectors that may be used to deliver a nucleic acid of certain aspects of the present disclosure are described below.
  • Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lenti viral vectors are well known in the art (see, for example, U.S. Patents 6,013,516 and 5,994,136).
  • Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences.
  • recombinant lentivirus capable of infecting a non-dividing cell — wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat — is described in U.S. Patent 5,994,136, incorporated herein by reference.
  • Expression cassettes included in vectors useful in the present disclosure in particular contain (in a 5'-to-3' direction) a eukaryotic transcriptional promoter operably linked to a protein-coding sequence, splice signals including intervening sequences, and a transcriptional termination/polyadenylation sequence.
  • the promoters and enhancers that control the transcription of protein encoding genes in eukaryotic cells are composed of multiple genetic elements. The cellular machinery is able to gather and integrate the regulatory information conveyed by each element, allowing different genes to evolve distinct, often complex patterns of transcriptional regulation.
  • a promoter used in the context of the present disclosure includes constitutive, inducible, and tissue-specific promoters. a. Promoter/Enhancers
  • the expression constructs provided herein comprise a promoter to drive expression of the antigen receptor.
  • a promoter generally comprises a sequence that functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as, for example, the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation.
  • these are located in the region 30110 bp- upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
  • a promoter To bring a coding sequence “under the control of’ a promoter, one positions the 5' end of the transcription initiation site of the transcriptional reading frame “downstream” of (i.e., 3' of) the chosen promoter.
  • the “upstream” promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • a promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
  • a promoter may be one naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.”
  • an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
  • a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment.
  • Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
  • promoters that are most commonly used in recombinant DNA construction include the piactamase (penicillinase), lactose and tryptophan (trp-) promoter systems.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein.
  • PCRTM nucleic acid amplification technology
  • control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
  • promoter and/or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression.
  • Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, (see, for example Sambrook et al. 1989, incorporated herein by reference).
  • the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to directhigh level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.
  • the promoter may be heterologous or endogenous.
  • any promoter/enhancer combination (as per, for example, the Eukaryotic Promoter Data Base EPDB, through world wide web at epd.isb-sib.ch/) could also be used to drive expression.
  • Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment.
  • Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • Non-limiting examples of promoters include early or late viral promoters, such as, SV40 early or late promoters, cytomegalovirus (CMV) immediate early promoters, Rous Sarcoma Virus (RSV) early promoters; eukaryotic cell promoters, such as, e. g., beta actin promoter, GADPH promoter, metallothionein promoter; and concatenated response element promoters, such as cyclic AMP response element promoters (ere), serum response element promoter (sre), phorbol ester promoter (TP A) and response element promoters (tre) near a minimal TATA box. It is also possible to use human growth hormone promoter sequences (e.
  • the human growth hormone minimal promoter described at Genbank, accession no. X05244, nucleotide 283- 341) or a mouse mammary tumor promoter (available from the ATCC, Cat. No. ATCC 45007).
  • the promoter is CMV IE, dectin-1, dectin-2, human CD11c, F4/80, SM22, RSV, SV40, Ad MLP, beta-actin, MHC class I or MHC class II promoter, however any other promoter that is useful to drive expression of the therapeutic gene is applicable to the practice of the present disclosure.
  • methods of the disclosure also concern enhancer sequences, z.e., nucleic acid sequences that increase a promoter’s activity and that have the potential to act in cis, and regardless of their orientation, even over relatively long distances (up to several kilobases away from the target promoter).
  • enhancer function is not necessarily restricted to such long distances as they may also function in close proximity to a given promoter.
