US20230338589A1 - In vivo immunoimaging of interferon-gamma - Google Patents

In vivo immunoimaging of interferon-gamma Download PDF

Info

Publication number
US20230338589A1
US20230338589A1 US18/144,382 US202318144382A US2023338589A1 US 20230338589 A1 US20230338589 A1 US 20230338589A1 US 202318144382 A US202318144382 A US 202318144382A US 2023338589 A1 US2023338589 A1 US 2023338589A1
Authority
US
United States
Prior art keywords
antibody
labeled
ifn
antibody conjugate
tumor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
US18/144,382
Inventor
Nerissa Viola
Heather GIBSON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wayne State University
Original Assignee
Wayne State University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wayne State University filed Critical Wayne State University
Priority to US18/144,382 priority Critical patent/US20230338589A1/en
Assigned to WAYNE STATE UNIVERSITY reassignment WAYNE STATE UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GIBSON, Heather, Viola, Nerissa
Publication of US20230338589A1 publication Critical patent/US20230338589A1/en
Pending legal-status Critical Current

Links

Images

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04Organic compounds
    • A61K51/08Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1021Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against cytokines, e.g. growth factors, VEGF, TNF, lymphokines or interferons
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00Preparations for testing in vivo
    • A61K49/001Preparation for luminescence or biological staining
    • A61K49/0013Luminescence
    • A61K49/0017Fluorescence in vivo
    • A61K49/005Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0058Antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04Organic compounds
    • A61K51/08Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/249Interferons
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16HHEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H20/00ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance
    • G16H20/10ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance relating to drugs or medications, e.g. for ensuring correct administration to patients
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16HHEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H20/00ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance
    • G16H20/40ICT specially adapted for therapies or health-improving plans, e.g. for handling prescriptions, for steering therapy or for monitoring patient compliance relating to mechanical, radiation or invasive therapies, e.g. surgery, laser therapy, dialysis or acupuncture
    • GPHYSICS
    • G16INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16HHEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H30/00ICT specially adapted for the handling or processing of medical images
    • G16H30/20ICT specially adapted for the handling or processing of medical images for handling medical images, e.g. DICOM, HL7 or PACS
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • the present disclosure generally relates to antibody conjugates, to methods for imaging antibody conjugates in vivo, methods and compositions relating to antibody conjugates, and to methods for assessing an effect of a composition or immune-mediated treatment for cancer, injury, or infection.
  • the present disclosure specifically relates to antibody conjugates which specifically recognize interferon gamma (IFN- ⁇ ), to methods for imaging IFN- ⁇ antibody conjugates in vivo, methods and compositions relating to IFN- ⁇ antibody conjugates, and to methods for assessing an effect of a composition or immune-mediated treatment for cancer, injury, or infection.
  • IFN- ⁇ interferon gamma
  • TAA tumor-associated antigens
  • the disclosure demonstrates that the in vivo immunoimaging of interferon gamma (IFN-y) identifies active T cells, such as activated tumor-infiltrating T cells, in vivo relative to agents that recognize other biomarkers.
  • IFN-y interferon gamma
  • Embodiments of the present disclosure are directed to methods of in vivo immunoimaging IFN- ⁇ as a marker of activate T cells in a subject including: (a) administering a labeled-antibody conjugate to a subject, wherein the labeled-antibody conjugate includes: an antibody that specifically recognizes IFN- ⁇ , and at least one detection label conjugated to the antibody, wherein the at least one detection label is a radionuclide; and (b) detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging.
  • Embodiments of the present disclosure are directed to labeled-antibody conjugates including: an antibody that specifically recognizes and binds to IFN- ⁇ , and at least one detection label conjugated to the antibody, wherein the at least one detection label is a radionuclide tracer.
  • Embodiments of the present disclosure are directed to therapeutic radionuclide-antibody conjugates including: 1) an antibody that specifically recognizes and binds to IFN- ⁇ , a bifunctional chelator conjugated to the antibody, and a radionuclide therapeutic agent complexed to the bifunctional chelator or 2) an antibody that specifically recognizes and binds to IFN- ⁇ , a linker conjugated to the antibody, and a radionuclide therapeutic agent conjugated to the antibody via the linker.
  • FIG. 1 C is a graph showing results of testing 89 Zr-anti-IFN- ⁇ receptor-localized IFN-y binding in vitro.
  • FIG. 1 D is a pair of images showing liver (L), heart (H), and spleen (S) removed from mice used in the experiments described for FIG. 1 C and imaged ex vivo;
  • TUBO cells were inoculated 10 days prior to the start of vaccinations, given on days 0 and 14. PET imaging was conducted on day 15 (Ctrl);
  • MIP whole body maximum intensity projections
  • FIG. 4 D is a graph showing tumor VOIs were calculated for each mouse studied for the experiment described in FIG. 4 C ;
  • FIG. 4 F is a graph showing tumor VOIs were calculated for each mouse studied for the experiment described in FIG. 4 E ;
  • FIG. 6 B is a graph showing weekly tumor growth rate, calculated by regression analysis of log tumor growth, versus 89 Zr-anti-IFN- ⁇ labeled-antibody conjugate uptake plotted for each mouse and evaluated by Pearson’s correlation;
  • FIG. 6 C is a graph showing tumor growth during passive immunotherapy with anti-neu mAb 7.16.4, given as 5 doses at 1.5 mg i.p. every 3-4 days as indicated beginning 15 days after tumor inoculation.
  • FIG. 6 D is a graph showing tumor VOIs calculated for 89 Zr-anti-IFN- ⁇ or 89 Zr-rat-IgG labeled-antibody conjugate in 7.16.4 treated TUBO-bearing mice;
  • FIG. 6 F is a graph showing PD-1 expression analyzed by flow cytometry on CD8+ tumor infiltrates from ( FIG. 6 F );
  • FIG. 7 B is a graph of the volumes-of-interest obtained from select tissues over time from 24-120 h p.i.;
  • FIG. 8 B is a graph of the volumes-of-interest obtained from select tissues over time from 4-72 h p.i.;
  • FIG. 9 A shows MIP images of 89 Zr-anti-IFN ⁇ detection in all control (left) and vaccinated (right) TUBO bearing mice;
  • FIG. 9 B shows MIP images of 89 Zr-anti-CD3 detection in all control (left) and vaccinated (right) TUBO bearing mice;
  • FIG. 10 A is a plot of flow cytometry results showing T cell detection in TUBO-bearing BALB/c mice. Tumors from untreated TUBO-bearing mice were dissociated and stained with CD45, to detect total leukocyte infiltrates, and the T cell receptor beta chain (TCR ⁇ ), to identify the T cell fraction, by flow cytometry;
  • TCR ⁇ T cell receptor beta chain
  • FIG. 10 B is a graph showing Spleen VOIs calculated for each TUBO-bearing mouse imaged with either 89 Zr-anti-IFN ⁇ or 89 Zr-anti-CD3;
  • FIG. 11 A shows an image demonstrating immunoblot detection of T cell markers in imaged TUBO-bearing mice.
  • Western blots from lysed decayed tumor tissues post-IFN-y PET were conducted to analyze presence of CD3 and CD8 protein in the Vx vs Ctrl mice.
  • Beta-tubulin is detected as a gel loading control;
  • FIG. 11 B shows an image demonstrating immunoblot detection of T cell markers in imaged TUBO-bearing mice.
  • Western blots from lysed decayed tumor tissues post-CD3 PET were conducted to analyze presence of CD3 and CD8 protein in the Vx vs Ctrl mice.
  • Beta-tubulin is detected as a gel loading control;
  • FIG. 12 A shows MIP images of 89 Zr-anti-IFN ⁇ detection in all control (left) and vaccinated (right) NeuT mice bearing spontaneous salivary tumors;
  • FIG. 12 B shows MIP images of 89 Zr-anti-CD3 detection in all control (left) and vaccinated (right) NeuT mice bearing spontaneous salivary tumors;
  • FIG. 13 shows 89 Zr-IFN- ⁇ PET of vaccinated mice for correlation to tumor growth.
  • the MIP images show 89 Zr-anti-IFN ⁇ detection in all vaccinated TUBO mice bearing tumors;
  • FIG. 14 A is a graph showing tissue biodistribution of anti-IFN-y PET labeled-antibody conjugate.
  • BALB/c mice were treated with 100 ⁇ g CpG intramuscular and co-injected with 89 Zr-IFN- ⁇ intravenous (mAb clone AN-18 or XMG1.2). Uptake in blood, heart, stomach, small intestine, large intestine, pancreas, brain, bone, muscle, and lymph node was analyzed by gamma counter 72 hours later;
  • FIG. 14 B is a graph showing tissue biodistribution of anti-IFN-y PET labeled-antibody conjugates.
  • BALB/c mice were treated with 100 ⁇ g CpG intramuscular and co-injected with 89 Zr-IFN- ⁇ intravenous (mAb clone AN-18 or XMG1.2). Uptake in lungs, liver, kidney, and spleen, was analyzed by gamma counter 72 hours later; and
  • FIG. 15 is an image showing results of injection of 100-150 ⁇ Ci anti-IFN-y PET labeled-diabody conjugate into a TUBO-bearing BALB/c mouse after Vx.
  • label refers to a chemical moiety that can be covalently attached to an antibody and that functions to provide a detectable signal, such as, e.g., radionuclide tracers.
  • antibody is used herein in its broadest sense and specifically refers to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity (see e.g., Miller et al. (2003) J Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein that is capable of recognizing and binding to a specific antigen, such as, e.g., IFN- ⁇ .
  • a target antigen may have numerous binding sites, also called epitopes, recognized by complementarity determining regions of antibodies.
  • each antibody that specifically binds to a different epitope has a different structure.
  • one antigen may have more than one corresponding antibody.
  • An antibody includes a full-length immunoglobulin molecule or an immunologically-active portion of a full-length immunoglobulin molecule, such as, e.g., a molecule that contains an antigen binding site that immunospecifically recognizes and binds to an antigen of a target of interest or part thereof, such as, e.g., IFN- ⁇ .
  • the immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
  • the immunoglobulins can be derived from any species. In some embodiments, however, the immunoglobulin is of rodent or human origin.
  • the antibody is an antibody fragment and in specific embodiments, the antibody fragment is a diabody.
  • the term “specifically recognizes and binds to” refers to antibodies that are capable of binding to an antigen of interest, such as, e.g., IFN- ⁇ , with sufficient binding affinity such that the antibody is useful in targeting the antigen of interest.
  • the antibodies may have a binding affinity K D of from about 10 -4 to about 10 -15 , or from about 10 -6 to about 10 -13 , or from about 10 -7 to about 10 -12 , or from about 10 -9 to about 10 -10 , for the antigen of interest.
  • the antibody is one that binds to IFN- ⁇ , it will preferentially bind to IFN- ⁇ as opposed to other antigens and/or extracellular components.
  • the antibody may not significantly cross-react with other extracellular components.
  • the extent of binding of the antibody to non- IFN- ⁇ antigens and/or other extracellular components is less than about 10% (such as, e.g., 0% to about 9%), as determined by standard techniques known to those of ordinary skill in the art, such as, e.g., by flow cytometric analysis.
  • IFN- ⁇ interferon gamma
  • IFN- ⁇ is a member of the interferon family, which exhibits similar antiviral and anti-proliferative properties characteristic of interferons-a and - ⁇ (IFN- ⁇ and IFN- ⁇ ). IFN- ⁇ was originally discovered as a product induced upon mitogenic activation of lymphocytes. The recombinant production of human IFN- ⁇ was first reported by Gray, Goeddel and co-workers (Gray et al., Nature 295:503-508, 1982), and is subject of U.S. Pat. Nos.
