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WO2025019228A1 - Fully human monoclonal antibodies and chimeric antigen receptors against cd276 for the treatment of solid tumors - Google Patents

Fully human monoclonal antibodies and chimeric antigen receptors against cd276 for the treatment of solid tumors Download PDF

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Publication number
WO2025019228A1
WO2025019228A1 PCT/US2024/037349 US2024037349W WO2025019228A1 WO 2025019228 A1 WO2025019228 A1 WO 2025019228A1 US 2024037349 W US2024037349 W US 2024037349W WO 2025019228 A1 WO2025019228 A1 WO 2025019228A1
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seq
antibody
amino acid
car
domain
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PCT/US2024/037349
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French (fr)
Inventor
Bradley ST. CROIX
Pradip BAJGAIN
Yang Feng
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Publication of WO2025019228A1 publication Critical patent/WO2025019228A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Definitions

  • This disclosure concerns human monoclonal antibodies that specifically bind CD276 and conjugates, such as chimeric antigen receptors (CARs), based on these antibodies.
  • CARs chimeric antigen receptors
  • the disclosure further concerns use of the monoclonal antibodies and conjugates, such as in the treatment of solid tumors.
  • CD276 (also known as B7-H3) is a single pass transmembrane protein that is highly expressed in the stroma of most solid tumors and is also frequently overexpressed by the tumor cells themselves.
  • Chimeric antigen receptors (CARs) targeting human CD276 have been previously evaluated in preclinical studies resulting in the initiation of several clinical trials. However, the CARs currently being tested in early phase trials were constructed using humanized forms of mouse derived anti-CD276 antibodies. The process of humanization can impact antibody function and may not completely eliminate immunogenicity, subjecting T cells engineered to express mouse antibody-derived CARs to elimination by the endogenous immune system. Thus, a need exists for the development of improved CD276-targeted CARs, such as those incorporating fully human monoclonal antibodies.
  • CD276-specific antibodies isolated from human antibody libraries.
  • the disclosed antibodies were used to develop chimeric antigen receptors (CARs) containing 100% human amino acid sequences, thereby eliminating the need for humanization.
  • CARs chimeric antigen receptors
  • the CD276- targeted CARs disclosed herein induce cell killing of several tumor cell lines, including pancreatic and neuroblastoma tumor cell lines.
  • CARs generated using antibody Y111 induced therapeutically effective anti-tumor responses in orthotopic pancreatic tumor and metastatic neuroblastoma models, and can produce better clinical responses in human solid tumor patients compared to CARs currently being tested in human clinical trials.
  • CD276-specific antibodies include the complementarity determining region (CDR) sequences (or the complete variable domains) of any one of antibodies Y111 , Y 422, Y868, Y117 and YE5 as disclosed herein.
  • CDR complementarity determining region
  • conjugates that include a disclosed monoclonal antibody (or the CDR sequences of a disclosed monoclonal antibody).
  • CARs CAR-expressing cells (such as T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), macrophages and induced pluripotent stem cells (iPSCs)), fusion proteins (such as Fc fusion proteins), immunoconjugates (such as immuno toxins), multi-specific antibodies (such as bispecific or trispecific antibodies), antibody-drug conjugates (ADCs), antibody-nanoparticle conjugates, and antibody-photon absorber conjugates (such as IR700 conjugates for immunoPET imaging) that include a monoclonal antibody disclosed herein.
  • nucleic acid molecules encoding a disclosed monoclonal antibody, CAR, immunoconjugate, multi- specific antibody, or fusion protein.
  • the nucleic acid molecule is part of a vector.
  • isolated cells that include a nucleic acid molecule or vector disclosed herein.
  • compositions that include a pharmaceutically acceptable carrier and a CD276- specific monoclonal antibody, CAR, CAR-expressing cell, immunoconjugate, ADC, multispecific antibody, antibody-nanoparticle conjugate, or fusion protein disclosed herein.
  • the composition is lyophilized.
  • Methods of detecting expression of CD276 in a sample are further provided.
  • the method includes contacting the sample with a CD276-specific monoclonal antibody disclosed herein and detecting binding of the monoclonal antibody to the sample.
  • Methods of diagnosing a subject as having a CD276-positive tumor are further provided.
  • the method includes contacting a sample obtained from the subject with a CD276-specific monoclonal antibody disclosed herein and detecting binding of the monoclonal antibody to the sample.
  • kits for treating a CD276-positive cancer in a subject include administering to the subject a monoclonal antibody, CAR, CAR-expressing cell, immunoconjugate, ADC, multi- specific antibody, antibody-nanoparticle conjugate, fusion protein, or composition disclosed herein.
  • the CD276-positive cancer is a solid tumor, such as, but not limited to, a pancreatic cancer, a neuroblastoma, a liver cancer, a kidney cancer, a bladder cancer, a cervical cancer, an esophageal cancer, a prostate cancer, a breast cancer, an ovarian cancer, a colon cancer, a lung cancer, a brain cancer, a pediatric cancer, a melanoma or a mesothelioma.
  • a pancreatic cancer such as, but not limited to, a pancreatic cancer, a neuroblastoma, a liver cancer, a kidney cancer, a bladder cancer, a cervical cancer, an esophageal cancer, a prostate cancer, a breast cancer, an ovarian cancer, a colon cancer, a lung cancer, a brain cancer, a pediatric cancer, a melanoma or a mesothelioma.
  • kits that include a monoclonal antibody, CAR, CAR-expressing cell, immunoconjugate, ADC, multi-specific antibody, antibody-nanoparticle conjugate, fusion protein, or composition disclosed herein; and a pharmaceutically acceptable carrier, buffer, cell culture media, cell culture plates or flasks, a solid support, a fluorescent label, a radioactive label, an enzymatic label, an enzymatic substrate, a secondary antibody, one or more check point inhibitors, one or more additional anti-cancer agents, one or more transfection reagents, and/or instructional materials.
  • FIG. 1 Fully-human CD276 antibodies bind to CD276 on the tumor cell surface.
  • Flow cytometry was used to assess the binding of CD276 antibodies to human pancreatic cancer cell line Panel.
  • Five anti-CD276 antibodies (Y422, Y868, Yl l l, Y117 and YE5) were produced in scFv- Fc format and purified for the binding assay.
  • Serially diluted antibodies at the indicated concentrations were incubated with Panel cells, followed by Alexa-647 labeled anti-human Fab secondary antibody.
  • a previously described anti-CD276 antibody (m276) in scFv format was included as positive control and a non-binding scFv was used as a negative control.
  • FIGS. 2A-2B Fully-human CD276 antibodies used to construct chimeric antigen receptors (CARs).
  • FIG. 2A Schematic of the overall structure of the CARs.
  • the CARs include an antibody single-chain variable fragment (scFv), CD28 derived hinge and transmembrane domains, and 4 IBB and CD3 zeta (CD3Q cytoplasmic signaling domains.
  • the Y111 CAR was also made with a CD28 costimulatory domain.
  • the scFv in all five CARs generated are in the VH-VL orientation.
  • the Y111 CAR was also made in a VL-VH orientation for comparison.
  • FIG. 2B Table listing the anti-CD276 antibodies used for construction of the CARs and their target specificity.
  • FIGS. 3A-3B Transduced T cells express CD276 CARs. Retrovirally transduced T cells were incubated with rCD276-Bio (biotin-tagged recombinant CD276 ectodomain), followed by allophycocyanin (APC)-labeled streptavidin. Flow cytometry was used to detect CAR expression on the T cell surface. Untransduced (UTD) cells were used as negative controls.
  • FIG. 3A Histograms showing CAR expression in T cells from a representative donor.
  • FIG. 3B Graph displaying average summary data for four independent donors.
  • FIGS. 4A-4B Expansion and viability of CAR transduced T cells.
  • T cells were maintained in T cell media (1 :1 mix of RPMI-1640 and Click’s media containing 10% FBS and 1% Glutamax) supplemented with recombinant human IL-2 (50 lU/mL). IL-2 was added every 2-3 days. Cells were collected, counted, and viability was measured on day 11 after transduction. Counting and viability measurements were performed using a Countess II automated cell counter. Fold expansion was calculated by dividing day 11 total cell numbers by the starting cell number on the day of transduction.
  • FIG. 4A Fold T cell expansion at day 11 post-transduction.
  • FIG. 4B T cell viability at day 11 post-transduction.
  • FIGS. 5A-5G Anti-tumor activity of CD276 CAR T cells against pancreatic cancer and neuroblastoma tumor cells.
  • Firefly-lucif erase labeled tumor cells 10,000 cells were seeded in opaque white 96-well plates in 100 (tL T cell media.
  • T cells resuspended in 100 pL T cell media were added at the indicated CAR+ T cell effector to target (E:T) ratios.
  • E:T CAR+ T cell effector to target
  • 10 pL D-Luciferin was added to each well such that the final concentration was 15 mg/mL. Plates were incubated at 37°C for 5 minutes after which signal intensity was captured using a Clariostar plate reader.
  • Percent killing was calculated as 100x[l-(sample luminescence)/(UTD luminescence)]. Shown is % killing in CD276 knockout (KO) NBEB cells (FIG. 5 A), neuroblastoma cell lines NBEB (FIG. 5B), LAN5 (FIG. 5C) and IMR5 (FIG. 5D), and pancreatic cancer cell lines Panel (FIG. 5E), HP AC (FIG. 5F) and MiaPaca (FIG. 5G).
  • KO CD276 knockout
  • FIG. 6 Study design used to test anti-tumor activity of CD276 CAR T cells in an orthotopic Panel pancreatic tumor model.
  • (Top) 250,000 firefly luciferase-labeled Panel cells were injected into the pancreas of 6- to 8-week-old female non-obese diabetic (NOD) scid gamma (NSG) mice. Two weeks later, mice were imaged using IVIS Spectrum to measure tumor burden and grouped into different treatment groups listed in the table (bottom). Mice were treated with CARs containing Y422, Y868, Yll l, Y117 or YE5 scFv.
  • NOD non-obese diabetic
  • CARs containing previously described anti-CD276 scFvs MGA271 and 376.96 were also included.
  • Five million CAR+ T cells were injected intravenously in the tail vein. Tumor burden was monitored by imaging weekly at first and then at specified time points later in the study. Body weight measurements were also taken at the time of imaging on a weekly basis. To image the tumor, 100 pL of 15 mg/mL D-luciferin was injected intraperitonially, and imaging was performed using an IVIS Spectrum imager after 10 minutes.
  • FIG. 7 Study design used to test anti-tumor activity of CD276 CAR T cells in an orthotopic Panel pancreatic tumor model. Shown is tumor luminescence data from the experiment described in FIG. 6. Data in the graph represent mean + standard error of mean (SEM) for 6-8 mice per group. CARs based on antibodies 376.96 and MGA271 have been described previously and these same variable domains were incorporated into the CARs of this study for comparison. The humanized MGA271 scFv sequence was used to make the CAR. The 376.96 scFv was used in its native (murine) form without humanization. Mice treated with the Yll 1-based CAR exhibited the greatest reduction in tumor size.
  • FIGS. 8A-8C Anti-tumor activity of CD276 CAR T cells in an orthotopic HP AC pancreatic tumor model.
  • FIG. 8A 250,000 firefly luciferase-labeled HP AC cells were injected into the pancreas of 6-8-week-old female NSG mice. Ten days later, mice were imaged using IVIS Spectrum to measure tumor burden and grouped into different treatment groups as shown in FIG. 8B. Five million CAR+ T cells were injected intravenously in the tail vein. Tumor burden was monitored by imaging weekly first and then at specified time points later in the study. To image the tumor, 100 pL of 15 mg/mL D-luciferin was injected intraperitonially, and imaging was performed after 10 minutes.
  • FIG. 8C Body weight measurements were taken at the time of imaging on a weekly basis. The graph shows average ( ⁇ SEM) body weight of mice.
  • FIGS. 9A-9B Anti-tumor activity of CD276 CAR T cells in an orthotopic HP AC pancreatic tumor model.
  • FIG. 9A IVIS images showing tumor luminescence for the experiment described in FIGS. 8A-8C. Five or six representative mice are shown for each treatment group.
  • FIG. 9B Graph showing tumor luminescence represented as average ⁇ SEM.
  • UTD untransfected. Mice treated with the Y 111 -based CAR exhibited the greatest reduction in tumor size.
  • FIGS. 10A-10B Anti-tumor activity of CD276 CAR T cells in an orthotopic HPAC pancreatic tumor.
  • FIG. 10A Graph showing long-term follow up of tumor size and a comparison of the disclosed CARs with MGA271 based CAR.
  • FIG. 10B Tumor luminescence in individual mice for all groups. The results demonstrated that Y 111 -based CAR T cells were the most effective at eliminating the pancreatic tumors.
  • FIG. 11 Production of cytolytic proteins by CAR T cells co-cultured with CD276 + Panel tumor cells.
  • Un-transduced (UTD) T cells, and T cells expressing a CD19 CAR or one of three CD276- specific CARs (Y868, Yll l, and Y117) were co-cultured with Panel pancreatic cancer cells for 16 hours at an effector-to-target ratio (E:T) of 1:2.
  • E:T effector-to-target ratio
  • T cells were then collected and labeled intracellularly with antibodies against granzyme B, perforin, and CD 107a.
  • UTD and CD19 CAR T cells were used as negative controls.
  • FIGS. 12A-12C Anti-tumor activity of CD276 CAR T cells in a metastatic neuroblastoma (IMR5) model.
  • FIG. 12A Schematic of the study design. Firefly-luciferase labeled IMR5 cells (IxlO 6 ) were injected into 6-8-week-old female NSG mice via tail vein (Day -35). Five weeks later, mice were imaged using IVIS Spectrum to measure tumor burden and grouped into different treatment groups. Two million Y868, Y111, Y117 or 376.96 CAR T cells were injected intravenously in the tail vein (Day 0). Tumor burden was monitored by imaging on Days -1, 7, 14, 21, 29, 40, 49 and 60.
  • FIG. 12B Graph quantifying tumor luminescence of untreated (UTD) mice and mice treated with CAR T cells, measured on Days -1, 7, 14, 21, 29, 40, 49 and 60. Data in the graph represent mean ⁇ standard error of mean (SEM) for 8 mice per group.
  • FIG. 12C Tumor luminescence images showing tumor burden in each animal.
  • FIGS. 13A-13B Long-term persistence and anti-tumor activity of Y111 CAR in the HP AC orthotopic pancreatic tumor model.
  • the long-term survivor mice from the HP AC study were rechallenged with 4xl0 6 firefly-luciferase labeled HP AC cells injected subcutaneously in the lower right abdomen.
  • Age matched mice that had never received tumor cells or CAR T cells were used as controls and were similarly injected with the tumor cells.
  • Mice were imaged using IVIS Spectrum to measure tumor burden on Days -7, 8, 46, 72, 101, 123, 143 and 182 after tumor re-challenge. (FIG.
  • FIG. 13 A Images of control and Yl ll CAR T cell-treated mice showing tumor burden.
  • FIG. 13B Graph of tumor luminescence (photons/second) in the age-matched control and Yl l l CAR T cell-treated mice.
  • FIGS. 14A-14B Optimization of Yl l l CAR.
  • FIGS. 14A-14B Optimization of Yl l l CAR.
  • FIG. 14A Schematic of the study design. Firefly-luciferase labeled Panel cells (2.5xl0 5 ) were injected into the pancreas of 6-8-week-old female NSG mice (Day -14). Two weeks later, mice were imaged using IVIS Lumina to measure tumor burden and grouped into different treatment groups.
  • T cells expressing (1) a Y111 -based CAR having CD28 hinge and transmembrane (HTM) regions, a 4- 1BB costimulatory domain, and a CD3 ⁇ signaling domain (Yll l-HL-28HTM-BBz); (2) a Y111- based CAR having CD28 hinge and transmembrane (HTM) regions, a CD28 costimulatory domain, and a CD3 ⁇ signaling domain (Yll l-HL-28HTM-28z); (3) a CD276-specific VHH single-domain antibody (B12)-based CAR having CD28 hinge and transmembrane (HTM) regions, a 4- IBB costimulatory domain, and a CD3 ⁇ signaling domain (B12-VHH-28HTM-BBz); and a B12-based CAR having CD28 hinge and transmembrane (HTM) regions, a CD28 costimulatory domain, and a CD3 ⁇ signaling domain (B12-VHH-28HTM-
  • FIG. 14B Graph of tumor luminescence (photons/second) measured on Days -1, 7, 14, 21, 28, 35, 42, 49 and 56 after CAR T cell treatment. To image the tumor, 100 pl of 15 mg/mL D-luciferin was injected intraperitonially and imaging was done after 10 minutes.
  • nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.
  • SEQ ID NO: 1 is the amino acid sequence of the Y111 VH domain.
  • SEQ ID NO: 2 is a nucleic acid sequence encoding the Y11 1 VH domain.
  • SEQ ID Nos: 3-8 are the CDR sequences of antibody Y111.
  • SEQ ID NO: 9 is the amino acid sequence of the Y422 VH domain.
  • SEQ ID NO: 10 is a nucleic acid sequence encoding the Y422 VH domain.
  • SEQ ID Nos: 11-16 are the CDR sequences of antibody Y422.
  • SEQ ID NO: 17 is the amino acid sequence of the Y868 VH domain.
  • SEQ ID NO: 18 is a nucleic acid sequence encoding the Y868 VH domain.
  • SEQ ID NOs: 19-24 are the CDR sequences of antibody Y868.
  • SEQ ID NO: 25 is the amino acid sequence of the Y 117 VH domain.
  • SEQ ID NO: 26 is a nucleic acid sequence encoding the Y117 VH domain.
  • SEQ ID NOs: 27-32 are the CDR sequences of antibody Y117.
  • SEQ ID NO: 33 is the amino acid sequence of the YE5 VH domain.
  • SEQ ID NO: 34 is a nucleic acid sequence encoding the YES VH domain.
  • SEQ ID NOs: 35-40 are the CDR sequences of antibody YE5.
  • SEQ ID NO: 41 is the amino acid sequence of a signal peptide.
  • SEQ ID NO: 42 is the amino acid sequence of a GS linker.
  • SEQ ID NO: 43 is the amino acid sequence of a CD28 hinge region.
  • SEQ ID NO: 44 is the amino acid sequence of a CD28 transmembrane domain.
  • SEQ ID NO: 45 is the amino acid sequence of a 41BB costimulatory domain.
  • SEQ ID NO: 46 is the amino acid sequence of a CD3 ⁇ intracellular signaling domain.
  • SEQ ID NO: 47 is the amino acid sequence of the Yl ll CAR (Yll l-HL-28HTM-BBz).
  • SEQ ID NO: 48 is a nucleic acid sequence encoding the Yl ll CAR (Y111-HL-28HTM- BBz).
  • SEQ ID NO: 49 is the amino acid sequence of the Y422 CAR.
  • SEQ ID NO: 50 is a nucleic acid sequence encoding the Y422 CAR.
  • SEQ ID NO: 51 is the amino acid sequence of the Y868 CAR.
  • SEQ ID NO: 52 is a nucleic acid sequence encoding the Y868 CAR.
  • SEQ ID NO: 53 is the amino acid sequence of the Y117 CAR.
  • SEQ ID NO: 54 is a nucleic acid sequence encoding the Y117 CAR.
  • SEQ ID NO: 55 is the amino acid sequence of the YE5 CAR.
  • SEQ ID NO: 56 is a nucleic acid sequence encoding the YE5 CAR.
  • SEQ ID NO: 57 is the amino acid sequence of a Yl l l scFv (VH-linker-VL orientation).
  • SEQ ID NO: 58 is a nucleic acid sequence encoding a Yl ll scFv (VH-linker-VL orientation).
  • SEQ ID NO: 59 is the amino acid sequence of a Yl l l scFv (VL-linker-VH orientation).
  • SEQ ID NO: 60 is a nucleic acid sequence encoding a Y111 scFv (VL-linker-VH orientation).
  • SEQ ID NO: 61 is the amino acid sequence of the Yl ll-HL-28HTM-28z CAR.
  • SEQ ID NO: 62 is a nucleic acid sequence encoding the Yl ll -HL-28HTM-28z CAR.
  • SEQ ID NO: 63 is the amino acid sequence of the Yl ll-LH-28HTM-BBz CAR.
  • SEQ ID NO: 64 is a nucleic acid sequence encoding the Y 11 l-LH-28HTM-BBz CAR.
  • SEQ ID NO: 65 is the amino acid sequence of a CD28 costimulatory domain.
  • SEQ ID NO: 66 is a nucleic acid sequence encoding a CD28 costimulatory domain.
  • SEQ ID NO: 67 is a nucleic acid sequence encoding a signal peptide.
  • SEQ ID NO: 68 is a nucleic acid sequence encoding a GS linker.
  • SEQ ID NO: 69 is a nucleic acid sequence encoding a CD28 hinge.
  • SEQ ID NO: 70 is a nucleic acid sequence encoding a CD28 transmembrane domain.
  • SEQ ID NO: 71 is a nucleic acid sequence encoding a 4- IBB costimulatory domain.
  • SEQ ID NO: 72 is a nucleic acid sequence encoding a CD3 ⁇ signaling domain.
  • an antigen includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.”
  • the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:
  • 4-1BB A costimulatory molecule expressed by T cell receptor (TCR)-activated lymphocytes, and by other cells including natural killer cells. Ligation of 4- IBB induces a signaling cascade that results in cytokine production, expression of anti-apoptotic molecules and an enhanced immune response.
  • An exemplary amino acid sequence of 4- IBB is set forth herein as SEQ ID NO: 45.
  • exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraprostatic, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
  • Antibody A polypeptide ligand comprising at least one variable region that recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen.
  • Mammalian immunoglobulin molecules are composed of a heavy (H) chain and a light (L) chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region, respectively. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody.
  • Antibody isotypes not found in mammals include IgX, IgY, IgW and IgNAR.
  • IgY is the primary antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE.
  • IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.
  • Antibody variable regions contain "framework” regions and hypervariable regions, known as “complementarity determining regions” or “CDRs.”
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the framework regions of an antibody serve to position and align the CDRs in three-dimensional space.
  • the amino acid sequence boundaries of a given CDR can be readily determined using any of a number of numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the “Kabat” numbering scheme), Chothia et al.
  • the Kabat, Paratome and IMGT databases are maintained online.
  • the AbRSA tool can be used to determine the CDR boundaries according to Kabat, IMGT or Chothia (online at aligncdr.labshare.cn/aligncdr/abrsa.php).
  • a “single-domain antibody” refers to an antibody having a single domain (a variable domain) that is capable of specifically binding an antigen, or an epitope of an antigen, in the absence of an additional antibody domain.
  • Single-domain antibodies include, for example, VH domain antibodies, VNAR antibodies, camelid VHH antibodies, and VL domain antibodies.
  • VNAR antibodies are produced by cartilaginous fish, such as nurse sharks, wobbegong sharks, spiny dogfish and bamboo sharks.
  • Camelid VHH antibodies are produced by several species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies that are naturally devoid of light chains.
  • a “monoclonal antibody” is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence of a single antibody has been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art. Monoclonal antibodies include humanized monoclonal antibodies.
  • a “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species.
  • a “humanized” antibody is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rabbit, rat, shark or synthetic) immunoglobulin.
  • the non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.”
  • all CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical.
  • a humanized immunoglobulin all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences.
  • a humanized antibody binds to the same antigen as the donor antibody that provides the CDRs.
  • Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.
  • ADC Antibody-drug conjugate
  • ADC A molecule that includes an antibody (or antigenbinding fragment of an antibody) conjugated to a drug, such as a cytotoxic agent.
  • ADCs can be used to specifically target a drug to cancer cells through specific binding of the antibody to a tumor antigen expressed on the cell surface.
  • exemplary drugs for use with ADCs include antimicrotubule agents (such as maytansinoids, auristatin E and auristatin F) and interstrand crosslinking agents (for example, pyrrolobenzodiazepines; PBDs).
  • the ADC is a bispecific ADC, which is comprised of two monoclonal antibodies or antigen-fragments thereof, each directed to a different antigen or epitope, conjugated to a drug.
  • the agent attached to the antibody is IRDye® 700 DX (IR700, Li-cor, Lincoln, NE), which can then be used with near infrared light NIR light to kill cancer cells to which the antibody binds (photoimmunotherapy; see for example US 8,524,239 and 10,538,590).
  • IR700 Li-cor, Lincoln, NE
  • amino-reactive IR700 can be covalently conjugated to an antibody using the NHS ester of 1R700.
  • Anti-microtubule agent A type of drug that blocks cell growth by stopping mitosis.
  • Anti-microtubule agents also referred to as “anti-mitotic agents,” are used to treat cancer.
  • Binding affinity Affinity of an antibody for an antigen.
  • affinity is calculated by a modification of the Scatchard method described by Frankel et al. , Mol. Immunol. , 16: 101-106, 1979.
  • binding affinity is measured by an antigen/antibody dissociation rate.
  • a high binding affinity is measured by a competition radioimmunoassay.
  • binding affinity is measured by ELISA.
  • antibody affinity is measured by flow cytometry or by surface plasmon reference.
  • An antibody that “specifically binds” an antigen is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens.
  • a monoclonal antibody (such as an anti-CD276 antibody provided herein) specifically binds to a target (such as a CD276) with a binding constant that is at least 10 3 M 1 greater, 10 4 M 1 greater or 10 5 M 1 greater than a binding constant for other molecules in a sample or subject.
  • an antibody e.g., monoclonal antibody has an equilibrium constant (KD) of 5 pM or less, such as 5,000 nM or less, 900 nM or less, 500 nM or less, 250 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less.
  • a monoclonal antibody binds to a target, such as CD276 with a binding affinity of at least about 1 x 10' 6 M, at least about 0.5 x 10" 6 M, at least about 1 x 10" 7 M, at least about 0.5 x 10" 7 M, at least about 1 x 10" 8 M, at least about 0.5 x 10" 8 M, at least about 1 x 10" 9 M, at least about 0.5 x 10" 9 M, or at least about 0.1 x 10' 9 .
  • a specific binding agent that binds to its target has a dissociation constant (Kd) of ⁇ 1000 nM, ⁇ 750 nM, 500 nM, ⁇ 250 nM, ⁇ 100 nM, ⁇ 50 nM, ⁇ 25 nM, ⁇ 10 nM, ⁇ 5 nM, ⁇ 2.5 nM, ⁇ 1 nM, ⁇ 0.5 nM, ⁇ 0.25 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g., 10’ 6 M or less, e.g., from 10' 6 M to 10 10 M, e.g., from 10 10 M to IO 12 M).
  • Kd dissociation constant
  • binding affinity is measured using the Octet system (Creative Biolabs), which is based on bio-layer interferometry (BLI) technology.
  • Kd is measured using surface plasmon resonance assays using a BIACORES-2000 or a BIACORES-3000 (BIAcore, Inc., Piscataway, N.J.).
  • Bispecific antibody A recombinant protein that includes antigen-binding fragments of two different monoclonal antibodies and is thereby capable of binding two different antigens or two different epitopes of the same antigen (such as CD276).
  • bispecific antibodies are used for cancer immunotherapy by simultaneously targeting, for example, both CTLs (such as a CTL receptor component such as CD3) or effector natural killer (NK) cells, and a tumor antigen (such as CD276).
  • CTLs such as a CTL receptor component such as CD3
  • NK effector natural killer
  • a tumor antigen such as CD276
  • a multi-specific antibody is a recombinant protein that includes antigen-binding fragments of at least two different monoclonal antibodies, such as two, three or four different monoclonal antibodies.
  • Brain cancer or tumor A type of cancer or tumor that develops from brain tissue.
  • Brain cancers include, but are not limited to, neuroblastoma, medulloblastoma, glioma, glioblastoma, meningioma, pituitary adenoma, astrocytoma, choroid plexus carcinoma, ependymoma and pineoblastoma.
  • Breast cancer A type of cancer that forms in tissues of the breast, usually the ducts and lobules.
  • Types of breast cancer include, for example, ductal carcinoma in situ, invasive ductal carcinoma, triple negative breast cancer, inflammatory breast cancer, metastatic breast cancer, medullary carcinoma, tubular carcinoma and mucinous carcinoma.
  • Triple negative breast cancer refers to a type of breast cancer in which the cancer cells do not express estrogen receptors, progesterone receptors or significant levels of HER2/neu protein.
  • Triple negative breast cancer is also called ER-negative PR-negative HER2/neu-negative breast cancer.
  • CD276 An immune checkpoint molecule that is expressed in the stroma of most or all solid tumors and may also be expressed by solid tumor cells. This protein is a member of the B7 superfamily of costimulatory molecules. CD276 is also known as B7H3.
  • CD276-positive cancer A cancer that expresses or overexpresses CD276.
  • Examples of CD276-positive cancers include, but are not limited to, liver cancers (such as hepatocellular carcinoma), pancreatic cancers, kidney cancers, bladder cancers, cervical cancers, esophageal cancers, prostate cancers, breast cancers, ovarian cancers, colon cancers, lung cancers, brain cancers (such as neuroblastoma or glioblastoma), pediatric cancers (such as osteosarcoma, neuroblastoma, rhabdomyosarcoma or Ewing’s sarcoma), melanoma and mesothelioma (see, for example, Seaman et al., Cancer Cell 31 (4):501-505, 2017).
  • a CD276-positive cancer refers to a cancer in which CD276 is expressed in the tumor stroma, and may also be expressed by the tumor cells.
  • Chemotherapeutic agent Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth.
  • a chemotherapeutic agent is an agent of use in treating a CD276-positive tumor.
  • a chemotherapeutic agent is a radioactive compound.
  • a skilled person can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch.
  • Combination chemotherapy is the administration of more than one agent to treat cancer.
  • One example is the administration of an antibody that binds CD276 used in combination with a radioactive or chemical compound.
  • a chemotherapeutic agent is a biologic, such as a therapeutic antibody (e.g., therapeutic monoclonal antibody), such as an anti-CD276 antibody provided herein, as well as other anti-cancer antibodies, such as anti-PDl or anti-PDLl (e.g., pembrolizumab and nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-EGFR (e.g., cetuximab), anti-VEGF (e.g., bevacizumab), or combinations thereof (e.g., anti-PD-1 and anti- CTLA-4).
  • a therapeutic antibody e.g., therapeutic monoclonal antibody
  • anti-CD276 antibody provided herein
  • other anti-cancer antibodies such as anti-PDl or anti-PDLl (e.g., pembrolizumab and nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-EG
  • Chimeric antigen receptor A chimeric molecule that includes an antigen-binding portion (such as a scFv) and a signaling domain, such as a signaling domain from a T cell receptor (for example, CD3Q.
  • CARs are comprised of an antigen-binding moiety, a transmembrane domain and an endodomain.
  • the endodomain typically includes a signaling chain having an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3 or FceRIy.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the endodomain further includes the intracellular portion of at least one additional costimulatory domain, such as CD28, 4-1BB (CD137), ICOS, 0X40 (CD134), CD27 and/or DAP10.
  • the CAR is multispecific (such as bispecific) or bicistronic.
  • a multispecific CAR is a single CAR molecule comprised of at least two antigen-binding domains (such as scFvs) that each bind a different antigen or a different epitope on the same antigen (see, for example, US 2018/0230225).
  • a bispecific CAR refers to a single CAR molecule having two antigen-binding domains that each bind a different antigen.
  • a bicistronic CAR refers to two complete CAR molecules, each containing an antigen-binding moiety that binds a different antigen.
  • a bicistronic CAR construct expresses two complete CAR molecules that are linked by a cleavage linker.
  • T cells or NK cells expressing a bispecific or bicistronic CAR can bind cells that express both of the antigens to which the binding moieties are directed (see, for example, Qin et al., Blood 130:810, 2017; and WO/2018/2I3337).
  • Colon cancer A type of cancer that develops in the colon or the rectum.
  • the most common type of colon cancer also known as “colorectal cancer” is colorectal adenocarcinoma, which accounts for approximately 95% of all colon cancers.
  • Adenocarcinomas develop in the cells lining the inside of the colon and/or rectum.
  • colorectal cancers include gastrointestinal carcinoid tumors, metastatic colorectal cancer, primary colorectal lymphoma (a type of non-Hodgkin’s lymphoma), gastrointestinal stromal tumors (classified as a sarcoma and arising from interstitial cells of Cajal), leiomyosarcoma (arising from smooth muscle cells) and colorectal melanoma.
  • Complementarity determining region A region of hypervariable amino acid sequence that defines the binding affinity and specificity of an antibody.
  • the light and heavy chains of a mammalian immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively.
  • a single-domain antibody contains three CDRs, referred to herein as CDR1, CDR2 and CDR3.
  • a “conjugate” is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to an effector molecule or a second protein (such as a second antibody).
  • the effector molecule can be, for example, a drug, toxin, therapeutic agent, detectable label, protein, nucleic acid, lipid, nanoparticle, photon absorber, carbohydrate or recombinant virus.
  • an antibody conjugate is often referred to as an “immunoconjugate.”
  • the conjugate includes an antibody linked to a drug (such as a cytotoxic agent)
  • the conjugate is often referred to as an “antibody-drug conjugate” or “ADC.”
  • ADC antibody-drug conjugate
  • Other antibody conjugates include, for example, multi-specific (such as bispecific or trispecific) antibodies and chimeric antigen receptors (CARs).
  • a monoclonal antibody that specifically binds CD276 can include at most about 1, at most about 2, at most about 5, and most about 10, or at most about 15 conservative substitutions and specifically bind the CD276 polypeptide.
  • the term “conservative variant” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibody specifically binds CD276. Non-conservative substitutions are those that reduce an activity or binding to CD276.
  • Placement in direct physical association includes both in solid and liquid form.
  • Cytotoxic agent Any drug or compound that kills cells.
  • Cytotoxicity The toxicity of a molecule, such as an immunotoxin, to the cells intended to be targeted, as opposed to the cells of the rest of an organism.
  • toxicity refers to toxicity of an immunotoxin to cells other than those that are the cells intended to be targeted by the targeting moiety of the immunotoxin
  • animal toxicity refers to toxicity of the immunotoxin to an animal by toxicity of the immunotoxin to cells other than those intended to be targeted by the immunotoxin.
  • Degenerate variant A polynucleotide encoding a polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide is unchanged.
  • Diagnostic Identifying the presence or nature of a pathologic condition, such as a CD276- positive cancer. Diagnostic methods differ in their sensitivity and specificity.
  • the "sensitivity” of a diagnostic assay is the percentage of diseased individuals who test positive (percent of true positives).
  • the "specificity” of a diagnostic assay is one minus the false positive rate, where the false positive rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • “Prognostic” is the probability of development (such as severity) of a pathologic condition, such cancer.
  • Drug Any compound used to treat, ameliorate or prevent a disease or condition in a subject.
  • the drug is an anti-cancer agent, for example a cytotoxic agent, such as an anti-mitotic or anti-microtubule agent.
  • Effector molecule The portion of a chimeric molecule that is intended to have a desired effect on a cell to which the chimeric molecule is targeted. Effector molecule is also known as an effector moiety (EM), therapeutic agent, diagnostic agent, or similar terms.
  • Therapeutic agents include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, photon absorbers, lipids, carbohydrates, or recombinant viruses.
  • Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides.
  • the molecule linked to a targeting moiety may be an encapsulation system, such as a liposome or micelle that contains a therapeutic composition such as a drug, a nucleic acid (such as an antisense nucleic acid), or another therapeutic moiety that can be shielded from direct exposure to the circulatory system.
  • a therapeutic composition such as a drug, a nucleic acid (such as an antisense nucleic acid), or another therapeutic moiety that can be shielded from direct exposure to the circulatory system.
  • Means of preparing liposomes attached to antibodies are well known (see, for example, U.S. Patent No. 4,957,735; and Connor el al., Pharm Ther 28:341-365, 1985). Diagnostic agents or moieties include radioisotopes and other detectable labels.
  • Detectable labels useful for such purposes are also well known and include radioactive isotopes such as 35 S, n C, 13 N, 15 O, 18 F, 19 F, " m Tc, 131 I, 3 H, 14 C, 15 N, 90 Y, "TC, i n In and 125 I, fluorophores, chemiluminescent agents, and enzymes.
  • radioactive isotopes such as 35 S, n C, 13 N, 15 O, 18 F, 19 F, " m Tc, 131 I, 3 H, 14 C, 15 N, 90 Y, "TC, i n In and 125 I, fluorophores, chemiluminescent agents, and enzymes.
  • Epitope An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic (that elicit a specific immune response). An antibody specifically binds a particular antigenic epitope on a polypeptide, such as CD276.
  • Framework region Amino acid sequences interposed between CDRs. Framework regions of an immunoglobulin molecule include variable light and variable heavy framework regions.
  • Fusion protein A protein comprising at least a portion of two different (heterologous) proteins.
  • Heterologous Originating from a separate genetic source or species.
  • Immune response A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus.
  • the response is specific for a particular antigen (an “antigen-specific response”).
  • an immune response is a T cell response, such as a CD4 + response or a CD8 + response.
  • the response is a B cell response, and results in the production of specific antibodies.
  • Immunoconjugate A covalent linkage of an effector molecule to an antibody or functional fragment thereof.
  • the effector molecule can be, for example, a detectable label, a photon absorber (such as IR700), or a toxin (to form an immunotoxin, such as an immunotoxin comprising Pseudomonas exotoxin or a variant thereof).
  • toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, or modified toxins thereof, or other toxic agents that directly or indirectly inhibit cell growth or kill cells.
  • PE and DT are highly toxic compounds that typically bring about death through liver toxicity.
  • PE and DT can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (such as the domain la of PE and the B chain of DT) and replacing it with a different targeting moiety, such as an antibody.
  • an antibody is joined to an effector molecule.
  • an antibody joined to an effector molecule is further joined to a lipid or other molecule, such as to increase its half-life in the body.
  • the linkage can be either by chemical or recombinant means.
  • the linkage is chemical, wherein a reaction between the antibody moiety and the effector molecule has produced a covalent bond formed between the two molecules to form one molecule.
  • a peptide linker short peptide sequence
  • immunoconjugates were originally prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are also sometimes referred to as “chimeric molecules.”
  • chimeric molecule refers to a targeting moiety, such as a ligand or an antibody, conjugated (coupled) to an effector molecule.