  • a specific initiation signal also may be used in the expression constructs provided in the present disclosure for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be “in-frame” with the reading frame of the desired coding sequence to ensure translation of the entire insert. The exogenous translational control signals and initiation codons can be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.
  • IRES elements are used to create multigene, or polycistronic, messages.
  • IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites.
  • IRES elements from two members of the picomavirus family polio and encephalomyocarditis
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
  • cleavage sequences could be used to co-express genes by linking open reading frames to form a single cistron.
  • An exemplary cleavage sequence is the F2A (Foot-and-mouth diease virus 2A) or a “2A-like” sequence (e.g., Thosea asigna virus 2A; T2A).
  • a vector in a host cell may contain one or more origins of replication sites (often termed “ori”), for example, a nucleic acid sequence corresponding to oriP of EBV as described above or a genetically engineered oriP with a similar or elevated function in programming, which is a specific nucleic acid sequence at which replication is initiated.
  • ori origins of replication sites
  • a replication origin of other extra-chromosomally replicating virus as described above or an autonomously replicating sequence (ARS) can be employed.
  • cells containing a construct of the present disclosure may be identified in vitro or in vivo by including a marker in the expression vector.
  • markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector.
  • a selection marker is one that confers a property that allows for selection.
  • a positive selection marker is one in which the presence of the marker allows for its selection, while a negative selection marker is one in which its presence prevents its selection.
  • An example of a positive selection marker is a drug resistance marker.
  • a drug selection marker aids in the cloning and identification of transformants
  • genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selection markers.
  • other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated.
  • screenable enzymes as negative selection markers such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized.
  • immunologic markers possibly in conjunction with FACS analysis.
  • the marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selection and screenable markers are well known to one of skill in the art.
  • nucleic acid such as DNA or RNA
  • introduction of a nucleic acid, such as DNA or RNA, into the immune cells of the current disclosure may use any suitable methods for nucleic acid delivery for transformation of a cell, as described herein or as would be known to one of ordinary skill in the art.
  • Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection, by injection, including microinjection); by electroporation; by calcium phosphate precipitation; by using DEAE-dextran followed by polyethylene glycol; by direct sonic loading; by liposome mediated transfection and receptor-mediated transfection; by microprojectile bombardment; by agitation with silicon carbide fibers; by Agrobacterium-mediated transformation; by desiccation/inhibition-mediated DNA uptake, and any combination of such methods.
  • organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.
  • Certain aspects of the present embodiments can be used to prevent or treat a disease or disorder associated with ZP4 signaling, including one in which killing of ZP4 -positive cells would ameliorate at least one symptom of the disease or disorder.
  • Signaling of ZP4 may be reduced by any suitable compositions to prevent cancer cell proliferation. In particular cases, such substances would be an anti-ZP4 antibody or anti-ZP4 CAR-expressing cells.
  • the present disclosure provides methods for immunotherapy comprising administering an effective amount of the compositions that comprise the antibodies, including at least CAR T cells, of the present disclosure.
  • a medical disease or disorder is treated by administration of a CAR-expressing cell population that elicits an immune response.
  • cancer is treated by administration of a CAR immune cell population that elicits an immune response.
  • methods for treating or delaying progression of cancer in an individual comprising administering to the individual an effective amount of an antigen-specific cell therapy.
  • the present methods may be applied for the treatment of immune disorders, solid cancers, and hematologic cancers, as examples.
  • the cancer may be a B cell malignancy, such as B cell acute lymphoblastic leukemia (ALL), diffuse, large B cell lymphoma, follicular lymphoma, marginal zone lymphoma, lymphoplasmacytic lymphoma, Burkitt lymphoma, and chronic lymphocytic leukemia.
  • ALL B cell acute lymphoblastic leukemia
  • diffuse large B cell lymphoma
  • follicular lymphoma follicular lymphoma
  • marginal zone lymphoma lymphoplasmacytic lymphoma
  • Burkitt lymphoma Burkitt lymphoma
  • chronic lymphocytic leukemia chronic lymphocytic leukemia