  • the recombinant human IFN- ⁇ of Gray and Goeddel as produced in E. coli consisted of 146 amino acids, the N-terminal position of the molecule commencing with the sequence CysTyrCys. It was later found that the native human IFN- ⁇ (i.e., that arising from mitogen induction of human peripheral blood lymphocytes and subsequent purification) is a polypeptide that lacks the CysTyrCys N-terminus assigned by Gray et al., supra.
  • IFN- ⁇ E. coli-derived recombinant human IFN- ⁇ (rhIFN- ⁇ ) was determined (Ealick et al., Science 252:698-702, 1991), showing that the protein exists as a tightly intertwined non-covalent homodimer, in which the two identical polypeptide chains are oriented in an antiparallel manner.
  • IFN- ⁇ is known to exhibit a broad range of biological activities, including antitumor, antimicrobial and immunoregulatory activities.
  • IFN- ⁇ A particular form of recombinant human IFN- ⁇ is commercially available as an immunomodulatory drug for the treatment of chronic granulomatous disease characterized by severe, recurrent infections of the skin, lymph nodes, liver, lungs, and bones due to phagocyte dysfunction (rhIFN- ⁇ -1b, Actimmune®, Genentech, Inc. South San Francisco, Calif.). IFN- ⁇ was subsequently approved for use in patients with severe, malignant osteopetrosis. IFN- ⁇ may refer to IFN- ⁇ of any species including both human and mouse IFN- ⁇ .
  • bifunctional chelator refers to a chemical moiety that attaches an antibody to a radionuclide.
  • Bifunctional chelators may include a chelator covalently attached to a first linking group.
  • Bifunctional chelators may function by complexing metal ions with the chelator and by covalently attaching the chelator to an antibody with the first linking group, as is known to those of ordinary skill in the art.
  • bifunctional chelators may be covalently attached to a primary amine group, a hydroxyl group, and/or a cysteine amino acid of an antibody.
  • Suitable chelators include: 1,4,7-triazacyclononane-N,N′,N′-triacetic acid (i.e., NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (i.e., DOTA), ethylenediaminetetraacetic acid (i.e., EDTA), diethylenetriaminepentaacetic acid (i.e., DTPA; CAS Reg. No.
  • 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid i.e., DOPA
  • N′- ⁇ 5-[Acetyl(hydroxy)amino]pentyl ⁇ -N-[5-( ⁇ 4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl ⁇ amino)pentyl]-N-hydroxysuccinamide i.e., deferoxamine or DFO; CAS Reg. No.
  • bifunctional chelators include 4-maleimidobutyramidobenzyl-DOTA (which can be prepared following the procedure of Axworthy et al (2000) Proc. Natl. Acad. Sci. USA 97(4):1802-1807), 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (i.e., MX-DTPA, see e.g., International Pub. No.
  • p-SCN-Bn-Deferoxamine i.e., p-SCN-Bn-DFO
  • p-SCN-Bn-DFO has the following structure:
  • the term “isolated” refers to an antibody that has been identified and separated and/or recovered from at least one contaminant component of its natural environment. Contaminant components may be materials that would interfere with diagnostic and/or therapeutic uses for the antibody, such as, e.g., enzymes, hormones, and/or other proteinaceous or nonproteinaceous solutes.
  • the isolated antibody will be purified to greater than about 95% (such as, e.g., about 96% to about 100%) by weight of antibody as determined by the Lowry method, or to greater than about 99% by weight of antibody.
  • the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, wherein the individual antibodies in the population are identical except for naturally occurring post-translational modifications. Monoclonal antibodies may be highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different antigenic determinants (such as, e.g., epitopes), each monoclonal antibody is directed against a single antigenic determinant on the antigen. Monoclonal antibodies may be synthesized uncontaminated by other antibodies.
  • monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, it is not intended to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present disclosure may be prepared by the Kohler hybridoma method (see e.g., Kohler et al. (1975) Nature 256:495), or they may be prepared by recombinant DNA methods (see e.g., U.S. Pat. No. 4,816,567 and U.S. Pat. No. 5,807,715, each of which is hereby incorporated by reference in its entirety).
  • monoclonal antibodies may be isolated from phage antibody libraries using standard techniques (see e.g., Clackson et al. (1991) Nature, 352:624-628 and Marks et al. (1991) J. Mol. Biol., 222:581-597).
  • antibody fragment and “fragment” refer to a portion of a full length antibody that includes a region capable of recognizing and binding to a specific antigen, such as, e.g., the antigen binding, variable, or hypervariable (also known as complementarity determining) region thereof.
  • a specific antigen such as, e.g., the antigen binding, variable, or hypervariable (also known as complementarity determining) region thereof.
  • antibody fragments include Fab, Fab′, F(ab′) 2 , F(ab) 2 , Fv, sFv, and scFv fragments; diabodies; linear antibodies; minibodies (see e.g., Olafsen et al. (2004) Protein Eng. Design & Sel.
  • antibody 17(4):315-323 fragments produced by a Fab expression library, anti-idiotypic (hereinafter, “anti-Id”) antibodies, complementary determining region (hereinafter, “CDR”) and epitope-binding fragments of any of the above that immunospecifically recognize and bind to IFN- ⁇ , single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • anti-Id anti-idiotypic
  • CDR complementary determining region
  • radionuclide tracers include radioisotopes, which by virtue of radioactive decay, are detectable. Radionuclide tracers may be detected by various imaging techniques, such as, e.g., PET and single-photon emission computed tomography (hereinafter, “SPECT”) imaging. In embodiments, radionuclide tracers have an energy of from about 20 to about 4,000 kiloelectronvolts (i.e., keV).
  • radionuclide tracers include radionuclides that emit gamma radiation, positrons, or a combination of gamma radiation and positrons.
  • radionuclide tracers have appropriate decay characteristics for optimizing image resolution and quantitative accuracy and/or have appropriate residualization.
  • radionuclide tracers may have a physical half-life (i.e., t 1 ⁇ 2 ) compatible with the time required for the antibody to achieve optimal specific: non-specific binding ratios.
  • radionuclide tracers suitable for PET imaging include 11 C (t 1 ⁇ 2 ⁇ 20 min), 13 N (t 1 ⁇ 2 ⁇ 10 min), 15 O (t 1 ⁇ 2 ⁇ 2 min), 18 F (t 1 ⁇ 2 ⁇ 1.83 h), 44 Sc (t 1 ⁇ 2 ⁇ 3.97 h), 45 Ti (t 1 ⁇ 2 ⁇ 3 h), 52 Mn (t 1 ⁇ 2 ⁇ 5.6 days), 64 Cu (t 1 ⁇ 2 ⁇ 12.7 h), 68 Ga (t 1 ⁇ 2 ⁇ 1.13 h), 76 Br (t 1 ⁇ 2 ⁇ 16.2 h), 82 Rb (t 1 ⁇ 2 ⁇ 1.27 min), 86 Y (t 1 ⁇ 2 ⁇ 14.7 h), 89 Zr (t 1 ⁇ 2 ⁇ 78.4 h), 124 I (t 1 ⁇ 2 ⁇ 100.3 h), or a combination of any two or more thereof.
  • radionuclide tracers suitable for SPECT imaging include 99m Tc (t 1 ⁇ 2 ⁇ 6 h), 111 In (t 1 ⁇ 2 ⁇ 2.80 days), 131 I (t 1 ⁇ 2 ⁇ 8 days), 177 Lu (t 1 ⁇ 2 ⁇ 6.64 days), 186 Re (t 1 ⁇ 2 ⁇ 3.7186 days), or a combination of any two or more thereof.
  • radionuclide refers to a radionuclide moiety that can be attached to an antibody that functions to deliver a cytotoxic dose of radiation, such as, e.g., a radionuclide therapeutic agent, to a target of interest, such as, e.g., a tumor.
  • Suitable radionuclide therapeutic agents include radionuclides that emit beta particle radiation, alpha particle radiation, Auger electron radiation, or a combination of any two or more thereof.
  • suitable radionuclide therapeutic agents include 125 I, 131 I, 177 Lu, 186 Re, 225 Ac, 225 Ra, or a combination of any two or more thereof.
  • immunoimaging refers to imaging IFN- ⁇ in vivo in a subject via detection of a labeled anti-IFN- ⁇ antibody conjugate to produce an image indicative of the location and/or level of IFN- ⁇ in the subject, wherein the location and/or level of IFN- ⁇ in the subject is indicative of activated T cells.
  • Embodiments of the present disclosure are directed toward antibody conjugates, including labeled-antibody conjugates and therapeutic radionuclide-antibody conjugates, to methods for imaging, to mouse models, and to methods for assessing the effect of a composition or immune-mediated treatment for cancer, injury or infection, particularly human cancer, human injury or human infection.
  • antibody conjugates include both labeled-antibody conjugates and therapeutic radionuclide-antibody conjugates.
  • labeled-antibody conjugates include an antibody that specifically recognizes and binds to IFN- ⁇ , and at least one detection label conjugated to the antibody, wherein the at least one detection label includes a radionuclide tracer.
  • Suitable antibodies that specifically recognize and bind to IFN- ⁇ may be prepared by standard techniques known to those of ordinary skill in the art.
  • the antibody is an isolated antibody.
  • the antibody is selected from a monoclonal antibody, an antibody fragment, or combination thereof.
  • the antibody is a monoclonal antibody expressed by a hybridoma cell line or by CHO cells, NS0 cells, Sp2/0 cells, HEK cells, BHK cells, or PER.C6 cells.
  • the antibody is an antibody fragment. In further embodiments, the antibody is a Fab fragment.
  • Antibody fragments that recognize specific epitopes can be produced by standard techniques known to those of ordinary skill in the art. For example, F(ab′) 2 fragments can be produced by pepsin digestion of the antibody molecule. (See e.g., U.S. Pat. Nos. 4,036,945 and 4,331,647 and references contained therein; see also Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959), Edelman et al., in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and Coligan et al.
  • Fab′ expression libraries can be constructed (see e.g., Huse et al., 1989, Science, 246:1274-1281) to allow rapid and easy identification of monoclonal Fab′ fragments with desired specificity.
  • a single chain Fv molecule (i.e., scFv) includes a V L domain and a V H domain.
  • the V L and V H domains may associate to form a target binding site.
  • V L and V H domains are covalently linked by a peptide linker (i.e., L).
  • a scFv molecule is denoted as either V L -L-V H if the V L domain is the N-terminal part of the scFv molecule, or as V H -L-V L if the V H domain is the N-terminal part of the scFv molecule.
  • scFv molecules can be produced by standard techniques known to those of ordinary skill in the art.
  • Single domain antibodies i.e., V HH
  • Single domain antibodies may be obtained, for example, from camels, alpacas or llamas by standard immunization techniques known to those of ordinary skill in the art.
  • the V HH has potent antigen-binding capacity and can interact with novel epitopes that may be inaccessible to conventional V H -V L pairs.
  • alpaca serum IgG contains about 50% camel heavy chain only IgG antibodies (i.e., HCAbs) (see e.g., Maass et al., 2007).
  • alpacas may be immunized with known antigens, such as, e.g., TNF- ⁇ , and V HH ’ S can be isolated that bind to and neutralize the target antigen (see e.g., Maass et al., 2007).