  • conjugated or “linked” refers to making two polypeptides into one contiguous polypeptide molecule.
  • Immunoliposome A liposome with antibodies or antibody fragments conjugated to its surface. Immunoliposomes can carry cytotoxic agents or other drugs to antibody-targeted cells, such as tumor cells.
  • Interstrand crosslinking agent A type of cytotoxic drug capable of binding covalently between two strands of DNA, thereby preventing DNA replication and/or transcription.
  • Isolated An “isolated” biological component, such as a nucleic acid, protein (including antibodies) or organelle, has been substantially separated or purified away from other biological components in the environment (such as a cell) in which the component occurs, for example other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • Label A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule.
  • labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.
  • a “labeled antibody” refers to incorporation of another molecule in the antibody.
  • the label is a detectable marker, such as the incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods).
  • labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as 35 S, “C, 13 N, 15 O, 18 F, 19 F, " m Tc, 131 1, 3 H, 14 C, 15 N, 90 Y, "Tc, 11 ‘in and I25 I), fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as
  • Linker is a peptide within an antibody binding fragment (such as an Fv fragment) which serves to indirectly bond the variable heavy chain to the variable light chain.
  • Linker can also refer to a peptide serving to link a targeting moiety, such as an antibody, to an effector molecule, such as a cytotoxin or a detectable label.
  • conjugating joining
  • bonding bonding
  • linking refer to making two polypeptides into one contiguous polypeptide molecule, or to covalently attaching a radionuclide or other molecule to a polypeptide, such as an antibody.
  • the linkage can be either by chemical or recombinant means.
  • “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.
  • liver cancer Any type of cancer occurring in liver tissue.
  • the most common type of liver cancer is hepatocellular carcinoma (HCC), which develops in hepatocytes.
  • HCC hepatocellular carcinoma
  • Other types of liver cancer include cholangiocarcinoma, which develops in the bile ducts; liver angiosarcoma, which is a rare form of liver cancer that begins in the blood vessels of the liver; and hepatoblastoma, which is a very rare type of liver cancer found most often in children.
  • Lung cancer Any cancer that forms in the lung. Most cancers that begin in the lung are carcinomas. The two primary types of lung carcinoma are small-cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). Subclasses of NSCLC include adenocarcinoma, squamous-cell carcinoma and large-cell carcinoma.
  • SCLC small-cell lung carcinoma
  • NSCLC non-small cell lung carcinoma
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • Ovarian cancer Cancer that forms in tissues of the ovary. Most ovarian cancers are either ovarian epithelial carcinomas (cancer that begins in the cells on the surface of the ovary) or malignant germ cell tumors (cancer that begins in egg cells). Another type of ovarian cancer is stromal cell cancer, which originates in cells that release hormones and connect the different structures of the ovaries.
  • Pancreatic cancer A disease in which malignant cells are found in the tissues of the pancreas.
  • Pancreatic tumors can be either exocrine tumors or neuroendocrine tumors, based on the cell origin of the cancer.
  • the vast majority (-94%) of pancreatic cancers are exocrine tumors.
  • Exocrine cancers include, for example, adenocarcinoma (the most common type of exocrine tumor), acinar cell carcinoma, intraductal papillary-mucinous neoplasm (1PMN), and mucinous cystadenocarcinoma.
  • the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).
  • Pancreatic neuroendocrine tumors also referred to as islet cell tumors, are classified by the type of hormones they produce.
  • Exemplary neuroendocrine tumors include gastrinoma, glucaganoma, insulinoma, somatostatinoma, VIPoma (vasoactive intestinal peptide) and nonfunctional islet cell tumor.
  • Pediatric cancer A cancer that develops in children ages 0 to 14.
  • the major types of pediatric cancers include, for example, neuroblastoma, acute lymphoblastic leukemia (ALL), embryonal rhabdomyosarcoma (ERMS), alveolar rhabdomyosarcoma (ARMS), Ewing’s sarcoma, desmoplastic small round cell tumor (DRCT), osteosarcoma, brain and other CNS tumors (such as neuroblastoma and medulloblastoma), Wilm’s tumor, non-Hodgkin lymphoma, and retinoblastoma.
  • ALL acute lymphoblastic leukemia
  • ERMS embryonal rhabdomyosarcoma
  • ARMS alveolar rhabdomyosarcoma
  • Ewing’s sarcoma desmoplastic small round cell tumor (DRCT), osteosarcoma
  • brain and other CNS tumors such as neuroblastoma and medul
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Photoimmunotherapy A targeted cancer therapy that utilizes an antigen- specific antibody-photoabsorber conjugate that can be activated by near- infrared light to kill targeted cells.
  • the photon absorber is typically based on phthalocyanine dye, such as a near infrared (NIR) phthalocyanine dye (for example, IRDye® 700DX, also know known as IR700).
  • NIR near infrared
  • the antibody for example, a CD276-specific antibody
  • binds to the appropriate cell surface antigen e.g., CD276
  • the photo- activatable dye induces lethal damage to cell membranes after NIR-light exposure.
  • NIR-light exposure e.g., 690 nm
  • Preventing refers to inhibiting the full development of a disease.
  • Treating refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number of size of metastases.
  • Treating refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer.
  • a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell.
  • a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.
  • Substantial purification denotes purification from other proteins or cellular components.
  • a substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure.
  • a substantially purified protein is 90% free of other proteins or cellular components.
  • PBD Pyrrolobenzodiazepine
  • a recombinant nucleic acid or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.
  • Sample A biological specimen containing genomic DNA, RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, semen, saliva, tissue biopsy, fine needle aspirate, surgical specimen, and autopsy material.
  • a sample includes a tumor biopsy.
  • Sequence identity The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide or nucleic acid molecule will possess a relatively high degree of sequence identity when aligned using standard methods.
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
  • Homologs and variants of an antibody that specifically binds a CD276 polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of the antibody using the NCBI Blast 2.0, gapped blastp set to default parameters.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence.
  • Small molecule A molecule, typically with a molecular weight less than about 1000 Daltons, or in some aspects, less than about 500 Daltons, wherein the molecule is capable of modulating, to some measurable extent, an activity of a target molecule.
  • Subject Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals.
  • a subject is a human subject with a CD276-positive cancer.
  • Synthetic Produced by artificial means in a laboratory, for example a synthetic nucleic acid or protein (for example, an antibody) can be chemically synthesized in a laboratory.
  • a synthetic nucleic acid or protein for example, an antibody
  • Therapeutically effective amount A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit or suppress growth of a tumor.
  • a therapeutically effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of a tumor, such as reduce a tumor size and/or volume by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, and/or reduce the number and/or size/volume of metastases by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, for example as compared to a size/volume/number prior to treatment or for example as compared to a size/volume/number with a different treatment (such as treatment with a different CD276 antibody or different CD276-targeted CAR).
  • a dosage will generally be used that will achieve target
  • Toxin A molecule that is cytotoxic for a cell.
  • Toxins include abrin, ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinum toxin, saporin, restrictocin or gelonin, or modified toxins thereof.
  • PE and DT are highly toxic compounds that typically bring about death through liver toxicity.
  • PE and DT can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (such as domain la of PE or the B chain of DT) and replacing it with a different targeting moiety, such as an antibody.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • the vector is a virus vector, such as a lentivirus vector.
  • CD276 monoclonal antibodies that specifically bind CD276 with high affinity are described.
  • the CD276 monoclonal antibodies were isolated from human scFv phage display libraries.
  • the monoclonal antibodies were used to develop CD276-targeted CARs, which are shown herein to induce a durable anti-tumor response in several animal models of cancer, and induce cell killing of multiple tumor cell lines.
  • the amino acid sequences of the VH domain and VL domain of the disclosed antibodies (Yl ll, Y422, Y868, Y117 and YE5) are provided below.
  • the CDR sequences (according to Kabat) are listed in Tables 1-5 below. Although the CDR locations were identified using Kabat, a skilled person understands that other numbering schemes, such as Chothia, Paratome or IMGT, can also be used to determine the boundaries of each CDR.
  • ADSVKGRFTISRDNAENTLYLEMNSLTADDTAVYYCVRDTYRFFDYWGQGTLVTVSS Y422 VL domain (SEQ ID NO: 10)
  • the monoclonal antibody includes a variable heavy (VH) domain and a variable light (VL) domain.
  • the monoclonal antibody includes at least a portion of the amino acid sequence set forth herein as SEQ ID NO: 1 and/or SEQ ID NO: 2, such as one or more (such as all three) CDR sequences from SEQ ID NO: 1 and/or SEQ ID NO: 2, as determined by any numbering scheme, such as 1MGT, Kabat or Chothia, or any combination thereof.
  • the monoclonal antibody includes at least a portion of the amino acid sequence set forth herein as SEQ ID NO: 9 and/or SEQ ID NO: 10, such as one or more (such as all three) CDR sequences from SEQ ID NO: 9 and/or SEQ ID NO: 10, as determined by any numbering scheme, such as IMGT, Kabat or Chothia, or any combination thereof.
  • the monoclonal antibody includes at least a portion of the amino acid sequence set forth herein as SEQ ID NO: 17 and/or SEQ ID NO: 18, such as one or more (such as all three) CDR sequences from SEQ ID NO: 17 and/or SEQ ID NO: 18, as determined by any numbering scheme, such as IMGT, Kabat or Chothia, or any combination thereof.
  • the monoclonal antibody includes at least a portion of the amino acid sequence set forth herein as SEQ ID NO: 25 and/or SEQ ID NO: 26, such as one or more (such as all three) CDR sequences from SEQ ID NO: 25 and/or SEQ ID NO: 26, as determined by any numbering scheme, such as IMGT, Kabat or Chothia, or any combination thereof.
  • the monoclonal antibody includes at least a portion of the amino acid sequence set forth herein as SEQ ID NO: 33 and/or SEQ ID NO: 34, such as one or more (such as all three) CDR sequences from SEQ ID NO: 33 and/or SEQ ID NO: 34, as determined by any numbering scheme, such as IMGT, Kabat or Chothia, or any combination thereof.
  • the VH domain of the monoclonal antibodies includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1 and/or the VL domain of the monoclonal antibody comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2.
  • the VH domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9 and/or the VL domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10.
  • the VH domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 17 and/or the VL domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 18.
  • the VH domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 25 and/or the VL domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 26.
  • the VH domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 33 and/or the VL domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 34.
  • the CDR sequences are determined using the IMGT, Kabat or Chothia numbering scheme, or a combination thereof. In particular examples, the CDR sequences are determined using Kabat.
  • the CDR1, CDR2 and CDR3 sequences of the VH domain of the monoclonal antibody respectively include SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and/or the CDR1, CDR2 and CDR3 sequences of the VL domain of the monoclonal antibody respectively include SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
  • the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively include SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; and/or the amino acid sequences of the LCDR1, LCDR2 and LCDR3 respectively include SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.
  • the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively include SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21; and/or the amino acid sequences of the LCDR1, LCDR2 and LCDR3 respectively include SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.
  • the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively include SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29; and/or the amino acid sequences of the LCDR1, LCDR2 and LCDR3 respectively include SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32.
  • amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively include SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37; and/or the amino acid sequences of the LCDR1, LCDR2 and LCDR3 respectively include SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40.
  • the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1 and includes the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 1; and/or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2 and includes the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 2.
  • the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 1 and the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 2.
  • the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 9 and includes the HCDR1, HCDR2 and HCDR3 sequence of SEQ ID NO: 9; and/or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 10 and includes the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 10.
  • the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 9 and/or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 10.
  • the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 17 and includes the HCDR1, HCDR2 and HCDR3 sequence of SEQ ID NO: 17; and/or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 18 and includes the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 18.
  • the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 17 and/or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 18.
  • the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 25 and includes the HCDR1, HCDR2 and HCDR3 sequence of SEQ ID NO: 25; and/or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 26 and includes the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 26.
  • the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 25 and/or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 26.
  • the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%identical to SEQ ID NO: 33 and includes the HCDR1, HCDR2 and HCDR3 sequence of SEQ ID NO: 33; and/or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 34 and includes the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 34.
  • the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 33 and/or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 34.
  • the monoclonal antibody is an IgG, IgA, or IgM molecule. In some examples, the monoclonal antibodies is an IgGl, IgG2, IgG3 or IgG4.
  • the monoclonal antibody is an antigen-binding fragment selected from an Fab fragment, an Fab’ fragment, an F(ab)’2 fragment, a single chain variable fragment (scFv) and a disulfide stabilized variable fragment (dsFv).
  • the monoclonal antibody is a fully human monoclonal antibody. In other aspects, the monoclonal antibody is a chimeric antibody.
  • the monoclonal antibody further includes a constant region, such as an IgG constant region, for example, an IgGl constant region, such as a human IgGl constant region.
  • the constant region includes at least one amino acid modification to increase the half-life, stability and/or function of the monoclonal antibody.
  • chimeric antigen receptors that include a CD276-specific monoclonal antibody disclosed herein.
  • the monoclonal antibody component of the CAR is an scFv.
  • the scFv is in the VH domain-linker- VL domain (VH-linker- VL) format (N- to C-terminal direction).
  • the scFv is in the VL domain-linker- VH domain (VL-linker-VH) format (N- to C-terminal direction).
  • the linker can be any suitable linker for separating the VH and VL domains.
  • the linker is a glycine-serine (GS) linker, such as GGGGSGGGGSGGGGS (SEQ ID NO: 42).
  • the CAR further includes a hinge region, a transmembrane domain, a costimulatory domain, a signaling domain, or any combination thereof.
  • the hinge region includes a CD28 hinge region;
  • the transmembrane domain includes a CD28 transmembrane domain;
  • the costimulatory domain includes a 4-1BB costimulatory domain or a CD28 costimulatory domain;
  • the signaling domain includes a CD3 signaling domain.
  • the CD28 hinge region includes the amino acid sequence of SEQ ID NO: 43; the CD28 transmembrane domain includes the amino acid sequence of SEQ ID NO: 44; the 4- IBB costimulatory domain includes the amino acid sequence of SEQ ID NO: 45 or the CD28 costimulatory domain includes the amino acid sequence of SEQ ID NO: 65; and/or the CD3(j signaling domain includes the amino acid sequence of SEQ ID NO: 46.
  • the amino acid sequence of the CAR includes residues 20-480 of SEQ ID NO: 47, residues 20-484 of SEQ ID NO: 49, residues 20-484 of SEQ ID NO: 51, residues 20-488 of SEQ ID NO: 53, residues 20-486 of SEQ ID NO: 55, residues 20-479 of SEQ ID NO: 61, or residues 20- 480 of SEQ ID NO: 63.
  • isolated cells expressing a CD276-specific CAR disclosed herein.
  • the isolated cell is an immune cell, such as a T cell, B cell, natural killer (NK) cell, macrophage, dendritic cell (DC), or an induced pluripotent stem cell (iPSC).
  • the immune cells are allogeneic cells, such as allogeneic cells obtained from a healthy donor. CARs and CAR-expressing cells are further described in section IV.
  • ADCs antibody-drug conjugates
  • the drug is a small molecule, for example an anti-cancer agent, anti-microtubule agent, an anti-mitotic agent and/or a cytotoxic agent.
  • ADCs are further described in section VI.
  • multi-specific antibodies that include a CD276- specific monoclonal antibody disclosed herein and at least one additional monoclonal antibody or antigenbinding fragment thereof.
  • the multi-specific antibody is a bispecific antibody.
  • the multi-specific antibody is a trispecific antibody.
  • the multispecific antibody includes a CD276- specific monoclonal antibody disclosed herein and a second antibody that binds T cells, such as CD3 on T cells, or that binds NK cells, such as CD 16 on NK cells. Multi-specific antibodies are further described in section VII.
  • antibody-nanoparticle conjugates that include a nanoparticle conjugated to a CD276-specific monoclonal antibody disclosed herein.
  • the nanoparticle includes a polymeric nanoparticle, nanosphere, nanocapsule, liposome, dendrimer, polymeric micelle, or niosome.
  • the nanoparticle includes a cytotoxic agent. Antibody-nanoparticle conjugates are further described in section VIII.
  • fusion proteins that include a CD276-specific monoclonal antibody disclosed herein and a heterologous protein or peptide.
  • the heterologous protein is an Fc protein, such as a human Fc protein (e.g., human IgGl Fc).
  • the fusion protein further includes a linker, such as protein linker.
  • the nucleotide sequence encoding the CAR includes or consists of SEQ ID NO: 48 or a degenerate variant thereof, SEQ ID NO: 50 or a degenerate variant thereof, SEQ ID NO: 52 or a degenerate variant thereof, SEQ ID NO: 54 or a degenerate variant thereof, SEQ ID NO: 56 or a degenerate variant thereof, SEQ ID NO: 62 or a degenerate variant thereof, or SEQ ID NO: 64 or a degenerate variant thereof.
  • Vectors that include the disclosed nucleic acid molecules are also provided.
  • the vector is an expression vector.
  • the vector is a viral vector.
  • Isolated cells that include a nucleic acid molecule or vector disclosed herein are further provided.
  • the isolated cell is a prokaryotic cell, such as an E. coli cell.
  • the isolated cell is a mammalian cell, such as a human cell. Nucleic acid molecules are further described in section IX.
  • compositions that include a pharmaceutically acceptable carrier and a CD276-specific monoclonal antibody, fusion protein, CAR, isolated cell (such as a CAR expressing cell, for example a CAR T cell, a CAR NK cell or a CAR macrophage), immunoconjugate, ADC, multispecific antibody, antibody-nanoparticle conjugate, isolated nucleic acid molecule or vector disclosed herein are further provided by the present disclosure.
  • Compositions are further described in section X.
  • kits that include a monoclonal antibody, CAR, CAR-expressing cell, immunoconjugate, ADC, multi-specific antibody, antibody-nanoparticle conjugate, fusion protein, or composition disclosed herein; and one or more of a pharmaceutically acceptable carrier, buffer, cell culture media, cell culture plates or flasks, a solid support, a fluorescent label, a radioactive label, an enzymatic label, an enzymatic substrate, a secondary antibody, one or more check point inhibitors, one or more additional anti-cancer agents, one or more transfection reagents, and instructional materials. Kits are further described in section XII.
  • CARs include a binding moiety, an extracellular hinge and spacer element, a transmembrane region and an endodomain that performs signaling functions (Cartellieri et al., J Biomed Biotechnol 2010:956304, 2010; Dai et al., J Natl Cancer Inst 108(7):djv439, 2016).
  • the binding moiety is an antigen binding fragment of a monoclonal antibody, such as a scFv, or a single-domain antibody.
  • the spacer/hinge region typically includes sequences from IgG subclasses, such as IgGl, IgG4, IgD and CD8 domains.
  • the transmembrane domain can be derived from a variety of different T cell proteins, such as CD3 ⁇ , CD4, CD8 or CD28.
  • the endodomain can consist of a signaling chain having an IT AM, such as CD3 ⁇ or FceRIy.
  • the endodomain further includes the intracellular portion of at least one additional costimulatory domain, such as CD28, 4-1BB (CD137, TNFRSF9), OX-40 (CD134), ICOS, CD27 and/or DAP10.
  • Immune cells such as T cells, B cells, NK cells, dendritic cells, or macrophages, or iPSCs expressing CARs can be used to target a specific cell type, such as a CD276-positive tumor cell.
  • the antibodies disclosed herein can be used to engineer immune cells or iPSCs that express a CAR containing the CD276-specific monoclonal antibody, thereby targeting the engineered cells to CD276-postive tumor cells.
  • Multispecific (such as bispecific) or bicistronic CARs are also contemplated by the present disclosure.
  • the multispecific or bispecific CAR includes a monoclonal antibody (such as an scFv) specific for CD276 and a monoclonal antibody specific for a different antigen (or a different epitope of CD276).
  • a bicistronic CAR includes two CAR molecules expressed from the same construct where one CAR molecule is a CD276-targeted CAR and the second CAR targets a second antigen, such as a second tumor antigen (see, for example, Qin et al. , Blood 130:810, 2017; and WO/2018/213337).
  • CARs that include a CD276-specific antibody, such as any one of the monoclonal antibodies disclosed herein.
  • isolated nucleic acid molecules and vectors encoding the CARs include bispecific and bicistronic CARs
  • host cells such as T cells, B cells, NK cells, DCs, macrophages or iPSCs expressing the CARs, bispecific CAR or bicistronic CARs.
  • T cells, B cells, NK cells, DCs, macrophages or iPSCs expressing CARs comprised of a CD276-specific monoclonal antibody can be used for the treatment of a CD276-positive cancer.
  • the CAR is a bispecific CAR.
  • the CAR is a bicistronic CAR.
  • the CAR includes a signal peptide sequence, for example, N-terminal to the antigen-binding domain.
  • the signal peptide sequence can be any suitable signal peptide sequence, such as a signal sequence from granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), immunoglobulin light chain kappa, or IL-2.
  • GMCSFR granulocyte-macrophage colony-stimulating factor receptor
  • IL-2 immunoglobulin light chain kappa
  • the signal peptide sequence includes the signal peptide of SEQ ID NO: 41. While the signal peptide sequence may facilitate expression of the CAR on the surface of the cell, the presence of the signal peptide sequence in an expressed CAR is not necessary for the CAR to function.
  • the signal peptide sequence may be cleaved off of the CAR. Accordingly, in some aspects, the CAR lacks a signal peptide sequence.
  • the CD276- targeted CAR includes the following features in an N-terminal to C-terminal direction: signal peptide, VH domain, linker sequence, VL domain, hinge region, transmembrane domain, costimulatory domain, and signaling domain.
  • the signal peptide includes or consists of the amino acid sequence of SEQ ID NO: 41.
  • the linker sequence includes or consists of the amino acid sequence of SEQ ID NO: 42.
  • the hinge region is a CD28 hinge region, such as a CD28 hinge region that includes or consists of the amino acid sequence of SEQ ID NO: 43.
  • the transmembrane domain is a CD28 transmembrane domain, such as a CD28 transmembrane domain that includes or consists of the amino acid sequence of SEQ ID NO: 44.
  • the costimulatory domain is a 4- IBB costimulatory domain, such as a 4-1BB costimulatory domain including or consisting of the amino acid sequence of SEQ ID NO: 45, or the costimulatory domain is a CD28 costimulatory domain, such as a CD28 costimulatory domain including or consisting of the amino acid sequence of SEQ ID NO: 65.
  • the signaling domain is a CD3 ⁇ signaling domain, such as a CD3 ⁇ signaling domain that includes or consists of the amino acid sequence of SEQ ID NO: 46.
  • Nucleotide and amino acid sequences of exemplary CARs that include an antigen-binding fragment (such as a scFv) from CD276-specific monoclonal antibodies Yl l 1, Y422, Y868, Y117 and YE5, are provided below, along with amino acid and nucleotide sequences of individual components of the CARs.
  • an antigen-binding fragment such as a scFv
  • CD28 hinge amino acid sequence (SEQ ID NO: 43)
  • CD28 transmembrane domain amino acid sequence (SEQ ID NO: 44)
  • CD28 transmembrane domain nucleotide sequence SEQ ID NO: 70
  • CD28 costimulatory domain amino acid sequence (SEQ ID NO: 65)
  • CD28 costimulatory domain nucleotide sequence (SEQ ID NO: 66)
  • ITAMs underlined SEQ ID NO: 46
  • CD3 signaling domain nucleotide sequence (SEQ ID NO: 72)
  • VH-linker-VL Ylll scFv (VH-linker-VL) nucleotide sequence (SEQ ID NO: 58)
  • VL-linker-VH Ylll scFv (VL-linker-VH) nucleotide sequence (SEQ ID NO: 60)
  • Ylll CAR (Ylll-HL-28HTM-BBz) amino acid sequence (SEQ ID NO: 47)
  • Ylll CAR (Ylll-HL-28HTM-BBz) nucleotide sequence (SEQ ID NO: 48)
  • Ylll CAR (Ylll-HL-28HTM-28z) amino acid sequence (SEQ ID NO: 61)
  • Ylll CAR (Ylll-HL-28HTM-28z) nucleotide sequence (SEQ ID NO: 62)
  • Ylll CAR (Ylll-LH-28HTM-BBz) amino acid sequence (SEQ ID NO: 63)
  • Ylll CAR (Ylll-LH-28HTM-BBz) nucleotide sequence (SEQ ID NO: 64)
  • CD276-specific monoclonal antibodies modified to enable their use with a universal CAR system.
  • Universal CAR systems have been developed to increase CAR flexibility and expand their use to additional antigens.
  • autologous immune cells such as T cells
  • This process is lengthy and expensive, limiting its use.
  • Universal CARs are based on a system in which the signaling components of the CAR are split from the antigen-binding portion of the molecule but come together using a “lock-key” system.
  • biotin-binding immune receptor (BBIR) CARs are comprised of an intracellular T cell signaling domain fused to an extracellular domain comprising avidin.
  • Biotinylated antigen-specific (such as CD276-specific) monoclonal antibodies can then bind the BBIR to direct immune cells to antigen-expressing cells.
  • SUPRA split, universal and programmable
  • the CAR includes the intracellular signaling domains fused to an extracellular leucine zipper, which is paired with an antigen-specific monoclonal antibody fused to a cognate leucine zipper.
  • the CD276-specific monoclonal antibody is fused to one component of a specific binding pair.
  • the monoclonal antibody is fused to a leucine zipper or biotin.
  • a sortase is a prokaryotic enzyme that modifies surface proteins by recognizing and cleaving a carboxyl-terminal sorting signal. Sortase catalyzes transpeptidation between a sortase recognition motif and a sortase acceptor motif.
  • antigen-specific CARs can be generated by contacting an antigen-specific antibody fused to a sortase recognition motif with a portion of a CAR molecule that includes the intracellular signaling domain(s), a transmembrane region and an extracellular portion that includes a sortase acceptor motif.
  • a CD276-specific monoclonal antibody is modified to include a sortase recognition motif (see, for example, PCT Publication No. WO 2016/014553).
  • the CD276- targeted CAR is expressed in allogeneic immune cells, such as allogeneic T cells, B cells, NK cells, DCs or macrophages from a healthy donor(s).
  • the allogeneic immune cells are genetically engineered to express the CD276-targeted CAR, for example by disrupting expression of the endogenous T cell receptor by insertion of the CAR (see, for example, MacLeod et al., Mol Ther 25(4): 949-961, 2017).
  • Gene editing can be performed using any appropriate gene editing system, such as CRISPR/Cas9, zinc finger nucleases or transcription activator-like effector nucleases (TALEN).
  • the disclosed monoclonal antibodies can be conjugated to a therapeutic agent or effector molecule.
  • Immunoconjugates include, but are not limited to, molecules in which there is a covalent linkage of a therapeutic agent to an antibody.
  • a therapeutic agent is an agent with a particular biological activity directed against a particular target molecule or a cell bearing a target molecule.
  • therapeutic agents can include various drugs, such as vinblastine, daunomycin and the like, cytotoxins such as native or modified Pseudomonas exotoxin or diphtheria toxin, encapsulating agents (such as liposomes) that contain pharmacological compositions, radioactive agents such as 125 1, 32 P, 14 C, 3 H and 35 S, photon absorbers such as IR700, and other labels, target moieties and ligands.
  • drugs such as vinblastine, daunomycin and the like
  • cytotoxins such as native or modified Pseudomonas exotoxin or diphtheria toxin
  • encapsulating agents such as liposomes
  • radioactive agents such as 125 1, 32 P, 14 C, 3 H and 35 S
  • photon absorbers such as IR700
  • the choice of a particular therapeutic agent depends on the target molecule or cell, and the desired biological effect.
  • the therapeutic agent can be a cytotoxin that is used to bring about the death of a particular target cell (such as a CD276-expressing cell).
  • the therapeutic agent can be conjugated to a non-lethal pharmacological agent or a liposome containing a non-lethal pharmacological agent.
  • nucleic acids encoding antibodies and conjugates and fusion proteins thereof.
  • Effector molecules can be linked to an antibody of interest using any number of known means. Both covalent and noncovalent attachment means may be used.
  • the procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector.
  • Polypeptides typically contain a variety of functional groups, such as carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule.
  • the antibody is derivatized to expose or attach additional reactive functional groups.
  • the derivatization may involve attachment of any of a number of linker molecules.
  • the linker can be any molecule used to join the antibody to the effector molecule.
  • the linker is capable of forming covalent bonds to both the antibody and to the effector molecule.
  • Suitable linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.
  • the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
  • immunoconjugates include linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.
  • the antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein).
  • the antibodies or portion thereof is derivatized such that the binding to the target antigen is not affected adversely by the derivatization or labeling.
  • the antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bispecific antibody or a diabody), an Fc protein, a detection agent, a photon absorber, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, such as to create bispecific antibodies).
  • Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate).
  • Such linkers are commercially available.
  • the antibody can be conjugated with a detectable marker, for example, a detectable marker capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopic examination).
  • a detectable marker include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI).
  • useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like.
  • Bioluminescent markers are also of use, such as luciferase, green fluorescent protein (GFP) and yellow fluorescent protein (YFP).
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • An antibody can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, P-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like.
  • an antibody or antigen binding fragment When an antibody or antigen binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable.
  • An antibody or antigen binding fragment may also be conjugated with biotin and detected through indirect measurement of avidin or streptavidin binding. The avidin itself can be conjugated with an enzyme or a fluorescent label.
  • An antibody may be labeled with a magnetic agent, such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium), and manganese. Paramagnetic particles, such as superparamagnetic iron oxide, are also of use as labels.
  • An antibody may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some aspects, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
  • An antibody can also be labeled with a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes.
  • the radiolabel may be used to detect expression of a target antigen by x-ray, emission spectra, or other diagnostic techniques.
  • labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3 H, 14 C, 15 N, 35 S, 90 Y, "Tc, i n In, 125 I, and 131 I.
  • an antibody disclosed herein can also be conjugated to a photon absorber.
  • the photon absorber is a phthalocyanine dye, such as, but not limited to, IRDye® 700DX (also known as “IR700”).
  • Antibody-photoabsorber conjugates can be used for photoimmunotherapy (for example to kill CD276-positive tumor cells).
  • An antibody can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, such as to increase serum half-life or to increase tissue binding.
  • PEG polyethylene glycol
  • methyl or ethyl group a methyl or ethyl group
  • carbohydrate group a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group.
  • Toxins can be employed with the monoclonal antibodies described herein to produce immunotoxins.
  • Exemplary toxins include ricin, abrin, diphtheria toxin and subunits thereof, as well as botulinum toxins A through F. These toxins are readily available from commercial sources (for example, Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of the toxins described herein (see, for example, see, U.S. Patent Nos. 5,079,163 and 4,689,401).
  • the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095).
  • Pseudomonas exotoxin refers to a full-length native (naturally occurring) PE or a PE that has been modified. Such modifications can include, but are not limited to, elimination of domain la, various amino acid deletions in domains lb, II and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus (for example, see Siegall et al. , J. Biol. Chem. 264: 14256-14261, 1989).
  • PE employed with the monoclonal antibodies described herein can include the native sequence, cytotoxic fragments of the native sequence, and conservatively modified variants of native PE and its cytotoxic fragments.
  • Cytotoxic fragments of PE include those which are cytotoxic with or without subsequent proteolytic or other processing in the target cell. Cytotoxic fragments of PE include PE40, PE38, and PE35.
  • Cytotoxic fragments of PE include PE40, PE38, and PE35.
  • PE-LR protease-resistant PE variants and PE variants with reduced immunogenicity
  • PE-LR protease-resistant PE variants and PE variants with reduced immunogenicity
  • PE-LR protease-resistant PE variants and PE variants with reduced immunogenicity
  • PE-LR protease-resistant PE variants and PE variants with reduced immunogenicity
  • PE-LR protease-resistant PE variants and PE variants with reduced immunogenicity
  • the PE is a variant that is resistant to lysosomal degradation, such as PE- LR (Weldon et al., Blood 113(161:3792-3800, 2009; PCT Publication No. WO 2009/0329541.
  • the PE is a variant designated PE-LR/6X (PCT Publication No. WO 2011/0320221.
  • the PE variant is PE with reducing immunogenicity.
  • the PE is a variant designated PE-LR/8M (PCT Publication No. WO 2011/0320221.
  • Modification of PE may occur in any previously described variant, including cytotoxic fragments of PE (for example, PE38, PE-LR and PE-LR/8M).
  • Modified PEs may include any substitution(s), such as for one or more amino acid residues within one or more T-cell epitopes and/or B cell epitopes of PE, or deletion of one or more T-cell and/or B-cell epitopes (see, for example, U.S. Patent Application Publication No. 2015/00997071.
  • Contemplated forms of PE also include deimmunized forms of PE, for example versions with domain II deleted (for example, PE24).
  • Deimmunized forms of PE are described in, for example, PCT Publication Nos. WO 2005/052006, WO 2007/016150, WO 2007/014743, WO 2007/031741, WO 2009/32954, WO 2011/32022, WO 2012/154530, and WO 2012/170617.
  • the antibodies described herein can also be used to target any number of different diagnostic or therapeutic compounds to cells expressing CD276 on their surface (e.g., CD276- positive tumor cells).
  • an antibody of the present disclosure can be attached directly or via a linker to a drug that is to be delivered directly to cells expressing CD276. This can be done for therapeutic, diagnostic or research purposes.
  • Therapeutic agents include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, photon absorbers, lipids, carbohydrates, and recombinant viruses.
  • Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides.
  • the molecule linked to an antibody can be an encapsulation system, such as a nanoparticle, liposome or micelle that contains a therapeutic composition such as a drug, a nucleic acid (for example, an antisense nucleic acid), or another therapeutic moiety that is shielded from direct exposure to the circulatory system.
  • a therapeutic composition such as a drug, a nucleic acid (for example, an antisense nucleic acid), or another therapeutic moiety that is shielded from direct exposure to the circulatory system.
  • Means of preparing liposomes attached to antibodies are known (see, for example, U.S. Patent No. 4,957,735; Connor et al., Pharm. Ther. 28:341-365, 1985).
  • Antibodies described herein can also be covalently or non-covalently linked to a detectable label.
  • Detectable labels suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels include magnetic beads, fluorescent dyes (for example, fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (for example, 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes (such as horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, latex, and the like) beads.
  • fluorescent dyes for example, fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like
  • radiolabels for example, 3 H, 125 1, 35 S, 14 C, or 32 P
  • enzymes such as horseradish peroxidase, alkaline
  • radiolabels may be detected using photographic film or scintillation counters
  • fluorescent markers may be detected using a photodetector to detect emitted illumination.
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • ADCs Antibody-Drug Conjugates
  • ADCs are compounds comprised of an antigen-specific antibody (such as a monoclonal antibody or antigen-binding fragment of an immunoglobulin provided herein that binds CD276) and a drug, for example a cytotoxic agent (such as an anti-microtubule agent or cross-linking agent).
  • a cytotoxic agent such as an anti-microtubule agent or cross-linking agent.
  • the drug can be much more potent than agents used for standard systemic therapy.
  • the most common cytotoxic drugs currently used with ADCs have an IC50 that is 100- to 1000-fold more potent than conventional chemotherapeutic agents.
  • cytotoxic drugs include antimicrotubule agents, such as maytansinoids and auristatins (such as auristatin E and auristatin F).
  • Other cytotoxins for use with ADCs include pyrrolobenzodiazepines (PBDs), which covalently bind the minor groove of DNA to form interstrand crosslinks.
  • PBDs pyrrolobenzodiazepines
  • ADCs comprise a 1:2 to 1:4 ratio of antibody to drug (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).
  • the antibody and drug can be linked by a cleavable or non-cleavable linker.
  • a linker that is stable in the circulation to prevent systemic release of the cytotoxic drug that could result in significant off-target toxicity.
  • Non-cleavable linkers prevent release of the cytotoxic agent before the ADC is internalized by the target cell. Once in the lysosome, digestion of the antibody by lysosomal proteases results in the release of the cytotoxic agent (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).
  • One method for site-specific and stable conjugation of a drug to a monoclonal antibody (or antigen-biding fragment fused to Fc) is via glycan engineering.
  • Monoclonal antibodies have one conserved N-linked oligosaccharide chain at the Asn297 residue in the CH2 domain of each heavy chain (Qasba et al., Biotechnol Prog 24:520-526, 2008).
  • Y289L-Gal-Tl U.S. Patent Application Publication Nos. 2007/0258986 and 2006/0084162
  • 2-keto-galactose is transferred to free GlcNAc residues on the antibody heavy chain to provide a chemical handle for conjugation.
  • the oligosaccharide chain attached to monoclonal antibodies can be classified into three groups based on the terminal galactose residues - fully galactosylated (two galactose residues; IgG- G2), one galactose residue (IgG-Gl) or completely degalactosylated (IgG-GO).
  • Treatment of a monoclonal antibody with [31,4-galactosidase converts the antibody to the IgG-GO glycoform.
  • the mutant [31,4-galactosyltransferase enzyme is capable of transferring 2-keto-galactose or 2-azido- galactose from their respective UDP derivatives to the GlcNAc residues on the IgG-Gl and IgG-GO glycoforms.
  • the chemical handle on the transferred sugar enables conjugation of a variety of molecules to the monoclonal antibody via the glycan residues (Qasba et al. , Biotechnol Prog 24:520-526, 2008).
  • ADCs that include a drug (such as a cytotoxic agent) conjugated to a monoclonal antibody that binds (such as specifically binds) CD276.
  • a drug such as a cytotoxic agent
  • the drug is a small molecule.
  • the drug is a cross-linking agent, an anti-microtubule agent and/or anti-mitotic agent, or any cytotoxic agent suitable for mediating killing of tumor cells.
  • cytotoxic agents include, but are not limited to, a PBD, an auristatin, a maytansinoid, dolastatin, calicheamicin, nemorubicin and its derivatives, PNU- 159682, anthracycline, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, elinafide, a combretastain, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a puromycin, a tubulysin, a hemiasterlin, a spliceostatin, or a pladienolide, as well as stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.
  • PBD auristatin
  • a maytansinoid dolastatin
  • the ADC includes a pyrrolobenzodiazepine (PBD).
  • PBD pyrrolobenzodiazepine
  • the natural product anthramycin (a PBD) was first reported in 1965 (Leimgruber et al., J Am Chem Soc, 87:5793-5795, 1965; Leimgruber et al. , J Am Chem Soc, 87:5791-5793, 1965). Since then, a number of PBDs, both naturally-occurring and synthetic analogues, have been reported (Gerratana, Med Res Rev 32(2):254-293, 2012; and U.S. Patent Nos.