  • Tumors for which the present treatment methods are useful include any malignant cell type, such as those found in a solid tumor or a hematological tumor.
  • Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.
  • Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like.
  • cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, ductal carcinoma, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung
  • cancer of the peritoneum gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer)
  • pancreatic cancer cervical cancer, ovarian cancer, liver cancer, bladder cancer
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;
  • Certain embodiments concern methods of treatment of leukemia.
  • Leukemia is a cancer of the blood or bone marrow and is characterized by an abnormal proliferation (production by multiplication) of blood cells, usually white blood cells (leukocytes). It is part of the broad group of diseases called hematological neoplasms. Leukemia is a broad term covering a spectrum of diseases. Leukemia is clinically and pathologically split into its acute and chronic forms.
  • activated CD4 and/or CD8 T cells in the individual are characterized by y-IFN producing CD4 and/or CD8 T cells and/or enhanced cytolytic activity relative to prior to the administration of the combination.
  • y-IFN may be measured by any means known in the art, including, e.g., intracellular cytokine staining (ICS) involving cell fixation, permeabilization, and staining with an antibody against y- IFN.
  • Cytolytic activity may be measured by any means known in the art, e.g., using a cell killing assay with mixed effector and target cells.
  • the subject can be administered nonmyeloablative lymphodepleting chemotherapy prior to the T cell therapy.
  • the nonmyeloablative lymphodepleting chemotherapy can be any suitable such therapy, which can be administered by any suitable route.
  • the nonmyeloablative lymphodepleting chemotherapy can comprise, for example, the administration of cyclophosphamide and fludarabine, particularly if the cancer is melanoma, which can be metastatic.
  • An exemplary route of administering cyclophosphamide and fludarabine is intravenously.
  • any suitable dose of cyclophosphamide and fludarabine can be administered. In particular aspects, around 60 mg/kg of cyclophosphamide is administered for two days after which around 25 mg/m 2 fludarabine is administered for five days.
  • a T cell growth factor that promotes the growth and activation of the autologous T cells is administered to the subject either concomitantly with the autologous T cells or subsequently to the autologous T cells.
  • the T cell growth factor can be any suitable growth factor that promotes the growth and activation of the autologous T cells.
  • suitable T-cell growth factors include interleukin (IL)-2, IL-7, IL-15, and/or IL-12, which can be used alone or in various combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2.
  • IL-12 is a particular T- cell growth factor.
  • Therapeutically effective amounts of immune cells can be administered by a number of routes, including parenteral administration, for example, intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection, or infusion.
  • parenteral administration for example, intravenous, intraperitoneal, intramuscular, intrasternal, or intraarticular injection, or infusion.
  • Intratumoral injection, or injection into the tumor vasculature is specifically contemplated for discrete, solid, accessible tumors. Local, regional or systemic administration also may be appropriate.
  • the volume to be administered will be about 4-10 ml (in particular 10 ml), while for tumors of ⁇ 4 cm, a volume of about 1 -3 ml will be used (in particular
  • Multiple injections delivered as single dose comprise about 0.1 to about 0.5 ml volumes.
  • the T cell population can be administered in treatment regimens consistent with the disease, for example a single or a few doses over one to several days to ameliorate a disease state or periodic doses over an extended time to inhibit disease progression and prevent disease recurrence.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
  • the therapeutically effective amount of T cells will be dependent on the subject being treated, the severity and type of the affliction, and the manner of administration.
  • doses that could be used in the treatment of human subjects range from at least 3.8* 10 4 , at least 3.8x10’, at least 3.8xl0 6 , at least 3.8x l0 7 , at least 3.8x l0 8 , at least 3.8xl0 9 , or at least 3.8x l0 10 T cells/m 2 .
  • the dose used in the treatment of human subjects ranges from about 3.8 IO 9 to about 3.8 IO 10 T cells/m 2 .
  • a therapeutically effective amount of T cells can vary from about 5* IO 6 cells per kg body weight to about 7.5* 10 8 cells per kg body weight, such as about 2* 10 7 cells to about 5 10 8 cells per kg body weight, or about 5 10 7 cells to about 2 10 8 cells per kg body weight.