  • PCR primers that amplify virtually all alpaca V HH coding sequences have been identified and may be used to construct alpaca V HH phage display libraries, which can be used for antibody fragment isolation by standard biopanning techniques well known to those of ordinary skill in the art. (See Maass et al., 2007).
  • an antibody fragment is produced by proteolytic hydrolysis of a full-length antibody and/or by expression in E. coli, CHO cells, or another host of DNA coding for the antibody fragment.
  • antibody fragments can be obtained by pepsin or papain digestion of full-length antibodies by standard techniques known to those of ordinary skill in the art.
  • an antibody fragment can be produced by enzymatic cleavage of antibodies with pepsin to provide an ⁇ 100 kDa fragment denoted F(ab′)2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce a ⁇ 50 kDa Fab′ monovalent fragment.
  • a thiol reducing agent and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce a ⁇ 50 kDa Fab′ monovalent fragment.
  • an enzymatic cleavage using papain produces two monovalent Fab fragments and an Fc fragment directly.
  • antibody fragments may include peptides coding for a single complementary determining region (i.e., CDR).
  • CDR complementary determining region
  • a CDR is a segment of the variable region of an antibody that is complementary in structure to the epitope to which the antibody binds and is more variable than the rest of the variable region. Accordingly, in embodiments, a CDR is referred to as a hypervariable region.
  • a variable region includes three CDRs.
  • CDR peptides can be produced by constructing genes encoding the CDR of an antibody of interest. Such genes may be prepared, for example, by using the polymerase chain reaction (i.e., PCR) to synthesize the variable region from RNA of antibody-producing cells.
  • the antibody conjugate is an anti-IFN- ⁇ diabody that specifically recognizes and binds to IFN- ⁇ .
  • a diabody included in a labeled-antibody conjugate is a noncovalent dimer of single-chain Fv (scFv) fragment that has the heavy chain variable (V H ) and light chain variable (V L ) regions connected by a linker, such as a peptide linker.
  • the diabody includes heavy chain variable region (V H ) having the amino acid sequence disclosed herein as SEQ ID NO:11 and light chain variable region (V L ) having the amino acid sequence disclosed herein as SEQ ID NO:12.
  • the linker used is too short to allow pairing between the two domains on the same chain, such that the linked V H - V L domains are forced to pair with complementary domains of another chain, creating two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).
  • An exemplary linker is SGGGGS (SEQ ID NO:13).
  • an scFv included in an anti-IFN- ⁇ diabody that specifically recognizes and binds to IFN- ⁇ has SEQ ID NO:14.
  • an scFv included in an anti-IFN- ⁇ diabody that specifically recognizes and binds to IFN- ⁇ has SEQ ID NO:15.
  • Variants of the scFv of SEQ ID NO:14 and/or SEQ ID NO:15 can also be included in an anti-IFN- ⁇ diabody in labeled-antibody conjugate according to embodiments.
  • a functional variant of the scFv of SEQ ID NO:14 has at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, sequence identity with the amino acid sequence set forth in SEQ ID NO:14 wherein the diabody including the variant or variants specifically recognizes and binds to IFN- ⁇ .
  • a functional variant of the scFv of SEQ ID NO:15 has at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, sequence identity with the amino acid sequence set forth in SEQ ID NO:15 wherein the diabody including the variant or variants specifically recognizes and binds to IFN- ⁇ .
  • the diabody including scFv of SEQ ID NO:14 and/or SEQ ID NO:15 or a variant of either thereof can be isolated or generated recombinantly or synthetically.
  • Mutations can be introduced using standard molecular biology techniques such as site-directed mutagenesis and PCR-mediated mutagenesis to produce a diabody including a variant of the scFv SEQ ID NO: 14 and/or SEQ ID NO:15.
  • site-directed mutagenesis and PCR-mediated mutagenesis to produce a diabody including a variant of the scFv SEQ ID NO: 14 and/or SEQ ID NO:15.
  • One of skill in the art will recognize that one or more amino acid mutations can be introduced without altering the functional properties of the diabody.
  • Variants of the diabody V H and V L domains of SEQ ID NO:11 and SEQ ID NO:12 can also be included in a labeled-antibody conjugate according to embodiments.
  • a functional variant of the diabody V H and V L domains of SEQ ID NO:11 and SEQ ID NO:12 has at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, sequence identity with the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:12 wherein the diabody including the variant or variants specifically recognizes and binds to IFN- ⁇ .
  • the diabody including V H and V L domains of SEQ ID NO:11 and SEQ ID NO:12 or a variant of either thereof can be isolated or generated recombinantly or synthetically.
  • Mutations can be introduced using standard molecular biology techniques such as site-directed mutagenesis and PCR-mediated mutagenesis to produce a diabody including a variant of V H and V L domains of SEQ ID NO:11 and SEQ ID NO:12.
  • site-directed mutagenesis and PCR-mediated mutagenesis to produce a diabody including a variant of V H and V L domains of SEQ ID NO:11 and SEQ ID NO:12.
  • PCR-mediated mutagenesis to produce a diabody including a variant of V H and V L domains of SEQ ID NO:11 and SEQ ID NO:12.
  • amino acid mutations can be introduced without altering the functional properties of the diabody.
  • Conservative amino acid substitutions can be made in the diabody sequences of SEQ ID NO:11, SEQ ID NO:12, and/or SEQ ID NO:14, to produce functional variants.
  • Conservative amino acid substitutions are art recognized substitutions of one amino acid for another amino acid having similar characteristics.
  • each amino acid can be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar, hydrophobic, and hydrophilic.
  • a conservative substitution is a substitution of one amino acid having a specified structural or functional characteristic for another amino acid having the same characteristic.
  • Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine, and valine; aromatic amino acids include phenylalanine, tyrosine, and tryptophan; polar amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, and tryptophan; and conservative substitutions include substitutions among amino acids within each group.
  • Amino acids can also be described in terms of steric effects or relative size, e.g., alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine, and valine are all typically considered to be small.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in a first amino acid or nucleotide sequence for optimal alignment with a second amino acid or nucleotide sequence using the default parameters of an alignment software program).
  • the amino acids at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the sequences are identical at that position.
  • the determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • a non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, PNAS USA 87:2264-68, e.g., as modified as in Karlin and Altschul, 1993, PNAS USA 90:5873-77. In calculating percent identity, only exact matches are typically counted.
  • cleaving antibodies such as, e.g., separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the antibody fragments specifically bind to the antigen that is recognized by the intact antibody.
  • the detection label or radionuclide therapeutic agent is conjugated directly, or indirectly, to the antibody to produce an antibody conjugate.
  • Suitable linkers, chelating agents and methods of use for conjugation of a detection label or radionuclide therapeutic agent to an antibody are well-known in the art, see for example, Shan S. Wong et al., Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation, Second Edition, CRC Press, 2011.
  • an antibody conjugate is an imaging agent which can be used in diagnostic procedures as well as for localization of activated T cells. Imaging can be performed by many procedures well-known to those having ordinary skill in the art, for example, by positron emission tomography (PET), single photon emission computed tomography (SPECT), Cerenkov imaging, photoacoustic imaging, ultrasound imaging, optical coherent tomography, optical imaging, including fluorescence imaging, and/or bioluminescence imaging, or a combination of any two or more thereof.
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • Cerenkov imaging photoacoustic imaging
  • ultrasound imaging optical coherent tomography
  • optical imaging including fluorescence imaging, and/or bioluminescence imaging, or a combination of any two or more thereof.
  • the labeled-antibody conjugates include at least one detection label conjugated to the antibody, wherein the at least one detection label includes a radionuclide tracer. In embodiments, the at least one detection label is a combination of a radionuclide tracer and a fluorophore.
  • the radionuclide tracer is selected from 11 C, 13 N, 15 O, 18 F, 44 Sc, 45 Ti, 52 Mn, 64 Cu, 68 Ga, 76 Br, 82 Rb, 86 Y, 89 Zr, 99m Tc, 111 In, 124 I, 131 I, 186 Re, 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 77 Br, 81 Rb, 81m Kr, 87m Sr, 89 Zr, 113m In, 123 I, 125 I, 127 Cs, 129 Cs, 132 I, 177 Lu, 186 Re, 197 Hg, 203 Pb, 206 Bi, 225 Ac, 225 Ra, or combination of any two or more thereof.
  • the radionuclide tracer is 89 Zr.
  • the radionuclide tracer is optionally conjugated to the antibody with a bifunctional chelator.
  • the radionuclide tracer is 11 C, 13 N, 18 F, 76 Br 123 I, 124 I, or 125 I
  • a bifunctional chelator is not required for conjugation to the antibody wherein 11 C, 13 N, 18 F, 76 Br 123 I, 124 I, or 125 I, can be directly conjugated to the antibody with standard techniques known to those of ordinary skill in the art.
  • the bifunctional chelator includes a chelator attached to a first linking group. Suitable chelators and first linking groups are known to those of ordinary skill in the art.
  • the bifunctional chelator includes a chelator selected from DFO, NOTA, DOTA, EDTA, DOPA, DTPA, NODA, or a combination of any two or more thereof.
  • the bifunctional chelator is p-SCN-Bn-DFO.
  • the bifunctional chelator is attached to the antibody.
  • the radionuclide tracer is complexed to the bifunctional chelator, wherein the bifunctional chelator is covalently attached to the antibody.
  • the bifunctional chelator is attached to the antibody by reacting the bifunctional chelator with the antibody by standard techniques known to those of ordinary skill in the art.
  • an ion of the radionuclide tracer is complexed to the bifunctional chelator by standard techniques known to those of ordinary skill in the art.
  • the bifunctional chelator p-SCN-Bn-DFO is reacted with the anti-IFN- ⁇ antibody at a ratio of about 5:1 in saline solution at a pH ⁇ 9 for about 1 hour at about 37° C. for covalent attachment thereof; and (2) the radionuclide tracer 89 Zr 4+ is reacted with the bifunctional chelator p-SCN-Bn-DFO covalently attached to the antibody at a pH ⁇ 7.0-7.2 for about 1 hour at room temperature to form a radiolabeled-antibody conjugate.
  • the labeled-antibody conjugates include at least one detection label conjugated to the antibody, wherein the at least one detection label includes a radionuclide tracer.
  • the at least one detection label also includes a fluorophore.
  • the fluorophore is a fluorescent dye.
  • the fluorophore emits fluorescence in the visible (i.e., 400-700 nm) or near-infrared (i.e., 700-1400 nm) region.
  • the antibody conjugates include therapeutic radionuclide-antibody conjugates.
  • therapeutic radionuclide-antibody conjugates include an antibody that specifically recognizes and binds IFN- ⁇ , a bifunctional chelator conjugated to the antibody, and a radionuclide therapeutic agent complexed to the bifunctional chelator.
  • the antibody is as described herein with regard to labeled-antibody conjugates.
  • the bifunctional chelator is as described herein with regard to labeled-antibody conjugates.
  • the therapeutic radionuclide-antibody conjugate includes a radionuclide therapeutic agent complexed to the bifunctional chelator.
  • the radionuclide therapeutic agent is selected from 125 I, 131 I, 177 Lu, 186 Re, 225 Ac, 225 Ra, or a combination of any two or more thereof.
  • the bifunctional chelator is attached to the antibody.
  • the radionuclide therapeutic agent is complexed to the bifunctional chelator, wherein the bifunctional chelator is covalently attached to the antibody.