  • PBD dimers recognize and bind to specific DNA sequences, and have been shown to be useful as cytotoxic agents. PBD dimers have been conjugated to antibodies and the resulting ADC shown to have anti-cancer properties (see, for example, US 2010/0203007).
  • Exemplary linkage sites on the PBD dimer include the fivemembered pyrrolo ring, the tether between the PBD units, and the N10-C11 imine group (see WO 2009/016516; US 2009/304710; US 2010/047257; US 2009/036431; US 2011/0256157; and WO 2011/130598).
  • the ADC includes an antibody conjugated to one or more maytansinoid molecules.
  • Maytansinoids are derivatives of maytansine and are mitotic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinoids are disclosed, for example, in U.S. Patent Nos.
  • the ADC includes an antibody conjugated to a dolastatin or auristatin, or an analog or derivative thereof (see U.S. Patent Nos. 5,635,483; 5,780,588; 5,767,237; and 6,124,431).
  • Auristatins are derivatives of the marine mollusk compound dolastatin- 10. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al., Antimicrob Agents and Chemother 45(12):3580-3584, 2001) and have anticancer (U.S. Patent No.
  • dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin F, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (Monomethyl Auristatin D or monomethyl dolastatin 10), MMAF (Monomethyl Auristatin F or N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE (Monomethyl Auristatin E or N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other auristatins (see, for example, U.S. Publication No. 2013/0129753).
  • the ADC includes an antibody conjugated to one or more calicheamicin molecules.
  • the calicheamicin family of antibiotics, and analogues thereof, can produce doublestranded DNA breaks at sub-picomolar concentrations (Hinman et al. , Cancer Res 53:3336-3342, 1993; Lode et al. , Cancer Res 58:2925-2928, 1998).
  • Exemplary methods for preparing ADCs with a calicheamicin drug moiety are described in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.
  • the ADC includes an anthracycline.
  • Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. It is believed that anthracyclines can operate to kill cells by a number of different mechanisms, including intercalation of the drug molecules into the DNA of the cell thereby inhibiting DNA-dependent nucleic acid synthesis; inducing production of free radicals which then react with cellular macromolecules to cause damage to the cells; and/or interactions of the drug molecules with the cell membrane.
  • Non-limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, daunorubicin, doxorubicin, epirubicin, nemorubicin, valrubicin and mitoxantrone, and derivatives thereof.
  • PNU- 159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al. , Clin Cancer Res 11(4): 1608- 1617, 2005).
  • Nemorubicin is a semisynthetic analog of doxorubicin with a 2-methoxymorpholino group on the glycoside amino of doxorubicin (Grand! et al. , Cancer Treat Rev 17:133, 1990; Ripamonti et al., Br J Cancer 65:703-707, 1992).
  • the ADC can further include a linker.
  • the linker is a bifunctional or multifunctional moiety that can be used to link one or more drug moieties to an antibody to form an ADC.
  • ADCs are prepared using a linker having reactive functionalities for covalently attaching to the drug and to the antibody. For example, a cysteine thiol of an antibody can form a bond with a reactive functional group of a linker or a drug-linker intermediate to make an ADC.
  • a linker has a functionality that can react with a free cysteine present on an antibody to form a covalent bond.
  • exemplary linkers with such reactive functionalities include maleimide, haloacetamides, > -haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.
  • a linker has a functionality that can react with an electrophilic group present on an antibody.
  • electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups.
  • a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit.
  • Non-limiting examples include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide.
  • the linker is a cleavable linker, which facilitates release of the drug.
  • cleavable linkers include acid-labile linkers (for example, comprising hydrazone), protease-sensitive linkers (for example, peptidase-sensitive), photolabile linkers, and disulfide- containing linkers (Chari et al. , Cancer Res 52:127-131, 1992; U.S. Patent No. 5,208,020).
  • the ADCs disclosed herein can be used for the treatment of a CD276-positive tumor alone or in combination with another therapeutic agent and/or in combination with any standard therapy for the treatment of a CD276-positive cancer. VII. Multi-specific Antibodies
  • Multi-specific antibodies are recombinant proteins comprised of two or more monoclonal antibodies or antigen-binding fragments of two or more different monoclonal antibodies.
  • bispecific antibodies are comprised of two different monoclonal antibodies or antigenbinding fragments thereof.
  • bispecific antibodies bind two different antigens (or two different epitopes of an antigen) and trispecific antibodies bind three different antigens (or three different epitopes of an antigen).
  • multi-specific, such as trispecific or bispecific, monoclonal antibodies that include a first CD276- specific monoclonal antibody.
  • the multi- specific monoclonal antibody further includes a second antibody that specifically binds a different epitope of CD276 or a different cell-surface antigen.
  • the multi-specific monoclonal antibody further includes a second antibody that specifically binds PD-1 (such as nivolumab, JTX- 4014 by Jounce Therapeutics, nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IB 1308), tislelizumab (BGB-A317), toripalimab (JS 001), dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, or AMP- 514).
  • the multi- specific monoclonal antibody further includes a second antibody that specifically binds CTLA-4 (such as ipilimumab or tremelimumab).
  • the multi- specific monoclonal antibody includes a first monoclonal antibody specific for CD276 (such as a CD276-specific scFv) and further includes a monoclonal antibody (or antigen-binding fragment such as an scFv) that specifically binds a component of the T cell receptor, such as CD3.
  • the multi- specific monoclonal antibody includes a first monoclonal antibody specific for CD276 (such as a CD276- specific scFv) and further includes a monoclonal antibody (or antigen-binding fragment such as an scFv) that specifically binds a NK cell activating receptor, such as CD16, Ly49, or CD94.
  • Multi- specific antibodies that include a CD276- specific antibody can be used for the treatment of a CD276- positive cancer.
  • methods of treating a subject with a CD276-positive cancer by administering to the subject a therapeutically effective amount of the CD276-targeting multi- specific antibody.
  • the monoclonal antibodies disclosed herein can be conjugated to a variety of different types of nanoparticles to deliver cytotoxic agents directly to CD276-expressing cells via binding of the antibody to CD276 expressed on the surface of cells.
  • the use of nanoparticles reduces off-target side effects and can also improve drug bioavailability and reduce the dose of a drug required to achieve a therapeutic effect.
  • Nanoparticle formulations can be tailored to suit the drug that is to be carried or encapsulated within the nanoparticle. For example, hydrophobic molecules can be incorporated inside the core of a nanoparticle, while hydrophilic drugs can be carried within an aqueous core protected by a polymeric or lipid shell.
  • nanoparticles include, but at not limited to, nanospheres, nanocapsules, liposomes, dendrimers, polymeric micelles, niosomes, and polymeric nanoparticles (Fay and Scott, Immunotherapy 3(3):381-394, 2011).
  • Liposomes are common types of nanoparticles used for drug delivery.
  • An antibody conjugated to a liposome is often referred to as an “immunoliposome.”
  • the liposomal component of an immunoliposome is typically a lipid vesicle of one or more concentric phospholipid bilayers.
  • the phospholipids are composed of a hydrophilic head group and two hydrophobic chains to enable encapsulation of both hydrophobic and hydrophilic drugs.
  • Conventional liposomes are rapidly removed from the circulation via macrophages of the reticuloendothelial system (RES). To generate long-circulating liposomes, the composition, size and charge of the liposome can be modulated.
  • RES reticuloendothelial system
  • the surface of the liposome may also be modified, such as with a glycolipid or sialic acid.
  • a glycolipid or sialic acid for example, the inclusion of polyethylene glycol (PEG) significantly increases circulation half-life.
  • PEG polyethylene glycol
  • Niosomes are non-ionic surfactant-based vesicles having a structure similar to liposomes.
  • the membranes of niosomes are composed only of nonionic surfactants, such as polyglyceryl- alkyl ethers or /V-palmitoylgl ucosamine.
  • Niosomes range from small, unilamellar to large, multilamellar particles. These nanoparticles are monodisperse, water-soluble, chemically stable, have low toxicity, are biodegradable and non-immunogenic, and increase bioavailability of encapsulated drugs.
  • Dendrimers include a range of branched polymer complexes. These nanoparticles are water-soluble, biocompatible and are sufficiently non-immunogenic for human use. Generally, dendrimers consist of an initiator core, surrounded by a layer of a selected polymer that is grafted to the core, forming a branched macromolecular complex. Dendrimers are typically produced using polymers such as poly(amidoamine) or poly(L-lysine). Dendrimers have been used for a variety of therapeutic and diagnostic applications, including for the delivery of DNA, RNA, bioimaging contrast agents, chemotherapeutic agents and other drugs.
  • Polymeric micelles are composed of aggregates of amphiphilic co-polymers (consisting of both hydrophilic and hydrophobic monomer units) assembled into hydrophobic cores, surrounded by a corona of hydrophilic polymeric chains exposed to the aqueous environment.
  • the polymers used to prepare polymeric micelles are heterobifunctional copolymers composed of a hydrophilic block of PEG, poly(vinyl pyrrolidone) and hydrophobic poly(L-lactide) or poly(L- lysine) that forms the particle core.
  • Polymeric micelles can be used to carry drugs that have poor solubility. These nanoparticles have been used to encapsulate a number of drugs, including doxorubicin and camptothecin. Cationic micelles have also been developed to carry DNA or RNA molecules.
  • Nanospheres consist of a solid matrix of polymer, while nanocapsules contain an aqueous core.
  • the formulation selected typically depends on the solubility of the therapeutic agent to be carried/encapsulated; poorly water-soluble drugs are more readily encapsulated within nanospheres, while water-soluble and labile drugs, such as DNA and proteins, are more readily encapsulated within nanocapsules.
  • the polymers used to produce these nanoparticles include, for example, poly(acrylamide), poly(ester), poly(alkylcyanoacrylates), poly(lactic acid) (PLA), poly(glycolic acids) (PGA), and poly(D,L-lactic-co-glycolic acid) (PLGA).
  • Antibodies can be conjugated to a suitable nanoparticle according to standard known methods. For example, conjugation can be either covalent or non-covalent.
  • the nanoparticle is a liposome
  • the antibody is attached to a sterically stabilized, long circulation liposome via a PEG chain. Coupling of antibodies or antibody fragments to a liposome can also involve thioester bonds, for example by reaction of thiols and maleimide groups.
  • Crosslinking agents can be used to create sulfhydryl groups for attachment of antibodies to nanoparticles (Paszko and Senge, Curr Med Chem 19(31)5239-5277, 2012).
  • Nucleic acid molecules for example, DNA, cDNA, mRNA, or RNA molecules
  • Nucleic acid molecules encoding these molecules can readily be produced using the amino acid sequences provided herein (such as the CDR sequences and the variable domain sequences), sequences available (such as framework or constant region sequences), and the genetic code.
  • the nucleic acid molecules can be expressed in a host cell (such as a mammalian cell or a bacterial cell) to produce a disclosed antibody, fusion protein or antibody conjugate (e.g. , CAR, immunotoxin, multi-specific antibody).
  • nucleotide sequence of a nucleic acid molecule encoding a CD276- specific monoclonal antibody (or portion thereof) disclosed herein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 1-40.
  • nucleotide sequence of the nucleic acid molecule encoding a disclosed antibody (or portion thereof) includes or consists of any one of SEQ ID NOs: 1-40.
  • a nucleic acid molecule encoding a CD276-targeted CAR is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 48, 50, 52, 54, 56, 62 and 64, or a degenerate variant thereof.
  • the nucleotide sequence encoding the CAR includes or consists of any one of SEQ ID NOs: 48, 50, 52, 54, 56, 62 and 64, or a degenerate variant thereof
  • the genetic code can be used to construct a variety of functionally equivalent nucleic acid sequences, such as nucleic acids that differ in their sequence, but which encode the same antibody sequence, or encode a conjugate or fusion protein including the antibody sequence.
  • Nucleic acid molecules encoding the antibodies, fusion proteins, CARs and other conjugates that specifically bind to CD276 can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by standard methods. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
  • Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4 th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: lohn Wiley and Sons, including supplements).
  • Nucleic acids can also be prepared by amplification methods.
  • Amplification methods include the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcriptionbased amplification system (TAS), and the self-sustained sequence replication system (3SR).
  • the nucleic acid molecules can be expressed in a recombinantly engineered cell such as in bacterial, plant, yeast, insect and mammalian cells.
  • the antibodies and conjugates can be expressed as individual proteins including the antibody (linked to an effector molecule or detectable marker as needed) or can be expressed as a fusion protein. Any suitable method of expressing and purifying antibodies and antigen binding fragments may be used; non-limiting examples are provided in Al- Rubeai (Ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011).
  • One or more DNA sequences encoding the antibodies, CARs, fusion proteins, or other conjugates can be expressed in vitro by DNA transfer into a suitable host cell.
  • the cell may be prokaryotic or eukaryotic.
  • Numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells, for example mammalian cells, such as the COS, CHO, HeLa and myeloma cell lines, can be used to express the disclosed antibodies and antigen binding fragments.
  • Methods of stable transfer meaning that the foreign DNA is continuously maintained in the host, may be used.
  • nucleic acids encoding the antibodies, CARs and other conjugates described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette.
  • the promoter can be any promoter of interest, including a cytomegalovirus promoter.
  • an enhancer such as a cytomegalovirus enhancer, is included in the construct.
  • the cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences useful for regulation of the expression of the DNA encoding the protein.
  • the expression cassettes can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signals for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • the vector can encode a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).
  • expression cassettes can contain, for example, a strong promoter to direct transcription, a ribosome binding site for translational initiation (e.g., internal ribosomal binding sequences), and a transcription/translation terminator.
  • a strong promoter to direct transcription e.g., a ribosome binding site for translational initiation (e.g., internal ribosomal binding sequences), and a transcription/translation terminator.
  • this can include a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site, and a transcription termination signal.
  • control sequences can include a promoter and/or an enhancer derived from, for example, an immunoglobulin gene, HTLV, SV40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and/or acceptor sequences (for example, CMV and/or HTLV splice acceptor and donor sequences).
  • the cassettes can be transferred into the chosen host cell by any suitable method such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.
  • Modifications can be made to a nucleic acid encoding an antibody described herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the antibody into a fusion protein. Such modifications include, for example, termination codons, sequences to create conveniently located restriction sites, and sequences to add a methionine at the amino terminus to provide an initiation site, or additional amino acids (such as poly His) to aid in purification steps.
  • the antibodies, CARs, fusion proteins, and other conjugates can be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009).
  • the antibodies, CARs, fusion proteins, and other conjugates need not be 100% pure.
  • the antibodies should be substantially free of endotoxin.
  • compositions include one or more of the disclosed monoclonal antibodies that bind (for example specifically bind) CD276 in a carrier.
  • Compositions comprising fusion proteins (such as scFv-Fc fusion proteins), ADCs, CARs (and immune cells expressing CARs), multi- specific (such as bispecific or trispecific) antibodies, antibody -nanoparticle conjugates, immunoliposomes and immunoconjugates are also provided, as are nucleic acid molecule and vectors encoding the antibodies or antibody conjugates.
  • the compositions can be prepared in unit dosage form for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome.
  • the antibody, fusion protein, ADC, CAR, CAR-expressing cell, multi-specific antibody, antibody-nanoparticle conjugate, immunoliposome or immunoconjugate can be formulated for systemic or local administration.
  • the compositions for administration can include a solution of the antibody, fusion protein, ADC, CAR, CAR-expressing cell (such as a T cell, B cell, DC, NK cell, macrophage or iPSC), multi- specific (such as bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome or immunoconjugate in a pharmaceutically acceptable carrier, such as an aqueous carrier.
  • aqueous carriers can be used, for example, buffered saline and the like.
  • compositions may be sterilized by conventional sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject’s needs.
  • An exemplary pharmaceutical composition for intravenous administration includes about 0.1 to 10 mg of an antibody (or fusion protein, ADC, CAR, multi-specific antibody, antibody- nanoparticle conjugate, or immunoconjugate), per subject per day. Dosages from 0. 1 up to about 100 mg per subject per day may be used, particularly if the agent is administered to a secluded site and not into the circulatory or lymph system, such as into a body cavity or into a lumen of an organ.
  • the composition can be a liquid formulation including one or more antibodies in a concentration range from about 0.
  • 1 mg/ml to about 20 mg/ml or from about 0.5 mg/ml to about 20 mg/ml, or from about 1 mg/ml to about 20 mg/ml, or from about 0.1 mg/ml to about 10 mg/ml, or from about 0.5 mg/ml to about 10 mg/ml, or from about 1 mg/ml to about 10 mg/ml.
  • Actual methods for preparing administrable compositions will be known or apparent to a skilled person and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21 st Edition (2005).
  • Monoclonal antibodies may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration.
  • the antibody solution can be added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of from 0.5 to 15 mg/kg of body weight.
  • Antibodies, Fc fusion proteins, ADCs, CARs (or CAR-expressing cells), multi-specific (such as bispecific or trispecific) antibodies, antibody- nanoparticle conjugates, immunoliposomes or immunoconjugates can be administered by slow infusion, rather than in an intravenous push or bolus.
  • a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level.
  • an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30-minute period if the previous dose was well tolerated.
  • Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems.
  • Particulate systems include, for example, microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
  • Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle.
  • Particles, microspheres, and microcapsules smaller than about 1 pm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.
  • Capillaries have a diameter of approximately 5 pm so that only nanoparticles are administered intravenously.
  • Microparticles are typically around 100 pm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).
  • Polymers can be used for ion-controlled release of the antibody-based compositions disclosed herein.
  • Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known (Langer, Accounts Chem. Res. 26:537-542, 1993).
  • the block copolymer, poloxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It is an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990).
  • hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al. , Int. J. Pharm. 112:215-224, 1994).
  • liposomes are used for controlled release as well as drug targeting of the lipid- capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent Nos.
  • the antibodies, compositions, CARs (and CAR-expressing immune cells or iPSCs), ADCs, multi- specific (such as bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes and immunoconjugates disclosed herein can be administered to slow or inhibit the growth of tumor cells or inhibit the metastasis of tumor cells, such as a CD276-positive solid tumor.
  • a therapeutically effective amount of a composition is administered to a subject in an amount sufficient to inhibit growth, replication or metastasis of cancer cells, or to inhibit a sign or a symptom of the cancer.
  • Suitable subjects may include those diagnosed with a solid tumor that expresses CD276, such as, but not limited to, pancreatic cancer, neuroblastoma, liver cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer, pediatric cancer, melanoma or mesothelioma.
  • a solid tumor that expresses CD276, such as, but not limited to, pancreatic cancer, neuroblastoma, liver cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer, pediatric cancer, melanoma or mesothelioma.
  • a method of treating a CD276-positive cancer in a subject by administering to the subject a therapeutically effective amount of a CD276-specific monoclonal antibody, immunoconjugate, CAR (or an immune cell or iPSC expressing a CAR), ADC, multispecific (such as bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome or composition disclosed herein.
  • Also provided herein is a method of inhibiting tumor growth or metastasis of a CD276-positive cancer in a subject by administering to the subject a therapeutically effective amount of a CD276-specific monoclonal antibody, immunoconjugate, CAR (such as an immune cell or iPSC expressing a CAR), ADC, multi- specific (such as bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome or composition disclosed herein.
  • a CD276-specific monoclonal antibody such as an immune cell or iPSC expressing a CAR
  • ADC multi- specific antibody
  • antibody-nanoparticle conjugate such as bispecific or trispecific
  • the CD276-positive cancer is a solid tumor, such as pancreatic cancer, neuroblastoma, liver cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer, pediatric cancer, melanoma or mesothelioma.
  • the tumor does not need to be completely eliminated or inhibited for the method to be effective.
  • the method can decrease tumor size (e.g., volume) or metastasis by a particular amount, for example by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100% as compared to the absence of the treatment.
  • the method can decrease tumor size (e.g., volume) or metastasis by a particular amount, for example by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100% as compared to treatment with a different CD276 antibody or CD276 CAR (e.g., one not disclosed herein).
  • tumor size e.g., volume
  • metastasis by a particular amount, for example by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100% as compared to treatment with a different CD276 antibody or CD276 CAR (e.g., one not disclosed herein).
  • the method includes administering to the subject a therapeutically effective amount of an isolated cell expressing a CD276-targeted CAR disclosed herein.
  • the isolated cell is an immune cell, such as a T cell, B cell, NK cell, macrophage, DC, or an iPSC.
  • a therapeutically effective amount of a CD276-specific monoclonal antibody, ADC, CAR (for example an immune cell or iPSC expressing a CAR), multi- specific (such as bispecific or trispecific) antibody, immunoconjugate, immunoliposome or composition disclosed herein will depend upon the severity of the disease, the type of disease, and the general state of the patient’s health.
  • a therapeutically effective amount of the antibody-based composition is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • Antibodies and conjugates thereof can be administered, for example, by intravenous infusion.
  • Doses of the antibody or conjugate thereof can vary, but generally range between about 0.5 mg/kg to about 50 mg/kg, such as a dose of about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg.
  • the dose of the antibody or conjugate can be from about 0.5 mg/kg to about 5 mg/kg, such as a dose of about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg or about 5 mg/kg.
  • the dose of CAR-expressing immune cells or iPSCs is 10 4 to 10 9 cells/kg body weight, such as 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges.
  • Exemplary doses are 10 6 cells/kg to about 10 8 cells/kg, such as from about 5 x 10 6 cells/kg to about 7.5 x 10 7 cells/kg, such as at about 2.5 x 10 7 cells/kg, or at about 5.0 x 10 7 cells/kg.
  • the antibody or conjugate is administered according to a dosing schedule determined by a medical practitioner. In some examples, the antibody or conjugate is administered weekly, every two weeks, every three weeks, or every four weeks.
  • a subject is administered DNA or RNA encoding a disclosed antibody to provide in vivo antibody production, for example using the cellular machinery of the subject.
  • Any suitable method of nucleic acid administration may be used; non-limiting examples are provided in U.S. Patent No. 5,643,578, U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637.
  • U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding proteins to an organism.
  • One approach to administration of nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid.
  • the nucleotide sequence encoding the disclosed antibody, or antigen binding fragments thereof can be placed under the control of a promoter to increase expression.
  • the methods include liposomal delivery of the nucleic acids. Such methods can be applied to the production of an antibody, or antigen binding fragments thereof.
  • a subject (such as a human subject with a CD276-positive tumor) is administered an effective amount of a viral vector that includes one or more nucleic acid molecules encoding a disclosed antibody.
  • the viral vector is designed for expression of the nucleic acid molecules encoding a disclosed antibody, and administration of the effective amount of the viral vector to the subject leads to expression of an effective amount of the antibody in the subject.
  • Nonlimiting examples of viral vectors that can be used to express a disclosed antibody or antigen binding fragment in a subject include those provided in Johnson et al., Nat. Med., 15(8):901 -906, 2009 and Gardner er al., Nature, 519(7541):87-91, 2015.
  • a nucleic acid encoding a disclosed antibody, or conjugate thereof is introduced directly into tissue.
  • the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOSTM Gene Gun.
  • the nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter.
  • the DNA is injected into muscle, although it can also be injected directly into other sites.
  • Dosages for injection are usually around 0.5 pg/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Patent No. 5,589,466).
  • Single or multiple administrations of a composition including a disclosed antibody or antibody conjugate, or nucleic acid molecule encoding such molecules can be administered depending on the dosage and frequency as required and tolerated by the patient.
  • the dosage can be administered once but may be applied periodically until either a desired result is achieved or until side effects warrant discontinuation of therapy. Generally, the dose is sufficient to inhibit growth or metastasis of a CD276-positive cancer without producing unacceptable toxicity to the patient.
  • the dosage normally lies within a range of circulating concentrations that include the EDso, with little or minimal toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the CD276-specific antibody, antibody conjugate, nucleic acid molecule encoding such molecules, or a composition including such molecules can be administered to subjects in various ways, including local and systemic administration, such as, e.g., by injection subcutaneously, intravenously, intra-arterially, intraperitoneally, intramuscularly, intradermally, or intrathecally.
  • the composition is administered by inhalation, such as by using an inhaler.
  • the antibody, antigen binding fragment, or nucleic acid molecule encoding such molecules, or a composition including such molecules is administered by a single subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal or intrathecal injection once a day.
  • the antibody, antigen binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules can also be administered by direct injection at or near the site of disease.
  • a further method of administration is by osmotic pump (e.g., an ALZET pump) or mini-pump (e.g., an ALZET mini-osmotic pump), which allows for controlled, continuous and/or slow-release delivery of the antibody, antibody conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules, over a pre-determined period.
  • the osmotic pump or mini-pump can be implanted subcutaneously, or near a target site.
  • a CD276-specific antibody provided herein is conjugated to IR700, and photoimmunotherapy is used to treat a CD276-positive cancer.
  • a method can include administering to the subject with a CD276-positive cancer a therapeutically effective amount of one or more CD276-specific antibody-IR700 conjugates, wherein the CD276-specific antibody specifically binds to CD276-expressing cells.
  • irradiation is performed at a wavelength of 660 to 740 nm (such as 660 to 710 nm, for example, 680 nm) and at a dose of at least 1 J cm' 2 (such as at least 1 to about 50 J cm' 2 ) thereby treating the CD276-positive cancer in the subject.
  • the CD276-positive tumor is irradiated at a wavelength of 660 to 740 nm (such as 660 to 710 nm, for example, 680 nm) at a dose of at least 1 J cm' 2 (such as at least 1 J cm' 2 , at least 4 J cm' 2, at least 10 J cm' 2 , at least 50 J cm' 2 , or at least 100 J cm' 2 ) thereby treating the cancer in the subject.
  • multiple rounds of treatment are performed, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 treatment cycles.
  • a therapeutically effective dose of a CD276-specific antibody-IR700 conjugates is at least 0.5 milligram per 60 kilogram (mg/kg), at least 5 mg/60 kg, at least 10 mg/60 kg, at least 20 mg/60 kg, at least 30 mg/60 kg, at least 50 mg/60 kg, for example 0.5 to 50 mg/60 kg, such as a dose of 1 mg/ 60 kg, 2 mg/60 kg, 5 mg/60 kg, 20 mg/60 kg, or 50 mg/60 kg, for example when administered intravenously.
  • a therapeutically effective dose of a CD276- specific antibody- IR700 conjugates is at least 10 pg/kg, such as at least 100 pg/kg, at least 500 pg/kg, or at least 500 pg/kg, for example 10 pg/kg to 1000 pg/kg, such as a dose of 100 pg/kg, 250 pg/kg, about 500 pg/kg, 750 pg/kg, or 1000 pg/kg, for example when administered i.p.
  • a therapeutically effective dose of an CD276- specific antibody-IR700 conjugates is at least 1 pg/ml, such as at least 500 pg/ml, such as between 20 pg/ml to 100 pg/ml, such as 10 pg/ml, 20 pg/ml, 30 pg/ml, 40 pg/ml, 50 pg/ml, 60 pg/ml, 70 pg/ml, 80 pg/ml, 90 pg/ml or 100 pg/ml administered in a topical solution.
  • the treatment methods further include administration of other anti-cancer agents or therapeutic treatments.
  • anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g. anti-androgens) and anti-angiogenesis agents.
  • chemotherapeutic agents such as, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g. anti-androgens) and anti-angiogenesis agents.
  • Other anti-cancer treatments include radiation therapy and other antibodies that specifically target cancer cells.
  • alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).
  • nitrogen mustards such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil
  • alkyl sulfonates such as busulfan
  • nitrosoureas such as carmustine, lomustine, semustine, streptozocin, or dacarbazine.
  • Non-limiting examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.
  • folic acid analogs such as methotrexate
  • pyrimidine analogs such as 5-FU or cytarabine
  • purine analogs such as mercaptopurine or thioguanine.
  • Non-limiting examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L- asparaginase).
  • vinca alkaloids such as vinblastine, vincristine, or vindesine
  • epipodophyllotoxins such as etoposide or teniposide
  • antibiotics such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C
  • enzymes such as L- asparaginase
  • miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide).
  • platinum coordination complexes such as cis-diamine-dichloroplatinum II also known as cisplatin
  • substituted ureas such as hydroxyurea
  • methyl hydrazine derivatives such as procarbazine
  • adrenocrotical suppressants such as mitotane and aminoglutethimide
  • hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testosterone propionate and fluoxymesterone).
  • adrenocorticosteroids such as prednisone
  • progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate
  • estrogens such as diethylstilbestrol and ethinyl estradiol
  • antiestrogens such as tamoxifen
  • androgens such as testosterone propionate and fluoxymesterone
  • Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP- 16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol.
  • Non- limiting examples of immunomodulators that can be used include AS- 101 (Wyeth- Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Jersey, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).
  • Exemplary biologies that can be used in combination with the disclosed methods include one or more monoclonal antibodies (mAbs) used to treat cancer, such as mAbs specific for EGFR (e.g., cetuximab), VEGF (e.g., bevacizumab), PD-1 (e.g., nivolumab, JTX-4014 by Jounce Therapeutics, nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IB 1308), tislelizumab (BGB-A317), toripalimab (JS 001, dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, or AMP-514), CD276 (e.g., atezolizumab, avelumab
  • the additional therapeutic agent administered is an anti-cancer monoclonal antibody, for example one or more of: 3F8, Abagovomab, Adecatumumab, Afutuzumab, Alacizumab , Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Apolizumab, Arcitumomab, Bavituximab, Bectumomab, Belimumab, Besilesomab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Capromab pendetide, Catumaxomab, CC49, Cetuximab, Citatuzumab communicatingox, Cixutumumab, Clivatuzumab tetraxetan,
  • the methods further include surgical treatment, for example surgical resection of the cancer or a portion of it.
  • the methods further include administration of radiotherapy, for example administration of radioactive material or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical resection.
  • the disclosed monoclonal antibodies can be used for in vivo imaging to detect a CD276-positive cancer.
  • the antibodies are labelled with a detectable moiety, such as a radioisotope, fluorescent label, or positron emitting radionuclides.
  • a detectable moiety such as a radioisotope, fluorescent label, or positron emitting radionuclides.
  • the monoclonal antibodies (or antigen-binding fragments thereof) disclosed herein can be conjugated to a positron emitting radionuclide for use in positron emission tomography (PET); this diagnostic process is often referred to as immuno PET.
  • PET positron emission tomography
  • the antibodies can be directly or indirectly labelled with a detectable moiety (e.g., by using a labeled secondary antibody that binds to the CD276 antibody), such as a radioisotope, enzyme, or fluorescent label.
  • a detectable moiety e.g., by using a labeled secondary antibody that binds to the CD276 antibody
  • a radioisotope, enzyme, or fluorescent label e.g., a labeled secondary antibody that binds to the CD276 antibody
  • the presence of CD276 is detected in a biological sample from a subject and can be used to identify a subject with a CD276-positive cancer.
  • the sample can be any sample, including, but not limited to, blood, serum, urine, semen, sputum, saliva, mucus, nasal wash, nasopharyngeal samples, oropharyngeal samples, tissue, cells, tissue biopsy, fine needle aspirate, surgical specimen, feces, cerebral spinal fluid (CSF), and bronchoalveolar lavage (BAL) fluid.
  • Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes.
  • the method of detection can include contacting a cell or sample, with an antibody or antibody conjugate (e.g., a conjugate including a detectable marker) that specifically binds to CD276 under conditions sufficient to form an immune complex, and detecting the immune complex (e.g., by detecting a detectable marker conjugated to the antibody or antigen binding fragment).
  • an antibody or antibody conjugate e.g., a conjugate including a detectable marker
  • detecting the immune complex e.g., by detecting a detectable marker conjugated to the antibody or antigen binding fragment.
  • a method of determining if a subject has a CD276-positive cancer by contacting a sample from the subject with a CD276- specific monoclonal antibody disclosed herein; and detecting binding of the monoclonal antibody to the sample.
  • An increase in binding of the monoclonal antibody to the sample as compared to binding of the monoclonal antibody to a control sample identifies the subject as having a CD276-positive cancer
  • a method of confirming a diagnosis of a CD276-positive cancer in a subject by contacting a sample from a subject diagnosed with a CD276-positive cancer with a CD276 monoclonal antibody disclosed herein; and detecting binding of the monoclonal antibody to the sample.
  • An increase in binding of the monoclonal antibody to the sample as compared to binding of the monoclonal antibody to a control sample confirms the diagnosis of a CD276-positive cancer in the subject.
  • the antibody or antigen binding fragment is directly labeled with a detectable marker.
  • the antibody that binds CD276 (the primary antibody) is unlabeled and a secondary antibody or other molecule that can bind the primary antibody is utilized for detection.
  • the secondary antibody that is chosen is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a human IgG, then the secondary antibody may be an anti-human-IgG.
  • Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.
  • Suitable labels for the antibody or secondary antibody include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin.
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
  • a non-limiting exemplary luminescent material is luminol; a non-limiting exemplary a magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125 I, 131 1, 35 S or 3 H.
  • CD276 can be assayed in a biological sample by a competition immunoassay utilizing CD276 standards labeled with a detectable substance and an unlabeled antibody that specifically binds CD276.
  • a competition immunoassay utilizing CD276 standards labeled with a detectable substance and an unlabeled antibody that specifically binds CD276.
  • the biological sample, the labeled CD276 standards and the antibody that specifically binds CD276 are combined and the amount of labeled CD276 standard bound to the unlabeled antibody is determined.
  • the amount of CD276 in the biological sample is inversely proportional to the amount of labeled CD276 standard bound to the antibody that specifically binds CD276.
  • the immunoassays and methods disclosed herein can be used for a number of purposes.
  • the antibody that specifically binds CD276 may be used to detect the production of CD276 in cells in cell culture.
  • the antibody can be used to detect the amount of CD276 in a biological sample, such as a sample obtained from a subject having or suspected or having a CD276-positive cancer.
  • kits for detecting CD276 in a biological sample can include a monoclonal antibody that specifically binds CD276, such as monoclonal antibody disclosed herein.
  • the monoclonal antibody is labeled (for example, with a fluorescent, radioactive, or an enzymatic label).
  • the antibody is present on a solid support, such as a bead or multi-well plate.
  • the kit further includes a detectably labeled secondary antibody that permits detection of the antibody that specifically binds CD276.
  • kits in one aspect, includes instructional materials disclosing means of use of an antibody that binds CD276.
  • the instructional materials may be written, in an electronic form or may be visual (such as video files).
  • the kits may also include additional components to facilitate the particular application for which the kit is designed.
  • the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like).
  • the kits may additionally include buffers and other reagents routinely used for the practice of a particular method.
  • the kits may additionally include materials to obtain a sample, such as a swab, syringe, needle, and the like. Such kits and appropriate contents are well known.
  • the diagnostic kit comprises an immunoassay.
  • the method of detecting CD276 in a biological sample generally includes the steps of contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to CD276.
  • the antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.
  • the monoclonal antibodies disclosed herein can also be utilized in immunoassays, such as, but not limited to radioimmunoassays (RIAs), ELISA, lateral flow assay (LFA), or immunohistochemical assays.
  • the antibodies can also be used for fluorescence activated cell sorting (FACS), such as for identifying/detecting CD276-positive cells.
  • FACS employs a plurality of color channels, low angle and obtuse light-scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells (see U.S. Patent No. 5,061,620). Any of the monoclonal antibodies that bind CD276, as disclosed herein, can be used in these assays.
  • the monoclonal antibodies can be used in a conventional immunoassay, including, without limitation, ELISA, RIA, LFA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation.
  • the disclosed antibodies can also be used in nanotechnology methods, such as microfluidic immunoassays, which can be used to capture CD276, or exosomes containing CD276.
  • Suitable samples for use with a microfluidic immunoassay or other nanotechnology method include but are not limited to, saliva, blood, and fecal samples. Microfluidic immunoassays are described in U.S. Patent Application No.
  • Example 1 Fully-human CD276 antibodies bind to CD276 on the tumor cell surface
  • Flow cytometry was used to assess binding of the CD276 antibodies to human pancreatic cancer cell line Panel. All five anti-CD276 antibodies were produced in scFv-Fc format and purified for the binding assay. Serially diluted antibodies at concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 pg/mL were incubated with Panel cells, followed by Alexa-647 labeled antihuman Fab secondary antibody. A previously described anti-CD276 antibody (m276) in scFv format was included as positive control and a non-binding scFv was used as a negative control. As shown in FIG. 1, all five antibodies exhibited significant binding to CD276-positive Panel cells.
  • the fully-human CD276 antibodies (Yll l, Y422, Y868, Y117 and YE5) were used to construct chimeric antigen receptors (CARs).
  • CARs chimeric antigen receptors
  • FIG. 2A A schematic of the overall structure of the CARs is shown in FIG. 2A.
  • the CARs include an antibody single-chain variable fragment (scFv), CD28 derived hinge and transmembrane domains, and 4 IBB and CD3 zeta (CD3Q cytoplasmic signaling domains.
  • the scFv in all five CARs generated are in the VH-VL orientation.
  • the CD276 specificity of each antibody is listed in FIG. 2B.
  • T cells were retrovirally transduced with nucleic acid encoding the CARs.
  • Transduced T cells were incubated with rCD276-Bio (biotin-tagged recombinant CD276 ectodomain), followed by APC labeled streptavidin. Flow cytometry was used to detect CAR expression on the T cell surface. Untransduced (UTD) cells were used as negative controls. Histograms of CAR expression in T cells from a representative donor are shown in FIG. 3 A. A summary of the binding data for four independent donors is shown in FIG. 3B. The results showed that Y868, Yl l l and Y117 based CARs had the greatest expression levels.
  • T cells were maintained in T cell media (1:1 mix of RPMI-1640 and Click’s media containing 10% FBS and 1% Glutamax) supplemented with recombinant human IL-2 (50 lU/mL). IL-2 was added every 2-3 days. Cells were collected, counted, and viability was measured on day 11 after transduction. Counting and viability measurements were performed using a Countess II automated cell counter. Fold expansion was calculated by dividing day 11 total cell numbers by starting cell number on the day of transduction. Fold T cell expansion at day 11 post-transduction is shown in FIG. 4A. T cell viability at day 11 post-transduction is shown in FIG. 4B. These results demonstrated that expression of the CD276-targeted CARs did not significantly alter T cell expansion or viability.