  • the exact amount of T cells is readily determined by one of skill in the art based on the age, weight, sex, and physiological condition of the subject. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • an effective amount of ZP4 CAR-expressing immune cells are delivered to an individual in need thereof, such as an individual that has cancer.
  • the cells then enhance the individual’s immune system to attack the cancer cells.
  • the individual is provided with one or more doses of the immune cells.
  • the duration between the administrations should be sufficient to allow time for propagation in the individual, and in specific embodiments the duration between doses is 1, 2, 3, 4, 5, 6, 7, or more days.
  • the cells that have been engineered to express a ZP4 CAR are provided to an individual in a therapeutically effective amount (in a range from 10 3 to 10 10 ) that ameliorates at least one symptom related to cancer cells in the individual.
  • a therapeutically effective amount may be from 10 3 to I O 10 , 10 3 to 10 9 , 10 3 to 10 8 , 10 3 to 10 7 , 10 3 to 10 6 , 10 3 to 10 5 , 10 3 to 10 4 , 10 4 to IO 10 , 10 4 to 10 9 , 10 4 to 10 8 , 10 4 to 10 7 , 10 4 to 10 6 , 10 4 to 10 5 , 10 5 to IO 10 , 10 5 to 10 9 , 10 5 to 10 8 , IO 5 to 10 7 , IO 5 to 10 6 , 10 6 to IO 10 , 10 6 to 10 9 , 10 6 to 10 8 , 10 6 to 10 7 , 10 7 to IO 10 , IO 7 to 10 9 , IO 7 to 10 8 , 10 8 to IO 10 , IO 8 to 10 9 , or 10 9 to IO 10 cells.
  • an individual having a certain cancer is provided once or multiple times a therapeutically effective amount of cells expressing ZP4 CARs.
  • compositions and formulations comprising CAR-expressing cells and a pharmaceutically acceptable carrier.
  • compositions and formulations as described herein can be prepared by mixing the active ingredients (such as an antibody or a polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22 nd edition, 2012), in the form of lyophilized formulations or aqueous solutions.
  • active ingredients such as an antibody or a polypeptide
  • optional pharmaceutically acceptable carriers Remington's Pharmaceutical Sciences 22 nd edition, 2012
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
  • sHASEGP soluble neutral-active hyaluronidase glycoproteins
  • rHuPH20 HYLENEX®, Baxter International, Inc.
  • a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
  • compositions and methods of the present embodiments involve a T cell population in combination with at least one additional therapy.
  • the additional therapy may be radiation therapy, surgery (e. ., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing.
  • the additional therapy may be in the form of adjuvant or neoadjuvant therapy.
  • the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent.
  • the additional therapy is the administration of side- effect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, etc.).
  • the additional therapy is radiation therapy.
  • the additional therapy is surgery.
  • the additional therapy is a combination of radiation therapy and surgery.
  • the additional therapy is gamma irradiation.
  • the additional therapy is therapy targeting PBK/AKT/mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or chemopreventative agent.
  • the additional therapy may be one or more of the chemotherapeutic agents known in the art.
  • An immune cell therapy may be administered before, during, after, or in various combinations relative to an additional cancer therapy, such as immune checkpoint therapy.
  • the administrations may be in intervals ranging from concurrently to minutes to days to weeks.
  • the immune cell therapy is provided to a patient separately from an additional therapeutic agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient.
  • an immune cell therapy is “A” and an anti-cancer therapy is “B”:
  • chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogue
  • DNA damaging factors include what are commonly known as y-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation, and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • immunotherapies may be used in combination or in conjunction with methods of the embodiments.
  • immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • Rituximab (RITUXAN®) is such an example.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells
  • ADCs Antibody-drug conjugates
  • MAbs monoclonal antibodies
  • cell-killing drugs may be used in combination therapies.
  • This approach combines the high specificity of MAbs against their antigen targets with highly potent cytotoxic drugs, resulting in “armed” MAbs that deliver the payload (drug) to tumor cells with enriched levels of the antigen.
  • Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in decreased toxicity and improved therapeutic index.
  • Exemplary ADC drugs inlcude ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine or T-DM1).
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG- 72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, erb b2 and p 155.
  • An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects.
  • Immune stimulating molecules also exist including: cytokines, such as IL -2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.