  • the bifunctional chelator is attached to the antibody by reacting the bifunctional chelator with the antibody. Then, in embodiments, an ion of the radionuclide therapeutic agent is complexed to the bifunctional chelator.
  • the therapeutic radionuclide-antibody conjugate includes a labeled-antibody conjugate attached to a therapeutic moiety.
  • the therapeutic moiety can be, without limitation, a small molecule drug, an oligonucleotide or polynucleotide, such as an antisense oligonucleotide or polynucleotide, an siRNA, an mRNA, an miRNA, an shRNA, a peptide or protein.
  • a therapeutic moiety is attached to, enclosed in or partially enclosed in, a particle.
  • An included particle can be selected from among a lipid particle; a polymer particle; an inorganic particle; and an inorganic/organic particle.
  • a mixture of particle types can also be included.
  • the particles can be of any shape, size, composition, or physiochemical characteristics compatible with administration to a subject.
  • the particles can be organic or inorganic particles, such as glass or metal and can be particles of a synthetic or naturally occurring polymer, such as polystyrene, polycarbonate, silicon, nylon, cellulose, agarose, dextran, and polyacrylamide.
  • the particle is a lipid particle including, but not limited to, liposomes, micelles, unilamellar or mulitlamellar vesicles; polymer particles such as hydrogel particles, polyglycolic acid particles or polylactic acid particles; inorganic particles such as calcium phosphate particles such as described in for example U.S. Pat. No. 5,648,097; and inorganic/organic particulate carriers such as described for example in U.S. Pat. No. 6,630,486. Further description of liposomes and methods relating to their preparation and use may be found in Liposomes: A Practical Approach (The Practical Approach Series, 264), V. P. Torchilin and V. Weissig (Eds.), Oxford University Press; 2nd ed., 2003.
  • An included particle is typically formulated such that the particle has a diameter, or longest dimension, in the range of about 1 nm - 10 microns.
  • an included particle is a nanoparticle, formulated such that the nanoparticle has a diameter, or longest dimension, in the range of about 1 nm — about 1000 nm. Further aspects of nanoparticles are described in S.M. Moghimi et al., FASEB J. 2005, 19, 311-30; Choi, et al., Mol Imaging. 2010 Dec; 9(6): 291-310; and Bogart et al., ACS Nano, 2014, 8 (4), pp 3107-3122.
  • methods for imaging include: (a) administering a labeled-antibody conjugate to a subject, wherein the labeled-antibody conjugate includes: an antibody that specifically recognizes and binds to IFN- ⁇ , and at least one detection label conjugated to the antibody, wherein the at least one detection label includes a radionuclide tracer; and (b) detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging.
  • the labeled-antibody conjugate is as described herein with regard to antibody conjugates.
  • the antibody and the at least one detection label are as described herein with regard to antibody conjugates.
  • the methods include administering a labeled-antibody conjugate to a subject. In embodiments, the methods include administering an effective amount of a labeled-antibody conjugate to a subject.
  • the labeled-antibody conjugate may be administered by any suitable route known to those of ordinary skill in the art. In embodiments, administration of the labeled-antibody conjugate is by intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intratumoral, perfusion through a regional catheter, and/or direct intralesional injection. In embodiments wherein the labeled-antibody conjugate is administered by injection, the administration may be by continuous infusion, by single bolus, and/or by multiple boluses.
  • administering the radiolabeled-antibody conjugate is non-immunogenic to the subject.
  • the subject is a mammal.
  • the subject is a mammal selected from humans, non-human primates, canines, felines, murines, bovines, equines, ovines, porcines, and lagomorphs.
  • the subject is a mouse or a human.
  • the labeled-antibody conjugate administered to a subject includes a radionuclide tracer. In some embodiments, the labeled-antibody conjugate administered to a subject includes a radionuclide tracer only, i.e. without a fluorophore.
  • the radionuclide tracer is selected from 11 C, 13 N, 15 O, 18 F, 44 Sc, 45 Ti, 52 Mn, 64 Cu, 68 Ga, 76 Br, 82 Rb, 86 Y, 89 Zr, 99m Tc, 111 In, 124 I, 131 I, 186 Re, 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 77 Br, 81 Rb, 81m Kr, 87m Sr, 89 Zr, 113m In, 123 I, 125 I, 127 Cs, 129 Cs, 132 I, 177 Lu, 186 Re, 197 Hg, 203 Pb. 206 Bi, 225 Ac, 225 Ra, or combination of any two or more thereof.
  • the radionuclide tracer is 89 Zr.
  • the labeled-antibody conjugate administered to a subject includes a fluorophore.
  • the fluorophore is a fluorescent dye.
  • the methods for imaging include detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging. In embodiments, the presence of the labeled-antibody conjugate is detected in real time. In embodiments, the presence of the labeled-antibody conjugate is detected non-invasively and/or minimally-invasively.
  • the presence of the labeled-antibody conjugate may be detected in vivo by PET imaging, SPECT imaging, or combination thereof.
  • the radionuclide tracer is selected from 11 C, 13 N, 15 O, 18 F, 64 Cu, 68 Ga, 76 Br, 82 Rb, 86 Y, 89 Zr, 124 I, or a combination of any two or more thereof, the presence of the labeled-antibody conjugate may be detected in vivo by PET imaging.
  • the presence of the labeled-antibody conjugate may be detected in vivo by SPECT imaging.
  • the presence of the labeled-antibody conjugate may be detected in vivo by optical imaging.
  • optical imaging techniques are known to those of ordinary skill in the art. (See e.g., Ntziachristos, Annu. Rev. Biomed. Eng. 2006, 8:1-33; Troyan, S. L. et al., Ann. Surg. Oncol . 16, 2943-2952 (2009); Luker, G. D. & Luker, K. E., J. Nucl. Med . 49, 1-4 (2008); Tromberg, B. J. et al., Med. Phys .
  • in vivo optical imaging is selected from confocal microscopy, planar imaging, fluorescence molecular tomography, complete projection tomography, fluorescence tomography direct imaging, two-photon in vivo imaging or a combination of any two or more thereof.
  • planar imaging is selected from epi-illumination (i.e., photographic) imaging, trans-illumination imaging, tomographic imaging, or a combination of any two or more thereof.
  • the fluorophore may be selected such that it emits fluorescence in the visible or near-infrared region.
  • fluorophores emitting fluorescence in the visible or near-infrared region may be selected from fluorescein, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red® (Molecular Probes, Inc., Eugene, OR), AlexaFluor® (Molecular Probes, Inc., Eugene), AlexaFluor 350, AlexaFluor 405, AlexaFluor 430, AlexaFluor 488, AlexaFluor 500, AlexaFluor 532, AlexaFluor 546, AlexaFluor 568, AlexaFluor 594, AlexaFluor 610, AlexaFluor 633, AlexaFluor 647
  • the presence of the labeled-antibody conjugate is detected by in vivo photoacoustic imaging.
  • photoacoustic dyes included in the conjugate may be selected from Methylene blue, Evan’s blue, Trypan blue, Patent blue, Indocyanine Green, IRDye800CW, DiR, Cy7, Cy7.5, and porphyrins or a combination of any two or more thereof.
  • the labeled antibody conjugate includes a combination of a radionuclide tracer and a fluorophore
  • the presence of the labeled-antibody conjugate may be detected in vivo by a combination of PET or SPECT and optical imaging.
  • the labeled antibody conjugate includes a combination of a radionuclide tracer and a fluorophore
  • the presence of the labeled-antibody conjugate may be detected in vivo by a combination of PET or SPECT and photoacoustic imaging.
  • the presence of the labeled-antibody conjugate may be detected in vivo by a combination of any two or more of: PET, SPECT, Cerenkov imaging, photoacoustic imaging, ultrasound imaging, optical coherent tomography, optical imaging, including fluorescence imaging, and/or bioluminescence imaging.
  • a method for imaging a subject includes 1) administering a labeled-antibody conjugate to a subject, wherein the labeled-antibody conjugate includes: an antibody that specifically binds to IFN- ⁇ , and a detection label conjugated to the antibody, wherein the detection label is a radionuclide tracer or fluorophore; and 2) detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging.
  • the subject has cancer.
  • the cancer is bladder cancer, brain tumors, breast cancer, cervical cancer, colorectal cancer, kidney cancer, leukemia, lung cancer, melanoma, myeloma, pancreatic cancer, gastric cancer, intestinal cancer, esophageal cancer, mesothelioma cancer, endometrial cancer, ovarian cancer, head and neck cancer, bone cancer, sarcoma, cholangiocarcinoma, gall bladder cancer, testicular cancer, thyroid cancer, or prostate cancer.
  • the subject has an inflammatory condition or a disease associated with inflammation, such as, but not limited to, infection, tissue injury, Alzheimer’s disease, obesity, stroke, heart attack, Hepatitis C, and Crohn’s disease.
  • a disease associated with inflammation such as, but not limited to, infection, tissue injury, Alzheimer’s disease, obesity, stroke, heart attack, Hepatitis C, and Crohn’s disease.
  • Combinations of a labeled-antibody conjugate and at least one therapeutic agent are administered according to aspects of the present invention.
  • a labeled-antibody conjugate and at least one additional active agent are administered to a subject to treat cancer in a subject in need thereof.
  • a labeled-antibody conjugate and at least two additional active agents are administered to a subject to treat cancer in a subject in need thereof.
  • additional active agent is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide).
  • the additional active agent can be, without limitation, a small molecule drug, an oligonucleotide or polynucleotide, such as an antisense oligonucleotide or polynucleotide, an siRNA, an mRNA, an miRNA, an shRNA, a peptide or protein.
  • An additional active agent can be a therapeutic agent or a diagnostic agent according to embodiments.
  • Additional active agents which are therapeutic agents included in aspects of methods and compositions of the present invention include, but are not limited to, antibiotics, antivirals, antineoplastic agents, analgesics, antipyretics, antidepressants, antipsychotics, anti-cancer agents, antihistamines, anti-osteoporosis agents, anti-osteonecrosis agents, antiinflammatory agents, anxiolytics, chemotherapeutic agents, diuretics, growth factors, hormones, non-steroidal antiinflammatory agents, steroids and vasoactive agents.
  • combination treatments include: (1) administration of pharmaceutical compositions that include a labeled antibody conjugate in combination with one or more additional active agents; (2) co-administration of a labeled antibody conjugate with one or more additional active agents wherein the labeled antibody conjugate and the one or more additional active agents are formulated in the same composition and (3) co-administration of a labeled antibody conjugate with one or more additional active agents wherein the labeled antibody conjugate and the one or more additional therapeutic agents have not been formulated in the same composition.
  • the labeled antibody conjugate and the one or more additional active agents may be administered at the same time or at different times.
  • An adjunct anti-cancer treatment can be administered in combination with a labeled antibody conjugate, such as prior to, simultaneous with or after administration of the labeled antibody conjugate, such as an anti-cancer radiation treatment of a subject or an affected area of a subject’s body.
  • An additional active agent can be an anti-cancer agent according to embodiments, such as a targeted therapy agent or chemotherapeutic.
  • Anti-cancer agents are described, for example, in Goodman et al., Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 8th Ed., Macmillan Publishing Co., 1990.