  • FIGS. 5A-5G show percent killing in CD276 knockout (KO) NBEB cells (FIG. 5A), neuroblastoma cell line NBEB (FIG. 5B), neuroblastoma cell line LAN5 (FIG. 5C) neuroblastoma cell line IMR5 (FIG. 5D), pancreatic cancer cell line Panel (FIG. 5E), pancreatic cancer cell line HP AC (FIG. 5F) and pancreatic cancer cell line MiaPaca (FIG. 5G).
  • the results show that the CD276-targeted CAR T cells effectively induced cytotoxicity of CD276-positive pancreatic and neuroblastoma tumor cells.
  • Example 4 CD276-targeted CAR T cells in a pancreatic (Panel) tumor model
  • mice Five million CAR+ T cells were injected intravenously in the tail vein. Tumor burden was monitored by imaging weekly for seven weeks (D7, D16, D21, D28, D35, D42 and D49) and then at specified time points later in this study (D63, D91, DI 12, D156 and D296). Body weight measurements were also taken at the time of imaging on a weekly basis. To image the tumor, 100 mL of 15 mg/mL D-luciferin was injected intraperitonially, and imaging was performed after 10 minutes. The results are shown in FIG. 7. Mice treated with the Y111-based CAR exhibited the greatest reduction in tumor size. At the conclusion of the study (day 296), no tumors had relapsed in mice treated with the Y111 CAR.
  • Example 5 CD276-targeted CAR T cells in a pancreatic (HPAC) tumor model
  • HPAC pancreatic tumor model Anti-tumor activity of CD276 CAR T cells in an orthotopic HPAC pancreatic tumor model was evaluated.
  • Firefly-luciferase labeled HPAC cells (250,000) were injected into the pancreas of 6-8-week-old female NSG mice (FIG. 8A).
  • mice were imaged using IVIS Spectrum to measure tumor burden and grouped into different treatment groups as shown in FIG. 8B.
  • mice were treated with CARs containing Y422, Y868, Y 111 , Y117 or YES scFv.
  • a CAR containing anti-CD276 scFv MGA271 was also included in this study for comparison.
  • Five million CAR+ T cells were injected intravenously in the tail vein. Tumor burden was monitored by imaging weekly for seven weeks (D7, D14, D21, D28, D35 and D42). Body weight measurements were also taken at the time of imaging on a weekly basis (FIG. 8C).
  • FIG. 9A Tumor luminescence was quantified weekly for seven weeks (FIG. 9B), followed by specified time points later in the study (up to D255; FIG. 10A).
  • FIG. 10B shows tumor luminescence in individual mice for all groups. The results demonstrate that Y111-based CAR T cells were the most effective at eliminating the pancreatic tumors. All treated mice maintained normal weight throughout the study.
  • Example 6 Activated CAR T cells product cytolytic proteins
  • This example describes production of cytolytic proteins by CAR T cells co-cultured with CD276 + Panel tumor cells.
  • Un-transduced (UTD) T cells, and T cells expressing a CD19 CAR or one of three CD276-specific CARs (Y868, Yll l, and Y117) were co-cultured with Panel pancreatic cancer cells for 16 hours at an effector-to-target ratio (E:T) of 1:2.
  • E:T effector-to-target ratio
  • T cells were then collected and labeled intracellularly with antibodies against granzyme B, perforin, and CD107a.
  • E:T effector-to-target ratio
  • Y868, Yl ll and Y117-based CAR T cells produced higher levels of perforin, granzyme B and CD107a than UTD and CD19 CAR T cells.
  • Example 7 Anti-tumor activity of CD276 CAR T cells in a metastatic neuroblastoma (IMR5) model
  • This example compares the anti-tumor activity of Y868, Yl l l and Y117 CAR T cells to T cells expressing a CAR containing mouse-derived anti-human CD276 monoclonal antibody 376.96 (see Li et al., Nat Commun 14:5920, 2023).
  • Firefly-luciferase labeled IMR5 cells (IxlO 6 ) were injected into 6-8-week-old female NSG mice via tail vein (Day -35). Five weeks later, mice were imaged using IVIS Spectrum to measure tumor burden and grouped into different treatment groups. Two million Y868, Yll 1, YI 17 or 376.96 CAR T cells were injected intravenously in the tail vein (Day 0) (FIG. 12A). Tumor burden was monitored by imaging on Days -1, 7, 14, 21, 29, 40, 49 and 60. To image the tumor, 100 pl of 15 mg/mL D-luciferin was injected intraperitonially and imaging was done after 10 minutes. Body weight measurements were also taken at the time of imaging. As shown in FIGS. 12B-12C, T cells expressing the Y 111 -based CAR significantly reduced tumor burden in treated mice and were more effective than the other CD276-targeted CARs. No significant changes in body weight were observed.
  • Example 8 Long-term persistence and anti-tumor activity of Ylll CAR in the HPAC orthotopic pancreatic tumor model
  • mice from the HPAC study were rechallenged with 4xl0 6 fireflyluciferase labeled HPAC cells injected subcutaneously in the lower right abdomen. Age matched mice that had never received tumor cells or CAR T cells were used as controls and were similarly injected with the tumor cells. Mice were imaged using IVIS Spectrum to measure tumor burden on Days -7, 8, 46, 72, 101, 123, 143 and 182 after CAR T cell treatment. As shown in FIGS. 13A- 13B, treatment with Y111 CAR T cells significantly reduced tumor burden in treated mice and the effect of treatment persisted throughout the study period (444 days after CAR T cell treatment).
  • Y 111 CAR Y 111 CAR
  • two variants were evaluated - one with the original 4- IBB costimulatory domain (Yl l l-HL-28HTM-BBz, SEQ ID NO: 47), and the other with a CD28 costimulatory domain (Yll l-HL-28HTM-28z SEQ ID NO: 61).
  • the two Yl l 1-based CAR constructs were also compared to two CAR constructs having a camel-derived single domain (VHH) CD276- specific antibody (B 12; described in Li et al., Nat Commun 14:5920, 2023 and WO 2021/081052).
  • VHH camel-derived single domain
  • the B 12-based CARs include a CD28 HTM, a CD3 ⁇ signaling domain and either a 4- IBB costimulatory domain (B12-VHH-28HTM-BBz) or a CD28 costimulatory domain (B12-VHH-28HTM-28z). All four constructs were compared in a pancreatic tumor model (Pane 1).
  • Firefly-luciferase labeled Panel cells (2.5xl0 5 ) were injected into the pancreas of 6-8- week-old female NSG mice (Day -14). Two weeks later, mice were imaged using IVIS Spectrum to measure tumor burden and grouped into the following four treatment groups: Yl l 1-HL-28HTM- BBz CAR T cells; Y1 ll-HL-28HTM-28z CAR T cells; B 12- VHH-28HTM-BBz CAR T cells; and B12-VHH-28HTM-28z CAR T cells. Five million CAR+ T cells were injected intravenously in the tail vein (Day 0). Tumor burden and body weight were monitored weekly (FIG. 14A).
  • SPR Surface plasmon resonance
  • Yl ll full IgG was also tested in an SPR assay to reflect multiple copies of scFv molecules on the surface of CAR T cells.
  • Human CD276-ECD-AP fusion protein was immobilized on a CM5 chip.
  • Yl l l IgG was tested at concentrations of 0.08, 0.4, 2, 10 and 50 nM. Data were fitted with 1:2 model to calculate the KD. The results showed that Yll 1 IgG binds human CD276 with an affinity of 21 nM.

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Abstract

CD276 (also known as B7H3) is highly expressed in the stroma of most solid tumors and is also frequently overexpressed by tumor cells, making CD276 an important target for anti-cancer therapy. Fully human monoclonal antibodies that bind CD276 are described. Chimeric antigen receptors (CARs) generated using the CD276 antibodies are also described. The monoclonal antibodies, CARs and other antibody conjugates can be used for the detection and treatment of CD276-positive cancers.

Description

FULLY HUMAN MONOCLONAL ANTIBODIES AND CHIMERIC ANTIGEN RECEPTORS AGAINST CD276 FOR THE TREATMENT OF SOLID TUMORS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 63/514,596, filed July 20, 2023, which is herein incorporated by reference in its entirety.
FIELD
This disclosure concerns human monoclonal antibodies that specifically bind CD276 and conjugates, such as chimeric antigen receptors (CARs), based on these antibodies. The disclosure further concerns use of the monoclonal antibodies and conjugates, such as in the treatment of solid tumors.
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
This invention was made with government support under ZIA BC010578 awarded by the National Institutes of Health. The government has certain rights in the invention.
INCORPORATION OF ELECTRONIC SEQUENCE LISTING
The electronic sequence listing, submitted herewith as an XML file named 4239-110066- O2.xml (80,868 bytes), created on June 9, 2024, is herein incorporated by reference in its entirety.
BACKGROUND
CD276 (also known as B7-H3) is a single pass transmembrane protein that is highly expressed in the stroma of most solid tumors and is also frequently overexpressed by the tumor cells themselves. Chimeric antigen receptors (CARs) targeting human CD276 have been previously evaluated in preclinical studies resulting in the initiation of several clinical trials. However, the CARs currently being tested in early phase trials were constructed using humanized forms of mouse derived anti-CD276 antibodies. The process of humanization can impact antibody function and may not completely eliminate immunogenicity, subjecting T cells engineered to express mouse antibody-derived CARs to elimination by the endogenous immune system. Thus, a need exists for the development of improved CD276-targeted CARs, such as those incorporating fully human monoclonal antibodies. SUMMARY
Disclosed herein are fully human CD276- specific antibodies isolated from human antibody libraries. The disclosed antibodies were used to develop chimeric antigen receptors (CARs) containing 100% human amino acid sequences, thereby eliminating the need for humanization. The CD276- targeted CARs disclosed herein induce cell killing of several tumor cell lines, including pancreatic and neuroblastoma tumor cell lines. CARs generated using antibody Y111 induced therapeutically effective anti-tumor responses in orthotopic pancreatic tumor and metastatic neuroblastoma models, and can produce better clinical responses in human solid tumor patients compared to CARs currently being tested in human clinical trials.
Provided herein are monoclonal antibodies that specifically bind CD276. The CD276- specific antibodies include the complementarity determining region (CDR) sequences (or the complete variable domains) of any one of antibodies Y111 , Y 422, Y868, Y117 and YE5 as disclosed herein.
Also provided herein are conjugates that include a disclosed monoclonal antibody (or the CDR sequences of a disclosed monoclonal antibody). In some aspects, provided are CARs, CAR- expressing cells (such as T cells, B cells, natural killer (NK) cells, dendritic cells (DCs), macrophages and induced pluripotent stem cells (iPSCs)), fusion proteins (such as Fc fusion proteins), immunoconjugates (such as immuno toxins), multi-specific antibodies (such as bispecific or trispecific antibodies), antibody-drug conjugates (ADCs), antibody-nanoparticle conjugates, and antibody-photon absorber conjugates (such as IR700 conjugates for immunoPET imaging) that include a monoclonal antibody disclosed herein.
Further provided are nucleic acid molecules encoding a disclosed monoclonal antibody, CAR, immunoconjugate, multi- specific antibody, or fusion protein. In some aspects, the nucleic acid molecule is part of a vector. Also provided are isolated cells that include a nucleic acid molecule or vector disclosed herein.
Also provided are compositions that include a pharmaceutically acceptable carrier and a CD276- specific monoclonal antibody, CAR, CAR-expressing cell, immunoconjugate, ADC, multispecific antibody, antibody-nanoparticle conjugate, or fusion protein disclosed herein. In some examples, the composition is lyophilized.
Methods of detecting expression of CD276 in a sample are further provided. In some aspects, the method includes contacting the sample with a CD276-specific monoclonal antibody disclosed herein and detecting binding of the monoclonal antibody to the sample. Methods of diagnosing a subject as having a CD276-positive tumor are further provided. In some aspects, the method includes contacting a sample obtained from the subject with a CD276-specific monoclonal antibody disclosed herein and detecting binding of the monoclonal antibody to the sample.
Also provided are methods of treating a CD276-positive cancer in a subject, and methods of inhibiting tumor growth or metastasis of a CD276-positive cancer in a subject. In some aspects, the methods include administering to the subject a monoclonal antibody, CAR, CAR-expressing cell, immunoconjugate, ADC, multi- specific antibody, antibody-nanoparticle conjugate, fusion protein, or composition disclosed herein. In some aspects, the CD276-positive cancer is a solid tumor, such as, but not limited to, a pancreatic cancer, a neuroblastoma, a liver cancer, a kidney cancer, a bladder cancer, a cervical cancer, an esophageal cancer, a prostate cancer, a breast cancer, an ovarian cancer, a colon cancer, a lung cancer, a brain cancer, a pediatric cancer, a melanoma or a mesothelioma.
Further provided herein are kits that include a monoclonal antibody, CAR, CAR-expressing cell, immunoconjugate, ADC, multi-specific antibody, antibody-nanoparticle conjugate, fusion protein, or composition disclosed herein; and a pharmaceutically acceptable carrier, buffer, cell culture media, cell culture plates or flasks, a solid support, a fluorescent label, a radioactive label, an enzymatic label, an enzymatic substrate, a secondary antibody, one or more check point inhibitors, one or more additional anti-cancer agents, one or more transfection reagents, and/or instructional materials.
The foregoing and other features of this disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Fully-human CD276 antibodies bind to CD276 on the tumor cell surface. Flow cytometry was used to assess the binding of CD276 antibodies to human pancreatic cancer cell line Panel. Five anti-CD276 antibodies (Y422, Y868, Yl l l, Y117 and YE5) were produced in scFv- Fc format and purified for the binding assay. Serially diluted antibodies at the indicated concentrations were incubated with Panel cells, followed by Alexa-647 labeled anti-human Fab secondary antibody. A previously described anti-CD276 antibody (m276) in scFv format was included as positive control and a non-binding scFv was used as a negative control.
FIGS. 2A-2B: Fully-human CD276 antibodies used to construct chimeric antigen receptors (CARs). (FIG. 2A) Schematic of the overall structure of the CARs. The CARs include an antibody single-chain variable fragment (scFv), CD28 derived hinge and transmembrane domains, and 4 IBB and CD3 zeta (CD3Q cytoplasmic signaling domains. The Y111 CAR was also made with a CD28 costimulatory domain. The scFv in all five CARs generated are in the VH-VL orientation. The Y111 CAR was also made in a VL-VH orientation for comparison. (FIG. 2B) Table listing the anti-CD276 antibodies used for construction of the CARs and their target specificity.
FIGS. 3A-3B: Transduced T cells express CD276 CARs. Retrovirally transduced T cells were incubated with rCD276-Bio (biotin-tagged recombinant CD276 ectodomain), followed by allophycocyanin (APC)-labeled streptavidin. Flow cytometry was used to detect CAR expression on the T cell surface. Untransduced (UTD) cells were used as negative controls. (FIG. 3A) Histograms showing CAR expression in T cells from a representative donor. (FIG. 3B) Graph displaying average summary data for four independent donors.
FIGS. 4A-4B: Expansion and viability of CAR transduced T cells. T cells were maintained in T cell media (1 :1 mix of RPMI-1640 and Click’s media containing 10% FBS and 1% Glutamax) supplemented with recombinant human IL-2 (50 lU/mL). IL-2 was added every 2-3 days. Cells were collected, counted, and viability was measured on day 11 after transduction. Counting and viability measurements were performed using a Countess II automated cell counter. Fold expansion was calculated by dividing day 11 total cell numbers by the starting cell number on the day of transduction. (FIG. 4A) Fold T cell expansion at day 11 post-transduction. (FIG. 4B) T cell viability at day 11 post-transduction.
FIGS. 5A-5G: Anti-tumor activity of CD276 CAR T cells against pancreatic cancer and neuroblastoma tumor cells. Firefly-lucif erase labeled tumor cells (10,000 cells) were seeded in opaque white 96-well plates in 100 (tL T cell media. Four hours later, T cells resuspended in 100 pL T cell media were added at the indicated CAR+ T cell effector to target (E:T) ratios. After 48 hours, 10 pL D-Luciferin was added to each well such that the final concentration was 15 mg/mL. Plates were incubated at 37°C for 5 minutes after which signal intensity was captured using a Clariostar plate reader. Percent killing was calculated as 100x[l-(sample luminescence)/(UTD luminescence)]. Shown is % killing in CD276 knockout (KO) NBEB cells (FIG. 5 A), neuroblastoma cell lines NBEB (FIG. 5B), LAN5 (FIG. 5C) and IMR5 (FIG. 5D), and pancreatic cancer cell lines Panel (FIG. 5E), HP AC (FIG. 5F) and MiaPaca (FIG. 5G).
FIG. 6: Study design used to test anti-tumor activity of CD276 CAR T cells in an orthotopic Panel pancreatic tumor model. (Top) 250,000 firefly luciferase-labeled Panel cells were injected into the pancreas of 6- to 8-week-old female non-obese diabetic (NOD) scid gamma (NSG) mice. Two weeks later, mice were imaged using IVIS Spectrum to measure tumor burden and grouped into different treatment groups listed in the table (bottom). Mice were treated with CARs containing Y422, Y868, Yll l, Y117 or YE5 scFv. CARs containing previously described anti-CD276 scFvs MGA271 and 376.96 were also included. Five million CAR+ T cells were injected intravenously in the tail vein. Tumor burden was monitored by imaging weekly at first and then at specified time points later in the study. Body weight measurements were also taken at the time of imaging on a weekly basis. To image the tumor, 100 pL of 15 mg/mL D-luciferin was injected intraperitonially, and imaging was performed using an IVIS Spectrum imager after 10 minutes.
FIG. 7: Study design used to test anti-tumor activity of CD276 CAR T cells in an orthotopic Panel pancreatic tumor model. Shown is tumor luminescence data from the experiment described in FIG. 6. Data in the graph represent mean + standard error of mean (SEM) for 6-8 mice per group. CARs based on antibodies 376.96 and MGA271 have been described previously and these same variable domains were incorporated into the CARs of this study for comparison. The humanized MGA271 scFv sequence was used to make the CAR. The 376.96 scFv was used in its native (murine) form without humanization. Mice treated with the Yll 1-based CAR exhibited the greatest reduction in tumor size. At the conclusion of the study (day 296), no tumors had relapsed in mice treated with the Yl ll CAR. ** = Yl ll vs. 376.96, p=0.0031 ; **** = Yl l l vs. MGA271, p<0.0001.
FIGS. 8A-8C: Anti-tumor activity of CD276 CAR T cells in an orthotopic HP AC pancreatic tumor model. (FIG. 8A) 250,000 firefly luciferase-labeled HP AC cells were injected into the pancreas of 6-8-week-old female NSG mice. Ten days later, mice were imaged using IVIS Spectrum to measure tumor burden and grouped into different treatment groups as shown in FIG. 8B. Five million CAR+ T cells were injected intravenously in the tail vein. Tumor burden was monitored by imaging weekly first and then at specified time points later in the study. To image the tumor, 100 pL of 15 mg/mL D-luciferin was injected intraperitonially, and imaging was performed after 10 minutes. (FIG. 8C) Body weight measurements were taken at the time of imaging on a weekly basis. The graph shows average (± SEM) body weight of mice.
FIGS. 9A-9B: Anti-tumor activity of CD276 CAR T cells in an orthotopic HP AC pancreatic tumor model. (FIG. 9A) IVIS images showing tumor luminescence for the experiment described in FIGS. 8A-8C. Five or six representative mice are shown for each treatment group. (FIG. 9B) Graph showing tumor luminescence represented as average ± SEM. UTD = untransfected. Mice treated with the Y 111 -based CAR exhibited the greatest reduction in tumor size.
FIGS. 10A-10B: Anti-tumor activity of CD276 CAR T cells in an orthotopic HPAC pancreatic tumor. (FIG. 10A) Graph showing long-term follow up of tumor size and a comparison of the disclosed CARs with MGA271 based CAR. (FIG. 10B) Tumor luminescence in individual mice for all groups. The results demonstrated that Y 111 -based CAR T cells were the most effective at eliminating the pancreatic tumors.
FIG. 11: Production of cytolytic proteins by CAR T cells co-cultured with CD276+ Panel tumor cells. Un-transduced (UTD) T cells, and T cells expressing a CD19 CAR or one of three CD276- specific CARs (Y868, Yll l, and Y117) were co-cultured with Panel pancreatic cancer cells for 16 hours at an effector-to-target ratio (E:T) of 1:2. T cells were then collected and labeled intracellularly with antibodies against granzyme B, perforin, and CD 107a. UTD and CD19 CAR T cells were used as negative controls. Dot plots on the top row illustrate granzyme B (x-axis) and perforin (y-axis) production and the histograms on the bottom row indicate CD107a expression.
FIGS. 12A-12C: Anti-tumor activity of CD276 CAR T cells in a metastatic neuroblastoma (IMR5) model. (FIG. 12A) Schematic of the study design. Firefly-luciferase labeled IMR5 cells (IxlO6) were injected into 6-8-week-old female NSG mice via tail vein (Day -35). Five weeks later, mice were imaged using IVIS Spectrum to measure tumor burden and grouped into different treatment groups. Two million Y868, Y111, Y117 or 376.96 CAR T cells were injected intravenously in the tail vein (Day 0). Tumor burden was monitored by imaging on Days -1, 7, 14, 21, 29, 40, 49 and 60. Body weight measurements were also taken at the time of imaging. To image the tumor, 100 pl of 15 mg/mL D-luciferin was injected intraperitonially and imaging was done after 10 minutes. (FIG. 12B) Graph quantifying tumor luminescence of untreated (UTD) mice and mice treated with CAR T cells, measured on Days -1, 7, 14, 21, 29, 40, 49 and 60. Data in the graph represent mean ± standard error of mean (SEM) for 8 mice per group. (FIG. 12C) Tumor luminescence images showing tumor burden in each animal.
FIGS. 13A-13B: Long-term persistence and anti-tumor activity of Y111 CAR in the HP AC orthotopic pancreatic tumor model. To test the long-term in vivo persistence and functional capacity of Y111 CAR T cells, the long-term survivor mice from the HP AC study were rechallenged with 4xl06 firefly-luciferase labeled HP AC cells injected subcutaneously in the lower right abdomen. Age matched mice that had never received tumor cells or CAR T cells were used as controls and were similarly injected with the tumor cells. Mice were imaged using IVIS Spectrum to measure tumor burden on Days -7, 8, 46, 72, 101, 123, 143 and 182 after tumor re-challenge. (FIG. 13 A) Images of control and Yl ll CAR T cell-treated mice showing tumor burden. (FIG. 13B) Graph of tumor luminescence (photons/second) in the age-matched control and Yl l l CAR T cell-treated mice.
FIGS. 14A-14B: Optimization of Yl l l CAR. (FIG. 14A) Schematic of the study design. Firefly-luciferase labeled Panel cells (2.5xl05) were injected into the pancreas of 6-8-week-old female NSG mice (Day -14). Two weeks later, mice were imaged using IVIS Lumina to measure tumor burden and grouped into different treatment groups. Included in this study were T cells expressing (1) a Y111 -based CAR having CD28 hinge and transmembrane (HTM) regions, a 4- 1BB costimulatory domain, and a CD3^ signaling domain (Yll l-HL-28HTM-BBz); (2) a Y111- based CAR having CD28 hinge and transmembrane (HTM) regions, a CD28 costimulatory domain, and a CD3^ signaling domain (Yll l-HL-28HTM-28z); (3) a CD276-specific VHH single-domain antibody (B12)-based CAR having CD28 hinge and transmembrane (HTM) regions, a 4- IBB costimulatory domain, and a CD3^ signaling domain (B12-VHH-28HTM-BBz); and a B12-based CAR having CD28 hinge and transmembrane (HTM) regions, a CD28 costimulatory domain, and a CD3^ signaling domain (B12-VHH-28HTM-28z). Five million CAR+ T cells were injected intravenously in the tail vein (Day 0). Tumor burden and body weight were monitored weekly. (FIG. 14B) Graph of tumor luminescence (photons/second) measured on Days -1, 7, 14, 21, 28, 35, 42, 49 and 56 after CAR T cell treatment. To image the tumor, 100 pl of 15 mg/mL D-luciferin was injected intraperitonially and imaging was done after 10 minutes.
SEQUENCE LISTING
The nucleic acid and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. In the accompanying sequence listing:
SEQ ID NO: 1 is the amino acid sequence of the Y111 VH domain.
SEQ ID NO: 2 is a nucleic acid sequence encoding the Y11 1 VH domain. SEQ ID NOs: 3-8 are the CDR sequences of antibody Y111.
SEQ ID NO: 9 is the amino acid sequence of the Y422 VH domain.
SEQ ID NO: 10 is a nucleic acid sequence encoding the Y422 VH domain. SEQ ID NOs: 11-16 are the CDR sequences of antibody Y422.
SEQ ID NO: 17 is the amino acid sequence of the Y868 VH domain.
SEQ ID NO: 18 is a nucleic acid sequence encoding the Y868 VH domain. SEQ ID NOs: 19-24 are the CDR sequences of antibody Y868. SEQ ID NO: 25 is the amino acid sequence of the Y 117 VH domain. SEQ ID NO: 26 is a nucleic acid sequence encoding the Y117 VH domain. SEQ ID NOs: 27-32 are the CDR sequences of antibody Y117. SEQ ID NO: 33 is the amino acid sequence of the YE5 VH domain. SEQ ID NO: 34 is a nucleic acid sequence encoding the YES VH domain. SEQ ID NOs: 35-40 are the CDR sequences of antibody YE5.
SEQ ID NO: 41 is the amino acid sequence of a signal peptide.
SEQ ID NO: 42 is the amino acid sequence of a GS linker.
SEQ ID NO: 43 is the amino acid sequence of a CD28 hinge region.
SEQ ID NO: 44 is the amino acid sequence of a CD28 transmembrane domain.
SEQ ID NO: 45 is the amino acid sequence of a 41BB costimulatory domain.
SEQ ID NO: 46 is the amino acid sequence of a CD3^ intracellular signaling domain.
SEQ ID NO: 47 is the amino acid sequence of the Yl ll CAR (Yll l-HL-28HTM-BBz).
SEQ ID NO: 48 is a nucleic acid sequence encoding the Yl ll CAR (Y111-HL-28HTM- BBz).
SEQ ID NO: 49 is the amino acid sequence of the Y422 CAR.
SEQ ID NO: 50 is a nucleic acid sequence encoding the Y422 CAR.
SEQ ID NO: 51 is the amino acid sequence of the Y868 CAR.
SEQ ID NO: 52 is a nucleic acid sequence encoding the Y868 CAR.
SEQ ID NO: 53 is the amino acid sequence of the Y117 CAR.
SEQ ID NO: 54 is a nucleic acid sequence encoding the Y117 CAR.
SEQ ID NO: 55 is the amino acid sequence of the YE5 CAR.
SEQ ID NO: 56 is a nucleic acid sequence encoding the YE5 CAR.
SEQ ID NO: 57 is the amino acid sequence of a Yl l l scFv (VH-linker-VL orientation).
SEQ ID NO: 58 is a nucleic acid sequence encoding a Yl ll scFv (VH-linker-VL orientation).
SEQ ID NO: 59 is the amino acid sequence of a Yl l l scFv (VL-linker-VH orientation).
SEQ ID NO: 60 is a nucleic acid sequence encoding a Y111 scFv (VL-linker-VH orientation).
SEQ ID NO: 61 is the amino acid sequence of the Yl ll-HL-28HTM-28z CAR.
SEQ ID NO: 62 is a nucleic acid sequence encoding the Yl ll -HL-28HTM-28z CAR.
SEQ ID NO: 63 is the amino acid sequence of the Yl ll-LH-28HTM-BBz CAR.
SEQ ID NO: 64 is a nucleic acid sequence encoding the Y 11 l-LH-28HTM-BBz CAR.
SEQ ID NO: 65 is the amino acid sequence of a CD28 costimulatory domain.
SEQ ID NO: 66 is a nucleic acid sequence encoding a CD28 costimulatory domain.
SEQ ID NO: 67 is a nucleic acid sequence encoding a signal peptide.
SEQ ID NO: 68 is a nucleic acid sequence encoding a GS linker.
SEQ ID NO: 69 is a nucleic acid sequence encoding a CD28 hinge.
SEQ ID NO: 70 is a nucleic acid sequence encoding a CD28 transmembrane domain. SEQ ID NO: 71 is a nucleic acid sequence encoding a 4- IBB costimulatory domain.
SEQ ID NO: 72 is a nucleic acid sequence encoding a CD3^ signaling domain.
DETAILED DESCRIPTION
I. Abbreviations
ADC antibody-drug conjugate
ADCC antibody-dependent cell-mediated cytotoxicity
APC allophycocyanin
B7H3 B7 homolog 3
CAR chimeric antigen receptor
CDR complementarity determining region
E:T effector to target ratio
NOD non-obese diabetic
NSG NOD scid gamma
PE Pseudomonas exotoxin
PET positron emission tomography scFv single-chain variable fragment
TM transmembrane
VH variable heavy
VL variable light
II. Summary of Terms
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin ’s genes XII. published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:
4-1BB: A costimulatory molecule expressed by T cell receptor (TCR)-activated lymphocytes, and by other cells including natural killer cells. Ligation of 4- IBB induces a signaling cascade that results in cytokine production, expression of anti-apoptotic molecules and an enhanced immune response. An exemplary amino acid sequence of 4- IBB is set forth herein as SEQ ID NO: 45.
Administration: To provide or give a subject an agent, such as a monoclonal antibody, CAR or CAR-expressing cell provided herein, by any effective route. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intraprostatic, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
Antibody: A polypeptide ligand comprising at least one variable region that recognizes and binds (such as specifically recognizes and specifically binds) an epitope of an antigen. Mammalian immunoglobulin molecules are composed of a heavy (H) chain and a light (L) chain, each of which has a variable region, termed the variable heavy (VH) region and the variable light (VL) region, respectively. Together, the VH region and the VL region are responsible for binding the antigen recognized by the antibody. There are five main heavy chain classes (or isotypes) of mammalian immunoglobulin, which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Antibody isotypes not found in mammals include IgX, IgY, IgW and IgNAR. IgY is the primary antibody produced by birds and reptiles and is functionally similar to mammalian IgG and IgE. IgW and IgNAR antibodies are produced by cartilaginous fish, while IgX antibodies are found in amphibians.
Antibody variable regions contain "framework" regions and hypervariable regions, known as “complementarity determining regions” or “CDRs.” The CDRs are primarily responsible for binding to an epitope of an antigen. The framework regions of an antibody serve to position and align the CDRs in three-dimensional space. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of numbering schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991; the “Kabat” numbering scheme), Chothia et al. (see Chothia and Lesk, J Mol Biol 196:901-917, 1987; Chothia et al., Nature 342:877, 1989; and Al-Lazikani et al., JMB 273,927-948, 1997; the “Chothia” numbering scheme), Kunik et al. (see Kunik et al., PLoS Comput Biol 8:el002388, 2012; and Kunik et al., Nucleic Acids Res 40(Web Server issue):W521-524, 2012; “Paratome CDRs”) and the ImMunoGeneTics (IMGT) database (see, Lefranc, Nucleic Acids Res 29:207-9, 2001; the “IMGT” numbering scheme). The Kabat, Paratome and IMGT databases are maintained online. In addition, the AbRSA tool can be used to determine the CDR boundaries according to Kabat, IMGT or Chothia (online at aligncdr.labshare.cn/aligncdr/abrsa.php).
A “single-domain antibody” refers to an antibody having a single domain (a variable domain) that is capable of specifically binding an antigen, or an epitope of an antigen, in the absence of an additional antibody domain. Single-domain antibodies include, for example, VH domain antibodies, VNAR antibodies, camelid VHH antibodies, and VL domain antibodies. VNAR antibodies are produced by cartilaginous fish, such as nurse sharks, wobbegong sharks, spiny dogfish and bamboo sharks. Camelid VHH antibodies are produced by several species including camel, llama, alpaca, dromedary, and guanaco, which produce heavy chain antibodies that are naturally devoid of light chains.
A “monoclonal antibody” is an antibody produced by a single clone of lymphocytes or by a cell into which the coding sequence of a single antibody has been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art. Monoclonal antibodies include humanized monoclonal antibodies.
A “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species.
A “humanized” antibody is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rabbit, rat, shark or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” In one aspect, all CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions.
Antibody-drug conjugate (ADC): A molecule that includes an antibody (or antigenbinding fragment of an antibody) conjugated to a drug, such as a cytotoxic agent. ADCs can be used to specifically target a drug to cancer cells through specific binding of the antibody to a tumor antigen expressed on the cell surface. Exemplary drugs for use with ADCs include antimicrotubule agents (such as maytansinoids, auristatin E and auristatin F) and interstrand crosslinking agents (for example, pyrrolobenzodiazepines; PBDs). In some cases, the ADC is a bispecific ADC, which is comprised of two monoclonal antibodies or antigen-fragments thereof, each directed to a different antigen or epitope, conjugated to a drug. In one example, the agent attached to the antibody is IRDye® 700 DX (IR700, Li-cor, Lincoln, NE), which can then be used with near infrared light NIR light to kill cancer cells to which the antibody binds (photoimmunotherapy; see for example US 8,524,239 and 10,538,590). For example, amino-reactive IR700 can be covalently conjugated to an antibody using the NHS ester of 1R700.
Anti-microtubule agent: A type of drug that blocks cell growth by stopping mitosis. Anti-microtubule agents, also referred to as “anti-mitotic agents,” are used to treat cancer.
Binding affinity: Affinity of an antibody for an antigen. In one aspect, affinity is calculated by a modification of the Scatchard method described by Frankel et al. , Mol. Immunol. , 16: 101-106, 1979. In another aspect, binding affinity is measured by an antigen/antibody dissociation rate. In another aspect, a high binding affinity is measured by a competition radioimmunoassay. In another aspect, binding affinity is measured by ELISA. In other aspects, antibody affinity is measured by flow cytometry or by surface plasmon reference. An antibody that “specifically binds” an antigen (such as CD276) is an antibody that binds the antigen with high affinity and does not significantly bind other unrelated antigens.
In some examples, a monoclonal antibody (such as an anti-CD276 antibody provided herein) specifically binds to a target (such as a CD276) with a binding constant that is at least 103 M 1 greater, 104 M 1 greater or 105 M 1 greater than a binding constant for other molecules in a sample or subject. In some examples, an antibody e.g., monoclonal antibody) has an equilibrium constant (KD) of 5 pM or less, such as 5,000 nM or less, 900 nM or less, 500 nM or less, 250 nM or less, 100 nM or less, 50 nM or less, 10 nM or less, 5 nM or less, or 1 nM or less. For example, a monoclonal antibody binds to a target, such as CD276 with a binding affinity of at least about 1 x 10'6 M, at least about 0.5 x 10"6 M, at least about 1 x 10"7 M, at least about 0.5 x 10"7 M, at least about 1 x 10"8 M, at least about 0.5 x 10"8 M, at least about 1 x 10"9 M, at least about 0.5 x 10"9 M, or at least about 0.1 x 10'9. In certain aspects, a specific binding agent that binds to its target has a dissociation constant (Kd) of <1000 nM, <750 nM, 500 nM, <250 nM, <100 nM, <50 nM, <25 nM, <10 nM, <5 nM, <2.5 nM, <1 nM, <0.5 nM, <0.25 nM, <0.01 nM, or <0.001 nM (e.g., 10’6M or less, e.g., from 10'6M to 10 10M, e.g., from 10 10 M to IO 12 M). In some examples, binding affinity is measured using the Octet system (Creative Biolabs), which is based on bio-layer interferometry (BLI) technology. In some examples, Kd is measured using surface plasmon resonance assays using a BIACORES-2000 or a BIACORES-3000 (BIAcore, Inc., Piscataway, N.J.). Bispecific antibody: A recombinant protein that includes antigen-binding fragments of two different monoclonal antibodies and is thereby capable of binding two different antigens or two different epitopes of the same antigen (such as CD276). In some aspects, bispecific antibodies are used for cancer immunotherapy by simultaneously targeting, for example, both CTLs (such as a CTL receptor component such as CD3) or effector natural killer (NK) cells, and a tumor antigen (such as CD276). Similarly, a multi-specific antibody is a recombinant protein that includes antigen-binding fragments of at least two different monoclonal antibodies, such as two, three or four different monoclonal antibodies.
Brain cancer or tumor: A type of cancer or tumor that develops from brain tissue. Brain cancers include, but are not limited to, neuroblastoma, medulloblastoma, glioma, glioblastoma, meningioma, pituitary adenoma, astrocytoma, choroid plexus carcinoma, ependymoma and pineoblastoma.
Breast cancer: A type of cancer that forms in tissues of the breast, usually the ducts and lobules. Types of breast cancer include, for example, ductal carcinoma in situ, invasive ductal carcinoma, triple negative breast cancer, inflammatory breast cancer, metastatic breast cancer, medullary carcinoma, tubular carcinoma and mucinous carcinoma. Triple negative breast cancer refers to a type of breast cancer in which the cancer cells do not express estrogen receptors, progesterone receptors or significant levels of HER2/neu protein. Triple negative breast cancer is also called ER-negative PR-negative HER2/neu-negative breast cancer.
CD276: An immune checkpoint molecule that is expressed in the stroma of most or all solid tumors and may also be expressed by solid tumor cells. This protein is a member of the B7 superfamily of costimulatory molecules. CD276 is also known as B7H3.
CD276-positive cancer: A cancer that expresses or overexpresses CD276. Examples of CD276-positive cancers include, but are not limited to, liver cancers (such as hepatocellular carcinoma), pancreatic cancers, kidney cancers, bladder cancers, cervical cancers, esophageal cancers, prostate cancers, breast cancers, ovarian cancers, colon cancers, lung cancers, brain cancers (such as neuroblastoma or glioblastoma), pediatric cancers (such as osteosarcoma, neuroblastoma, rhabdomyosarcoma or Ewing’s sarcoma), melanoma and mesothelioma (see, for example, Seaman et al., Cancer Cell 31 (4):501-505, 2017). In some instances, a CD276-positive cancer refers to a cancer in which CD276 is expressed in the tumor stroma, and may also be expressed by the tumor cells.