  • cytokines such as IL -2, IL-4, IL-12, GM-CSF, gamma-IFN
  • chemokines such as MIP-1, MCP-1, IL-8
  • growth factors such as FLT3 ligand.
  • immunotherapies include immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds); cytokine therapy, e.g., interferons a, 0, and y, IL-1, GM-CSF, and TNF; gene therapy, e.g., TNF, IL-1, IL-2, and p53; and monoclonal antibodies, e.g., anti-CD20, anti -ganglioside GM2, and anti- p 185. It is contemplated that one or more anti-cancer therapies may be employed with the antibody therapies described herein.
  • immune adjuvants e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds
  • cytokine therapy e.g., interferons a, 0, and y, IL-1, GM-CSF, and TNF
  • gene therapy e
  • the immunotherapy may be an immune checkpoint inhibitor.
  • Immune checkpoints either turn up a signal (e.g., co-stimulatory molecules) or turn down a signal.
  • Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3 -dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA).
  • the immune checkpoint inhibitors target the PD-1 axis and/or CTLA-4.
  • the immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligand or receptors, or, in particular, are antibodies, such as human antibodies.
  • Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used.
  • alternative and/or equivalent names may be in use for certain antibodies mentioned in the present disclosure.
  • Such alternative and/or equivalent names are interchangeable in the context of the present disclosure.
  • lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.
  • the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners.
  • the PD-1 ligand binding partners are PDL1 and/or PDL2.
  • a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partners.
  • PDL1 binding partners are PD-1 and/or B7-1.
  • the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners.
  • a PDL2 binding partner is PD-1.
  • the antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • the PD-1 binding antagonist is an anti-PD-1 antibody (e.g, a human antibody, a humanized antibody, or a chimeric antibody).
  • the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011.
  • the PD-1 binding antagonist is an immunoadhesin (e.g, an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence).
  • the PD-1 binding antagonist is AMP-224.
  • Nivolumab also known as MDX- 1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody that may be used.
  • Pembrolizumab also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an exemplary anti-PD-1 antibody.
  • CT-011 also known as hBAT or hBAT-1
  • AMP-224 also known as B7-DCIg, is a PDL2- Fc fusion soluble receptor.
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • CD 152 cytotoxic T-lymphocyte-associated protein 4
  • the complete cDNA sequence of human CTLA-4 has the Genbank accession number LI 5006.
  • CTLA- 4 is found on the surface of T cells and acts as an “off’ switch when bound to CD80 or CD86 on the surface of antigen-presenting cells.
  • CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells.
  • CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells.
  • CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal.
  • Intracellular CTLA4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.
  • the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e. ., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
  • an anti-CTLA-4 antibody e. ., a human antibody, a humanized antibody, or a chimeric antibody
  • an antigen binding fragment thereof e. ., an immunoadhesin, a fusion protein, or oligopeptide.
  • Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art.
  • art recognized anti-CTLA-4 antibodies can be used.
  • An exemplary anti- CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen binding fragments and variants thereof.
  • the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab.
  • the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1 , CDR2 and CDR3 domains of the VL region of ipilimumab.
  • the antibody competes for binding with and/or binds to the same epitope on CTLA-4 as the above-mentioned antibodies.
  • the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs’ surgery).
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may 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 may be of varying dosages as well.
  • agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment.
  • additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
  • cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments.
  • Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin.
  • FAKs focal adhesion kinase
  • An article of manufacture or a kit comprising immune cells, antibodies, reagents, buffers, or a combination thereof is also provided herein.
  • the article of manufacture or kit can further comprise a package insert comprising instructions for using the immune cells to treat or delay progression of cancer in an individual or to enhance immune function of an individual having cancer.
  • Any of the antigen-specific immune cells described herein may be included in the article of manufacture or kits.