  • Anti-cancer agents illustratively include acivicin, aclarubicin, acodazole, acronine, adozelesin, aldesleukin, alitretinoin, allopurinol, altretamine, ambomycin, ametantrone, amifostine, aminoglutethimide, amsacrine, anastrozole, anthramycin, arsenic trioxide, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene, bisnafide dimesylate, bizelesin, bleomycin, brequinar, bropirimine, busulfan, cactinomycin, calusterone, capecitabine, caracemide, carbetimer, carboplatin, carmustine, carubicin, carzelesin, cedef
  • the additional active agent is an anti-cancer immunotherapy agent.
  • the anti-cancer immunotherapy agent is an immune checkpoint inhibitor, a cytokine, a vaccine, an adoptive cell transfer therapy, or any therapeutic wherein the mechanism of action of the therapeutic is an increased number of tumor-infiltrating lymphocytes in the subject and/or an increased activation state of a tumor-infiltrating lymphocyte population in the subject.
  • the immune checkpoint inhibitor is a PD-1 inhibitor, PD-L1 inhibitor, or CTLA-4 inhibitor.
  • the immune checkpoint inhibitor selected from atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab, pembrolizumab, and an antigen-binding fragment of any one of the foregoing.
  • the subject is human and the antibody specifically binds to human IFN- ⁇ .
  • an anti-cancer immunotherapy agent is administered to a subject and a labeled antibody conjugate is administered simultaneously or subsequently followed by detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging to image IFN- ⁇ and thereby monitor the effectiveness of the treatment with the anti-cancer immunotherapy agent.
  • the subject received an anti-cancer immunotherapy prior to administering and detecting the presence of the labeled-antibody conjugate, and wherein the mechanism of action of the immunotherapy results in an increased number of tumor-infiltrating lymphocytes in the subject and/or an increased activation state of a tumor-infiltrating lymphocyte population in the subject; or wherein administering and detecting the presence of the labeled-antibody conjugate is performed prior to administration of a therapy to determine the state of active immunity in the subject.
  • efficacy of the anti-cancer immunotherapy is monitored non-invasively and in real-time in the subject.
  • significant binding of the labeled-antibody conjugate to the IFN- ⁇ antigen indicates the presence of activated lymphocytes and/or human cancer cells. In embodiments, significant binding of the labeled-antibody conjugate to the IFN- ⁇ antigen indicates the presence of a localized immune response in the subject.
  • PET imaging is quantitative wherein a correlation is determined between uptake of the radionuclide tracer quantified by radioactivity measurements of excised tissues and uptake estimated noninvasively by PET. Thus, efficacy of the anti-cancer immunotherapy is monitored non-invasively and in real-time in the subject.
  • an anti-inflammation immunotherapy agent is administered to a subject and a labeled antibody conjugate is administered simultaneously or subsequently followed by detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging to image IFN- ⁇ and thereby monitor the effectiveness of the treatment with the anti-inflammation immunotherapy agent.
  • the subject received an anti-inflammation immunotherapy prior to administering and detecting the presence of the labeled-antibody conjugate, and wherein the mechanism of action of the immunotherapy results in an increased number of activated lymphocytes in the subject and/or an increased activation state of an activated lymphocyte population in the subject; or wherein administering and detecting the presence of the labeled-antibody conjugate is performed prior to administration of a therapy to determine the state of active immunity in the subject.
  • efficacy of the anti-inflammation immunotherapy is monitored non-invasively and in real-time in the subject.
  • significant binding of the labeled-antibody conjugate to the IFN- ⁇ antigen indicates the presence of activated lymphocytes. In embodiments, significant binding of the labeled-antibody conjugate to the IFN- ⁇ antigen indicates the presence of a localized immune response in the subject.
  • PET imaging is quantitative wherein a correlation is determined between uptake of the radionuclide tracer quantified by radioactivity measurements of excised tissues and uptake estimated noninvasively by PET. In embodiments, PET imaging is quantitative wherein uptake of the radionuclide tracer is quantified by analysis of acquired images.
  • methods for imaging a subject include administering a labeled-antibody conjugate to a subject, wherein the labeled-antibody conjugate comprises: an antibody that specifically binds to IFN- ⁇ , and a detection label conjugated to the antibody, wherein the detection label is a radionuclide tracer or fluorophore; and detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging.
  • a labeled-antibody conjugate is administered to a subject prior to immunotherapy treatment, wherein the labeled-antibody conjugate includes an antibody that specifically binds to IFN- ⁇ , and a detection label conjugated to the antibody, wherein the detection label is a radionuclide tracer or fluorophore; and the presence of the labeled-antibody conjugate in the subject is detected in vivo by imaging to establish a baseline reading indicative of a baseline level of IFN- ⁇ to determine the state of active immunity in the subject.
  • An immunotherapy is then administered wherein the mechanism of action of the immunotherapy results in an increased number of activated lymphocytes in the subject and/or an increased activation state of an activated lymphocyte population in the subject.
  • the labeled-antibody conjugate is then administered to a subject again after the administration of the immunotherapy to establish a post-treatment reading indicative of a subsequent level of IFN- ⁇ to determine the state of active immunity in the subject.
  • the steps of administration of the immunotherapy and administration/detection of the labeled-antibody conjugate may be performed 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times, over a period of treatment time in the range of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, 1, 2, 3, 4, 5, 6, 7, or more days, 1, 2, 3, 4, 5, 6, 7, 8, or more weeks, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more months, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more years, depending on the course of the disease and the course of treatment.
  • a labeled-antibody conjugate is administered to a subject prior to immunotherapy treatment of cancer in a subject having or suspected of having cancer, wherein the labeled-antibody conjugate includes an antibody that specifically binds to IFN- ⁇ , and a detection label conjugated to the antibody, wherein the detection label is a radionuclide tracer or fluorophore; and the presence of the labeled-antibody conjugate in the subject is detected in vivo by imaging to establish a baseline reading indicative of a baseline level of IFN- ⁇ to determine the state of active immunity in the subject.
  • An anti-cancer immunotherapy is then administered wherein the mechanism of action of the anti-cancer immunotherapy results in an increased number of tumor-infiltrating lymphocytes in the subject and/or an increased activation state of a tumor-infiltrating lymphocyte population in the subject.
  • the labeled-antibody conjugate is then administered to a subject again after the administration of the anti-cancer immunotherapy to establish a post-treatment reading indicative of a subsequent level of IFN- ⁇ to determine the state of active immunity in the subject.
  • the steps of administration of the anti-cancer immunotherapy and administration/detection of the labeled-antibody conjugate may be performed 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times, over a period of treatment time in the range of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, 1, 2, 3, 4, 5, 6, 7, or more days, 1, 2, 3, 4, 5, 6, 7, 8, or more weeks, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more months, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more years, depending on the course of the disease and the course of treatment.
  • the disclosure discloses mouse models.
  • the mouse models include: a mouse having a human cancer xenograft and a labeled-antibody conjugate, wherein the labeled-antibody conjugate includes: an antibody that specifically recognizes and binds to IFN- ⁇ , and at least one detection label conjugated to the antibody, wherein the at least one detection label is selected from a radionuclide tracer or a fluorophore.
  • the labeled-antibody conjugate is as described herein with regard to antibody conjugates and methods of imaging.
  • the mouse model has a human cancer xenograft.
  • the human cancer xenograft is transplanted into the mouse.
  • the human cancer xenograft is established from human cancer cells, human cancer tissues, or combination thereof.
  • the human cancer xenograft includes human cancer cells and/or human cancer tissues of primary cancer and/or metastatic cancer.
  • the human cancer xenograft includes human cancer cells and/or human cancer tissues of primary cancer and/or metastatic cancer selected from brain cancer, melanoma, colon cancer, kidney cancer, prostate cancer, breast cancer, gastric cancer, pancreatic cancer, ovarian cancer and testicular cancer.
  • the mouse model having a human cancer xenograft is immunodeficient and further includes engrafted human hematopoetic stem cells such that the mouse has a “humanized” immune system which produces human IFN- ⁇ .
  • the methods include determining the effect of the composition or treatment on the growth of a human cancer xenograft in the mouse.
  • the effect of the composition or treatment on the growth of the human cancer xenograft is determined by imaging.
  • suitable imaging techniques include those described herein with regard to imaging methods.
  • the effect of the composition or treatment is determined by comparing the growth of the human cancer xenograft in the mouse administered the composition or treatment to growth of a human cancer xenograft in a mouse not administered the composition or treatment.
  • the mouse models include: a mouse having an injury or infection and a labeled-antibody conjugate, wherein the labeled-antibody conjugate includes: an antibody that specifically recognizes and binds to IFN- ⁇ , and at least one detection label conjugated to the antibody, wherein the at least one detection label is selected from a radionuclide tracer or a fluorophore.
  • the labeled-antibody conjugate is as described herein with regard to antibody conjugates and methods of imaging immune-mediated treatment for injury or infection.
  • the mouse models include: an immune competent mouse having a murine cancer, such as an allograft or induced murine tumor, and a labeled-antibody conjugate, wherein the labeled-antibody conjugate includes: an antibody that specifically recognizes and binds to murine IFN- ⁇ , and at least one detection label conjugated to the antibody, wherein the at least one detection label is selected from a radionuclide tracer or a fluorophore.
  • the labeled-antibody conjugate is as described herein with regard to antibody conjugates and methods of imaging.
  • mice Female BALB/c mice (6-8 week old) were purchased from Charles River Laboratories (Wilmington, MA). Heterozygous BALB/NeuT (NeuT) mice were in-house bred. NeuT male mice, which express a transforming rat neu, develop atypical ductal hyperplasia in 1-2 parotid glands by 6 weeks of age which progresses to multifocal acinic cell adenocarcinoma in situ at about 19 weeks of age.
  • p-SCN-Bn-Desferrioxamine (DFO, Macrocyclics, Plano, TX) was incubated with 145-2C11 (anti-CD3 ⁇ , Armenian hamster, IgG, eBioscience, San Diego, CA), or AN-18 (anti-IFNy, rat IgG1, kappa, eBioscience, San Diego, CA) at a ratio of 1:5 (Ab:DFO) in phosphate buffered saline at pH ⁇ 9 for 1 h at 37° C. and purified via centrifugation with molecular weight column filter (MWCO: 30 kDa, GE Vivaspin 500).
  • DFO molecular weight column filter
  • Tumor-bearing animals were injected intravenously (i.v.). with the radiolabeled antibody ( ⁇ 180-240 ⁇ Ci/mouse, 42.8 - 57.1 ⁇ g in 150 ⁇ L sterile saline) in the lateral tail vein. Imaging was acquired from 4-120 h post-injection (p.i.). The animals were anesthetized with 3-5% isoflurane (Baxter Healthcare, Deerfield, IL) in air for induction, then lowered to 1.5-2% for maintenance. Images were acquired with a microPET R4 camera (Siemens Medical Solutions, USA formerly Concorde Microsystems).
  • VOI 3-dimensional volumes-of-interest
  • IFN- ⁇ was systemically induced in na ⁇ ve BALB/c mice by intramuscular (i.m.) injection of 100 ⁇ g CpG-ODN (CpG-oligodeoxynucleotides, Integrated DNA Technologies, Coralville, IA). Within an hour of treatment, the mice were injected with 89 Zr-anti-IFN- ⁇ in the lateral tail vein for imaging (180-240 ⁇ Ci/mouse, 49.9 ⁇ 7.2 ⁇ g). PET imaging was conducted at 72 h p.i. Labeled-antibody conjugate uptake within the spleen and peripheral immune tissues were analyzed and compared against a control, untreated group.