Chemotherapeutic agent: Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth. In one aspect, a chemotherapeutic agent is an agent of use in treating a CD276-positive tumor. In one aspect, a chemotherapeutic agent is a radioactive compound. A skilled person can readily identify a chemotherapeutic agent of use (see for example, Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed., © 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (edsf: Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, D.S., Knobf, M.F., Durivage, H.J. (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy is the administration of more than one agent to treat cancer. One example is the administration of an antibody that binds CD276 used in combination with a radioactive or chemical compound. In one example, a chemotherapeutic agent is a biologic, such as a therapeutic antibody (e.g., therapeutic monoclonal antibody), such as an anti-CD276 antibody provided herein, as well as other anti-cancer antibodies, such as anti-PDl or anti-PDLl (e.g., pembrolizumab and nivolumab), anti-CTLA4 (e.g., ipilimumab), anti-EGFR (e.g., cetuximab), anti-VEGF (e.g., bevacizumab), or combinations thereof (e.g., anti-PD-1 and anti- CTLA-4).
Chimeric antigen receptor (CAR): A chimeric molecule that includes an antigen-binding portion (such as a scFv) and a signaling domain, such as a signaling domain from a T cell receptor (for example, CD3Q. Typically, CARs are comprised of an antigen-binding moiety, a transmembrane domain and an endodomain. The endodomain typically includes a signaling chain having an immunoreceptor tyrosine-based activation motif (ITAM), such as CD3 or FceRIy. In some instances, the endodomain further includes the intracellular portion of at least one additional costimulatory domain, such as CD28, 4-1BB (CD137), ICOS, 0X40 (CD134), CD27 and/or DAP10. In some examples, the CAR is multispecific (such as bispecific) or bicistronic. A multispecific CAR is a single CAR molecule comprised of at least two antigen-binding domains (such as scFvs) that each bind a different antigen or a different epitope on the same antigen (see, for example, US 2018/0230225). For example, a bispecific CAR refers to a single CAR molecule having two antigen-binding domains that each bind a different antigen. A bicistronic CAR refers to two complete CAR molecules, each containing an antigen-binding moiety that binds a different antigen. In some cases, a bicistronic CAR construct expresses two complete CAR molecules that are linked by a cleavage linker. T cells or NK cells expressing a bispecific or bicistronic CAR can bind cells that express both of the antigens to which the binding moieties are directed (see, for example, Qin et al., Blood 130:810, 2017; and WO/2018/2I3337).
Colon cancer: A type of cancer that develops in the colon or the rectum. The most common type of colon cancer (also known as “colorectal cancer”) is colorectal adenocarcinoma, which accounts for approximately 95% of all colon cancers. Adenocarcinomas develop in the cells lining the inside of the colon and/or rectum. Other types of colorectal cancers include gastrointestinal carcinoid tumors, metastatic colorectal cancer, primary colorectal lymphoma (a type of non-Hodgkin’s lymphoma), gastrointestinal stromal tumors (classified as a sarcoma and arising from interstitial cells of Cajal), leiomyosarcoma (arising from smooth muscle cells) and colorectal melanoma.
Complementarity determining region (CDR): A region of hypervariable amino acid sequence that defines the binding affinity and specificity of an antibody. The light and heavy chains of a mammalian immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. A single-domain antibody contains three CDRs, referred to herein as CDR1, CDR2 and CDR3.
Conjugate: In the context of the present disclosure, a “conjugate” is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to an effector molecule or a second protein (such as a second antibody). The effector molecule can be, for example, a drug, toxin, therapeutic agent, detectable label, protein, nucleic acid, lipid, nanoparticle, photon absorber, carbohydrate or recombinant virus. An antibody conjugate is often referred to as an “immunoconjugate.” When the conjugate includes an antibody linked to a drug (such as a cytotoxic agent), the conjugate is often referred to as an “antibody-drug conjugate” or “ADC.” Other antibody conjugates include, for example, multi-specific (such as bispecific or trispecific) antibodies and chimeric antigen receptors (CARs).
Conservative variant: A protein containing conservative amino acid substitutions that do not substantially affect or decrease the affinity of a protein, such as an antibody to CD276. For example, a monoclonal antibody that specifically binds CD276 can include at most about 1, at most about 2, at most about 5, and most about 10, or at most about 15 conservative substitutions and specifically bind the CD276 polypeptide. The term “conservative variant” also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibody specifically binds CD276. Non-conservative substitutions are those that reduce an activity or binding to CD276.
Conservative amino acid substitution tables providing functionally similar amino acids are well known. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
Contacting: Placement in direct physical association; includes both in solid and liquid form.
Cytotoxic agent: Any drug or compound that kills cells.
Cytotoxicity: The toxicity of a molecule, such as an immunotoxin, to the cells intended to be targeted, as opposed to the cells of the rest of an organism. In contrast, the term “toxicity” refers to toxicity of an immunotoxin to cells other than those that are the cells intended to be targeted by the targeting moiety of the immunotoxin, and the term “animal toxicity” refers to toxicity of the immunotoxin to an animal by toxicity of the immunotoxin to cells other than those intended to be targeted by the immunotoxin.
Degenerate variant: A polynucleotide encoding a polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the polypeptide is unchanged.
Diagnostic: Identifying the presence or nature of a pathologic condition, such as a CD276- positive cancer. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of true positives). The "specificity" of a diagnostic assay is one minus the false positive rate, where the false positive rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis. "Prognostic" is the probability of development (such as severity) of a pathologic condition, such cancer.
Drug: Any compound used to treat, ameliorate or prevent a disease or condition in a subject. In some aspects herein, the drug is an anti-cancer agent, for example a cytotoxic agent, such as an anti-mitotic or anti-microtubule agent.
Effector molecule: The portion of a chimeric molecule that is intended to have a desired effect on a cell to which the chimeric molecule is targeted. Effector molecule is also known as an effector moiety (EM), therapeutic agent, diagnostic agent, or similar terms. Therapeutic agents (or drugs) include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, photon absorbers, lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides. Alternatively, the molecule linked to a targeting moiety, such as an anti-CD276 antibody, may be an encapsulation system, such as a liposome or micelle that contains a therapeutic composition such as a drug, a nucleic acid (such as an antisense nucleic acid), or another therapeutic moiety that can be shielded from direct exposure to the circulatory system. Means of preparing liposomes attached to antibodies are well known (see, for example, U.S. Patent No. 4,957,735; and Connor el al., Pharm Ther 28:341-365, 1985). Diagnostic agents or moieties include radioisotopes and other detectable labels. Detectable labels useful for such purposes are also well known and include radioactive isotopes such as 35S, nC, 13N, 15O, 18F, 19F, "mTc, 131I, 3H, 14C, 15N, 90Y, "TC, i nIn and 125I, fluorophores, chemiluminescent agents, and enzymes.
Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic (that elicit a specific immune response). An antibody specifically binds a particular antigenic epitope on a polypeptide, such as CD276.
Framework region: Amino acid sequences interposed between CDRs. Framework regions of an immunoglobulin molecule include variable light and variable heavy framework regions.
Fusion protein: A protein comprising at least a portion of two different (heterologous) proteins.
Heterologous: Originating from a separate genetic source or species.
Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one aspect, the response is specific for a particular antigen (an “antigen-specific response”). In one aspect, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another aspect, the response is a B cell response, and results in the production of specific antibodies.
Immunoconjugate: A covalent linkage of an effector molecule to an antibody or functional fragment thereof. The effector molecule can be, for example, a detectable label, a photon absorber (such as IR700), or a toxin (to form an immunotoxin, such as an immunotoxin comprising Pseudomonas exotoxin or a variant thereof). Specific, non-limiting examples of toxins include, but are not limited to, abrin, ricin, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, or modified toxins thereof, or other toxic agents that directly or indirectly inhibit cell growth or kill cells. For example, PE and DT are highly toxic compounds that typically bring about death through liver toxicity. PE and DT, however, can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (such as the domain la of PE and the B chain of DT) and replacing it with a different targeting moiety, such as an antibody. In one aspect, an antibody is joined to an effector molecule. In another aspect, an antibody joined to an effector molecule is further joined to a lipid or other molecule, such as to increase its half-life in the body. The linkage can be either by chemical or recombinant means. In one aspect, the linkage is chemical, wherein a reaction between the antibody moiety and the effector molecule has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule. Because immunoconjugates were originally prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are also sometimes referred to as “chimeric molecules.” The term “chimeric molecule,” as used herein, therefore refers to a targeting moiety, such as a ligand or an antibody, conjugated (coupled) to an effector molecule. The term “conjugated” or “linked” refers to making two polypeptides into one contiguous polypeptide molecule.
Immunoliposome: A liposome with antibodies or antibody fragments conjugated to its surface. Immunoliposomes can carry cytotoxic agents or other drugs to antibody-targeted cells, such as tumor cells.
Interstrand crosslinking agent: A type of cytotoxic drug capable of binding covalently between two strands of DNA, thereby preventing DNA replication and/or transcription.
Isolated: An “isolated” biological component, such as a nucleic acid, protein (including antibodies) or organelle, has been substantially separated or purified away from other biological components in the environment (such as a cell) in which the component occurs, for example other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
Label: A detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. In one example, a “labeled antibody” refers to incorporation of another molecule in the antibody. For example, the label is a detectable marker, such as the incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as 35S, “C, 13N, 15O, 18F, 19F, "mTc, 1311, 3H, 14C, 15N, 90Y, "Tc, 11 ‘in and I25I), fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some aspects, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
Linker: In some cases, a linker is a peptide within an antibody binding fragment (such as an Fv fragment) which serves to indirectly bond the variable heavy chain to the variable light chain. “Linker” can also refer to a peptide serving to link a targeting moiety, such as an antibody, to an effector molecule, such as a cytotoxin or a detectable label. The terms “conjugating,” “joining,” “bonding” or “linking” refer to making two polypeptides into one contiguous polypeptide molecule, or to covalently attaching a radionuclide or other molecule to a polypeptide, such as an antibody. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.
Liver cancer: Any type of cancer occurring in liver tissue. The most common type of liver cancer is hepatocellular carcinoma (HCC), which develops in hepatocytes. Other types of liver cancer include cholangiocarcinoma, which develops in the bile ducts; liver angiosarcoma, which is a rare form of liver cancer that begins in the blood vessels of the liver; and hepatoblastoma, which is a very rare type of liver cancer found most often in children.
Lung cancer: Any cancer that forms in the lung. Most cancers that begin in the lung are carcinomas. The two primary types of lung carcinoma are small-cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). Subclasses of NSCLC include adenocarcinoma, squamous-cell carcinoma and large-cell carcinoma.
Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
Ovarian cancer: Cancer that forms in tissues of the ovary. Most ovarian cancers are either ovarian epithelial carcinomas (cancer that begins in the cells on the surface of the ovary) or malignant germ cell tumors (cancer that begins in egg cells). Another type of ovarian cancer is stromal cell cancer, which originates in cells that release hormones and connect the different structures of the ovaries.
Pancreatic cancer: A disease in which malignant cells are found in the tissues of the pancreas. Pancreatic tumors can be either exocrine tumors or neuroendocrine tumors, based on the cell origin of the cancer. The vast majority (-94%) of pancreatic cancers are exocrine tumors. Exocrine cancers include, for example, adenocarcinoma (the most common type of exocrine tumor), acinar cell carcinoma, intraductal papillary-mucinous neoplasm (1PMN), and mucinous cystadenocarcinoma. In some examples, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). Pancreatic neuroendocrine tumors, also referred to as islet cell tumors, are classified by the type of hormones they produce. Exemplary neuroendocrine tumors include gastrinoma, glucaganoma, insulinoma, somatostatinoma, VIPoma (vasoactive intestinal peptide) and nonfunctional islet cell tumor.
Pediatric cancer: A cancer that develops in children ages 0 to 14. The major types of pediatric cancers include, for example, neuroblastoma, acute lymphoblastic leukemia (ALL), embryonal rhabdomyosarcoma (ERMS), alveolar rhabdomyosarcoma (ARMS), Ewing’s sarcoma, desmoplastic small round cell tumor (DRCT), osteosarcoma, brain and other CNS tumors (such as neuroblastoma and medulloblastoma), Wilm’s tumor, non-Hodgkin lymphoma, and retinoblastoma.
Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of the antibodies and other compositions disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (such as powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Photoimmunotherapy: A targeted cancer therapy that utilizes an antigen- specific antibody-photoabsorber conjugate that can be activated by near- infrared light to kill targeted cells. The photon absorber is typically based on phthalocyanine dye, such as a near infrared (NIR) phthalocyanine dye (for example, IRDye® 700DX, also know known as IR700). The antibody (for example, a CD276-specific antibody) binds to the appropriate cell surface antigen (e.g., CD276) and the photo- activatable dye induces lethal damage to cell membranes after NIR-light exposure. NIR-light exposure (e.g., 690 nm) induces highly selective, necrotic cancer cell death within minutes without damage to adjoining cells (see, for example, U.S. Application No. 2018/0236076).
Preventing, treating or ameliorating a disease: “Preventing” a disease refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number of size of metastases. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer.
Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell. In one aspect, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation. Substantial purification denotes purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific, non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components.
Pyrrolobenzodiazepine (PBD): A class of sequence-selective DNA minor-groove binding crosslinking agents originally discovered in Streptomyces species. PBDs are significantly more potent than systemic chemotherapeutic drugs. The mechanism of action of PBDs is associated with their ability to form an adduct in the minor groove of DNA, thereby interfering with DNA processing. In the context of the present disclosure, PBDs include naturally produced and isolated PBDs, chemically synthesized naturally occurring PBDs, and chemically synthesized non-naturally occurring PBDs. PBDs also include monomeric, dimeric and hybrid PBDs (for a review see Gerratana, Med Res Rev 32(2):254-293, 2012).
Recombinant: A recombinant nucleic acid or protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.
Sample (or biological sample): A biological specimen containing genomic DNA, RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, tissue, cells, urine, semen, saliva, tissue biopsy, fine needle aspirate, surgical specimen, and autopsy material. In one example, a sample includes a tumor biopsy.
Sequence identity: The similarity between amino acid or nucleic acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide or nucleic acid molecule will possess a relatively high degree of sequence identity when aligned using standard methods.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins and Sharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research 16: 10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988. Altschul et al., Nature Genet. 6: 119, 1994, presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
Homologs and variants of an antibody that specifically binds a CD276 polypeptide are typically characterized by possession of at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of the antibody using the NCBI Blast 2.0, gapped blastp set to default parameters. For comparisons of amino acid sequences of greater than about 30 amino acids, the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). When aligning short peptides (fewer than around 30 amino acids), the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologs could be obtained that fall outside of the ranges provided.
Small molecule: A molecule, typically with a molecular weight less than about 1000 Daltons, or in some aspects, less than about 500 Daltons, wherein the molecule is capable of modulating, to some measurable extent, an activity of a target molecule.
Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals. In one aspect, a subject is a human subject with a CD276-positive cancer.
Synthetic: Produced by artificial means in a laboratory, for example a synthetic nucleic acid or protein (for example, an antibody) can be chemically synthesized in a laboratory.
Therapeutically effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to inhibit or suppress growth of a tumor. In one aspect, a therapeutically effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of a tumor, such as reduce a tumor size and/or volume by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, and/or reduce the number and/or size/volume of metastases by at least 10%, at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, or even 100%, for example as compared to a size/volume/number prior to treatment or for example as compared to a size/volume/number with a different treatment (such as treatment with a different CD276 antibody or different CD276-targeted CAR). When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in tumors) that has been shown to achieve a desired in vitro effect.
Toxin: A molecule that is cytotoxic for a cell. Toxins include abrin, ricin, Pseudomonas exotoxin (PE), diphtheria toxin (DT), botulinum toxin, saporin, restrictocin or gelonin, or modified toxins thereof. For example, PE and DT are highly toxic compounds that typically bring about death through liver toxicity. PE and DT, however, can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (such as domain la of PE or the B chain of DT) and replacing it with a different targeting moiety, such as an antibody.
Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art. In some aspects, the vector is a virus vector, such as a lentivirus vector.
III. Monoclonal Antibodies Specific for CD276
Monoclonal antibodies that specifically bind CD276 with high affinity are described. The CD276 monoclonal antibodies were isolated from human scFv phage display libraries. The monoclonal antibodies were used to develop CD276-targeted CARs, which are shown herein to induce a durable anti-tumor response in several animal models of cancer, and induce cell killing of multiple tumor cell lines. The amino acid sequences of the VH domain and VL domain of the disclosed antibodies (Yl ll, Y422, Y868, Y117 and YE5) are provided below. The CDR sequences (according to Kabat) are listed in Tables 1-5 below. Although the CDR locations were identified using Kabat, a skilled person understands that other numbering schemes, such as Chothia, Paratome or IMGT, can also be used to determine the boundaries of each CDR.
Ylll VH domain (SEQ ID NO: 1)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGRIIPILGIANY AQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSNPGGAFDIWGQGTMVTVSS
Ylll VL domain (SEQ ID NO: 2)
DIQLTQSPSTLSASVGDRVTITCRASQSVRSNLAWYHQKPGKAPKLLIYKASTLESGVPSRI SGSGSGTEFTLTISSLQPDDFATYFCQQYAYYPTFGQGTRLEIK
Table 1. Ylll CDR Sequences
Figure imgf000026_0001
Y422 VH domain (SEQ ID NO: 9)
EVQLVESGGGLVQPGGSLTLSCAASGFTFSSYAMSWVRQAPGQGLEWVSAISGSGGSTYY
ADSVKGRFTISRDNAENTLYLEMNSLTADDTAVYYCVRDTYRFFDYWGQGTLVTVSS Y422 VL domain (SEQ ID NO: 10)
QSVLTQPPSVSGAPGQTVTISCSGSTSNIGARYPVHWYQQFPGTAPTLLIYGNNNRPSGVPD
RFSGSMSGTSASLAITGLQAEDEADYYCQSFDNSLSGVLFGGGTQVTVL
Table 2. Y422 CDR Sequences
Figure imgf000027_0001
Y868 VH domain (SEQ ID NO: 17)
QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYY ADSVKGRFTISRDNAKNSLYLQVNSLRAEDTALYYCVRDTYRFFDYWGQGTLVTVSS
Y868 VL domain (SEQ ID NO: 18)
QSVLTQPPSVSGAPGQRVTISCSGSTSNIGARYPVHWYQQFPGTVPKLLIYGNNNRPSGVPD
RFSGSKSGTSASLVITGLQAGDEADYYCQSFDNSLSGVLFGGGTKVTVL
Table 3. Y868 CDR Sequences
Figure imgf000027_0002
Y117 VH domain (SEQ ID NO: 25)
QVQLVQSGAEVKQPGASVKVSCKASGYTFTDYAITWVRQAPGQGLEWVGWINTDTGNPT
YAQAFTGRLVFSLDTSVSTAYLQISSLKAEDTAVYYCARVTPSGSLVHYDFWGQGTLVTV SS
Y117 VL domain (SEQ ID NO: 26)
QSVLTQPPSVSGAPGQRVT1SCTGSRSNTGAGFDVHWYQQLPGTAPKLLIYGNNNRPSGVP
DRFSGSRSGTSASLAINGLQAEDEADYYCQSYDYNLSGVIFGGGTKLTAL
Table 4. Y117 CDR Sequences
Figure imgf000028_0001
YES VH domain (SEQ ID NO: 33)
QVQLQQSGPGLVKSSQTLSLTCAISGDSVSSNNAAWNWIRQSPSRGLEWLGRTYYRSKWY
NNYAESVKSRITISPDTSMNHFSLQLNSVTPEDTAVYYCARSRGFFDYWGQGTLVTVSS
YE5 VL domain (SEQ ID NO: 34)
QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNTNRPSGVP
DRFSASRSATSASLAITGLQAEDEADYYCQSHDSSLSAYVFGSGTKVTVL
Table 5. YE5 CDR Sequences
Figure imgf000028_0002
Provided herein are monoclonal antibodies that bind, such as specifically bind, CD276 (such as human CD276). In some aspects, the monoclonal antibody includes a variable heavy (VH) domain and a variable light (VL) domain. In some examples, the monoclonal antibody includes at least a portion of the amino acid sequence set forth herein as SEQ ID NO: 1 and/or SEQ ID NO: 2, such as one or more (such as all three) CDR sequences from SEQ ID NO: 1 and/or SEQ ID NO: 2, as determined by any numbering scheme, such as 1MGT, Kabat or Chothia, or any combination thereof. In other examples, the monoclonal antibody includes at least a portion of the amino acid sequence set forth herein as SEQ ID NO: 9 and/or SEQ ID NO: 10, such as one or more (such as all three) CDR sequences from SEQ ID NO: 9 and/or SEQ ID NO: 10, as determined by any numbering scheme, such as IMGT, Kabat or Chothia, or any combination thereof. In other examples, the monoclonal antibody includes at least a portion of the amino acid sequence set forth herein as SEQ ID NO: 17 and/or SEQ ID NO: 18, such as one or more (such as all three) CDR sequences from SEQ ID NO: 17 and/or SEQ ID NO: 18, as determined by any numbering scheme, such as IMGT, Kabat or Chothia, or any combination thereof. In other examples, the monoclonal antibody includes at least a portion of the amino acid sequence set forth herein as SEQ ID NO: 25 and/or SEQ ID NO: 26, such as one or more (such as all three) CDR sequences from SEQ ID NO: 25 and/or SEQ ID NO: 26, as determined by any numbering scheme, such as IMGT, Kabat or Chothia, or any combination thereof. In other examples, the monoclonal antibody includes at least a portion of the amino acid sequence set forth herein as SEQ ID NO: 33 and/or SEQ ID NO: 34, such as one or more (such as all three) CDR sequences from SEQ ID NO: 33 and/or SEQ ID NO: 34, as determined by any numbering scheme, such as IMGT, Kabat or Chothia, or any combination thereof.
In some aspects, the VH domain of the monoclonal antibodies includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1 and/or the VL domain of the monoclonal antibody comprises the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 2. In other aspects, the VH domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 9 and/or the VL domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 10. In other aspects, the VH domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 17 and/or the VL domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 18. In other aspects, the VH domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 25 and/or the VL domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 26. In other aspects, the VH domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 33 and/or the VL domain of the monoclonal antibody includes the CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 34. In some examples, the CDR sequences are determined using the IMGT, Kabat or Chothia numbering scheme, or a combination thereof. In particular examples, the CDR sequences are determined using Kabat.
In some aspects, the CDR1, CDR2 and CDR3 sequences of the VH domain of the monoclonal antibody respectively include SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and/or the CDR1, CDR2 and CDR3 sequences of the VL domain of the monoclonal antibody respectively include SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. In other aspects, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively include SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13; and/or the amino acid sequences of the LCDR1, LCDR2 and LCDR3 respectively include SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. In other aspects, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively include SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21; and/or the amino acid sequences of the LCDR1, LCDR2 and LCDR3 respectively include SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24. In other aspects, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively include SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29; and/or the amino acid sequences of the LCDR1, LCDR2 and LCDR3 respectively include SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32. In other aspects, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively include SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37; and/or the amino acid sequences of the LCDR1, LCDR2 and LCDR3 respectively include SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40.
In some examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1 and includes the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 1; and/or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 2 and includes the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 2. In specific non-limiting examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 1 and the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 2.
In other examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 9 and includes the HCDR1, HCDR2 and HCDR3 sequence of SEQ ID NO: 9; and/or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 10 and includes the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 10. In specific non-limiting examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 9 and/or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 10.
In other examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 17 and includes the HCDR1, HCDR2 and HCDR3 sequence of SEQ ID NO: 17; and/or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 18 and includes the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 18. In specific non-limiting examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 17 and/or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 18.
In other examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 25 and includes the HCDR1, HCDR2 and HCDR3 sequence of SEQ ID NO: 25; and/or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 26 and includes the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 26. In specific non-limiting examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 25 and/or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 26.
In other examples, the amino acid sequence of the VH domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%identical to SEQ ID NO: 33 and includes the HCDR1, HCDR2 and HCDR3 sequence of SEQ ID NO: 33; and/or the amino acid sequence of the VL domain is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 34 and includes the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 34. In specific non-limiting examples, the amino acid sequence of the VH domain includes or consists of SEQ ID NO: 33 and/or the amino acid sequence of the VL domain includes or consists of SEQ ID NO: 34.
In some aspects, the monoclonal antibody is an IgG, IgA, or IgM molecule. In some examples, the monoclonal antibodies is an IgGl, IgG2, IgG3 or IgG4.
In some aspects, the monoclonal antibody is an antigen-binding fragment selected from an Fab fragment, an Fab’ fragment, an F(ab)’2 fragment, a single chain variable fragment (scFv) and a disulfide stabilized variable fragment (dsFv). In some aspects, the monoclonal antibody is a fully human monoclonal antibody. In other aspects, the monoclonal antibody is a chimeric antibody.
In some aspects, the monoclonal antibody further includes a constant region, such as an IgG constant region, for example, an IgGl constant region, such as a human IgGl constant region. In some examples, the constant region includes at least one amino acid modification to increase the half-life, stability and/or function of the monoclonal antibody.
Also provided herein are chimeric antigen receptors (CARs) that include a CD276-specific monoclonal antibody disclosed herein. In some aspects, the monoclonal antibody component of the CAR is an scFv. In some examples, the scFv is in the VH domain-linker- VL domain (VH-linker- VL) format (N- to C-terminal direction). In other examples, the scFv is in the VL domain-linker- VH domain (VL-linker-VH) format (N- to C-terminal direction). The linker can be any suitable linker for separating the VH and VL domains. In some examples, the linker is a glycine-serine (GS) linker, such as GGGGSGGGGSGGGGS (SEQ ID NO: 42).
In some aspects, the CAR further includes a hinge region, a transmembrane domain, a costimulatory domain, a signaling domain, or any combination thereof. In some examples of the CAR, the hinge region includes a CD28 hinge region; the transmembrane domain includes a CD28 transmembrane domain; the costimulatory domain includes a 4-1BB costimulatory domain or a CD28 costimulatory domain; and/or the signaling domain includes a CD3 signaling domain. In specific examples, the CD28 hinge region includes the amino acid sequence of SEQ ID NO: 43; the CD28 transmembrane domain includes the amino acid sequence of SEQ ID NO: 44; the 4- IBB costimulatory domain includes the amino acid sequence of SEQ ID NO: 45 or the CD28 costimulatory domain includes the amino acid sequence of SEQ ID NO: 65; and/or the CD3(j signaling domain includes the amino acid sequence of SEQ ID NO: 46. In particular non-limiting examples, the amino acid sequence of the CAR includes residues 20-480 of SEQ ID NO: 47, residues 20-484 of SEQ ID NO: 49, residues 20-484 of SEQ ID NO: 51, residues 20-488 of SEQ ID NO: 53, residues 20-486 of SEQ ID NO: 55, residues 20-479 of SEQ ID NO: 61, or residues 20- 480 of SEQ ID NO: 63.
Further provided are isolated cells expressing a CD276- specific CAR disclosed herein. In some aspects, the isolated cell is an immune cell, such as a T cell, B cell, natural killer (NK) cell, macrophage, dendritic cell (DC), or an induced pluripotent stem cell (iPSC). In some examples, the immune cells are allogeneic cells, such as allogeneic cells obtained from a healthy donor. CARs and CAR-expressing cells are further described in section IV.
Also provided herein are immunoconjugates that include a CD276-specific monoclonal antibody disclosed herein and an effector molecule. In some aspects, the effector molecule is a toxin, such as a Pseudomonas exotoxin or a variant thereof, for example PE38. In other aspects, the effector molecule is a detectable label, such as a fluorophore, an enzyme or a radioisotope. In other aspects, the effector molecule is a photon absorber, such as IR700. Immunoconjugates that include a photon absorber can be used, for example, for photoimmunotherapy or in vivo diagnostic imaging. Immunoconjugates are further described in section V.
Further provided herein are antibody-drug conjugates (ADCs) that include a drug conjugated to a CD276-specific monoclonal antibody disclosed herein. In some aspects, the drug is a small molecule, for example an anti-cancer agent, anti-microtubule agent, an anti-mitotic agent and/or a cytotoxic agent. ADCs are further described in section VI.
Also provided herein are multi-specific antibodies that include a CD276- specific monoclonal antibody disclosed herein and at least one additional monoclonal antibody or antigenbinding fragment thereof. In some aspects, the multi-specific antibody is a bispecific antibody. In other aspects, the multi-specific antibody is a trispecific antibody. In some examples, the multispecific antibody includes a CD276- specific monoclonal antibody disclosed herein and a second antibody that binds T cells, such as CD3 on T cells, or that binds NK cells, such as CD 16 on NK cells. Multi-specific antibodies are further described in section VII.
Further provided herein are antibody-nanoparticle conjugates that include a nanoparticle conjugated to a CD276-specific monoclonal antibody disclosed herein. In some aspects, the nanoparticle includes a polymeric nanoparticle, nanosphere, nanocapsule, liposome, dendrimer, polymeric micelle, or niosome. In some aspects, the nanoparticle includes a cytotoxic agent. Antibody-nanoparticle conjugates are further described in section VIII.
Also provided herein are fusion proteins that include a CD276-specific monoclonal antibody disclosed herein and a heterologous protein or peptide. In some aspects, the heterologous protein is an Fc protein, such as a human Fc protein (e.g., human IgGl Fc). In some examples, the fusion protein further includes a linker, such as protein linker.
Further provided herein are nucleic acid molecules that encode a monoclonal antibody, fusion protein, CAR, immunoconjugate, or multiple-specific antibody disclosed herein. In some aspects, the nucleic acid molecule is operably linked to a promoter. In some aspects in which the nucleic acid molecule encodes a CAR, the nucleotide sequence encoding the CAR is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 48 or a degenerate variant thereof; at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 50 or a degenerate variant thereof; at least 90% identical to SEQ ID NO: 52 or a degenerate variant thereof; at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 54 or a degenerate variant thereof; at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 56, or a degenerate variant thereof; at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 62, or a degenerate variant thereof; or at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 64, or a degenerate variant thereof. In some examples, the nucleotide sequence encoding the CAR includes or consists of SEQ ID NO: 48 or a degenerate variant thereof, SEQ ID NO: 50 or a degenerate variant thereof, SEQ ID NO: 52 or a degenerate variant thereof, SEQ ID NO: 54 or a degenerate variant thereof, SEQ ID NO: 56 or a degenerate variant thereof, SEQ ID NO: 62 or a degenerate variant thereof, or SEQ ID NO: 64 or a degenerate variant thereof.
Vectors that include the disclosed nucleic acid molecules are also provided. In some examples, the vector is an expression vector. In other examples, the vector is a viral vector. Isolated cells that include a nucleic acid molecule or vector disclosed herein are further provided. In some examples, the isolated cell is a prokaryotic cell, such as an E. coli cell. In other examples, the isolated cell is a mammalian cell, such as a human cell. Nucleic acid molecules are further described in section IX.
Compositions that include a pharmaceutically acceptable carrier and a CD276-specific monoclonal antibody, fusion protein, CAR, isolated cell (such as a CAR expressing cell, for example a CAR T cell, a CAR NK cell or a CAR macrophage), immunoconjugate, ADC, multispecific antibody, antibody-nanoparticle conjugate, isolated nucleic acid molecule or vector disclosed herein are further provided by the present disclosure. Compositions are further described in section X.
Also provided are methods of detecting expression of CD276 in a sample. In some aspects, the method includes contacting the sample with a CD276-specific monoclonal antibody disclosed herein and detecting binding of the monoclonal antibody to the sample, thereby detecting expression of CD276 in the sample. Further provided are methods of diagnosing a subject as having a CD276-positive tumor. In some aspects, the method includes contacting the sample with a CD276- specific monoclonal antibody disclosed herein and detecting binding of the monoclonal antibody to the sample, thereby diagnosing the subject as having a CD276-positive tumor. In some examples of the disclosed methods, the monoclonal antibody is directly labeled. In other examples, the method includes contacting the monoclonal antibody with a detection antibody, and detecting the binding of the detection antibody to the monoclonal antibody, thereby detecting expression of CD276 in the sample or diagnosing the subject as having a CD276-positive cancer. In some examples, the sample is obtained from a subject suspected of having a CD276-positive cancer. In particular examples, the sample is a tumor biopsy. Methods for diagnosis and detection are described in section XII.
Further provided are methods of treating a CD276-positive cancer in a subject and/or methods of inhibiting tumor growth or metastasis of a CD276-positive cancer in a subject. In some aspects, the method includes administering to the subject a therapeutically effective amount of a CD276- specific monoclonal antibody, CAR, isolated cell (such as a CAR expressing immune cell, for example a CAR T cell, a CAR B cell, a CAR NK cell, a CAR DC, or a CAR macrophage; or an iPSC), immunoconjugate, ADC, multi-specific antibody, antibody-nanoparticle conjugate, isolated nucleic acid molecule, vector, or composition disclosed herein. In some examples of the disclosed methods, the CD276-positive cancer is a solid tumor. In particular examples, the solid tumor is a pancreatic cancer, a neuroblastoma, a liver cancer, a kidney cancer, a bladder cancer, a cervical cancer, an esophageal cancer, a prostate cancer, a breast cancer, an ovarian cancer, a colon cancer, a lung cancer, a brain cancer, a pediatric cancer, a melanoma or a mesothelioma. Therapeutic methods are further described in section XI.
Also provided herein are kits that include a monoclonal antibody, CAR, CAR-expressing cell, immunoconjugate, ADC, multi-specific antibody, antibody-nanoparticle conjugate, fusion protein, or composition disclosed herein; and one or more of a pharmaceutically acceptable carrier, buffer, cell culture media, cell culture plates or flasks, a solid support, a fluorescent label, a radioactive label, an enzymatic label, an enzymatic substrate, a secondary antibody, one or more check point inhibitors, one or more additional anti-cancer agents, one or more transfection reagents, and instructional materials. Kits are further described in section XII.
IV. Chimeric Antigen Receptors (CARs)
The disclosed anti-CD276 monoclonal antibodies (including antigen-binding fragments) can be used to produce CARs (also known as chimeric T cell receptors, artificial T cell receptors or chimeric immunoreceptors) and/or immune cells, such as T lymphocytes (such as CTLs), B cells, natural killer (NK) cells, dendritic cells or macrophages, engineered to express CARs. Induced pluripotent stem cells (iPSCs) can also be used to express CARs. Generally, CARs include a binding moiety, an extracellular hinge and spacer element, a transmembrane region and an endodomain that performs signaling functions (Cartellieri et al., J Biomed Biotechnol 2010:956304, 2010; Dai et al., J Natl Cancer Inst 108(7):djv439, 2016). In many instances, the binding moiety is an antigen binding fragment of a monoclonal antibody, such as a scFv, or a single-domain antibody. The spacer/hinge region typically includes sequences from IgG subclasses, such as IgGl, IgG4, IgD and CD8 domains. The transmembrane domain can be derived from a variety of different T cell proteins, such as CD3^, CD4, CD8 or CD28. Several different endodomains have been used to generate CARs. For example, the endodomain can consist of a signaling chain having an IT AM, such as CD3^ or FceRIy. In some instances, the endodomain further includes the intracellular portion of at least one additional costimulatory domain, such as CD28, 4-1BB (CD137, TNFRSF9), OX-40 (CD134), ICOS, CD27 and/or DAP10.
Immune cells, such as T cells, B cells, NK cells, dendritic cells, or macrophages, or iPSCs expressing CARs can be used to target a specific cell type, such as a CD276-positive tumor cell. Thus, the antibodies disclosed herein can be used to engineer immune cells or iPSCs that express a CAR containing the CD276-specific monoclonal antibody, thereby targeting the engineered cells to CD276-postive tumor cells.
Multispecific (such as bispecific) or bicistronic CARs are also contemplated by the present disclosure. In some aspects, the multispecific or bispecific CAR includes a monoclonal antibody (such as an scFv) specific for CD276 and a monoclonal antibody specific for a different antigen (or a different epitope of CD276). Similarly, a bicistronic CAR includes two CAR molecules expressed from the same construct where one CAR molecule is a CD276-targeted CAR and the second CAR targets a second antigen, such as a second tumor antigen (see, for example, Qin et al. , Blood 130:810, 2017; and WO/2018/213337).
Accordingly, provided herein are CARs that include a CD276-specific antibody, such as any one of the monoclonal antibodies disclosed herein. Also provided are isolated nucleic acid molecules and vectors encoding the CARs (including bispecific and bicistronic CARs), and host cells, such as T cells, B cells, NK cells, DCs, macrophages or iPSCs expressing the CARs, bispecific CAR or bicistronic CARs. T cells, B cells, NK cells, DCs, macrophages or iPSCs expressing CARs comprised of a CD276-specific monoclonal antibody can be used for the treatment of a CD276-positive cancer. In some aspects herein, the CAR is a bispecific CAR. In other aspects herein, the CAR is a bicistronic CAR.
In some aspects, the CAR includes a signal peptide sequence, for example, N-terminal to the antigen-binding domain. The signal peptide sequence can be any suitable signal peptide sequence, such as a signal sequence from granulocyte-macrophage colony-stimulating factor receptor (GMCSFR), immunoglobulin light chain kappa, or IL-2. In some examples, the signal peptide sequence includes the signal peptide of SEQ ID NO: 41. While the signal peptide sequence may facilitate expression of the CAR on the surface of the cell, the presence of the signal peptide sequence in an expressed CAR is not necessary for the CAR to function. Upon expression of the CAR on the cell surface, the signal peptide sequence may be cleaved off of the CAR. Accordingly, in some aspects, the CAR lacks a signal peptide sequence. In some aspects, the CD276- targeted CAR includes the following features in an N-terminal to C-terminal direction: signal peptide, VH domain, linker sequence, VL domain, hinge region, transmembrane domain, costimulatory domain, and signaling domain. In some examples, the signal peptide includes or consists of the amino acid sequence of SEQ ID NO: 41. In some examples, the linker sequence includes or consists of the amino acid sequence of SEQ ID NO: 42. In some examples, the hinge region is a CD28 hinge region, such as a CD28 hinge region that includes or consists of the amino acid sequence of SEQ ID NO: 43. In some examples, the transmembrane domain is a CD28 transmembrane domain, such as a CD28 transmembrane domain that includes or consists of the amino acid sequence of SEQ ID NO: 44. In some examples, the costimulatory domain is a 4- IBB costimulatory domain, such as a 4-1BB costimulatory domain including or consisting of the amino acid sequence of SEQ ID NO: 45, or the costimulatory domain is a CD28 costimulatory domain, such as a CD28 costimulatory domain including or consisting of the amino acid sequence of SEQ ID NO: 65. In some examples, the signaling domain is a CD3^ signaling domain, such as a CD3^ signaling domain that includes or consists of the amino acid sequence of SEQ ID NO: 46.