  • Suitable containers include, for example, bottles, vials, bags and syringes.
  • the container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy).
  • the container holds the formulation and the label on, or associated with, the container may indicate directions for use.
  • the article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • the article of manufacture further includes one or more of another agent (e.g., a chemotherapeutic agent, and anti -neoplastic agent).
  • Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.
  • CAR-T cells have the potential to cure patients with advanced cancer, as demonstrated by their extraordinary success in hematological malignancies, which resulted in the FDA approval of five CAR-T therapies since 2017. Broadening their application toward solid tumors like breast cancer, however, has proven challenging.
  • One major hurdle is finding a target that is highly expressed by cancer cells but not vital tissues.
  • the inventors sought to find such a target for triple negative breast cancer (TNBC) and identified Zona Pellucida 4 (ZP4) as a promising and novel target for TNBC.
  • ZP4 is a component of the human zona pellucida (ZP) matrix, an extracellular structure that surrounds the oocyte and functions in folliculogenesis, species-specific fertilization, and early development. ZP4 expression is restricted to the ovaries, with no expression reported in any other normal tissues.
  • the mRNA expression data was first analyzed of 54 normal human tissues from the Genotype-Tissue Expression (GTEx) project, and ZP4 had no detectable mRNA expression in any GTEx tissues except for a small number of ovary samples (FIG. 1A). Further investigation of the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data analysis demonstrated significant ZP4 expression in a subset of breast cancer samples belonging to the basal/TNBC subgroup (FIG. 1C). Similar observation was made in the The Cancer Genome Atlas (TCGA) and Clinical Proteomic Tumor Analysis Consortium (CPTAC) (proteomic) cohort. Based on this analysis, ZP4 expressing tumors correspond to approximately 10% of basal/TNBC samples.
  • GTEx Genotype-Tissue Expression
  • the inventors have confirmed variable levels of ZP4 expression in TNBC PDX models by mRNA sequencing, proteomics and IHC staining (FIG. 2). Having RNA sequencing and proteomics data from these models provides a unique opportunity to study the antitumor activity of ZP4 CART cells against variable ZP4 tumor expression levels and also understand the inhibitory mechanisms that affect CAR-T cells in the breast TME.
  • ZP4 was overexpressed in TNBC cell line SUMI 59 and used as a target in a cytotoxicity assay.
  • ZP4 CAR- T cells killed ZP4+ SUMI 59 cells (FIG. 3C-D).
  • CD 19 truncated CD 19 reporter separated by a T2As sequence was also included so that we could enrich the CAR T cells with magnetic bead selection.
  • ZP4 CAR expression was determined by flow cytometry with a goat anti-human Fey fragment specific antibody. Following transduction, we detected stable ZP4 CAR expression on the surface of activated healthy donor T cells (Figure 9B). Analysis of T cell phenotype via flow cytometry did not reveal significant differences in CD4/CD8 distribution or differentiation status. All three CARs are predominantly CD8+ ( Figure 9C), and have a predominantly effector memory (Tern) phenotype ( Figure 9D).
  • cytotoxic potential of ZP4 CAR T cells was measured in a standard 4-6 hour chromium-51 ( 51 Cr)- release cytotoxicity assay using ZP4+ MDA-MB-231 cells at effectortarget (E:T) ratios of 40: 1, 20: 1, 10: 1, and 5: 1. Effector cells (NT or ZP4 CAR T cells) were co-incubated with 51 Cr-labeled target cells in triplicate wells. At the end of the incubation period, supernatants were harvested, and radioactivity counted in a gamma counter. To determine cytotoxic activity, we calculate the percentage of specific lysis. Clone 108, clone 128, and clone 164 CAR T cells each demonstrated antigen-specific cytotoxic killing of ZP4+ target cells, killing at least 80% of cells at the 40: 1 E:T ratio ( Figure 9E).
  • the polyfunctionality of our ZP4 CAR T cells was measured using Isoplexis, a method of performing multiplex cytokine analysis at a single-cell resolution and determining how many of our cells are producing multiple cytokines.
  • the polyfunctional strength index (PSI) is the percentages of poly functional cells per sample and the cytokine secretion intensities of each of those cells.
  • PSI polyfunctional strength index
  • polyfunctionality is a meaningful indicator of potency.
  • zl28 CAR T cells have the highest PSI, indicating that this could be a more potent product (Figure 10G). This could potentially explain why z!28 CAR T cells outperform the other CARs over multiple stimulations and will be investigated further.
  • zl28 CAR T cells demonstrated the greatest degree of in vivo persistence, as visualized via IVIS ( Figure 11 A) and quantified as average radiance ( Figure 1 IB).
  • IVIS IVIS
  • Figure 1 IB quantified as average radiance