  • mice were injected i.v. in the lateral tail vein with 89 Zr-anti-IFN- ⁇ (20-30 ⁇ Ci, 4.7-7.1 ⁇ g) in 100-150 ⁇ L saline followed by euthanasia via CO 2 asphyxiation after 72 h p.i., the time point identified at which tumor-to-background is sufficient.
  • 80 ⁇ g of cold AN-18 was co-injected with the probe in a separate cohort of mice.
  • Select organs were harvested post-sacrifice, weighed and measured for bound radioactivity with a gamma counter (Perkin Elmer 2480 Wizard 2). The %ID/g was calculated as the % of activity bound to the tissue normalized against total administered activity per gram of tissue weight.
  • TUBO cells (4.5 ⁇ 10 5 ) were plated into 6 well plates in triplicate and allowed to adhere overnight. In separate wells, cells were either left untreated or incubated with recombinant IFN- ⁇ (10 ng/mL) for 15 minutes before being washed 3 times with PBS. 89 Zr-anti-IFN- ⁇ (150 ng, 0.63 nCi) was added and cells were incubated for 1 h at 37° C. Following the incubation period, the media was collected and the cells were rinsed with 1 ⁇ phosphate buffered saline (PBS) twice. Surface-bound activity was removed by washing the cells in 100 mM acetic acid + 100 mM glycine (1:1, pH 3.5) at 4° C.
  • PBS phosphate buffered saline
  • the cells were then lysed with 1 M NaOH. All washes (media plus PBS, acid and alkaline) were collected in separate tubes and measured for bound activity using a gamma counter (Perkin Elmer). The %-internalized activity was calculated as the ratio of the activity of the lysate and the total activity collected from the media, PBS, acid and base washes, normalized to 450,000 cells counted using a Countess II Automated Cell Counter (Thermo Fisher).
  • the HER2/neu DNA vaccine consists of 20 ⁇ g of pGM-CSF (encoding murine GM-CSF) and 50 ⁇ g pE2TM (encoding the extracellular and transmembrane regions of human HER2) in 50 ⁇ L PBS, which is injected i.m. into each gastrocnemius followed immediately by application of electrode gel and square wave electroporation using a BTX830 (BTX Harvard Apparatus, Holliston, MA).
  • mice were depleted of Tregs by intraperitoneal (i.p.) injection of 500 ⁇ g anti-CD25 mAb PC61 10 days prior to the first vaccination.
  • Tumor tissue was snap frozen in liquid nitrogen.
  • Total tumor RNA was collected by Trizol preparation (Thermo Fisher, Waltham, MA) after homogenization.
  • cDNA was synthesized with ProtoScript II reverse transcriptase (New England Biolabs, MA).
  • Real-time qPCR was conducted with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA) using 10 ng cDNA/well and 500 nM primers specific to the indicated gene (Life Tech, Carlsbad, CA).
  • CD3 forward primer CACTCTGGGCTTGCTGATGG, SEQ ID NO:1
  • CD3 reverse primer TCATAGTCTGGGTTGGAACAGG
  • CD8 forward primer GCTGGTAGTCTGCATCCTGCTTC, SEQ ID NO:3
  • CD8 reverse primer TTGCTAGCAGGCTATCAGTGTTGTG, SEQ ID NO:4
  • IFN- ⁇ forward primer GAGCTCATTGAATGCTTGGC, SEQ ID NO:5
  • IFN- ⁇ reverse primer GCGTCATTGAATCACACCTG, SEQ ID NO:6
  • PD-1 forward primer CGTCCCTCAGTCAAGAGGAG, SEQ ID NO:7
  • PD-1 reverse primer GTCCCTAGAAGTGCCCAACA, SEQ ID NO:8
  • GAPDH forward primer AAGCTCACTGGCATGGCCTTC, SEQ ID NO:9
  • GAPDH reverse primer TGCTTCACCACCTTCTTGATGTC, SEQ ID NO:9
  • GAPDH reverse primer TGCTTCACCACCT
  • Relative mRNA quantities are calculated by 2 - ⁇ CT compared to GAPDH.
  • Tumor tissue was homogenized in standard RIPA buffer with protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was measured by BCA assay (ThermoFisher). High protein binding plates (ThermoFisher) were coated with 3 ⁇ g/mL anti-mouse-IFN-y mAb clone AN-18 (eBioscience) in coating buffer (0.1 M Na 2 HPO 4 , pH to 9.0) and washed prior to addition of samples or standard curve using recombinant mouse IFN- ⁇ (Peprotech, Rocky Hill, NJ) in duplicate.
  • coating buffer 0.1 M Na 2 HPO 4 , pH to 9.0
  • IFN- ⁇ was detected with biotin-conjugated anti-mouse IFN- ⁇ clone R4-6A2 (eBioscience), avidin-HRP (ThermoFisher), and TMB substrate (ThermoFisher).
  • Serum HER2- and neu-specific IgG were quantified by flow cytometry with a BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ), using HER2 over-expressing SKOV3 cells or neu transfected 3T3/NKB cells. Serum was incubated with the appropriate cell line at a 1:100 dilution, followed by washes and detection with PE-conjugated anti-mouse IgG secondary antibody and additional washes prior to detection by flow cytometry. Standard curves for HER2 and neu were run using antibody clones TA-1 and 7.16.4, respectively. Regression analysis was conducted using standard curves of anti-HER2 mAb TA-1 (Calbiochem, Burlington, MA) or anti-neu mAb 7.16.4 (Calbiochem, Burlington, MA).
  • HER2- and neu-specific IFN- ⁇ production was measured by ELISPOT assay.
  • recombinant HER2 or neu (10 ⁇ g/mL, Sino Biologicals, Beijing, China) were incubated with splenocytes for 48 h in round-bottom wells, followed by transfer to anti-IFN-y coated (clone AN-18, eBioscience) ELISPOT plates (Millipore Sigma, Burlington, MA) for an additional 48 h.
  • mice bearing TUBO tumors were injected i.p. 5 times every 3-4 days with sterile-filtered ascites containing 1 mg anti-neu mAb 7.16.4 diluted in PBS to a final volume of 300 ⁇ L.
  • IHC was performed using anti-CD8 (clone D4W2Z, Cell Signaling) antibody. After euthanasia, tumors were harvested and fixed in formalin before being embedded in paraffin. Blocks were sectioned into 4 ⁇ m sections using a Sakura Accu-Cut SRM microtome (Catalog#: SRM- 200 CV) and adhered onto positively charged slides (Histomax Plus, VWR). Slides were then incubated for 12 minutes at 65° C. and deparrafinized in washes of xylene and graded alcohols. Antigen retrieval was performed in PT module buffer (TA-250-PM4X, Fisher) for CD8 (1:200). Primary antibody incubations were performed for 1 h at room temperature in a humidified chamber.
  • PT module buffer TA-250-PM4X, Fisher
  • CD8 T cell enumeration was conducted by a blinded board certified pathologist. Each tumor sample was screened for hotspots of CD8 lymphocytes using a Nikon Eclipse Ci microscope at 100x magnification. The number of CD8 + T lymphocytes were counted in the three regions of highest infiltration at 400x magnification with a 0.55 mm field diameter, and an average was calculated.
  • tissue sections were subjected to xylenes, graduated alcohol and distilled water washes. They were then stained with hematoxylin (TA-125-MH, Fisher) for 5 minutes, rinsed with an acid wash for 1 minute and a bluing agent for 15 seconds.
  • TUBO tumors from untreated BALB/c mice were dissociated using the GentleMACs Dissociator and mouse tumor dissociation kit (Miltenyi, Germany) following the manufacturer protocol.
  • Cells were stained with a combination of either: FITC-conjugated CD45 mAb (clone 30-F11), APC-conjugated TCR ⁇ mAb (clone H57-597), and eFluor780-conjugated viability dye or FITC-conjugated CD45 mAb (clone 30-F11), PE-Cy7-conjugated CD8 mAb (clone 53-6.7), APC-conjugated PD-1 mAb (J43), and eFluor780-conjugated viability dye.
  • mice treated with CpG-ODN to stimulate IFN- ⁇ whole-body PET images were acquired 72 h p.i., a time point identified to exhibit reliable labeled-antibody conjugate uptake in the tumor, with low liver and blood pool background ( FIG. 7 A , FIG. 7 B , FIG. 8 A , FIG. 8 B ).
  • IFN- ⁇ is a soluble protein
  • the mechanism of localized IFN- ⁇ imaging was investigated. Plated TUBO tumor cells were exposed to IFN- ⁇ and/or 89 Zr-anti-IFN- ⁇ labeled-antibody conjugate in quintuplicate followed by analysis of membrane binding and internalization ( FIG. 1 C ). TUBO cells incubated with 89 Zr-anti-IFN- ⁇ alone show limited labeled-antibody conjugate surface binding (1.13 ⁇ 0.28%) and internalization (0.29 ⁇ 0.13%).
  • the spleen, liver, heart and spleen tissues were removed and scanned ex vivo ( FIG. 1 D ).
  • the notable low accumulation of the isotype control labeled-antibody conjugate after vaccination supports the specificity of the IFN- ⁇ labeled-antibody conjugate and suggests increased 89 Zr-anti-IFN- ⁇ uptake is not simply due vascular permeability and retention effects post-ITx.
  • mice Total T cell presence in the tumor microenvironment was assessed in separate groups of mice via immunoPET imaging of CD3+ tumor infiltrating lymphocytes using 89 Zr-anti-CD3 ( FIG. 2 E , FIG. 2 F , and FIG. 9 B ).
  • T cell-homing secondary lymphoid tissue (Ctrl: 17.06 ⁇ 3.56 %ID/g, Vx: 18.36 ⁇ 1.49 %ID/g, FIG. 10 B ), which can act as a labeled-antibody conjugate “sink.”
  • limited splenic accumulation was observed with the IFN- ⁇ PET probe (Ctrl: 3.58 ⁇ 0.81 %ID/g, p ⁇ 0.0001, Vx: 4.97 ⁇ 0.97 %ID/g, p ⁇ 0.0001).
  • HER2/neu-specific serum IgG FIG. 3 E and FIG. 3 F
  • splenic T cell responses FIG. 3 G and FIG. 3 H
  • TUBO tumors constitutively express the cell surface oncogene neu, which is foreign in wild-type BALB/c mice.
  • SC splenocytes
  • Neu transgenic mice are engineered to express a transforming rat neu under direction of the mouse mammary tumor virus promoter, allowing immune system recognition of neu as a self-antigen.
  • Male NeuT mice which develop 1-2 spontaneous neu+ salivary tumors between 30-40 weeks of age, were used.
  • Tregs were depleted using anti-CD25 mAb clone PC61 to enhance ITx response given NeuT mice are immune tolerant to rat neu, followed by two HER2/neu DNA vaccinations as detailed in FIGS. 4 A and 4 B .
  • T cell infiltration and IFN- ⁇ production were conducted by qPCR in tumor tissue samples ( FIG. 5 A , FIG. 5 B , and FIG. 5 C ).
  • Peripheral immune response to the vaccine was evaluated by measuring serum anti-HER2 and anti-neu IgG as well as spleen-resident HER2- and neu-responsive IFN- ⁇ -producing T cells. Tolerance to HER2/neu in NeuT mice was apparent with a comparatively lower ITx response vs. wild-type BALB/c mice bearing TUBO tumors in FIGS. 2 .