Nucleotide and amino acid sequences of exemplary CARs that include an antigen-binding fragment (such as a scFv) from CD276-specific monoclonal antibodies Yl l 1, Y422, Y868, Y117 and YE5, are provided below, along with amino acid and nucleotide sequences of individual components of the CARs.
Signal peptide amino acid sequence (SEQ ID NO: 41)
MEFGLSWLFLVAILKGVQC
Signal peptide nucleotide sequence (SEQ ID NO: 67)
ATGGAGTTTGGGCTGAGCTGGCTGTTCCTCGTAGCGATCTTGAAGGGTGTCCAGTGT
GS linker amino acid sequence (SEQ ID NO: 42)
GGGGSGGGGSGGGGS
GS linker nucleotide sequence (SEQ ID NO: 68)
GGCGGTGGCGGATCAGGAGGTGGAGGTTCTGGTGGAGGTGGATCA
CD28 hinge amino acid sequence (SEQ ID NO: 43)
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP CD28 hinge nucleotide sequence (SEQ ID NO: 69)
ATCGAAGTGATGTACCCTCCCCCCTATCTGGACAACGAGAAATCCAACGGCACAATTA
TCCACGTCAAGGGCAAGCACCTGTGCCCGTCTCCCCTGTTCCCAGGTCCTTCTAAACCT
CD28 transmembrane domain amino acid sequence (SEQ ID NO: 44)
FWVLVVVGGVLACYSLLVTVAFIIFWV
CD28 transmembrane domain nucleotide sequence (SEQ ID NO: 70)
TTCTGGGTGCTTGTGGTGGTCGGAGGTGTCCTGGCCTGCTACAGTTTGCTGGTGACCGT
GGCCTTTATAATCTTTTGGGTG
4-IBB costimulatory domain amino acid sequence (SEQ ID NO: 45)
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
4- IBB costimulatory domain nucleotide sequence (SEQ ID NO: 71)
AAACGCGGCAGGAAGAAGCTCCTTTATATCTTCAAGCAACCCTTTATGAGGCCGGTGC
AGACTACCCAAGAGGAGGACGGGTGCTCGTGTAGATTCCCAGAAGAGGAGGAAGGCG GCTGTGAACTG
CD28 costimulatory domain amino acid sequence (SEQ ID NO: 65)
RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
CD28 costimulatory domain nucleotide sequence (SEQ ID NO: 66)
CGGTCAAAGAGATCCCGACTGCTGCACTCCGACTATATGAATATGACCCCACGGAGAC
CTGGGCCAACAAGGAAGCACTACCAGCCCTACGCCCCTCCACGGGATTTTGCCGCGTA TAGAAGC
CD3 signaling domain amino acid sequence (ITAMs underlined; SEQ ID NO: 46)
RVKFSRSADAPAYOOGONQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY
NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMOALPPR
CD3 signaling domain nucleotide sequence (SEQ ID NO: 72)
CGAGTCAAATTTAGCCGGTCTGCCGACGCTCCAGCTTACCAGCAGGGGCAGAACCAGC
TCTATAACGAGCTCAATCTGGGCAGAAGGGAGGAGTACGACGTGCTGGACAAACGCC
GCGGGAGGGACCCAGAAATGGGCGGGAAGCCTCGGAGAAAGAACCCACAAGAAGGA
CTGTATAACGAGCTACAGAAGGATAAGATGGCCGAGGCTTATAGCGAAATCGGGATG
AAGGGCGAAAGGCGCAGGGGAAAAGGGCACGACGGACTATACCAAGGCCTCAGCAC
AGCTACAAAGGACACCTACGACGCTCTTCACATGCAGGCCTTGCCACCAAGG Ylll scFv (VH-linker-VL) amino acid sequence (SEQ ID NO: 57)
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGRIIPILGIANY
AQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSNPGGAFDIWGQGTMVTVSSGG
GGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCRASQSVRSNLAWYHQKPGKAPKLLI
YKASTLESGVPSRISGSGSGTEFTLTISSLQPDDFATYFCQQYAYYPTFGQGTRLEIK
Ylll scFv (VH-linker-VL) nucleotide sequence (SEQ ID NO: 58)
CAGGTCCAATTGGTGCAGTCAGGGGCAGAAGTCAAGAAGCCAGGTGCCAGCGTTAAA
GTGAGCTGCAAAGCCAGTGGCTATACCTTTACAAGCTACGGAATTAGTTGGGTTAGGC
AGGCCCCTGGGCAGGGACTAGAGTGGATGGGTAGAATTATTCCAATACTGGGTATTGC
TAACTACGCACAGAAGTTTCAGGGCCGGGTGACTATTACAGCCGATAAATCTACTTCC
ACTGCGTATATGGAACTTTCCTCCCTGCGCTCTGAGGATACAGCTGTCTACTATTGTGC
GCGATCTAATCCCGGCGGCGCCTTTGATATTTGGGGGCAGGGTACAATGGTTACCGTG
TCTAGTGGCGGTGGCGGATCAGGAGGTGGAGGTTCTGGTGGAGGTGGATCAGATATCC
AGCTGACCCAATCTCCTTCCACACTAAGCGCCTCAGTGGGGGACAGGGTAACTATTAC
CTGTCGAGCGAGCCAATCCGTCCGGTCCAATCTCGCCTGGTATCACCAGAAGCCGGGC
AAGGCACCCAAACTACTGATTTATAAGGCCTCGACACTGGAGTCGGGGGTGCCTTCCC
GGATTAGCGGTTCGGGAAGCGGAACAGAGTTTACACTCACTATTTCCAGCTTACAGCC
CGACGACTTTGCCACCTACTTTTGTCAGCAGTACGCCTATTACCCAACTTTCGGACAGG
GCACCAGATTGGAGATTAAA
Ylll scFv (VL-linker-VH) amino acid sequence (SEQ ID NO: 59)
DIQLTQSPSTLSASVGDRVTITCRASQSVRSNLAWYHQKPGKAPKLLIYKASTLESGVPSRI
SGSGSGTEFTLTISSLQPDDFATYFCQQYAYYPTFGQGTRLEIKGGGGSGGGGSGGGGSQV
QLVQSGAEVKKPGASVKVSCKASGYTFTSYG1SWVRQAPGQGLEWMGR1IP1LG1ANYAQ
KFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSNPGGAFDIWGQGTMVTVSS
Ylll scFv (VL-linker-VH) nucleotide sequence (SEQ ID NO: 60)
GATATCCAGCTGACCCAATCTCCTTCCACACTAAGCGCCTCAGTGGGGGACAGGGTAA
CTATTACCTGTCGAGCGAGCCAATCCGTCCGGTCCAATCTCGCCTGGTATCACCAGAA
GCCGGGCAAGGCACCCAAACTACTGATTTATAAGGCCTCGACACTGGAGTCGGGGGTG
CCTTCCCGGATTAGCGGTTCGGGAAGCGGAACAGAGTTTACACTCACTATTTCCAGCTT
ACAGCCCGACGACTTTGCCACCTACTTTTGTCAGCAGTACGCCTATTACCCAACTTTCG
GACAGGGCACCAGATTGGAGATTAAAGGCGGTGGCGGATCAGGAGGTGGAGGTTCTG
GTGGAGGTGGATCACAGGTCCAATTGGTGCAGTCAGGGGCAGAAGTCAAGAAGCCAG
GTGCCAGCGTTAAAGTGAGCTGCAAAGCCAGTGGCTATACCTTTACAAGCTACGGAAT
TAGTTGGGTTAGGCAGGCCCCTGGGCAGGGACTAGAGTGGATGGGTAGAATTATTCCA
ATACTGGGTATTGCTAACTACGCACAGAAGTTTCAGGGCCGGGTGACTATTACAGCCG
ATAAATCTACTTCCACTGCGTATATGGAACTTTCCTCCCTGCGCTCTGAGGATACAGCT
GTCTACTATTGTGCGCGATCTAATCCCGGCGGCGCCTTTGATATTTGGGGGCAGGGTAC
AATGGTTACCGTGTCTAGT
Ylll CAR (Ylll-HL-28HTM-BBz) amino acid sequence (SEQ ID NO: 47)
MEFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAP
GQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSNPG
GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCRASQSVR
SNLAWYHQKPGKAPKLLIYKASTLESGVPSRISGSGSGTEFTLTISSLQPDDFATYFCQQYA YYPTFGQGTRLEIKGSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVV
GGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE
LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL
YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
Ylll CAR (Ylll-HL-28HTM-BBz) nucleotide sequence (SEQ ID NO: 48)
ATGGAGTTTGGGCTGAGCTGGCTGTTCCTCGTAGCGATCTTGAAGGGTGTCCAGTGTCA
GGTCCAATTGGTGCAGTCAGGGGCAGAAGTCAAGAAGCCAGGTGCCAGCGTTAAAGT
GAGCTGCAAAGCCAGTGGCTATACCTTTACAAGCTACGGAATTAGTTGGGTTAGGCAG
GCCCCTGGGCAGGGACTAGAGTGGATGGGTAGAATTATTCCAATACTGGGTATTGCTA
ACTACGCACAGAAGTTTCAGGGCCGGGTGACTATTACAGCCGATAAATCTACTTCCAC
TGCGTATATGGAACTTTCCTCCCTGCGCTCTGAGGATACAGCTGTCTACTATTGTGCGC
GATCTAATCCCGGCGGCGCCTTTGATATTTGGGGGCAGGGTACAATGGTTACCGTGTCT
AGTGGCGGTGGCGGATCAGGAGGTGGAGGTTCTGGTGGAGGTGGATCAGATATCCAG
CTGACCCAATCTCCTTCCACACTAAGCGCCTCAGTGGGGGACAGGGTAACTATTACCT
GTCGAGCGAGCCAATCCGTCCGGTCCAATCTCGCCTGGTATCACCAGAAGCCGGGCAA
GGCACCCAAACTACTGATTTATAAGGCCTCGACACTGGAGTCGGGGGTGCCTTCCCGG
ATTAGCGGTTCGGGAAGCGGAACAGAGTTTACACTCACTATTTCCAGCTTACAGCCCG
ACGACTTTGCCACCTACTTTTGTCAGCAGTACGCCTATTACCCAACTTTCGGACAGGGC
ACCAGATTGGAGATTAAAGGATCCATCGAAGTGATGTACCCTCCCCCCTATCTGGACA
ACGAGAAATCCAACGGCACAATTATCCACGTCAAGGGCAAGCACCTGTGCCCGTCTCC
CCTGTTCCCAGGTCCTTCTAAACCTTTCTGGGTGCTTGTGGTGGTCGGAGGTGTCCTGG
CCTGCTACAGTTTGCTGGTGACCGTGGCCTTTATAATCTTTTGGGTGAAACGCGGCAGG
AAGAAGCTCCTTTATATCTTCAAGCAACCCTTTATGAGGCCGGTGCAGACTACCCAAG
AGGAGGACGGGTGCTCGTGTAGATTCCCAGAAGAGGAGGAAGGCGGCTGTGAACTGC
GAGTCAAATTTAGCCGGTCTGCCGACGCTCCAGCTTACCAGCAGGGGCAGAACCAGCT
CTATAACGAGCTCAATCTGGGCAGAAGGGAGGAGTACGACGTGCTGGACAAACGCCG
CGGGAGGGACCCAGAAATGGGCGGGAAGCCTCGGAGAAAGAACCCACAAGAAGGAC
TGTATAACGAGCTACAGAAGGATAAGATGGCCGAGGCTTATAGCGAAATCGGGATGA
AGGGCGAAAGGCGCAGGGGAAAAGGGCACGACGGACTATACCAAGGCCTCAGCACA GCTACAAAGGACACCTACGACGCTCTTCACATGCAGGCCTTGCCACCAAGGTGA
Ylll CAR (Ylll-HL-28HTM-28z) amino acid sequence (SEQ ID NO: 61)
MEFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAP
GQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSNPG
GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCRASQSVR
SNLAWYHQKPGKAPKLLIYKASTLESGVPSRISGSGSGTEFTLTISSLQPDDFATYFCQQYA
YYPTFGQGTRLEIKGSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVV
GGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR
SRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL
YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
Ylll CAR (Ylll-HL-28HTM-28z) nucleotide sequence (SEQ ID NO: 62)
ATGGAGTTTGGGCTGAGCTGGCTGTTCCTCGTAGCGATCTTGAAGGGTGTCCAGTGTCA
GGTCCAATTGGTGCAGTCAGGGGCAGAAGTCAAGAAGCCAGGTGCCAGCGTTAAAGT
GAGCTGCAAAGCCAGTGGCTATACCTTTACAAGCTACGGAATTAGTTGGGTTAGGCAG
GCCCCTGGGCAGGGACTAGAGTGGATGGGTAGAATTATTCCAATACTGGGTATTGCTA
ACTACGCACAGAAGTTTCAGGGCCGGGTGACTATTACAGCCGATAAATCTACTTCCAC TGCGTATATGGAACTTTCCTCCCTGCGCTCTGAGGATACAGCTGTCTACTATTGTGCGC
GATCTAATCCCGGCGGCGCCTTTGATATTTGGGGGCAGGGTACAATGGTTACCGTGTCT
AGTGGCGGTGGCGGATCAGGAGGTGGAGGTTCTGGTGGAGGTGGATCAGATATCCAG
CTGACCCAATCTCCTTCCACACTAAGCGCCTCAGTGGGGGACAGGGTAACTATTACCT
GTCGAGCGAGCCAATCCGTCCGGTCCAATCTCGCCTGGTATCACCAGAAGCCGGGCAA
GGCACCCAAACTACTGATTTATAAGGCCTCGACACTGGAGTCGGGGGTGCCTTCCCGG
ATTAGCGGTTCGGGAAGCGGAACAGAGTTTACACTCACTATTTCCAGCTTACAGCCCG
ACGACTTTGCCACCTACTTTTGTCAGCAGTACGCCTATTACCCAACTTTCGGACAGGGC
ACCAGATTGGAGATTAAAGGATCCATCGAAGTGATGTACCCTCCCCCCTATCTGGACA
ACGAGAAATCCAACGGCACAATTATCCACGTCAAGGGCAAGCACCTGTGCCCGTCTCC
CCTGTTCCCAGGTCCTTCTAAACCTTTCTGGGTGCTTGTGGTGGTCGGAGGTGTCCTGG
CCTGCTACAGTTTGCTGGTGACCGTGGCCTTTATAATCTTTTGGGTGCGGTCAAAGAGA
TCCCGACTGCTGCACTCCGACTATATGAATATGACCCCACGGAGACCTGGGCCAACAA
GGAAGCACTACCAGCCCTACGCCCCTCCACGGGATTTTGCCGCGTATAGAAGCCGAGT
CAAATTTAGCCGGTCTGCCGACGCTCCAGCTTACCAGCAGGGGCAGAACCAGCTCTAT
AACGAGCTCAATCTGGGCAGAAGGGAGGAGTACGACGTGCTGGACAAACGCCGCGGG
AGGGACCCAGAAATGGGCGGGAAGCCTCGGAGAAAGAACCCACAAGAAGGACTGTAT
AACGAGCTACAGAAGGATAAGATGGCCGAGGCTTATAGCGAAATCGGGATGAAGGGC
GAAAGGCGCAGGGGAAAAGGGCACGACGGACTATACCAAGGCCTCAGCACAGCTACA
AAGGACACCTACGACGCTCTTCACATGCAGGCCTTGCCACCAAGG
Ylll CAR (Ylll-LH-28HTM-BBz) amino acid sequence (SEQ ID NO: 63)
MEFGLSWLFLVAILKGVQCQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAP
GQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSNPG
GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQLTQSPSTLSASVGDRVTITCRASQSVR
SNLAWYHQKPGKAPKLLIYKASTLESGVPSRISGSGSGTEFTLTISSLQPDDFATYFCQQYA
YYPTFGQGTRLEIKGSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVV
GGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE
LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL
YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
Ylll CAR (Ylll-LH-28HTM-BBz) nucleotide sequence (SEQ ID NO: 64)
TGGAGTTTGGGCTGAGCTGGCTGTTCCTCGTAGCGATCTTGAAGGGTGTCCAGTGTGAT
ATCCAGCTGACCCAATCTCCTTCCACACTAAGCGCCTCAGTGGGGGACAGGGTAACTA
TTACCTGTCGAGCGAGCCAATCCGTCCGGTCCAATCTCGCCTGGTATCACCAGAAGCC
GGGCAAGGCACCCAAACTACTGATTTATAAGGCCTCGACACTGGAGTCGGGGGTGCCT
TCCCGGATTAGCGGTTCGGGAAGCGGAACAGAGTTTACACTCACTATTTCCAGCTTAC
AGCCCGACGACTTTGCCACCTACTTTTGTCAGCAGTACGCCTATTACCCAACTTTCGGA
CAGGGCACCAGATTGGAGATTAAAGGCGGTGGCGGATCAGGAGGTGGAGGTTCTGGT
GGAGGTGGATCACAGGTCCAATTGGTGCAGTCAGGGGCAGAAGTCAAGAAGCCAGGT
GCCAGCGTTAAAGTGAGCTGCAAAGCCAGTGGCTATACCTTTACAAGCTACGGAATTA
GTTGGGTTAGGCAGGCCCCTGGGCAGGGACTAGAGTGGATGGGTAGAATTATTCCAAT
ACTGGGTATTGCTAACTACGCACAGAAGTTTCAGGGCCGGGTGACTATTACAGCCGAT
AAATCTACTTCCACTGCGTATATGGAACTTTCCTCCCTGCGCTCTGAGGATACAGCTGT
CTACTATTGTGCGCGATCTAATCCCGGCGGCGCCTTTGATATTTGGGGGCAGGGTACA
ATGGTTACCGTGTCTAGTGGATCCATCGAAGTGATGTACCCTCCCCCCTATCTGGACAA
CGAGAAATCCAACGGCACAATTATCCACGTCAAGGGCAAGCACCTGTGCCCGTCTCCC
CTGTTCCCAGGTCCTTCTAAACCTTTCTGGGTGCTTGTGGTGGTCGGAGGTGTCCTGGC
CTGCTACAGTTTGCTGGTGACCGTGGCCTTTATAATCTTTTGGGTGAAACGCGGCAGGA
AGAAGCTCCTTTATATCTTCAAGCAACCCTTTATGAGGCCGGTGCAGACTACCCAAGA GGAGGACGGGTGCTCGTGTAGATTCCCAGAAGAGGAGGAAGGCGGCTGTGAACTGCG
AGTCAAATTTAGCCGGTCTGCCGACGCTCCAGCTTACCAGCAGGGGCAGAACCAGCTC
TATAACGAGCTCAATCTGGGCAGAAGGGAGGAGTACGACGTGCTGGACAAACGCCGC
GGGAGGGACCCAGAAATGGGCGGGAAGCCTCGGAGAAAGAACCCACAAGAAGGACT
GTATAACGAGCTACAGAAGGATAAGATGGCCGAGGCTTATAGCGAAATCGGGATGAA
GGGCGAAAGGCGCAGGGGAAAAGGGCACGACGGACTATACCAAGGCCTCAGCACAGC
TACAAAGGACACCTACGACGCTCTTCACATGCAGGCCTTGCCACCAAGG
Y422 CAR amino acid sequence (SEQ ID NO: 49)
MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLTLSCAASGFTFSSYAMSWVRQAP
GQGLEWVSAISGSGGSTYYADSVKGRFTISRDNAENTLYLEMNSLTADDTAVYYCVRDTY
RFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSGAPGQTVTISCSGSTSNIG
ARYPVHWYQQFPGTAPTLLIYGNNNRPSGVPDRFSGSMSGTSASLAITGLQAEDEADYYC
QSFDNSLSGVLFGGGTQVTVLGSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP
FWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE
EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR
KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL
PPR
Y422 CAR nucleotide sequence (SEQ ID NO: 50)
ATGGAGTTTGGGCTGAGCTGGCTCTTCCTCGTGGCGATTCTCAAGGGAGTGCAGTGTG AAGTACAACTCGTGGAGAGCGGGGGCGGACTCGTTCAACCCGGGGGGAGTCTTACACT GTCTTGTGCCGCAAGTGGATTCACGTTCTCTAGCTACGCCATGTCATGGGTACGGCAGG
CCCCCGGTCAAGGCTTGGAATGGGTTAGCGCAATCTCAGGTTCCGGAGGGTCTACCTA
CTATGCCGACTCTGTTAAGGGCCGGTTTACAATAAGTCGGGACAACGCCGAAAACACC CTTTACCTGGAGATGAATAGCCTCACTGCGGATGACACCGCAGTGTATTACTGTGTCA GAGATACTTACAGATTCTTCGATTATTGGGGTCAGGGTACACTCGTGACAGTCTCTTCA
GGGGGAGGTGGGTCTGGAGGAGGTGGTTCCGGGGGTGGAGGAAGCCAGTCCGTACTC
ACCCAACCCCCTAGCGTTAGTGGTGCACCAGGGCAAACAGTTACCATATCATGTAGCG
GTTCAACGTCCAACATTGGGGCGCGCTACCCTGTACATTGGTACCAGCAGTTCCCCGG
GACGGCACCAACTCTCCTTATTTACGGAAACAATAACCGCCCCAGTGGGGTGCCCGAT
CGCTTTAGTGGCTCCATGTCAGGGACATCCGCCAGTCTCGCAATCACCGGCCTTCAGGC
GGAGGACGAGGCAGACTATTATTGCCAATCATTCGACAATTCTCTCAGCGGCGTTCTCT TCGGGGGCGGCACCCAGGTCACAGTACTCGGATCCATCGAAGTGATGTACCCTCCCCC CTATCTGGACAACGAGAAATCCAACGGCACAATTATCCACGTCAAGGGCAAGCACCTG
TGCCCGTCTCCCCTGTTCCCAGGTCCTTCTAAACCTTTCTGGGTGCTTGTGGTGGTCGG
AGGTGTCCTGGCCTGCTACAGTTTGCTGGTGACCGTGGCCTTTATAATCTTTTGGGTGA
AACGCGGCAGGAAGAAGCTCCTTTATATCTTCAAGCAACCCTTTATGAGGCCGGTGCA GACTACCCAAGAGGAGGACGGGTGCTCGTGTAGATTCCCAGAAGAGGAGGAAGGCGG CTGTGAACTGCGAGTCAAATTTAGCCGGTCTGCCGACGCTCCAGCTTACCAGCAGGGG
CAGAACCAGCTCTATAACGAGCTCAATCTGGGCAGAAGGGAGGAGTACGACGTGCTG
GACAAACGCCGCGGGAGGGACCCAGAAATGGGCGGGAAGCCTCGGAGAAAGAACCC ACAAGAAGGACTGTATAACGAGCTACAGAAGGATAAGATGGCCGAGGCTTATAGCGA
AATCGGGATGAAGGGCGAAAGGCGCAGGGGAAAAGGGCACGACGGACTATACCAAG GCCTCAGCACAGCTACAAAGGACACCTACGACGCTCTTCACATGCAGGCCTTGCCACC AAGGTGA Y868 CAR amino acid sequence (SEQ ID NO: 51)
MEFGLSWLFLVAILKGVQCQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP
GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNAKNSLYLQVNSLRAEDTALYYCVRDTY
RFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSGAPGQRVTISCSGSTSNIG
ARYPVHWYQQFPGTVPKLLIYGNNNRPSGVPDRFSGSKSGTSASLVITGLQAGDEADYYC
QSFDNSLSGVLFGGGTKVTVLGSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP
FWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPE
EEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR
KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL
PPR
Y868 CAR nucleotide sequence (SEQ ID NO: 52)
ATGGAGTTTGGGCTGAGCTGGCTGTTCCTCGTAGCGATCTTGAAGGGTGTCCAGTGTCA
GGTGCAGCTCGTCGAGTCGGGCGGAGGGCTCGTGCAACCAGGTGGCTCCCTGCGCCTT
AGTTGCGCAGCGTCAGGTTTCACCTTTTCAAGTTACGCTATGTCTTGGGTCCGACAAGC
TCCCGGGAAGGGACTAGAGTGGGTGAGCGCTATTTCAGGAAGCGGAGGCTCAACCTA
CTACGCCGATTCCGTTAAGGGGCGGTTCACAATCTCACGTGATAACGCCAAGAATAGC
CTGTATCTTCAAGTCAACTCCCTGAGAGCTGAAGACACCGCCCTTTACTATTGTGTTAG
GGATACTTACAGGTTCTTCGATTATTGGGGACAGGGGACGCTGGTTACTGTATCCAGC
GGCGGTGGCGGATCAGGAGGTGGAGGTTCTGGTGGAGGTGGATCACAGAGCGTTCTG
ACTCAGCCTCCAAGTGTGTCGGGCGCTCCGGGTCAGCGCGTGACTATCTCGTGTTCAG
GCAGTACTTCTAATATTGGGGCTCGGTACCCAGTTCACTGGTACCAGCAATTCCCTGGC
ACCGTCCCAAAATTATTAATATACGGTAATAACAATAGACCTAGTGGGGTTCCAGATC
GATTTAGTGGCAGCAAGTCAGGCACTTCCGCCTCGTTGGTGATCACAGGCTTACAGGC
AGGAGACGAGGCCGACTACTATTGTCAGTCCTTTGATAATTCACTGAGTGGTGTACTGT
TCGGGGGCGGAACAAAGGTGACGGTGTTGGGATCCATCGAAGTGATGTACCCTCCCCC
CTATCTGGACAACGAGAAATCCAACGGCACAATTATCCACGTCAAGGGCAAGCACCTG
TGCCCGTCTCCCCTGTTCCCAGGTCCTTCTAAACCTTTCTGGGTGCTTGTGGTGGTCGG
AGGTGTCCTGGCCTGCTACAGTTTGCTGGTGACCGTGGCCTTTATAATCTTTTGGGTGA
AACGCGGCAGGAAGAAGCTCCTTTATATCTTCAAGCAACCCTTTATGAGGCCGGTGCA
GACTACCCAAGAGGAGGACGGGTGCTCGTGTAGATTCCCAGAAGAGGAGGAAGGCGG
CTGTGAACTGCGAGTCAAATTTAGCCGGTCTGCCGACGCTCCAGCTTACCAGCAGGGG
CAGAACCAGCTCTATAACGAGCTCAATCTGGGCAGAAGGGAGGAGTACGACGTGCTG
GACAAACGCCGCGGGAGGGACCCAGAAATGGGCGGGAAGCCTCGGAGAAAGAACCC
ACAAGAAGGACTGTATAACGAGCTACAGAAGGATAAGATGGCCGAGGCTTATAGCGA
AATCGGGATGAAGGGCGAAAGGCGCAGGGGAAAAGGGCACGACGGACTATACCAAG
GCCTCAGCACAGCTACAAAGGACACCTACGACGCTCTTCACATGCAGGCCTTGCCACC
AAGGTGA
Y117 CAR amino acid sequence (SEQ ID NO: 53)
MEFGLSWLFLVAILKGVQCQVQLVQSGAEVKQPGASVKVSCKASGYTFTDYAITWVRQA
PGQGLEWVGWINTDTGNPTYAQAFTGRLVFSLDTSVSTAYLQISSLKAEDTAVYYCARVT
PSGSLVHYDFWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSGAPGQRVTISCTG
SRSNTGAGFDVHWYQQLPGTAPKLLIYGNNNRPSGVPDRFSGSRSGTSASLAINGLQAEDE
ADYYCQSYDYNLSGVIFGGGTKLTALGSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFP
GPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCS
CRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG
GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR Y117 CAR nucleotide sequence (SEQ ID NO: 54)
ATGGAGTTTGGGCTGAGCTGGCTCTTCCTCGTGGCGATTCTCAAGGGAGTGCAGTGTC
AGGTCCAGCTCGTGCAGAGCGGCGCGGAGGTGAAACAGCCTGGCGCTAGCGTGAAAG
TAAGTTGTAAGGCGAGCGGTTACACTTTTACCGACTACGCGATCACTTGGGTGAGGCA
GGCTCCGGGTCAGGGCCTGGAGTGGGTAGGCTGGATCAATACAGACACCGGGAACCC
CACCTACGCCCAGGCCTTTACTGGCAGACTGGTCTTCAGCCTGGATACCTCCGTGTCAA
CTGCGTACCTCCAGATCTCCTCCCTGAAGGCCGAGGATACTGCGGTATACTATTGTGCC
CGGGTCACGCCCTCTGGCAGTCTGGTGCACTACGACTTTTGGGGGCAGGGGACCCTCG
TGACAGTCAGCAGTGGGGGAGGTGGGTCTGGAGGAGGTGGTTCCGGGGGTGGAGGAA
GCCAGAGCGTTTTAACACAGCCGCCTTCCGTGTCCGGGGCACCCGGACAGCGCGTTAC
GATTTCTTGCACCGGAAGTCGGTCCAATACGGGTGCCGGGTTCGACGTTCACTGGTATC
AGCAGCTCCCCGGAACTGCACCCAAACTACTGATCTACGGGAACAACAATAGGCCCTC
AGGCGTGCCTGACCGGTTTTCTGGCTCTAGATCTGGCACGAGCGCCAGCTTGGCGATA
AACGGGCTGCAAGCAGAGGACGAAGCCGACTACTACTGCCAAAGCTACGATTACAAT
CTGAGTGGAGTGATATTCGGCGGTGGAACTAAGCTTACCGCGCTCGGATCCATCGAAG
TGATGTACCCTCCCCCCTATCTGGACAACGAGAAATCCAACGGCACAATTATCCACGT
CAAGGGCAAGCACCTGTGCCCGTCTCCCCTGTTCCCAGGTCCTTCTAAACCTTTCTGGG
TGCTTGTGGTGGTCGGAGGTGTCCTGGCCTGCTACAGTTTGCTGGTGACCGTGGCCTTT
ATAATCTTTTGGGTGAAACGCGGCAGGAAGAAGCTCCTTTATATCTTCAAGCAACCCTT
TATGAGGCCGGTGCAGACTACCCAAGAGGAGGACGGGTGCTCGTGTAGATTCCCAGA
AGAGGAGGAAGGCGGCTGTGAACTGCGAGTCAAATTTAGCCGGTCTGCCGACGCTCCA
GCTTACCAGCAGGGGCAGAACCAGCTCTATAACGAGCTCAATCTGGGCAGAAGGGAG
GAGTACGACGTGCTGGACAAACGCCGCGGGAGGGACCCAGAAATGGGCGGGAAGCCT
CGGAGAAAGAACCCACAAGAAGGACTGTATAACGAGCTACAGAAGGATAAGATGGCC
GAGGCTTATAGCGAAATCGGGATGAAGGGCGAAAGGCGCAGGGGAAAAGGGCACGA
CGGACTATACCAAGGCCTCAGCACAGCTACAAAGGACACCTACGACGCTCTTCACATG
CAGGCCTTGCCACCAAGGTGA
YE5 CAR amino acid sequence (SEQ ID NO: 55)
MEFGLSWLFLVAILKGVQCQVQLQQSGPGLVKSSQTLSLTCATSGDSVSSNNAAWNWTRQ SPSRGLEWLGRTYYRSKWYNNYAESVKSRITISPDTSMNHFSLQLNSVTPEDTAVYYCARS RGFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSGAPGQRVTISCTGSSSNI GAGYDVHWYQQLPGTAPKLLIYGNTNRPSGVPDRFSASRSATSASLAITGLQAEDEADYY CQSHDSSLSAYVFGSGTKVTVLGSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSK PFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP
EEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR
Y55 CAR nucleotide sequence (SEQ ID NO: 56)
ATGGAGTTTGGGCTGAGCTGGCTGTTCCTCGTAGCGATCTTGAAGGGTGTCCAGTGTCA
GGTGCAGCTGCAGCAGTCAGGCCCTGGTCTAGTTAAAAGTAGTCAGACCCTGTCTCTC
ACGTGTGCGATATCAGGAGACTCGGTGAGTAGTAACAACGCAGCTTGGAATTGGATTC
GACAATCCCCTAGCCGCGGCCTGGAGTGGCTGGGGCGCACTTACTACCGCTCGAAGTG
GTACAATAATTACGCCGAGAGCGTAAAATCAAGGATCACAATCAGCCCAGATACCTCA
ATGAATCACTTCAGCTTGCAACTCAACTCAGTGACACCTGAGGACACAGCCGTGTATT
ACTGTGCTAGGTCTAGGGGCTTTTTTGACTACTGGGGCCAAGGCACCCTTGTCACTGTG TCAAGCGGCGGTGGCGGATCAGGAGGTGGAGGTTCTGGTGGAGGTGGATCACAGAGC
GTGCTCACTCAGCCGCCAAGCGTCAGTGGCGCCCCCGGCCAGAGGGTCACAATCAGCT
GTACAGGGTCCTCCTCGAATATCGGGGCAGGGTACGACGTGCACTGGTACCAGCAGCT
CCCAGGGACCGCACCCAAGCTACTGATTTACGGGAACACCAACCGACCCTCAGGGGTT
CCCGACAGGTTTAGCGCCTCTCGGAGCGCCACCAGCGCTAGCTTAGCTATCACCGGTC
TCCAGGCCGAGGACGAGGCTGATTATTACTGCCAGTCTCACGATTCTTCCCTAAGTGCC
TACGTTTTTGGGAGCGGCACTAAGGTGACAGTGCTCGGATCCATCGAAGTGATGTACC
CTCCCCCCTATCTGGACAACGAGAAATCCAACGGCACAATTATCCACGTCAAGGGCAA
GCACCTGTGCCCGTCTCCCCTGTTCCCAGGTCCTTCTAAACCTTTCTGGGTGCTTGTGGT
GGTCGGAGGTGTCCTGGCCTGCTACAGTTTGCTGGTGACCGTGGCCTTTATAATCTTTT
GGGTGAAACGCGGCAGGAAGAAGCTCCTTTATATCTTCAAGCAACCCTTTATGAGGCC
GGTGCAGACTACCCAAGAGGAGGACGGGTGCTCGTGTAGATTCCCAGAAGAGGAGGA
AGGCGGCTGTGAACTGCGAGTCAAATTTAGCCGGTCTGCCGACGCTCCAGCTTACCAG
CAGGGGCAGAACCAGCTCTATAACGAGCTCAATCTGGGCAGAAGGGAGGAGTACGAC
GTGCTGGACAAACGCCGCGGGAGGGACCCAGAAATGGGCGGGAAGCCTCGGAGAAAG
AACCCACAAGAAGGACTGTATAACGAGCTACAGAAGGATAAGATGGCCGAGGCTTAT
AGCGAAATCGGGATGAAGGGCGAAAGGCGCAGGGGAAAAGGGCACGACGGACTATA
CCAAGGCCTCAGCACAGCTACAAAGGACACCTACGACGCTCTTCACATGCAGGCCTTG
CCACCAAGGTGA
Also provided herein are CD276-specific monoclonal antibodies modified to enable their use with a universal CAR system. Universal CAR systems have been developed to increase CAR flexibility and expand their use to additional antigens. Currently, for each patient who receives CAR immune cell therapy, autologous immune cells (such as T cells) must be cultured, expanded, and modified to express an antigen-specific CAR. This process is lengthy and expensive, limiting its use. Universal CARs are based on a system in which the signaling components of the CAR are split from the antigen-binding portion of the molecule but come together using a “lock-key” system. For example, biotin-binding immune receptor (BBIR) CARs are comprised of an intracellular T cell signaling domain fused to an extracellular domain comprising avidin. Biotinylated antigen-specific (such as CD276-specific) monoclonal antibodies can then bind the BBIR to direct immune cells to antigen-expressing cells. Another example is the split, universal and programmable (SUPRA) CAR system. In the SUPRA system, the CAR includes the intracellular signaling domains fused to an extracellular leucine zipper, which is paired with an antigen-specific monoclonal antibody fused to a cognate leucine zipper. For a review of universal CAR systems, see, for example, Zhao el al., J Hematol Oncol 11 (1): 132, 2018; and Cho et al., Cell 173:1426-1438, 2018. In some aspects herein, the CD276-specific monoclonal antibody is fused to one component of a specific binding pair. In some examples, the monoclonal antibody is fused to a leucine zipper or biotin.
Another type of universal CAR can be generated using a sortase enzyme. A sortase is a prokaryotic enzyme that modifies surface proteins by recognizing and cleaving a carboxyl-terminal sorting signal. Sortase catalyzes transpeptidation between a sortase recognition motif and a sortase acceptor motif. Thus, antigen- specific CARs can be generated by contacting an antigen-specific antibody fused to a sortase recognition motif with a portion of a CAR molecule that includes the intracellular signaling domain(s), a transmembrane region and an extracellular portion that includes a sortase acceptor motif. In the presence of the sortase enzyme, the two components become covalently attached to form a complete antigen-specific CAR. Accordingly, in some aspects herein, a CD276-specific monoclonal antibody is modified to include a sortase recognition motif (see, for example, PCT Publication No. WO 2016/014553).
In some aspects, the CD276- targeted CAR is expressed in allogeneic immune cells, such as allogeneic T cells, B cells, NK cells, DCs or macrophages from a healthy donor(s). In some examples, the allogeneic immune cells are genetically engineered to express the CD276-targeted CAR, for example by disrupting expression of the endogenous T cell receptor by insertion of the CAR (see, for example, MacLeod et al., Mol Ther 25(4): 949-961, 2017). Gene editing can be performed using any appropriate gene editing system, such as CRISPR/Cas9, zinc finger nucleases or transcription activator-like effector nucleases (TALEN).