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des anticorps ZP4 et des récepteurs antigéniques chimériques (CAR) spécifiques de ZP4. L'invention porte en outre sur des cellules immunitaires exprimant les récepteurs CAR spécifiques de ZP4 et sur des méthodes de traitement du cancer par administration des cellules immunitaires de récepteur CAR spécifiques de ZP4.
PCT/US2023/078519 2022-11-03 2023-11-02 Anticorps anti-zp4 et récepteurs antigéniques chimériques et leurs méthodes d'utilisation WO2024097877A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263382221P 2022-11-03 2022-11-03
US63/382,221 2022-11-03

Publications (1)

Publication Number Publication Date
WO2024097877A1 true WO2024097877A1 (fr) 2024-05-10

Family

ID=90931614

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/078519 WO2024097877A1 (fr) 2022-11-03 2023-11-02 Anticorps anti-zp4 et récepteurs antigéniques chimériques et leurs méthodes d'utilisation

Country Status (1)

Country Link
WO (1) WO2024097877A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100248267A1 (en) * 2007-08-01 2010-09-30 Japan Science And Technology Agency Method for measurement of concentration of antigen
US20160369005A1 (en) * 2013-06-06 2016-12-22 Igenica Biotherapeutics, Inc. Anti-c10orf54 antibodies and uses thereof
US20190389956A1 (en) * 2016-07-14 2019-12-26 Institute Of Biophysics, Chinese Academy Of Sciences Type i interferon receptor antibody and use thereof
US20200062832A1 (en) * 2018-08-21 2020-02-27 Quidel Corporation DbpA ANTIBODIES AND USES THEREOF
US20200316120A1 (en) * 2018-09-28 2020-10-08 Immpact-Bio Ltd. METHODS FOR IDENTIFYING ACTIVATING ANTIGEN RECEPTOR (aCAR)/INHIBITORY CHIMERIC ANTIGEN RECEPTOR (iCAR) PAIRS FOR USE IN CANCER THERAPIES
WO2022040506A2 (fr) * 2020-08-21 2022-02-24 Yale University Compositions de nanocorps et leurs procédés d'utilisation

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100248267A1 (en) * 2007-08-01 2010-09-30 Japan Science And Technology Agency Method for measurement of concentration of antigen
US20160369005A1 (en) * 2013-06-06 2016-12-22 Igenica Biotherapeutics, Inc. Anti-c10orf54 antibodies and uses thereof
US20190389956A1 (en) * 2016-07-14 2019-12-26 Institute Of Biophysics, Chinese Academy Of Sciences Type i interferon receptor antibody and use thereof
US20200062832A1 (en) * 2018-08-21 2020-02-27 Quidel Corporation DbpA ANTIBODIES AND USES THEREOF
US20200316120A1 (en) * 2018-09-28 2020-10-08 Immpact-Bio Ltd. METHODS FOR IDENTIFYING ACTIVATING ANTIGEN RECEPTOR (aCAR)/INHIBITORY CHIMERIC ANTIGEN RECEPTOR (iCAR) PAIRS FOR USE IN CANCER THERAPIES
WO2022040506A2 (fr) * 2020-08-21 2022-02-24 Yale University Compositions de nanocorps et leurs procédés d'utilisation

Similar Documents

Publication Publication Date Title
US20220380429A1 (en) T cells expressing membrane-anchored il-12 for the treatment of cancer
US20210317209A1 (en) Anti-cd79b antibodies and chimeric antigen receptors and methods of use thereof
US20220370495A1 (en) Immune cells for adoptive cell therapies
US20240123007A1 (en) Hla-restricted vcx/y peptides and t cell receptors and use thereof
KR20200085780A (ko) 암의 치료에서 car-t 또는 car-nk 세포를 사용한 lilrb4 표적화
US20220118015A1 (en) Modified il-12 t cell therapy for the treatment of cancer
EP4087618A1 (fr) Procédé de modification de cellules tueuses naturelles pour cibler des tumeurs positives cd70
US10821134B2 (en) BK virus specific T cells
US20210393753A1 (en) Fgl2 neutraling cell therapy and methods of use thereof
US20220372092A1 (en) Hla-restricted vcx/y peptides and t cell receptors and use thereof
US20230183371A1 (en) Anti-cd79b antibodies and chimeric antigen receptors and methods of use thereof
WO2024097877A1 (fr) Anticorps anti-zp4 et récepteurs antigéniques chimériques et leurs méthodes d'utilisation
US20230106973A1 (en) Methods for expansion of tumor infiltrating lymphocytes and use thereof
US20240254192A1 (en) Chimeric polypeptides and methods of use
WO2023107898A1 (fr) Ciblage double de malignités pédiatriques par cellules car-t sécrétant des activateurs de cellules immunitaires innés bispécifiques (bices)
CN117642172A (zh) 嵌合多肽及使用方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23887006

Country of ref document: EP

Kind code of ref document: A1