  • the ratio of anti-neu to anti-HER2 T cells detected was ⁇ 5.1% in NeuT mice, which is two-fold lower relative to that detected in non-immune tolerant BALB/c mice bearing TUBO (10.0%, FIG. 3 G and FIG. 3 H ).
  • IFN- ⁇ PET Imaging is an Indicator of Immune Activation Status In situ
  • IFN- ⁇ PET was then evaluated in a setting where tumor-infiltrating T cells are present but have become exhausted.
  • TUBO-bearing mice were treated with passive ITx, mAb 7.16.4 to rat neu. This mAb has been shown to inhibit neu signaling in addition to initiating host anti-tumor immunity.
  • 1 mg doses of 7.16.4 were given i.p. at 3-4 day intervals for a total of 5 treatments, which reduced and stabilized tumor growth ( FIG. 6 C ).
  • CD8 T cell infiltration was evaluated by IHC, see Table 2 below.
  • NOTA-PEG 4 -Tz was made (57 ⁇ 12% yield) by attaching NOTA-Bz-NCS to tetrazine (Tz)-PEG 4 -NH 2 using equimolar ratios in DMSO/TEA solvent for 1 h at room temperature (RT). Purification was done through C 18 RP-HPLC. 18 F-labeling proceeded using 18 F-AlF-NOTA method. Briefly, non-carrier added 18 F[F - ] will be incubated with 40 nmol AlCl 3 in 0.4 M KHCO 3 at pH ⁇ 4 for 10 min.
  • Dbs (MW: ⁇ 50 kDa, NeoClone, WI) is accomplished via inverse electron demand Diels-Alder bioorthogonal click reaction using Tz and transcyclooctene (TCO), which affords a rapid, mild method of labeling a protein, conserving its configuration.
  • TCO transcyclooctene
  • the Db fragment of anti-IFN ⁇ clone AN18 was attached to TCO-NHS, which was then conjugated to 18 F-AlF-NOTA-Tz at RT in PBS, pH ⁇ 7 for 15 min.
  • the labeled product was purified through a spin column (MWCO: 10 kDa).
  • Radio-instant thin layer chromatography and size exclusion chromatography indicated >95% purities with an overall decay-corrected radiochemical yield of 6-10%.
  • the specific activity was 3.8-4 mCi/mg.
  • 100-150 ⁇ Ci Db tracer was injected into a TUBO-bearing BALB/c mouse after Vx ( FIG. 15 ).
  • Tumor uptake is detected with an ROI of ⁇ 8.0 %ID/g at 3 h p.i., similar to results obtained with full-length mAb ( FIG. 2 C ).
  • the clearance appears to be through the gall bladder and intestines, a clear shift from the hepatic sink typically observed in full mAbs. This validated labeling method will be maintained throughout the proposed studies.
  • a cold 19 F-IFN- ⁇ Db compound will be prepared as reference standard for SEC quality control. Retention of affinity post-conjugation is evaluated through binding affinity via surface plasmon resonance.
  • IFN- ⁇ PET methods of the present invention measure active anti-tumor immunity, providing a predictive tool for non-invasive in situ tumor evaluation.
  • This approach is highly specific to the tumor compared to total T cell imaging due to the fact that IFN- ⁇ is secreted by CTLs within the tumor.
  • Imaging CD3, targets the general T cell population that are not only localized in the tumor but also in other lymphoid tissues.
  • IFN- ⁇ PET may be more sensitive for determining response to ITx when compared to peripheral immune evaluation.
  • IFN- ⁇ PET has the potential to serve as a universal non-invasive measurement of immune activity in situ for a variety of cancers with virtually any ITx modality with no need for knowledge of specific antigens or cumbersome ex vivo antigen recall assays. Additionally, the utility of IFN- ⁇ PET allows for cancer immune monitoring, and examination of localized inflammatory conditions such as injury, infection, or autoimmune disease.
  • PET imaging of released, soluble IFN ⁇ displayed higher sensitivity of active anti-tumor immunity detection versus total CD3 imaging.
  • a major drawback to the use of general T cell surface markers for PET imaging is labeled-antibody conjugate uptake in normal secondary lymphoid tissues with dense T cell content, such as the spleen, thymus and lymph nodes, which may limit tumor detection.
  • these results indicate non-invasive in situ tumor evaluation by PET more clearly depicts responses to immunotherapy when compared to peripheral immune evaluation by ELISPOT.
  • IFN- ⁇ PET also does not require knowledge of specific tumor antigens or cumbersome ex vivo antigen recall assays to determine the extent of immunotherapy response.
  • a mAb-based labeled-antibody conjugate may locally neutralize IFN- ⁇ activity, which could benefit an anti-tumor response by reducing PD-L1 upregulation.
  • Item 1 A method for in vivo immunoimaging IFN- ⁇ as a marker of active T cells in a subject, comprising: administering a labeled-antibody conjugate to a subject, wherein the labeled-antibody conjugate comprises: an antibody that specifically binds to IFN- ⁇ , and a detection label conjugated to the antibody, wherein the detection label is a radionuclide tracer or fluorophore; and detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging.
  • Item 2 The method of item 1, wherein the subject has cancer.
  • Item 3 The method of item 2, wherein the subject received an immunotherapy prior to administering and detecting the presence of the labeled-antibody conjugate, and wherein the mechanism of action of the immunotherapy results in an increased number of tumor-infiltrating lymphocytes in the subject and/or an increased activation state of a tumor-infiltrating lymphocyte population in the subject; or wherein administering and detecting the presence of the labeled-antibody conjugate is performed prior to administration of a therapy to determine the state of active immunity in the subject.
  • Item 4 The method of item 3, wherein the immunotherapy is an immune checkpoint inhibitor, a cytokine, a vaccine, or an adoptive cell transfer therapy.
  • Item 5 The method of any one of the preceding items, wherein the subject is human and the antibody specifically binds to human IFN- ⁇ .
  • Item 6 The method of any one of the preceding items, wherein the antibody is selected from a monoclonal antibody, an antibody fragment, or combination thereof.
  • Item 7 The method of item 6, wherein the antibody fragment is a Fab fragment.
  • Item 8 The method of item 6, wherein the antibody fragment is a diabody.
  • Item 9 The method of any one of the preceding items, wherein the radionuclide tracer is conjugated to the antibody or antibody fragment with a bifunctional chelator or linker.
  • the bifunctional chelator comprises a chelator selected from N′- ⁇ 5-[Acetyl(hydroxy)amino]pentyl ⁇ -N-[5-( ⁇ 4-[5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl ⁇ amino)pentyl]-N-hydroxysuccinamide (DFO), 1,4,7-triazacyclononane-N,N′,N′-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (i.e., DOPA), diethylenetriaminepentaacetic acid (DTPA), 2,2′-(7-(1-carboxy-4-((2,5
  • Item 11 The method of item 9 or item 10, wherein the bifunctional chelator is p-SCN-Bn-DFO.
  • Item 12 The method of any one of the preceding items, wherein the radionuclide tracer is selected from the group consisting of: 11 C, 13 N, 15 O, 18 F, 64 Cu, 68 Ga, 76 Br, 82 Rb, 86 Y, 89 Zr, 99m Tc, 111 IN 124 I, 131 I, 186 Re, 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 77 Br, 81 Rb, 81m Kr, 87m Sr, 113m In, 123 I, 125 I, 127 Cs, 129 Cs, 132 I, 197 Hg, 203 Pb, 206 Bi, and combination of any two or more thereof.
  • the radionuclide tracer is selected from the group consisting of: 11 C, 13 N, 15 O, 18 F, 64 Cu, 68 Ga, 76 Br, 82 Rb, 86 Y, 89 Zr, 99m Tc, 111 IN 124 I, 131 I,
  • Item 13 The method of item 12, wherein the radionuclide tracer is 89 Zr or 18 F.
  • Item 14 The method of any one of the preceding items, wherein the imaging comprises positron emission tomography (PET) imaging, single photon emission computed tomography (SPECT) imaging, or a both PET and SPECT.
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • Item 15 The method of item 14, wherein the imaging comprises PET.
  • Item 16 The method of any one of the preceding items, wherein the labeled-antibody conjugate further comprises a fluorophore.
  • Item 17 The method of any one of the preceding items, wherein specific binding of the labeled-antibody conjugate to IFN- ⁇ indicates the presence of activated T cells and/or human cancer cells.
  • Item 18 The method of any one of the preceding items, wherein the presence of the labeled-antibody conjugate is detected in real time.
  • a labeled-antibody conjugate comprising: an antibody that specifically binds to IFN- ⁇ , and a detection label conjugated to the antibody, wherein the detection label is a radionuclide tracer.
  • Item 20 The labeled-antibody conjugate of item 19, wherein the antibody is isolated.
  • Item 21 The labeled-antibody conjugate of item 19 or item 20, wherein the antibody is selected from a monoclonal antibody, an antibody fragment, or combination thereof.
  • Item 22 The labeled-antibody conjugate of item 21, wherein the antibody fragment is a Fab fragment.
  • Item 23 The labeled-antibody conjugate of item 21, wherein the antibody fragment is a diabody.
  • Item 24 The labeled-antibody conjugate of any one of items 19 to 23, wherein the radionuclide tracer is conjugated to the antibody with a bifunctional chelator or linker.
  • Item 25 The labeled-antibody conjugate of item 24, wherein the bifunctional chelator comprises a chelator selected from N′- ⁇ 5-[Acetyl(hydroxy)amino]pentyl ⁇ -N-[5-( ⁇ 4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl ⁇ amino)pentyl]-N-hydroxysuccinamide (DFO), 1,4,7-triazacyclononane-N,N′,N′-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (i.e., DOPA), diethylenetriaminepentaacetic acid (DTPA), 2,2′-(7-(1-carbox
  • Item 26 The labeled-antibody conjugate of item 24 or item 25, wherein the bifunctional chelator is p-SCN-Bn-DFO or NOTA.
  • Item 27 The labeled-antibody conjugate of any one of items 19 to 26, wherein the radionuclide tracer is selected from 11 C, 13 N, 15 O, 18 F, 64 Cu, 68 Ga, 76 Br, 82 Rb, 86 Y, 89 Zr, 99m Tc, 111 In, 124 I, 131 I, 186 Re, 43 K, 52 Fe, 57 Co, 67 Cu, 67 Ga, 77 Br, 81 Rb, 81m Kr, 87m Sr, 113m In, 123 I, 125 I, 127 Cs, 129 Cs, 132 I, 197 Hg, 203 Pb, 206 Bi, or a combination of any two or more thereof.
  • the radionuclide tracer is selected from 11 C, 13 N, 15 O, 18 F, 64 Cu, 68 Ga, 76 Br, 82 Rb, 86 Y, 89 Zr, 99m Tc, 111 In, 124 I, 131 I, 186 Re
  • Item 28 The labeled-antibody conjugate of any one of items 19 to 27, wherein the radionuclide tracer is 89 Zr or 18 F.
  • Item 29 The labeled-antibody conjugate of any one of items 19 to 28, wherein the antibody specifically binds to human IFN- ⁇ .
  • V H Diabody heavy chain variable region
  • V L Diabody light chain variable region
  • linker SGGGGS SEQ ID NO: 13
  • scFv SEQ ID NO: 14 which can be included in an anti-IFN-g diabody

Abstract

Methods for in vivo immunoimaging including: (a) administering a labeled-antibody conjugate to a subject, wherein the labeled-antibody conjugate includes: an antibody that specifically recognizes and binds to IFN-γ, and a detection label conjugated to the antibody, wherein the detection label is a radionuclide tracer or fluorophore; and (b) detecting the presence of the radiolabeled-antibody conjugate in the subject in vivo by imaging. Embodiments of the present disclosure are directed to labeled-antibody conjugates and therapeutic radionuclide-antibody conjugates.