V. Immunoconjugates
The disclosed monoclonal antibodies can be conjugated to a therapeutic agent or effector molecule. Immunoconjugates include, but are not limited to, molecules in which there is a covalent linkage of a therapeutic agent to an antibody. A therapeutic agent is an agent with a particular biological activity directed against a particular target molecule or a cell bearing a target molecule. One of skill will appreciate that therapeutic agents can include various drugs, such as vinblastine, daunomycin and the like, cytotoxins such as native or modified Pseudomonas exotoxin or diphtheria toxin, encapsulating agents (such as liposomes) that contain pharmacological compositions, radioactive agents such as 1251, 32P, 14C, 3H and 35S, photon absorbers such as IR700, and other labels, target moieties and ligands.
The choice of a particular therapeutic agent depends on the target molecule or cell, and the desired biological effect. Thus, for example, the therapeutic agent can be a cytotoxin that is used to bring about the death of a particular target cell (such as a CD276-expressing cell). Conversely, where it is desired to invoke a non-lethal biological response, the therapeutic agent can be conjugated to a non-lethal pharmacological agent or a liposome containing a non-lethal pharmacological agent.
With the therapeutic agents and antibodies described herein, one of skill can readily construct a variety of clones containing functionally equivalent nucleic acids, such as nucleic acids which differ in sequence, but which encode the same effector moiety or antibody sequence. Thus, the present disclosure provides nucleic acids encoding antibodies and conjugates and fusion proteins thereof.
Effector molecules can be linked to an antibody of interest using any number of known means. Both covalent and noncovalent attachment means may be used. The procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups, such as carboxylic acid (COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules. The linker can be any molecule used to join the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
In some circumstances, it is desirable to free the effector molecule from the antibody when the immunoconjugate has reached its target site. Therefore, in these circumstances, immunoconjugates include linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.
In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, a skilled person will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.
The antibodies disclosed herein can be derivatized or linked to another molecule (such as another peptide or protein). In general, the antibodies or portion thereof is derivatized such that the binding to the target antigen is not affected adversely by the derivatization or labeling. For example, the antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bispecific antibody or a diabody), an Fc protein, a detection agent, a photon absorber, a pharmaceutical agent, and/or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, such as to create bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate). Such linkers are commercially available.
The antibody can be conjugated with a detectable marker, for example, a detectable marker capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as computed tomography (CT), computed axial tomography (CAT) scans, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging (NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non- limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. Bioluminescent markers are also of use, such as luciferase, green fluorescent protein (GFP) and yellow fluorescent protein (YFP). An antibody can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, P-galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody or antigen binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. An antibody or antigen binding fragment may also be conjugated with biotin and detected through indirect measurement of avidin or streptavidin binding. The avidin itself can be conjugated with an enzyme or a fluorescent label.
An antibody may be labeled with a magnetic agent, such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium), and manganese. Paramagnetic particles, such as superparamagnetic iron oxide, are also of use as labels. An antibody may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some aspects, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. An antibody can also be labeled with a radiolabeled amino acid. The radiolabel may be used for both diagnostic and therapeutic purposes. For instance, the radiolabel may be used to detect expression of a target antigen by x-ray, emission spectra, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: 3H, 14C, 15N, 35S, 90Y, "Tc, i nIn, 125I, and 131I.
An antibody disclosed herein can also be conjugated to a photon absorber. In some aspects, the photon absorber is a phthalocyanine dye, such as, but not limited to, IRDye® 700DX (also known as “IR700”). Antibody-photoabsorber conjugates can be used for photoimmunotherapy (for example to kill CD276-positive tumor cells).
An antibody can also be derivatized with a chemical group such as polyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrate group. These groups may be useful to improve the biological characteristics of the antibody, such as to increase serum half-life or to increase tissue binding.
Toxins can be employed with the monoclonal antibodies described herein to produce immunotoxins. Exemplary toxins include ricin, abrin, diphtheria toxin and subunits thereof, as well as botulinum toxins A through F. These toxins are readily available from commercial sources (for example, Sigma Chemical Company, St. Louis, MO). Contemplated toxins also include variants of the toxins described herein (see, for example, see, U.S. Patent Nos. 5,079,163 and 4,689,401). In one aspect, the toxin is Pseudomonas exotoxin (PE) (U.S. Patent No. 5,602,095). As used herein, "Pseudomonas exotoxin" refers to a full-length native (naturally occurring) PE or a PE that has been modified. Such modifications can include, but are not limited to, elimination of domain la, various amino acid deletions in domains lb, II and III, single amino acid substitutions and the addition of one or more sequences at the carboxyl terminus (for example, see Siegall et al. , J. Biol. Chem. 264: 14256-14261, 1989).
PE employed with the monoclonal antibodies described herein can include the native sequence, cytotoxic fragments of the native sequence, and conservatively modified variants of native PE and its cytotoxic fragments. Cytotoxic fragments of PE include those which are cytotoxic with or without subsequent proteolytic or other processing in the target cell. Cytotoxic fragments of PE include PE40, PE38, and PE35. For additional description of PE and variants thereof, see for example, U.S. Patent Nos. 4,892,827; 5,512,658; 5,602,095; 5,608,039; 5,821,238; and 5,854,044; U.S. Patent Application Publication No. 2015/0099707; PCT Publication Nos. WO 99/51643 and WO 2014/052064; Pai etal., Proc. Natl. Acad. Set. USA 88:3358-3362, 1991; Kondo et al., J. Biol. Chem. 263:9470-9475, 1988; and Pastan et al., Biochim. Biophys. Acta 1333:C1-C6, 1997. Also contemplated herein are protease-resistant PE variants and PE variants with reduced immunogenicity, such as, but not limited to PE-LR, PE-6X, PE-8X, PE-LR/6X and PE-LR/8X (see, for example, Weldon et al., Blood 113(161:3792-3800, 2009; Onda et al., Proc Natl Acad Sci USA 105(32):11311-11316, 2008; and PCT Publication Nos. WO 2007/016150, WO 2009/032954 and WO 2011/0320221.
In some examples, the PE is a variant that is resistant to lysosomal degradation, such as PE- LR (Weldon et al., Blood 113(161:3792-3800, 2009; PCT Publication No. WO 2009/0329541. In other examples, the PE is a variant designated PE-LR/6X (PCT Publication No. WO 2011/0320221. In other examples, the PE variant is PE with reducing immunogenicity. In yet other examples, the PE is a variant designated PE-LR/8M (PCT Publication No. WO 2011/0320221.
Modification of PE may occur in any previously described variant, including cytotoxic fragments of PE (for example, PE38, PE-LR and PE-LR/8M). Modified PEs may include any substitution(s), such as for one or more amino acid residues within one or more T-cell epitopes and/or B cell epitopes of PE, or deletion of one or more T-cell and/or B-cell epitopes (see, for example, U.S. Patent Application Publication No. 2015/00997071.
Contemplated forms of PE also include deimmunized forms of PE, for example versions with domain II deleted (for example, PE24). Deimmunized forms of PE are described in, for example, PCT Publication Nos. WO 2005/052006, WO 2007/016150, WO 2007/014743, WO 2007/031741, WO 2009/32954, WO 2011/32022, WO 2012/154530, and WO 2012/170617.
The antibodies described herein can also be used to target any number of different diagnostic or therapeutic compounds to cells expressing CD276 on their surface (e.g., CD276- positive tumor cells). Thus, an antibody of the present disclosure can be attached directly or via a linker to a drug that is to be delivered directly to cells expressing CD276. This can be done for therapeutic, diagnostic or research purposes. Therapeutic agents include such compounds as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, photon absorbers, lipids, carbohydrates, and recombinant viruses. Nucleic acid therapeutic and diagnostic moieties include antisense nucleic acids, derivatized oligonucleotides for covalent cross-linking with single or duplex DNA, and triplex forming oligonucleotides.
Alternatively, the molecule linked to an antibody can be an encapsulation system, such as a nanoparticle, liposome or micelle that contains a therapeutic composition such as a drug, a nucleic acid (for example, an antisense nucleic acid), or another therapeutic moiety that is shielded from direct exposure to the circulatory system. Means of preparing liposomes attached to antibodies are known (see, for example, U.S. Patent No. 4,957,735; Connor et al., Pharm. Ther. 28:341-365, 1985). Antibodies described herein can also be covalently or non-covalently linked to a detectable label. Detectable labels suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels include magnetic beads, fluorescent dyes (for example, fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (for example, 3H, 1251, 35S, 14C, or 32P), enzymes (such as horseradish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (such as polystyrene, polypropylene, latex, and the like) beads.
Means of detecting such labels are known. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
VI. Antibody-Drug Conjugates (ADCs)
ADCs are compounds comprised of an antigen-specific antibody (such as a monoclonal antibody or antigen-binding fragment of an immunoglobulin provided herein that binds CD276) and a drug, for example a cytotoxic agent (such as an anti-microtubule agent or cross-linking agent). Because ADCs are capable of specifically targeting cells expressing a particular antigen, the drug can be much more potent than agents used for standard systemic therapy. For example, the most common cytotoxic drugs currently used with ADCs have an IC50 that is 100- to 1000-fold more potent than conventional chemotherapeutic agents. Common cytotoxic drugs include antimicrotubule agents, such as maytansinoids and auristatins (such as auristatin E and auristatin F). Other cytotoxins for use with ADCs include pyrrolobenzodiazepines (PBDs), which covalently bind the minor groove of DNA to form interstrand crosslinks. In many instances, ADCs comprise a 1:2 to 1:4 ratio of antibody to drug (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012).
The antibody and drug can be linked by a cleavable or non-cleavable linker. However, in some instances, it is desirable to have a linker that is stable in the circulation to prevent systemic release of the cytotoxic drug that could result in significant off-target toxicity. Non-cleavable linkers prevent release of the cytotoxic agent before the ADC is internalized by the target cell. Once in the lysosome, digestion of the antibody by lysosomal proteases results in the release of the cytotoxic agent (Bander, Clinical Advances in Hematology & Oncology 10(8; suppl 10):3-7, 2012). One method for site-specific and stable conjugation of a drug to a monoclonal antibody (or antigen-biding fragment fused to Fc) is via glycan engineering. Monoclonal antibodies have one conserved N-linked oligosaccharide chain at the Asn297 residue in the CH2 domain of each heavy chain (Qasba et al., Biotechnol Prog 24:520-526, 2008). Using a mutant > 1,4- galactosyltransferase enzyme (Y289L-Gal-Tl; U.S. Patent Application Publication Nos. 2007/0258986 and 2006/0084162), 2-keto-galactose is transferred to free GlcNAc residues on the antibody heavy chain to provide a chemical handle for conjugation.
The oligosaccharide chain attached to monoclonal antibodies can be classified into three groups based on the terminal galactose residues - fully galactosylated (two galactose residues; IgG- G2), one galactose residue (IgG-Gl) or completely degalactosylated (IgG-GO). Treatment of a monoclonal antibody with [31,4-galactosidase converts the antibody to the IgG-GO glycoform. The mutant [31,4-galactosyltransferase enzyme is capable of transferring 2-keto-galactose or 2-azido- galactose from their respective UDP derivatives to the GlcNAc residues on the IgG-Gl and IgG-GO glycoforms. The chemical handle on the transferred sugar enables conjugation of a variety of molecules to the monoclonal antibody via the glycan residues (Qasba et al. , Biotechnol Prog 24:520-526, 2008).
Provided herein are ADCs that include a drug (such as a cytotoxic agent) conjugated to a monoclonal antibody that binds (such as specifically binds) CD276. In some aspects, the drug is a small molecule. In some examples, the drug is a cross-linking agent, an anti-microtubule agent and/or anti-mitotic agent, or any cytotoxic agent suitable for mediating killing of tumor cells. Exemplary cytotoxic agents include, but are not limited to, a PBD, an auristatin, a maytansinoid, dolastatin, calicheamicin, nemorubicin and its derivatives, PNU- 159682, anthracycline, vinca alkaloid, taxane, trichothecene, CC1065, camptothecin, elinafide, a combretastain, a dolastatin, a duocarmycin, an enediyne, a geldanamycin, an indolino-benzodiazepine dimer, a puromycin, a tubulysin, a hemiasterlin, a spliceostatin, or a pladienolide, as well as stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.
In some aspects, the ADC includes a pyrrolobenzodiazepine (PBD). The natural product anthramycin (a PBD) was first reported in 1965 (Leimgruber et al., J Am Chem Soc, 87:5793-5795, 1965; Leimgruber et al. , J Am Chem Soc, 87:5791-5793, 1965). Since then, a number of PBDs, both naturally-occurring and synthetic analogues, have been reported (Gerratana, Med Res Rev 32(2):254-293, 2012; and U.S. Patent Nos. 6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; and 7,557,099). As one example, PBD dimers recognize and bind to specific DNA sequences, and have been shown to be useful as cytotoxic agents. PBD dimers have been conjugated to antibodies and the resulting ADC shown to have anti-cancer properties (see, for example, US 2010/0203007). Exemplary linkage sites on the PBD dimer include the fivemembered pyrrolo ring, the tether between the PBD units, and the N10-C11 imine group (see WO 2009/016516; US 2009/304710; US 2010/047257; US 2009/036431; US 2011/0256157; and WO 2011/130598).
In some aspects, the ADC includes an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine and are mitotic inhibitors which act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Patent No. 4,151,042). Synthetic maytansinoids are disclosed, for example, in U.S. Patent Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533.
In some aspects, the ADC includes an antibody conjugated to a dolastatin or auristatin, or an analog or derivative thereof (see U.S. Patent Nos. 5,635,483; 5,780,588; 5,767,237; and 6,124,431). Auristatins are derivatives of the marine mollusk compound dolastatin- 10. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al., Antimicrob Agents and Chemother 45(12):3580-3584, 2001) and have anticancer (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob Agents Chemother 42:2961-2965, 1998). Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin F, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (Monomethyl Auristatin D or monomethyl dolastatin 10), MMAF (Monomethyl Auristatin F or N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE (Monomethyl Auristatin E or N-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other auristatins (see, for example, U.S. Publication No. 2013/0129753).
In some aspects, the ADC includes an antibody conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics, and analogues thereof, can produce doublestranded DNA breaks at sub-picomolar concentrations (Hinman et al. , Cancer Res 53:3336-3342, 1993; Lode et al. , Cancer Res 58:2925-2928, 1998). Exemplary methods for preparing ADCs with a calicheamicin drug moiety are described in U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.
In some aspects, the ADC includes an anthracycline. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. It is believed that anthracyclines can operate to kill cells by a number of different mechanisms, including intercalation of the drug molecules into the DNA of the cell thereby inhibiting DNA-dependent nucleic acid synthesis; inducing production of free radicals which then react with cellular macromolecules to cause damage to the cells; and/or interactions of the drug molecules with the cell membrane. Non-limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, daunorubicin, doxorubicin, epirubicin, nemorubicin, valrubicin and mitoxantrone, and derivatives thereof. For example, PNU- 159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al. , Clin Cancer Res 11(4): 1608- 1617, 2005). Nemorubicin is a semisynthetic analog of doxorubicin with a 2-methoxymorpholino group on the glycoside amino of doxorubicin (Grand! et al. , Cancer Treat Rev 17:133, 1990; Ripamonti et al., Br J Cancer 65:703-707, 1992).
In some aspects, the ADC can further include a linker. In some examples, the linker is a bifunctional or multifunctional moiety that can be used to link one or more drug moieties to an antibody to form an ADC. In some aspects, ADCs are prepared using a linker having reactive functionalities for covalently attaching to the drug and to the antibody. For example, a cysteine thiol of an antibody can form a bond with a reactive functional group of a linker or a drug-linker intermediate to make an ADC.
In some examples, a linker has a functionality that can react with a free cysteine present on an antibody to form a covalent bond. Exemplary linkers with such reactive functionalities include maleimide, haloacetamides, > -haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.
In some examples, a linker has a functionality that can react with an electrophilic group present on an antibody. Examples of such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some cases, a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Non-limiting examples include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide.
In some examples, the linker is a cleavable linker, which facilitates release of the drug. Examples of cleavable linkers include acid-labile linkers (for example, comprising hydrazone), protease-sensitive linkers (for example, peptidase-sensitive), photolabile linkers, and disulfide- containing linkers (Chari et al. , Cancer Res 52:127-131, 1992; U.S. Patent No. 5,208,020). The ADCs disclosed herein can be used for the treatment of a CD276-positive tumor alone or in combination with another therapeutic agent and/or in combination with any standard therapy for the treatment of a CD276-positive cancer. VII. Multi-specific Antibodies
Multi-specific antibodies are recombinant proteins comprised of two or more monoclonal antibodies or antigen-binding fragments of two or more different monoclonal antibodies. For example, bispecific antibodies are comprised of two different monoclonal antibodies or antigenbinding fragments thereof. Thus, bispecific antibodies bind two different antigens (or two different epitopes of an antigen) and trispecific antibodies bind three different antigens (or three different epitopes of an antigen).
Provided herein are multi- specific, such as trispecific or bispecific, monoclonal antibodies that include a first CD276- specific monoclonal antibody. In some aspects, the multi- specific monoclonal antibody further includes a second antibody that specifically binds a different epitope of CD276 or a different cell-surface antigen. In some aspects, the multi-specific monoclonal antibody further includes a second antibody that specifically binds PD-1 (such as nivolumab, JTX- 4014 by Jounce Therapeutics, nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IB 1308), tislelizumab (BGB-A317), toripalimab (JS 001), dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, or AMP- 514). In some aspects, the multi- specific monoclonal antibody further includes a second antibody that specifically binds CTLA-4 (such as ipilimumab or tremelimumab).
In some aspects disclosed herein, the multi- specific monoclonal antibody includes a first monoclonal antibody specific for CD276 (such as a CD276-specific scFv) and further includes a monoclonal antibody (or antigen-binding fragment such as an scFv) that specifically binds a component of the T cell receptor, such as CD3. In other aspects, the multi- specific monoclonal antibody includes a first monoclonal antibody specific for CD276 (such as a CD276- specific scFv) and further includes a monoclonal antibody (or antigen-binding fragment such as an scFv) that specifically binds a NK cell activating receptor, such as CD16, Ly49, or CD94.
Also provided are isolated nucleic acid molecules and vectors encoding the multi-specific antibodies, and host cells comprising the nucleic acid molecules or vectors. Multi- specific antibodies that include a CD276- specific antibody can be used for the treatment of a CD276- positive cancer. Thus, provided herein are methods of treating a subject with a CD276-positive cancer by administering to the subject a therapeutically effective amount of the CD276-targeting multi- specific antibody. VIII. Antibody-Nanoparticle Conjugates
The monoclonal antibodies disclosed herein can be conjugated to a variety of different types of nanoparticles to deliver cytotoxic agents directly to CD276-expressing cells via binding of the antibody to CD276 expressed on the surface of cells. The use of nanoparticles reduces off-target side effects and can also improve drug bioavailability and reduce the dose of a drug required to achieve a therapeutic effect. Nanoparticle formulations can be tailored to suit the drug that is to be carried or encapsulated within the nanoparticle. For example, hydrophobic molecules can be incorporated inside the core of a nanoparticle, while hydrophilic drugs can be carried within an aqueous core protected by a polymeric or lipid shell. Examples of nanoparticles include, but at not limited to, nanospheres, nanocapsules, liposomes, dendrimers, polymeric micelles, niosomes, and polymeric nanoparticles (Fay and Scott, Immunotherapy 3(3):381-394, 2011).
Liposomes are common types of nanoparticles used for drug delivery. An antibody conjugated to a liposome is often referred to as an “immunoliposome.” The liposomal component of an immunoliposome is typically a lipid vesicle of one or more concentric phospholipid bilayers. In some cases, the phospholipids are composed of a hydrophilic head group and two hydrophobic chains to enable encapsulation of both hydrophobic and hydrophilic drugs. Conventional liposomes are rapidly removed from the circulation via macrophages of the reticuloendothelial system (RES). To generate long-circulating liposomes, the composition, size and charge of the liposome can be modulated. The surface of the liposome may also be modified, such as with a glycolipid or sialic acid. For example, the inclusion of polyethylene glycol (PEG) significantly increases circulation half-life. Liposomes for use as drug delivery agents, including for preparation of immunoliposomes, have been described (see, for example, Paszko and Senge, Curr Med Chem 19(31)5239-5277, 2012; Immordino et al., Int J Nanomedicine l(3):297-315, 2006; U.S. Patent Application Publication Nos. 2011/0268655; 2010/00329981).
Niosomes are non-ionic surfactant-based vesicles having a structure similar to liposomes. The membranes of niosomes are composed only of nonionic surfactants, such as polyglyceryl- alkyl ethers or /V-palmitoylgl ucosamine. Niosomes range from small, unilamellar to large, multilamellar particles. These nanoparticles are monodisperse, water-soluble, chemically stable, have low toxicity, are biodegradable and non-immunogenic, and increase bioavailability of encapsulated drugs.
Dendrimers include a range of branched polymer complexes. These nanoparticles are water-soluble, biocompatible and are sufficiently non-immunogenic for human use. Generally, dendrimers consist of an initiator core, surrounded by a layer of a selected polymer that is grafted to the core, forming a branched macromolecular complex. Dendrimers are typically produced using polymers such as poly(amidoamine) or poly(L-lysine). Dendrimers have been used for a variety of therapeutic and diagnostic applications, including for the delivery of DNA, RNA, bioimaging contrast agents, chemotherapeutic agents and other drugs.
Polymeric micelles are composed of aggregates of amphiphilic co-polymers (consisting of both hydrophilic and hydrophobic monomer units) assembled into hydrophobic cores, surrounded by a corona of hydrophilic polymeric chains exposed to the aqueous environment. In many cases, the polymers used to prepare polymeric micelles are heterobifunctional copolymers composed of a hydrophilic block of PEG, poly(vinyl pyrrolidone) and hydrophobic poly(L-lactide) or poly(L- lysine) that forms the particle core. Polymeric micelles can be used to carry drugs that have poor solubility. These nanoparticles have been used to encapsulate a number of drugs, including doxorubicin and camptothecin. Cationic micelles have also been developed to carry DNA or RNA molecules.
Polymeric nanoparticles include both nanospheres and nanocapsules. Nanospheres consist of a solid matrix of polymer, while nanocapsules contain an aqueous core. The formulation selected typically depends on the solubility of the therapeutic agent to be carried/encapsulated; poorly water-soluble drugs are more readily encapsulated within nanospheres, while water-soluble and labile drugs, such as DNA and proteins, are more readily encapsulated within nanocapsules. The polymers used to produce these nanoparticles include, for example, poly(acrylamide), poly(ester), poly(alkylcyanoacrylates), poly(lactic acid) (PLA), poly(glycolic acids) (PGA), and poly(D,L-lactic-co-glycolic acid) (PLGA).
Antibodies can be conjugated to a suitable nanoparticle according to standard known methods. For example, conjugation can be either covalent or non-covalent. In some aspects in which the nanoparticle is a liposome, the antibody is attached to a sterically stabilized, long circulation liposome via a PEG chain. Coupling of antibodies or antibody fragments to a liposome can also involve thioester bonds, for example by reaction of thiols and maleimide groups. Crosslinking agents can be used to create sulfhydryl groups for attachment of antibodies to nanoparticles (Paszko and Senge, Curr Med Chem 19(31)5239-5277, 2012).
IX. Nucleic Acid Molecules
Nucleic acid molecules (for example, DNA, cDNA, mRNA, or RNA molecules) encoding the amino acid sequences of the disclosed antibodies, fusion proteins, CARs and other conjugates that specifically bind to CD276, are provided. Nucleic acid molecules encoding these molecules can readily be produced using the amino acid sequences provided herein (such as the CDR sequences and the variable domain sequences), sequences available (such as framework or constant region sequences), and the genetic code. In some aspects, the nucleic acid molecules can be expressed in a host cell (such as a mammalian cell or a bacterial cell) to produce a disclosed antibody, fusion protein or antibody conjugate (e.g. , CAR, immunotoxin, multi-specific antibody).
In some aspects, the nucleotide sequence of a nucleic acid molecule encoding a CD276- specific monoclonal antibody (or portion thereof) disclosed herein is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 1-40. In some examples, the nucleotide sequence of the nucleic acid molecule encoding a disclosed antibody (or portion thereof) includes or consists of any one of SEQ ID NOs: 1-40.
In some aspects, a nucleic acid molecule encoding a CD276-targeted CAR is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 48, 50, 52, 54, 56, 62 and 64, or a degenerate variant thereof. In some examples, the nucleotide sequence encoding the CAR includes or consists of any one of SEQ ID NOs: 48, 50, 52, 54, 56, 62 and 64, or a degenerate variant thereof
The genetic code can be used to construct a variety of functionally equivalent nucleic acid sequences, such as nucleic acids that differ in their sequence, but which encode the same antibody sequence, or encode a conjugate or fusion protein including the antibody sequence.
Nucleic acid molecules encoding the antibodies, fusion proteins, CARs and other conjugates that specifically bind to CD276 can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by standard methods. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: lohn Wiley and Sons, including supplements).
Nucleic acids can also be prepared by amplification methods. Amplification methods include the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcriptionbased amplification system (TAS), and the self-sustained sequence replication system (3SR).
The nucleic acid molecules can be expressed in a recombinantly engineered cell such as in bacterial, plant, yeast, insect and mammalian cells. The antibodies and conjugates can be expressed as individual proteins including the antibody (linked to an effector molecule or detectable marker as needed) or can be expressed as a fusion protein. Any suitable method of expressing and purifying antibodies and antigen binding fragments may be used; non-limiting examples are provided in Al- Rubeai (Ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011).
One or more DNA sequences encoding the antibodies, CARs, fusion proteins, or other conjugates can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. Numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells, for example mammalian cells, such as the COS, CHO, HeLa and myeloma cell lines, can be used to express the disclosed antibodies and antigen binding fragments. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, may be used.
The expression of nucleic acids encoding the antibodies, CARs and other conjugates described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette. The promoter can be any promoter of interest, including a cytomegalovirus promoter. Optionally, an enhancer, such as a cytomegalovirus enhancer, is included in the construct. The cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences useful for regulation of the expression of the DNA encoding the protein. For example, the expression cassettes can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signals for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The vector can encode a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).
To obtain high level expression of a cloned gene, expression cassettes can contain, for example, a strong promoter to direct transcription, a ribosome binding site for translational initiation (e.g., internal ribosomal binding sequences), and a transcription/translation terminator. For E. coli, this can include a promoter such as the T7, trp, lac, or lambda promoters, a ribosome binding site, and a transcription termination signal. For eukaryotic cells, the control sequences can include a promoter and/or an enhancer derived from, for example, an immunoglobulin gene, HTLV, SV40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and/or acceptor sequences (for example, CMV and/or HTLV splice acceptor and donor sequences). The cassettes can be transferred into the chosen host cell by any suitable method such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.
Modifications can be made to a nucleic acid encoding an antibody described herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the antibody into a fusion protein. Such modifications include, for example, termination codons, sequences to create conveniently located restriction sites, and sequences to add a methionine at the amino terminus to provide an initiation site, or additional amino acids (such as poly His) to aid in purification steps.
Once expressed, the antibodies, CARs, fusion proteins, and other conjugates can be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009). The antibodies, CARs, fusion proteins, and other conjugates need not be 100% pure. Once purified, partially or to homogeneity as desired, if to be used prophylactically, the antibodies should be substantially free of endotoxin.
Methods for expression of antibodies, CARs, fusion proteins, and conjugates, and/or refolding to an appropriate active form, from mammalian cells, and bacteria such as E. coli have been described and are applicable to the antibodies disclosed herein. See, e.g., Greenfield (Ed.), Antibodies: A Laboratory Manual, 2nd ed. New York: Cold Spring Harbor Laboratory Press, 2014, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009, and Ward et al., Nature 341(6242):544- 546, 1989.
X. Compositions and Administration
Compositions are provided that include one or more of the disclosed monoclonal antibodies that bind (for example specifically bind) CD276 in a carrier. Compositions comprising fusion proteins (such as scFv-Fc fusion proteins), ADCs, CARs (and immune cells expressing CARs), multi- specific (such as bispecific or trispecific) antibodies, antibody -nanoparticle conjugates, immunoliposomes and immunoconjugates are also provided, as are nucleic acid molecule and vectors encoding the antibodies or antibody conjugates. The compositions can be prepared in unit dosage form for administration to a subject. The amount and timing of administration are at the discretion of the treating clinician to achieve the desired outcome. The antibody, fusion protein, ADC, CAR, CAR-expressing cell, multi-specific antibody, antibody-nanoparticle conjugate, immunoliposome or immunoconjugate can be formulated for systemic or local administration. The compositions for administration can include a solution of the antibody, fusion protein, ADC, CAR, CAR-expressing cell (such as a T cell, B cell, DC, NK cell, macrophage or iPSC), multi- specific (such as bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome or immunoconjugate in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventional sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject’s needs.
An exemplary pharmaceutical composition for intravenous administration includes about 0.1 to 10 mg of an antibody (or fusion protein, ADC, CAR, multi-specific antibody, antibody- nanoparticle conjugate, or immunoconjugate), per subject per day. Dosages from 0. 1 up to about 100 mg per subject per day may be used, particularly if the agent is administered to a secluded site and not into the circulatory or lymph system, such as into a body cavity or into a lumen of an organ. In some aspects, the composition can be a liquid formulation including one or more antibodies in a concentration range from about 0. 1 mg/ml to about 20 mg/ml, or from about 0.5 mg/ml to about 20 mg/ml, or from about 1 mg/ml to about 20 mg/ml, or from about 0.1 mg/ml to about 10 mg/ml, or from about 0.5 mg/ml to about 10 mg/ml, or from about 1 mg/ml to about 10 mg/ml. Actual methods for preparing administrable compositions will be known or apparent to a skilled person and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21st Edition (2005).
Monoclonal antibodies (or antibody conjugates, or nucleic acid molecules encoding such molecules) may be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. The antibody solution can be added to an infusion bag containing 0.9% sodium chloride, USP, and in some cases administered at a dosage of from 0.5 to 15 mg/kg of body weight. Considerable experience is available in the administration of antibody drugs, which have been marketed in the U.S. since the approval of RITUXAN™ in 1997. Antibodies, Fc fusion proteins, ADCs, CARs (or CAR-expressing cells), multi-specific (such as bispecific or trispecific) antibodies, antibody- nanoparticle conjugates, immunoliposomes or immunoconjugates can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30-minute period if the previous dose was well tolerated.
Controlled release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, A. J., Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Technomic Publishing Company, Inc., Lancaster, PA, (1995). Particulate systems include, for example, microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the therapeutic protein, such as a cytotoxin or a drug, as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 pm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 pm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 pm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, (1992).
Polymers can be used for ion-controlled release of the antibody-based compositions disclosed herein. Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known (Langer, Accounts Chem. Res. 26:537-542, 1993). For example, the block copolymer, poloxamer 407, exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It is an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992; and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al. , Int. J. Pharm. 112:215-224, 1994). In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid- capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA (1993)). Numerous additional systems for controlled delivery of therapeutic proteins are known (see U.S. Patent Nos. 5,055,303; 5,188,837; 4,235,871 ; 4,501 ,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206; 5,271,961; 5,254,342 and 5,534,496). XL Therapeutic Methods
The antibodies, compositions, CARs (and CAR-expressing immune cells or iPSCs), ADCs, multi- specific (such as bispecific or trispecific) antibodies, antibody-nanoparticle conjugates, immunoliposomes and immunoconjugates disclosed herein can be administered to slow or inhibit the growth of tumor cells or inhibit the metastasis of tumor cells, such as a CD276-positive solid tumor. In these applications, a therapeutically effective amount of a composition is administered to a subject in an amount sufficient to inhibit growth, replication or metastasis of cancer cells, or to inhibit a sign or a symptom of the cancer. Suitable subjects may include those diagnosed with a solid tumor that expresses CD276, such as, but not limited to, pancreatic cancer, neuroblastoma, liver cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer, pediatric cancer, melanoma or mesothelioma.
Provided herein is a method of treating a CD276-positive cancer in a subject by administering to the subject a therapeutically effective amount of a CD276-specific monoclonal antibody, immunoconjugate, CAR (or an immune cell or iPSC expressing a CAR), ADC, multispecific (such as bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome or composition disclosed herein. Also provided herein is a method of inhibiting tumor growth or metastasis of a CD276-positive cancer in a subject by administering to the subject a therapeutically effective amount of a CD276-specific monoclonal antibody, immunoconjugate, CAR (such as an immune cell or iPSC expressing a CAR), ADC, multi- specific (such as bispecific or trispecific) antibody, antibody-nanoparticle conjugate, immunoliposome or composition disclosed herein. In some aspects, the CD276-positive cancer is a solid tumor, such as pancreatic cancer, neuroblastoma, liver cancer, kidney cancer, bladder cancer, cervical cancer, esophageal cancer, prostate cancer, breast cancer, ovarian cancer, colon cancer, lung cancer, brain cancer, pediatric cancer, melanoma or mesothelioma.
The tumor does not need to be completely eliminated or inhibited for the method to be effective. For example, the method can decrease tumor size (e.g., volume) or metastasis by a particular amount, for example by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100% as compared to the absence of the treatment. In one example, the method can decrease tumor size (e.g., volume) or metastasis by a particular amount, for example by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or even 100% as compared to treatment with a different CD276 antibody or CD276 CAR (e.g., one not disclosed herein).
Also provided herein is a method of preventing or reducing the risk of allograft rejection in a subject who has received an allograft. In some aspects, the method includes administering to the subject a therapeutically effective amount of an isolated cell expressing a CD276-targeted CAR disclosed herein. In some examples, the isolated cell is an immune cell, such as a T cell, B cell, NK cell, macrophage, DC, or an iPSC.
A therapeutically effective amount of a CD276-specific monoclonal antibody, ADC, CAR (for example an immune cell or iPSC expressing a CAR), multi- specific (such as bispecific or trispecific) antibody, immunoconjugate, immunoliposome or composition disclosed herein will depend upon the severity of the disease, the type of disease, and the general state of the patient’s health. A therapeutically effective amount of the antibody-based composition is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer. Antibodies and conjugates thereof can be administered, for example, by intravenous infusion. Doses of the antibody or conjugate thereof can vary, but generally range between about 0.5 mg/kg to about 50 mg/kg, such as a dose of about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg. In some aspects, the dose of the antibody or conjugate can be from about 0.5 mg/kg to about 5 mg/kg, such as a dose of about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg or about 5 mg/kg. In some aspects, the dose of CAR-expressing immune cells or iPSCs is 104 to 109 cells/kg body weight, such as 105 to 106 cells/kg body weight, including all integer values within those ranges. Exemplary doses are 106 cells/kg to about 108 cells/kg, such as from about 5 x 106 cells/kg to about 7.5 x 107 cells/kg, such as at about 2.5 x 107 cells/kg, or at about 5.0 x 107 cells/kg.
The antibody or conjugate is administered according to a dosing schedule determined by a medical practitioner. In some examples, the antibody or conjugate is administered weekly, every two weeks, every three weeks, or every four weeks.
In some aspects, a subject is administered DNA or RNA encoding a disclosed antibody to provide in vivo antibody production, for example using the cellular machinery of the subject. Any suitable method of nucleic acid administration may be used; non-limiting examples are provided in U.S. Patent No. 5,643,578, U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637. U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding proteins to an organism. One approach to administration of nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibody, or antigen binding fragments thereof, can be placed under the control of a promoter to increase expression. The methods include liposomal delivery of the nucleic acids. Such methods can be applied to the production of an antibody, or antigen binding fragments thereof.
In several aspects, a subject (such as a human subject with a CD276-positive tumor) is administered an effective amount of a viral vector that includes one or more nucleic acid molecules encoding a disclosed antibody. The viral vector is designed for expression of the nucleic acid molecules encoding a disclosed antibody, and administration of the effective amount of the viral vector to the subject leads to expression of an effective amount of the antibody in the subject. Nonlimiting examples of viral vectors that can be used to express a disclosed antibody or antigen binding fragment in a subject include those provided in Johnson et al., Nat. Med., 15(8):901 -906, 2009 and Gardner er al., Nature, 519(7541):87-91, 2015.
In one aspect, a nucleic acid encoding a disclosed antibody, or conjugate thereof, is introduced directly into tissue. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOS™ Gene Gun. The nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter.
Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 pg/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Patent No. 5,589,466).
Single or multiple administrations of a composition including a disclosed antibody or antibody conjugate, or nucleic acid molecule encoding such molecules, can be administered depending on the dosage and frequency as required and tolerated by the patient. The dosage can be administered once but may be applied periodically until either a desired result is achieved or until side effects warrant discontinuation of therapy. Generally, the dose is sufficient to inhibit growth or metastasis of a CD276-positive cancer without producing unacceptable toxicity to the patient.
Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. The dosage normally lies within a range of circulating concentrations that include the EDso, with little or minimal toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
The CD276-specific antibody, antibody conjugate, nucleic acid molecule encoding such molecules, or a composition including such molecules, can be administered to subjects in various ways, including local and systemic administration, such as, e.g., by injection subcutaneously, intravenously, intra-arterially, intraperitoneally, intramuscularly, intradermally, or intrathecally. In some aspects, the composition is administered by inhalation, such as by using an inhaler. In one aspect, the antibody, antigen binding fragment, or nucleic acid molecule encoding such molecules, or a composition including such molecules, is administered by a single subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal or intrathecal injection once a day. The antibody, antigen binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules, can also be administered by direct injection at or near the site of disease. A further method of administration is by osmotic pump (e.g., an ALZET pump) or mini-pump (e.g., an ALZET mini-osmotic pump), which allows for controlled, continuous and/or slow-release delivery of the antibody, antibody conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules, over a pre-determined period. The osmotic pump or mini-pump can be implanted subcutaneously, or near a target site.
In one example, a CD276- specific antibody provided herein is conjugated to IR700, and photoimmunotherapy is used to treat a CD276-positive cancer. For example, such a method can include administering to the subject with a CD276-positive cancer a therapeutically effective amount of one or more CD276-specific antibody-IR700 conjugates, wherein the CD276-specific antibody specifically binds to CD276-expressing cells. Following administration of the conjugate, irradiation is performed at a wavelength of 660 to 740 nm (such as 660 to 710 nm, for example, 680 nm) and at a dose of at least 1 J cm'2 (such as at least 1 to about 50 J cm'2) thereby treating the CD276-positive cancer in the subject. In some examples, the CD276-positive tumor is irradiated at a wavelength of 660 to 740 nm (such as 660 to 710 nm, for example, 680 nm) at a dose of at least 1 J cm'2 (such as at least 1 J cm'2, at least 4 J cm'2, at least 10 J cm'2, at least 50 J cm'2, or at least 100 J cm'2) thereby treating the cancer in the subject. In some examples, multiple rounds of treatment are performed, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 treatment cycles. In particular examples, a therapeutically effective dose of a CD276-specific antibody-IR700 conjugates is at least 0.5 milligram per 60 kilogram (mg/kg), at least 5 mg/60 kg, at least 10 mg/60 kg, at least 20 mg/60 kg, at least 30 mg/60 kg, at least 50 mg/60 kg, for example 0.5 to 50 mg/60 kg, such as a dose of 1 mg/ 60 kg, 2 mg/60 kg, 5 mg/60 kg, 20 mg/60 kg, or 50 mg/60 kg, for example when administered intravenously. In another example, a therapeutically effective dose of a CD276- specific antibody- IR700 conjugates is at least 10 pg/kg, such as at least 100 pg/kg, at least 500 pg/kg, or at least 500 pg/kg, for example 10 pg/kg to 1000 pg/kg, such as a dose of 100 pg/kg, 250 pg/kg, about 500 pg/kg, 750 pg/kg, or 1000 pg/kg, for example when administered i.p. In one example, a therapeutically effective dose of an CD276- specific antibody-IR700 conjugates is at least 1 pg/ml, such as at least 500 pg/ml, such as between 20 pg/ml to 100 pg/ml, such as 10 pg/ml, 20 pg/ml, 30 pg/ml, 40 pg/ml, 50 pg/ml, 60 pg/ml, 70 pg/ml, 80 pg/ml, 90 pg/ml or 100 pg/ml administered in a topical solution. In some aspects, the treatment methods further include administration of other anti-cancer agents or therapeutic treatments. Any suitable anti-cancer agent can be administered in combination with the compositions disclosed herein. Exemplary anti-cancer agents include, but are not limited to, chemotherapeutic agents, such as, for example, mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormones (e.g. anti-androgens) and anti-angiogenesis agents. Other anti-cancer treatments include radiation therapy and other antibodies that specifically target cancer cells.
Non-limiting examples of alkylating agents include nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).
Non-limiting examples of antimetabolites include folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.
Non-limiting examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L- asparaginase).
Non-limiting examples of miscellaneous agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide).
Non-limiting examples of hormones and antagonists include adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testosterone propionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU, Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin, Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, such as docetaxel), Velban, Vincristine, VP- 16, while some more newer drugs include Gemcitabine (Gemzar), Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, Rituxan STI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin and calcitriol. Non- limiting examples of immunomodulators that can be used include AS- 101 (Wyeth- Ayerst Labs.), bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte macrophage colony stimulating factor; Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor; Genentech).
Exemplary biologies that can be used in combination with the disclosed methods include one or more monoclonal antibodies (mAbs) used to treat cancer, such as mAbs specific for EGFR (e.g., cetuximab), VEGF (e.g., bevacizumab), PD-1 (e.g., nivolumab, JTX-4014 by Jounce Therapeutics, nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IB 1308), tislelizumab (BGB-A317), toripalimab (JS 001, dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, or AMP-514), CD276 (e.g., atezolizumab, avelumab, durvalumab, cosibelimab, KN035 (envafolimab), BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, or MEDI-4737), CD25 (e.g., daclizumab or basiliximab), CD20 (e.g., Tositumomab (Bexxar®); Rituximab (Rituxan, Mabthera); Ibritumomab tiuxetan (Zevalin, for example in combination with yttrium- 90 or indium- 111 therapy); Ofatumumab (Arzerra®), veltuzumab, obinutuzumab, ublituximab, ocaratuzumab), CD22 (e.g., namatumab, inotuzumab ozogamicin, moxetumomab pasudotox) or CTLA4 (e.g., ipilimumab, tremelimumab). In some examples, the additional therapeutic agent administered is an anti-cancer monoclonal antibody, for example one or more of: 3F8, Abagovomab, Adecatumumab, Afutuzumab, Alacizumab , Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Apolizumab, Arcitumomab, Bavituximab, Bectumomab, Belimumab, Besilesomab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Capromab pendetide, Catumaxomab, CC49, Cetuximab, Citatuzumab bogatox, Cixutumumab, Clivatuzumab tetraxetan, Conatumumab, Dacetuzumab, Detumomab, Ecromeximab, Eculizumab, Edrecolomab, Epratuzumab, Ertumaxomab, Etaracizumab, Farletuzumab, Figitumumab, Galiximab, Gemtuzumab ozogamicin, Girentuximab, Glembatumumab vedotin, Ibritumomab tiuxetan, Igovomab, Imciromab, Intetumumab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Labetuzumab, Lexatumumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Mitumomab, Morolimumab, Nacolomab tafenatox, Naptumomab estafenatox, Necitumumab, Nimotuzumab, Nofetumomab merpentan, Ofatumumab, Olaratumab, Oportuzumab monatox, Oregovomab, Panitumumab, Pemtumomab, Pertuzumab, Pintumomab, Pritumumab, Ramucirumab, Rilotumumab, Rituximab, Robatumumab, Satumomab pendetide, Sibrotuzumab, Sonepcizumab, Tacatuzumab tetraxetan, Taplitumomab paptox, Tenatumomab, TGN1412, Ticilimumab (tremelimumab), Tigatuzumab, TNX-650, Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Veltuzumab, Volociximab, Votumumab, Zalutumumab, or combinations thereof In a specific example, the disclosed methods are used in combination with a therapeutic PD-1 mAb, such as one or more of nivolumab, JTX-4014 by Jounce Therapeutics, nivolumab, pembrolizumab, pidilizumab, cemiplimab, spartalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IB 1308), tislelizumab (BGB-A317), toripalimab (JS 001, dostarlimab (TSR-042, WBP-285), 1NCMGA00012 (MGA012), AMP-224, and AMP-514.
In some examples, the methods further include surgical treatment, for example surgical resection of the cancer or a portion of it. In some examples, the methods further include administration of radiotherapy, for example administration of radioactive material or energy (such as external beam therapy) to the tumor site to help eradicate the tumor or shrink it prior to surgical resection.
XII. Methods for Diagnosis and Detection
Methods are also provided for the detection of the presence of CD276 in vitro or in vivo. For example, the disclosed monoclonal antibodies can be used for in vivo imaging to detect a CD276-positive cancer. To use the disclosed antibodies as diagnostic reagents in vivo, the antibodies are labelled with a detectable moiety, such as a radioisotope, fluorescent label, or positron emitting radionuclides. As one example, the monoclonal antibodies (or antigen-binding fragments thereof) disclosed herein can be conjugated to a positron emitting radionuclide for use in positron emission tomography (PET); this diagnostic process is often referred to as immuno PET.
To use the disclosed antibodies as diagnostic reagents in vitro, the antibodies can be directly or indirectly labelled with a detectable moiety (e.g., by using a labeled secondary antibody that binds to the CD276 antibody), such as a radioisotope, enzyme, or fluorescent label. In some examples, the presence of CD276 is detected in a biological sample from a subject and can be used to identify a subject with a CD276-positive cancer. The sample can be any sample, including, but not limited to, blood, serum, urine, semen, sputum, saliva, mucus, nasal wash, nasopharyngeal samples, oropharyngeal samples, tissue, cells, tissue biopsy, fine needle aspirate, surgical specimen, feces, cerebral spinal fluid (CSF), and bronchoalveolar lavage (BAL) fluid. Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes. The method of detection can include contacting a cell or sample, with an antibody or antibody conjugate (e.g., a conjugate including a detectable marker) that specifically binds to CD276 under conditions sufficient to form an immune complex, and detecting the immune complex (e.g., by detecting a detectable marker conjugated to the antibody or antigen binding fragment). Provided herein is a method of determining if a subject has a CD276-positive cancer by contacting a sample from the subject with a CD276- specific monoclonal antibody disclosed herein; and detecting binding of the monoclonal antibody to the sample. An increase in binding of the monoclonal antibody to the sample as compared to binding of the monoclonal antibody to a control sample identifies the subject as having a CD276-positive cancer.
In another aspect, provided is a method of confirming a diagnosis of a CD276-positive cancer in a subject by contacting a sample from a subject diagnosed with a CD276-positive cancer with a CD276 monoclonal antibody disclosed herein; and detecting binding of the monoclonal antibody to the sample. An increase in binding of the monoclonal antibody to the sample as compared to binding of the monoclonal antibody to a control sample confirms the diagnosis of a CD276-positive cancer in the subject.
In one aspect, the antibody or antigen binding fragment is directly labeled with a detectable marker. In another aspect, the antibody that binds CD276 (the primary antibody) is unlabeled and a secondary antibody or other molecule that can bind the primary antibody is utilized for detection. The secondary antibody that is chosen is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a human IgG, then the secondary antibody may be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.
Suitable labels for the antibody or secondary antibody include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. Non-limiting examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin. Non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplary luminescent material is luminol; a non-limiting exemplary a magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125I, 1311, 35S or 3H.
In an alternative aspect, CD276 can be assayed in a biological sample by a competition immunoassay utilizing CD276 standards labeled with a detectable substance and an unlabeled antibody that specifically binds CD276. In this assay, the biological sample, the labeled CD276 standards and the antibody that specifically binds CD276 are combined and the amount of labeled CD276 standard bound to the unlabeled antibody is determined. The amount of CD276 in the biological sample is inversely proportional to the amount of labeled CD276 standard bound to the antibody that specifically binds CD276. The immunoassays and methods disclosed herein can be used for a number of purposes. In one aspect, the antibody that specifically binds CD276 may be used to detect the production of CD276 in cells in cell culture. In another aspect, the antibody can be used to detect the amount of CD276 in a biological sample, such as a sample obtained from a subject having or suspected or having a CD276-positive cancer.
In one aspect, a kit is provided for detecting CD276 in a biological sample, such as a tissue biopsy, fine needle aspirate, core biopsy, blood, serum, urine, semen, CSF, nasopharyngeal, oropharyngeal, sputum, or saliva sample. Kits for detecting CD276-positive cells can include a monoclonal antibody that specifically binds CD276, such as monoclonal antibody disclosed herein. In a further aspect, the monoclonal antibody is labeled (for example, with a fluorescent, radioactive, or an enzymatic label). In some examples, the antibody is present on a solid support, such as a bead or multi-well plate. In some examples, the kit further includes a detectably labeled secondary antibody that permits detection of the antibody that specifically binds CD276.
In one aspect, a kit includes instructional materials disclosing means of use of an antibody that binds CD276. The instructional materials may be written, in an electronic form or may be visual (such as video files). The kits may also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kit may additionally contain means of detecting a label (such as enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like). The kits may additionally include buffers and other reagents routinely used for the practice of a particular method. The kits may additionally include materials to obtain a sample, such as a swab, syringe, needle, and the like. Such kits and appropriate contents are well known.
In one aspect, the diagnostic kit comprises an immunoassay. Although the details of the immunoassays may vary with the particular format employed, the method of detecting CD276 in a biological sample generally includes the steps of contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to CD276. The antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.
The monoclonal antibodies disclosed herein can also be utilized in immunoassays, such as, but not limited to radioimmunoassays (RIAs), ELISA, lateral flow assay (LFA), or immunohistochemical assays. The antibodies can also be used for fluorescence activated cell sorting (FACS), such as for identifying/detecting CD276-positive cells. FACS employs a plurality of color channels, low angle and obtuse light-scattering detection channels, and impedance channels, among other more sophisticated levels of detection, to separate or sort cells (see U.S. Patent No. 5,061,620). Any of the monoclonal antibodies that bind CD276, as disclosed herein, can be used in these assays. Thus, the monoclonal antibodies can be used in a conventional immunoassay, including, without limitation, ELISA, RIA, LFA, FACS, tissue immunohistochemistry, Western blot or immunoprecipitation. The disclosed antibodies can also be used in nanotechnology methods, such as microfluidic immunoassays, which can be used to capture CD276, or exosomes containing CD276. Suitable samples for use with a microfluidic immunoassay or other nanotechnology method, include but are not limited to, saliva, blood, and fecal samples. Microfluidic immunoassays are described in U.S. Patent Application No. 2017/0370921, 2018/0036727, 2018/0149647, 2018/0031549, 2015/0158026 and 2015/0198593; and in Lin et al., JALA June 2010, pages 254-274; Lin et al., Anal Chem 92: 9454-9458, 2020; and Herr et al., Proc Natl Acad Sci USA 104(13): 5268-5273, 2007).
EXAMPLES
The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.
Example 1: Fully-human CD276 antibodies bind to CD276 on the tumor cell surface
Five anti-CD276 antibodies (Yll l, Y422, Y868, Y117 and YE5) were isolated from human scFv phage libraries. The amino acid sequences of the VH and VL domains of the monoclonal antibodies are set forth herein as follows (see also section IV of the description):
Figure imgf000072_0001
Flow cytometry was used to assess binding of the CD276 antibodies to human pancreatic cancer cell line Panel. All five anti-CD276 antibodies were produced in scFv-Fc format and purified for the binding assay. Serially diluted antibodies at concentrations of 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 pg/mL were incubated with Panel cells, followed by Alexa-647 labeled antihuman Fab secondary antibody. A previously described anti-CD276 antibody (m276) in scFv format was included as positive control and a non-binding scFv was used as a negative control. As shown in FIG. 1, all five antibodies exhibited significant binding to CD276-positive Panel cells.
Example 2: Generation and characterization of CD276-targeted CARs
The fully-human CD276 antibodies (Yll l, Y422, Y868, Y117 and YE5) were used to construct chimeric antigen receptors (CARs). A schematic of the overall structure of the CARs is shown in FIG. 2A. The CARs include an antibody single-chain variable fragment (scFv), CD28 derived hinge and transmembrane domains, and 4 IBB and CD3 zeta (CD3Q cytoplasmic signaling domains. The scFv in all five CARs generated are in the VH-VL orientation. The CD276 specificity of each antibody is listed in FIG. 2B.
To test the CD276-targeted CARs, T cells were retrovirally transduced with nucleic acid encoding the CARs. Transduced T cells were incubated with rCD276-Bio (biotin-tagged recombinant CD276 ectodomain), followed by APC labeled streptavidin. Flow cytometry was used to detect CAR expression on the T cell surface. Untransduced (UTD) cells were used as negative controls. Histograms of CAR expression in T cells from a representative donor are shown in FIG. 3 A. A summary of the binding data for four independent donors is shown in FIG. 3B. The results showed that Y868, Yl l l and Y117 based CARs had the greatest expression levels.
Expansion and viability of CAR transduced T cells was next evaluated. T cells were maintained in T cell media (1:1 mix of RPMI-1640 and Click’s media containing 10% FBS and 1% Glutamax) supplemented with recombinant human IL-2 (50 lU/mL). IL-2 was added every 2-3 days. Cells were collected, counted, and viability was measured on day 11 after transduction. Counting and viability measurements were performed using a Countess II automated cell counter. Fold expansion was calculated by dividing day 11 total cell numbers by starting cell number on the day of transduction. Fold T cell expansion at day 11 post-transduction is shown in FIG. 4A. T cell viability at day 11 post-transduction is shown in FIG. 4B. These results demonstrated that expression of the CD276-targeted CARs did not significantly alter T cell expansion or viability.
Example 3: Anti-tumor activity of CD276-targeted CARs in cancer cells lines
In vitro anti-tumor activity of the CD276-targeted CAR T cells against pancreatic cancer cells and neuroblastoma tumor cells was tested. Firefly-luciferase labeled tumor cells (10,000 cells) were seeded in opaque white 96- well plates in 100 pL T cell media. Four hours later, T cells resuspended in 100 pL T cell media were added at CAR+ T cell effector to target (E:T) ratios of 5:1, 2.5:1, 1.25: 1, 0.25:1. After 48 hours, 10 pL D-Luciferin was added to each well such that the final concentration was 15 mg/mL. Plates were incubated at 37°C for 5 minutes after which signal intensity was captured using a Clariostar plate reader. Percent killing was calculated as 100x[l- (sample luminescence)/(UTD luminescence)]. FIGS. 5A-5G show percent killing in CD276 knockout (KO) NBEB cells (FIG. 5A), neuroblastoma cell line NBEB (FIG. 5B), neuroblastoma cell line LAN5 (FIG. 5C) neuroblastoma cell line IMR5 (FIG. 5D), pancreatic cancer cell line Panel (FIG. 5E), pancreatic cancer cell line HP AC (FIG. 5F) and pancreatic cancer cell line MiaPaca (FIG. 5G). The results show that the CD276-targeted CAR T cells effectively induced cytotoxicity of CD276-positive pancreatic and neuroblastoma tumor cells.
Example 4: CD276-targeted CAR T cells in a pancreatic (Panel) tumor model
Anti-tumor activity of CD276 CAR T cells in an orthotopic pancreatic tumor model (Panel) was tested. Firefly-luciferase labeled Panel cells (250,000) were injected into the pancreas of 6- to 8-week-old female NSG mice. Two weeks later, mice were imaged using IVIS Spectrum to measure tumor burden and grouped into different treatment groups (FIG. 6). Mice were treated with CARs containing Y422, Y868, Y111, Y117 or YE5 scFv (6-8 mice per group). CARs containing previously described anti-CD276 scFvs MGA271 and 376-96 were also included in this study for comparison. The MGA271 scFv sequence was humanized before using to make the CAR. The 376.96 scFv was used in its native (murine) form without humanization.
Five million CAR+ T cells were injected intravenously in the tail vein. Tumor burden was monitored by imaging weekly for seven weeks (D7, D16, D21, D28, D35, D42 and D49) and then at specified time points later in this study (D63, D91, DI 12, D156 and D296). Body weight measurements were also taken at the time of imaging on a weekly basis. To image the tumor, 100 mL of 15 mg/mL D-luciferin was injected intraperitonially, and imaging was performed after 10 minutes. The results are shown in FIG. 7. Mice treated with the Y111-based CAR exhibited the greatest reduction in tumor size. At the conclusion of the study (day 296), no tumors had relapsed in mice treated with the Y111 CAR.
Example 5: CD276-targeted CAR T cells in a pancreatic (HPAC) tumor model
Anti-tumor activity of CD276 CAR T cells in an orthotopic HPAC pancreatic tumor model was evaluated. Firefly-luciferase labeled HPAC cells (250,000) were injected into the pancreas of 6-8-week-old female NSG mice (FIG. 8A). Ten days later, mice were imaged using IVIS Spectrum to measure tumor burden and grouped into different treatment groups as shown in FIG. 8B.
Mice were treated with CARs containing Y422, Y868, Y 111 , Y117 or YES scFv. A CAR containing anti-CD276 scFv MGA271 was also included in this study for comparison. Five million CAR+ T cells were injected intravenously in the tail vein. Tumor burden was monitored by imaging weekly for seven weeks (D7, D14, D21, D28, D35 and D42). Body weight measurements were also taken at the time of imaging on a weekly basis (FIG. 8C).
To image the tumors, 100 pL of 15 mg/mL D-luciferin was injected intraperitonially and imaging was performed after 10 minutes. IVIS images of tumor luminescence in representative mice are shown in FIG. 9A. Tumor luminescence was quantified weekly for seven weeks (FIG. 9B), followed by specified time points later in the study (up to D255; FIG. 10A). FIG. 10B shows tumor luminescence in individual mice for all groups. The results demonstrate that Y111-based CAR T cells were the most effective at eliminating the pancreatic tumors. All treated mice maintained normal weight throughout the study.
Example 6: Activated CAR T cells product cytolytic proteins
This example describes production of cytolytic proteins by CAR T cells co-cultured with CD276+ Panel tumor cells. Un-transduced (UTD) T cells, and T cells expressing a CD19 CAR or one of three CD276-specific CARs (Y868, Yll l, and Y117) were co-cultured with Panel pancreatic cancer cells for 16 hours at an effector-to-target ratio (E:T) of 1:2. T cells were then collected and labeled intracellularly with antibodies against granzyme B, perforin, and CD107a. As shown in FIG. 11, Y868, Yl ll and Y117-based CAR T cells produced higher levels of perforin, granzyme B and CD107a than UTD and CD19 CAR T cells.
Example 7 : Anti-tumor activity of CD276 CAR T cells in a metastatic neuroblastoma (IMR5) model
This example compares the anti-tumor activity of Y868, Yl l l and Y117 CAR T cells to T cells expressing a CAR containing mouse-derived anti-human CD276 monoclonal antibody 376.96 (see Li et al., Nat Commun 14:5920, 2023).
Firefly-luciferase labeled IMR5 cells (IxlO6) were injected into 6-8-week-old female NSG mice via tail vein (Day -35). Five weeks later, mice were imaged using IVIS Spectrum to measure tumor burden and grouped into different treatment groups. Two million Y868, Yll 1, YI 17 or 376.96 CAR T cells were injected intravenously in the tail vein (Day 0) (FIG. 12A). Tumor burden was monitored by imaging on Days -1, 7, 14, 21, 29, 40, 49 and 60. To image the tumor, 100 pl of 15 mg/mL D-luciferin was injected intraperitonially and imaging was done after 10 minutes. Body weight measurements were also taken at the time of imaging. As shown in FIGS. 12B-12C, T cells expressing the Y 111 -based CAR significantly reduced tumor burden in treated mice and were more effective than the other CD276-targeted CARs. No significant changes in body weight were observed.
Example 8: Long-term persistence and anti-tumor activity of Ylll CAR in the HPAC orthotopic pancreatic tumor model
To test the long-term in vivo persistence and functional capacity of Y111 CAR T cells, the long-term survivor mice from the HPAC study (Example 5) were rechallenged with 4xl06 fireflyluciferase labeled HPAC cells injected subcutaneously in the lower right abdomen. Age matched mice that had never received tumor cells or CAR T cells were used as controls and were similarly injected with the tumor cells. Mice were imaged using IVIS Spectrum to measure tumor burden on Days -7, 8, 46, 72, 101, 123, 143 and 182 after CAR T cell treatment. As shown in FIGS. 13A- 13B, treatment with Y111 CAR T cells significantly reduced tumor burden in treated mice and the effect of treatment persisted throughout the study period (444 days after CAR T cell treatment).
Example 9: Optimization of the Ylll CAR
To test whether the Y 111 CAR could be further optimized to improve efficacy, two variants were evaluated - one with the original 4- IBB costimulatory domain (Yl l l-HL-28HTM-BBz, SEQ ID NO: 47), and the other with a CD28 costimulatory domain (Yll l-HL-28HTM-28z SEQ ID NO: 61). The two Yl l 1-based CAR constructs were also compared to two CAR constructs having a camel-derived single domain (VHH) CD276- specific antibody (B 12; described in Li et al., Nat Commun 14:5920, 2023 and WO 2021/081052). The B 12-based CARs include a CD28 HTM, a CD3^ signaling domain and either a 4- IBB costimulatory domain (B12-VHH-28HTM-BBz) or a CD28 costimulatory domain (B12-VHH-28HTM-28z). All four constructs were compared in a pancreatic tumor model (Pane 1).
Firefly-luciferase labeled Panel cells (2.5xl05) were injected into the pancreas of 6-8- week-old female NSG mice (Day -14). Two weeks later, mice were imaged using IVIS Spectrum to measure tumor burden and grouped into the following four treatment groups: Yl l 1-HL-28HTM- BBz CAR T cells; Y1 ll-HL-28HTM-28z CAR T cells; B 12- VHH-28HTM-BBz CAR T cells; and B12-VHH-28HTM-28z CAR T cells. Five million CAR+ T cells were injected intravenously in the tail vein (Day 0). Tumor burden and body weight were monitored weekly (FIG. 14A). To image the tumor, 100 pl of 15 mg/mL D-luciferin was injected intraperitonially and imaging was performed after 10 minutes. As shown in FIG. 14B, both of the Y 111-based CARs significantly reduced tumor burden in treated mice, with B12-VHH-28HTM-BBz being slightly more effective. No significantly changes in body weight were observed. Example 10: Ylll binding studies
Surface plasmon resonance (SPR) was used to measure the binding of Y 111 Fab and Yl l l IgG to human or mouse CD276 extracellular domain (ECD) using Biacore X100. In a first study, human CD276 ECD protein was immobilized to a CM5 chip. Y111 Fab was in the mobile phase at concentrations of 0.32, 1.6, 8, 40 and 200 nM. The data were fitted with a 1:1 binding model to calculate the KD value. The results showed that Yl l l Fab binds human CD276 protein with an affinity of 20 nM.
Yl ll full IgG was also tested in an SPR assay to reflect multiple copies of scFv molecules on the surface of CAR T cells. Human CD276-ECD-AP fusion protein was immobilized on a CM5 chip. Yl l l IgG was tested at concentrations of 0.08, 0.4, 2, 10 and 50 nM. Data were fitted with 1:2 model to calculate the KD. The results showed that Yll 1 IgG binds human CD276 with an affinity of 21 nM.
In an additional study, the affinity of Yl ll IgG and 376.96 IgG for mouse CD276 ECD was evaluated by SPR. Mouse CD276-ECD protein was immobilized on a CM5 chip. Each IgG was analyzed at concentrations of 62.5, 125, 250, 500, 1000 nM. Data were fitted with a 1:2 model to calculate KD. Both Yll l and 376.96 bound mouse CD276 with low affinity (366 nM and 2.04 .M, respectively).
To determine whether Yl l l and 376.96 bind the same epitope on CD276, a competition binding study was performed. Human CD276-ECD-AP fusion protein was coated on an ELISA plate. Biotin-labeled Yl ll IgG was bound at various concentrations from 0.0001 to 10 nM in the presence of competing IgGs. Competing IgGs were not labelled and were added at a constant concentration of 10 nM. Binding of biotin- Y 111 was detected with streptavidin-HRP. The results showed that binding of biotin- Y 111 was reduced by unlabeled Yll l, but it was not changed by unlabeled 376.96 IgG, indicating that Y111 and 376.96 bind different epitopes on CD276.
It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

1. A monoclonal antibody that specifically binds CD276, comprising a variable heavy (VH) domain and a variable light (VL) domain, wherein: the VH domain comprises the heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3 sequences of SEQ ID NO: 1, and the VL domain comprises the light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 sequence of SEQ ID NO: 2; the VH domain comprises the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 9, and the VL domain comprises the LCDR1, LCDR2, and LCDR3 sequence of SEQ ID NO: 10; the VH domain comprises the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 17, and the VL domain comprises the LCDR1, LCDR2, and LCDR3 sequence of SEQ ID NO: 18; the VH domain comprises the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 25, and the VL domain comprises the LCDR1, LCDR2, and LCDR3 sequence of SEQ ID NO: 26; or the VH domain comprises the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 33, and the VL domain comprises the LCDR1, LCDR2, and LCDR3 sequence of SEQ ID NO: 34.
2. The monoclonal antibody of claim 1, wherein the CDR sequences are determined using Kabat, IMGT, or Chothia numbering conventions.
3. The monoclonal antibody of claim 1 or claim 2, wherein: the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16; the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24; the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32; or the amino acid sequences of the HCDR1, HCDR2 and HCDR3 respectively comprise SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40.
4. The monoclonal antibody of any one of claims 1-3, wherein: the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 1 and comprises the HCDR1, HCDR2 and HCDR3 sequence of SEQ ID NO: 1; and the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 2 and comprises the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 2.
5. The monoclonal antibody of any one of claims 1-3, wherein: the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 9 and comprises the HCDR1, HCDR2 and HCDR3 sequence of SEQ ID NO: 9, and the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 10 and comprises the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 10; the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 17 and comprises the HCDR1, HCDR2 and HCDR3 sequence of SEQ ID NO: 17, and the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 18 and comprises the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 18; the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 25 and comprises the HCDR1, HCDR2 and HCDR3 sequence of SEQ ID NO: 25, and the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 26 and comprises the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 26; or the amino acid sequence of the VH domain is at least 90% identical to SEQ ID NO: 33 and comprises the HCDR1, HCDR2 and HCDR3 sequence of SEQ ID NO: 33, and the amino acid sequence of the VL domain is at least 90% identical to SEQ ID NO: 34 and comprises the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 34.
6. The monoclonal antibody of any one of claims 1-5, wherein: the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 1 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 2; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 9 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 10; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 17 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 18; the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 25 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 26; or the amino acid sequence of the VH domain comprises or consists of SEQ ID NO: 33 and the amino acid sequence of the VL domain comprises or consists of SEQ ID NO: 34.
7. The monoclonal antibody of any one of claims 1-6, wherein the monoclonal antibody is an IgG, IgA, or IgM molecule.
8. The monoclonal antibody of any one of claims 1-6, wherein the monoclonal antibody is a Fab fragment, a Fab’ fragment, a F(ab)!2 fragment, a single chain variable fragment (scFv) or a disulfide stabilized variable fragment (dsFv).
9. The monoclonal antibody of any one of claims 1-8, which is a fully human monoclonal antibody.
10. A chimeric antigen receptor (CAR) comprising the monoclonal antibody of any one of claims 1-9.
11. The CAR of claim 10, wherein the monoclonal antibody is an scFv.
12. The CAR of claim 11, wherein the scFv is in the VH-linker-VL format.
13. The CAR of claim 11, wherein the scFv is in the VL-linker-VH format.
14. The CAR of any one of claims 11-13, wherein the amino acid sequence of the scFv comprises or consists of SEQ ID NO: 57 or SEQ ID NO: 58.
15. The CAR of any one of claims 10-14, further comprising a hinge region, a transmembrane domain, a costimulatory domain, a signaling domain, or any combination thereof.
16. The CAR of claim 15, wherein: the hinge region comprises a CD28 hinge region; the transmembrane domain comprises a CD28 transmembrane domain; the costimulatory domain comprises a 4- IBB or CD28 costimulatory domain; and/or the signaling domain comprises a CD3(^ signaling domain.
17. The CAR of claim 16, wherein: the CD28 hinge region comprises SEQ ID NO: 43; the CD28 transmembrane domain comprises SEQ ID NO: 44; the 4- IBB costimulatory domain comprises SEQ ID NO: 45 or the CD28 costimulatory domain comprises SEQ ID NO: 65; and/or the CD3^ signaling domain comprises SEQ ID NO: 46.
18. The CAR of any one of claims 10-17, wherein the amino acid sequence of the CAR comprises residues 20-480 of SEQ ID NO: 47, residues 20-484 of SEQ ID NO: 49, residues 20-484 of SEQ ID NO: 51, residues 20-488 of SEQ ID NO: 53, residues 20-486 of SEQ ID NO: 55, residues 20-479 of SEQ ID NO: 61, or residues 20-480 of SEQ ID NO: 63.
19. An isolated cell expressing the CAR of any one of claims 10-18.
20. The isolated cell of claim 19, which is a T cell, B cell, natural killer (NK) cell, macrophage, dendritic cell (DC), or induced pluripotent stem cell (iPSC).
21. An immunoconjugate comprising the monoclonal antibody of any one of claims 1-9 and an effector molecule.
22. The immunoconjugate of claim 21, wherein the effector molecule is a toxin, a photon absorber, or a detectable label.
23. The immunoconjugate of claim 22, wherein the toxin comprises a Pseudomonas exotoxin or a variant thereof.
24. The immunoconjugate of claim 22, wherein the detectable label comprises a fluorophore, an enzyme or a radioisotope.
25. An antibody-drug conjugate (ADC) comprising a drug conjugated to the monoclonal antibody of any one of claims 1-9.
26. The ADC of claim 25, wherein the drug is a small molecule.
27. The ADC of claim 25 or claim 26, wherein the drug is an anti-microtubule agent, an anti-mitotic agent and/or a cytotoxic agent.
28. A multi-specific antibody comprising the antibody of any of claims 1-9 and at least one additional monoclonal antibody or antigen-binding fragment thereof.
29. The multi- specific antibody of claim 28, which is a bispecific antibody or a trispecific antibody.
30. An antibody -nanoparticle conjugate, comprising a nanoparticle conjugated to the antibody of any one of claims 1-9.
31. The antibody-nanoparticle conjugate of claim 30, wherein the nanoparticle comprises a polymeric nanoparticle, nanosphere, nanocapsule, liposome, dendrimer, polymeric micelle, or niosome.
32. The antibody-nanoparticle conjugate of claim 30 or claim 31, wherein the nanoparticle comprises a cytotoxic agent.
33. A fusion protein comprising the antibody of any one of claims 1-9 and a heterologous protein or peptide.
34. The fusion protein of claim 33, wherein the heterologous protein is an Fc protein.
35. An isolated nucleic acid molecule encoding the monoclonal antibody of any one of claims 1-9, the CAR of any one of claims 10-18, the immunoconjugate of any one of claims 21-24, the multi- specific antibody of any one of claims 28-29 or the fusion protein of any one of claims
36. The isolated nucleic acid molecule of claim 35, wherein the nucleic acid molecule encodes a CAR and the nucleotide sequence encoding the CAR is: at least 90% identical to SEQ ID NO: 48 or a degenerate variant thereof; at least 90% identical to SEQ ID NO: 50 or a degenerate variant thereof; at least 90% identical to SEQ ID NO: 52 or a degenerate variant thereof; at least 90% identical to SEQ ID NO: 54 or a degenerate variant thereof; at least 90% identical to SEQ ID NO: 56, or a degenerate variant thereof; at least 90% identical to SEQ ID NO: 62, or a degenerate variant thereof; or at least 90% identical to SEQ ID NO: 64, or a degenerate variant thereof
37. The isolated nucleic acid molecule of claim 36, wherein the nucleotide sequence encoding the CAR comprises or consists of:
SEQ ID NO: 48 or a degenerate variant thereof;
SEQ ID NO: 50 or a degenerate variant thereof;
SEQ ID NO: 52 or a degenerate variant thereof;
SEQ ID NO: 54 or a degenerate variant thereof;
SEQ ID NO: 56 or a degenerate variant thereof;
SEQ ID NO: 62, or a degenerate variant thereof; or
SEQ ID NO: 64, or a degenerate variant thereof.
38. The isolated nucleic acid molecule of any one of claims 35-37 operably linked to a promoter.
39. A vector comprising the isolated nucleic acid molecule of any one of claims 35-38.
40. An isolated cell comprising the isolated nucleic acid molecule of any one of claims 35-38 or the vector of claim 39.
41. A composition comprising a pharmaceutically acceptable carrier and the monoclonal antibody of any one of claims 1-9, the CAR of any one of claims 10-18, the isolated cell of any one of claims 19, 20 and 40, the immunoconjugate of any one of claims 21-24, the ADC of any one of claims 25-27, the multi-specific antibody of any one of claims 28-29, the antibody-nanoparticle conjugate of any one of claims 30-32, the fusion protein of any one of claims 33-34, the isolated nucleic acid molecule of any one of claims 35-38, or the vector of claim 39.
42. A method of detecting expression of CD276 in a sample, comprising contacting the sample with the monoclonal antibody of any of claims 1-9; and detecting binding of the monoclonal antibody to the sample, thereby detecting expression of CD276 in the sample.
43. A method of diagnosing a subject as having a CD276-positive tumor, comprising contacting a sample obtained from the subject with the monoclonal antibody of any of claims 1-9; and detecting binding of the monoclonal antibody to the sample, thereby diagnosing the subject as having a CD276-positive tumor.
44. The method of claim 42 or claim 43, wherein the monoclonal antibody is directly labeled.
45. The method of claim 42 or claim 43, further comprising: contacting the monoclonal antibody with a detection antibody, and detecting the binding of the detection antibody to the monoclonal antibody, thereby detecting expression of CD276 in the sample or diagnosing the subject as having a CD276-positive cancer.
46. The method of any one of claims 42-45, wherein the sample is obtained from a subject suspected of having a CD276-positive cancer.
47. The method of any one of claims 42-46, wherein the sample is a tumor biopsy.
48. A method of treating a CD276-positive cancer in a subject, comprising administering to the subject the monoclonal antibody of any one of claims 1-9, the CAR of any one of claims 10-18, the isolated cell of any one of claims 19, 20 and 40, the immunoconjugate of any one of claims 21-24, the ADC of any one of claims 25-27, the multi- specific antibody of any one of claims 28-29, the antibody-nanoparticle conjugate of any one of claims 30-32, the fusion protein of any one of claims 30-34, or the composition of claim 41, thereby treating the CD276-positive cancer in the subject.
49. A method of inhibiting tumor growth or metastasis of a CD276-positive cancer in a subject, comprising administering to the subject the monoclonal antibody of any one of claims 1-9, the CAR of any one of claims 10-18, the isolated cell of any one of claims 19, 20 and 40, the immunoconjugate of any one of claims 21-24, the ADC of any one of claims 25-27, the multispecific antibody of any one of claims 28-29, the antibody-nanoparticle conjugate of any one of claims 30-32, the fusion protein of any one of claims 33-34, or the composition of claim 41, thereby inhibiting tumor growth or metastasis of the CD276-positive cancer in the subject.
50. The method of claim 48 or claim 49, wherein the CD276-positive cancer is a solid tumor.
51. The method of claim 50, wherein the solid tumor is a pancreatic cancer, a neuroblastoma, a liver cancer, a kidney cancer, a bladder cancer, a cervical cancer, an esophageal cancer, a prostate cancer, a breast cancer, an ovarian cancer, a colon cancer, a lung cancer, a brain cancer, a pediatric cancer, melanoma or mesothelioma.
52. A method of reducing the risk of allograft rejection in a subject who has received an allograft, comprising administering to the subject a therapeutically effective amount of the isolated cell of claim 19 or claim 20, thereby reducing the risk of allograft rejection in the subject.
53. A kit, comprising: the monoclonal antibody of any one of claims 1-9, the CAR of any one of claims 10-18, the isolated cell of any one of claims 19, 20 and 40, the immunoconjugate of any one of claims 21-24, the ADC of any one of claims 25-27, the multi- specific antibody of any one of claims 28-29, the antibody-nanoparticle conjugate of any one of claims 30-32, the fusion protein of any one of claims 33-34, the isolated nucleic acid molecule of any one of claims 35-38, the vector of claim 39, or the composition of claim 41; and one or more of a pharmaceutically acceptable carrier, buffer, cell culture media, cell culture plates or flasks, a solid support, a fluorescent label, a radioactive label, an enzymatic label, an enzymatic substrate, a secondary antibody, one or more check point inhibitors, one or more additional anti-cancer agents, one or more transfection reagents, and instructional materials.
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