Description

    REFERENCE TO RELATED APPLICATION
  • This application is a divisional of U.S. Pat. Application Serial No. 16/640,456, filed Feb. 20, 2020, which is a U.S. national stage application of PCT/US2018/047742, filed Aug. 23, 2018, which claims priority from U.S. Provisional Pat. Application Serial No. 62/549,231, filed Aug. 23, 2017; the entire content of each application is incorporated herein by reference.
  • SEQUENCE LISTING
  • The instant application contains a Sequence Listing which has been submitted electronically as a file in XML format and is hereby incorporated by reference in its entirety. Said XML format file, created on May 8, 2023, is named 47WAY13103VA_Sequence_list.xml and is 21,726 bytes in size.
  • FIELD OF THE INVENTION
  • The present disclosure generally relates to antibody conjugates, to methods for imaging antibody conjugates in vivo, methods and compositions relating to antibody conjugates, and to methods for assessing an effect of a composition or immune-mediated treatment for cancer, injury, or infection. The present disclosure specifically relates to antibody conjugates which specifically recognize interferon gamma (IFN-γ), to methods for imaging IFN-γ antibody conjugates in vivo, methods and compositions relating to IFN-γ antibody conjugates, and to methods for assessing an effect of a composition or immune-mediated treatment for cancer, injury, or infection.
  • BACKGROUND OF THE INVENTION
  • During adaptive cancer immunotherapy activated T cells infiltrating a tumor are the principal components of treatment providing a “search-and-destroy” mechanism through recognition of specific tumor-associated antigens (TAA). Recent emerging tumor-targeted immunotherapy strategies have met with positive and durable outcomes in the clinic, and yet at least half of cancer patients remain non-responsive thereby creating urgency to monitor and gauge an immunotherapy’s success in a timely manner. The primary focus of these efforts lies in techniques to measure peripheral immune responses. These assays are typically restricted to one antigen, are non-standardized and may not reflect dynamic activity occurring within a tumor. There is an ongoing need for antibody conjugates that identify the presence of activated tumor-infiltrating T cells in vivo and methods of use thereof. Image-guided focal analysis of intratumoral immune activity according to aspects of the present invention, provides non-invasive, real-time efficacy predictions to aid in assessment of the effectiveness of an immunotherapy.
  • SUMMARY OF THE INVENTION
  • In particular, the disclosure demonstrates that the in vivo immunoimaging of interferon gamma (IFN-y) identifies active T cells, such as activated tumor-infiltrating T cells, in vivo relative to agents that recognize other biomarkers.
  • Embodiments of the present disclosure are directed to methods of in vivo immunoimaging IFN-γ as a marker of activate T cells in a subject including: (a) administering a labeled-antibody conjugate to a subject, wherein the labeled-antibody conjugate includes: an antibody that specifically recognizes IFN-γ, and at least one detection label conjugated to the antibody, wherein the at least one detection label is a radionuclide; and (b) detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging.
  • Embodiments of the present disclosure are directed to labeled-antibody conjugates including: an antibody that specifically recognizes and binds to IFN-γ, and at least one detection label conjugated to the antibody, wherein the at least one detection label is a radionuclide tracer.
  • Embodiments of the present disclosure are directed to therapeutic radionuclide-antibody conjugates including: 1) an antibody that specifically recognizes and binds to IFN-γ, a bifunctional chelator conjugated to the antibody, and a radionuclide therapeutic agent complexed to the bifunctional chelator or 2) an antibody that specifically recognizes and binds to IFN-γ, a linker conjugated to the antibody, and a radionuclide therapeutic agent conjugated to the antibody via the linker.
  • It is to be understood that both the foregoing general description and the following detailed description describe various embodiments and are intended to provide an overview or framework for understanding the nature and character of the claimed subject matter. The accompanying drawings are included to provide a further understanding of the various embodiments and are incorporated into and constitute a part of this specification. The drawings illustrate the various embodiments described herein, and together with the description, serve to explain the principles and operations of the claimed subject matter.
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1A is a graph showing that BALB/c mice treated with CpG-ODN and imaged with the 89Zr-anti-IFN-γ labeled-antibody conjugate 72 h post-injection (p.i.) displayed higher uptake in the spleen compared to control (Ctrl) untreated cohorts (n=3 each);
  • FIG. 1B is a graph showing that tissue distribution of 89Zr-anti-IFN-γ at 72 h p.i. demonstrated lower probe accumulation in the spleen upon competitive saturation with 10× cold AN-18 mAb (n=4 each);
  • FIG. 1C is a graph showing results of testing 89Zr-anti-IFN-γ receptor-localized IFN-y binding in vitro. TUBO cells were incubated with 89Zr-anti-IFN-γ alone (n=5), or with recombinant IFN-y (rIFN-y) and washed before addition of 89Zr-anti-IFN-γ (n=5). Activity was measured by a gamma counter and adjusted;
  • FIG. 1D is a pair of images showing liver (L), heart (H), and spleen (S) removed from mice used in the experiments described for FIG. 1C and imaged ex vivo;
  • FIG. 2A is a graph showing results of Positron Emission Tomography (PET) evaluation of immunotherapy response in orthotopic TUBO mammary tumors by measurement of tumor volume of untreated control mice, Ctrl, n=11. TUBO cells were inoculated 10 days prior to the start of vaccinations, given on days 0 and 14. PET imaging was conducted on day 15 (Ctrl);
  • FIG. 2B is a graph showing results of PET evaluation of immunotherapy response in orthotopic TUBO mammary tumors by measurement of tumor volume of vaccinated mice, Vx, n=12. TUBO cells were inoculated 10 days prior to the start of vaccinations, given on days 0 and 14. PET imaging was conducted on day 21 (Vx);
  • FIG. 2C shows representative whole body maximum intensity projections (MIP, top row) and planar (bottom row) images of control (Ctrl, left panels, n=6) and vaccinated (Vx, right panels, n=6) mice with 89Zr-anti-IFN-γ labeled-antibody conjugate. White circle = tumor, L = liver, H = heart;
  • FIG. 2D is a graph showing tumor VOIs measured for each mouse from experiments described in FIG. 2C with a 89Zr-anti-IFN-γ labeled-antibody conjugate (left, n=6, untreated control; n=6, vaccinated control) or a 89Zr labeled rat IgG isotype control (right) included for each treatment group (n=3, untreated control; n=6, vaccinated control);
  • FIG. 2E shows representative MIP images (top panels) and planar sections (bottom panels) of 89Zr-anti-CD3 PET in control (left, n=5) and vaccinated mice (middle, n=6). White circle = tumor, L = liver, S = spleen, Th = Thymus;
  • FIG. 2F is a graph showing tumor VOIs measured for each mouse from experiments described in FIG. 2E (Ctrl, Vx, left (n=5) and middle (n=6)) and including results from a non-specific 89Zr labeled Armenian hamster IgG isotype control was used to measure tumor VOI in a separate group of untreated mice (IgG, right, n = 3);
  • FIG. 3A is a graph showing results of mRNA analysis of TUBO mammary tumors removed after imaging. For this, total RNA was obtained from Ctrl (n=11) and Vx (n=13) tumor tissue analyzed by qPCR with primers specific to CD3. Cultured TUBO cells serve as control (n=2);
  • FIG. 3B is a graph showing results of mRNA analysis of TUBO mammary tumors removed after imaging. For this, total RNA was obtained from Ctrl (n=11) and Vx (n=13) tumor tissue a analyzed by qPCR with primers specific to CD8. Cultured TUBO cells serve as control (n=2);
  • FIG. 3C is a graph showing results of mRNA analysis of TUBO mammary tumors removed after imaging. For this, total RNA was obtained from Ctrl (n=11) and Vx (n=13) tumor tissue a analyzed by qPCR with primers specific to IFN-γ. Cultured TUBO cells serve as control (n=2);
  • FIG. 3D is a graph showing results of an IFN-γ ELISA conducted with protein lysates of TUBO tumor segments of control Ctrl (n=10) and Vx (n=11) mice;
  • FIG. 3E is a graph showing results of measurement of HER2-specific IgG in serum of TUBO-bearing mice by flow cytometry (Ctrl: n=13, Vx: n=14);
  • FIG. 3F is a graph showing results of measurement of neu-specific IgG in serum of TUBO-bearing mice by flow cytometry (Ctrl: n=13, Vx: n=14);
  • FIG. 3G is a graph showing results of measurement of HER2-responsive T cells by IFN-γ ELISPOT (Ctrl: n=6, Vx: n=7);
  • FIG. 3H is a graph showing results of measurement of neu-responsive T cells by IFN-γ ELISPOT (Ctrl: n=6, Vx: n=7);
  • FIG. 4A is a graph showing results of PET detection of anti-tumor immunity in spontaneous tumor-bearing NeuT mice in which control, untreated mice (Ctrl, n=6) were imaged by PET after palpable tumors were permitted to grow 31 days;
  • FIG. 4B is a graph showing results of PET detection of anti-tumor immunity in spontaneous tumor-bearing vaccinated NeuT mice (Vx, n=7), which, upon detection of palpable spontaneous salivary tumors, regulatory T cells (Treg) were depleted 10 d prior to the first vaccination. These mice received two HER2/neu DNA vaccinations 14 d apart. PET imaging was conducted 7 days after the final vaccination;
  • FIG. 4C shows representative whole body maximum intensity projections (MIP, top row) and planar (bottom row) images of control (left panels, n=3) and HER2/neu DNA-vaccinated (right panels, n=4) NeuT mice with 89Zr-anti-IFN-γ PET labeled-antibody conjugate. White circle = tumor, L = liver;
  • FIG. 4D is a graph showing tumor VOIs were calculated for each mouse studied for the experiment described in FIG. 4C;
  • FIG. 4E shows representative whole body maximum intensity projections (MIP, top row) and planar (bottom row) images of control (left panels, n=3) and HER2/neu DNA-vaccinated (right panels, n=4) NeuT mice with CD3 PET labeled-antibody conjugate. White circle = tumor, L = liver, S = spleen;
  • FIG. 4F is a graph showing tumor VOIs were calculated for each mouse studied for the experiment described in FIG. 4E;
  • FIG. 5A is a graph showing results of mRNA analysis of spontaneous tumors from NeuT mice removed after imaging. For this, total RNA was obtained from Ctrl (n=3) and Vx (n=3) tumor tissue and analyzed by qPCR with primers specific to CD3;
  • FIG. 5B is a graph showing results of mRNA analysis of spontaneous tumors from NeuT mice removed after imaging. For this, total RNA was obtained from Ctrl (n=3) and Vx (n=3) tumor tissue and analyzed by qPCR with primers specific to CD8;
  • FIG. 5C is a graph showing results of mRNA analysis of spontaneous tumors from NeuT mice after imaging. For this, total RNA was obtained from Ctrl (n n=3) and Vx (n=3) tumor tissue and analyzed by qPCR with primers specific to IFN-y;
  • FIG. 5D is a graph showing results of measurement of HER2-specific IgG in serum of NeuT mice by flow cytometry (Ctrl: n=5, Vx: n=8);
  • FIG. 5E is a graph showing results of measurement of neu-specific IgG in serum of NeuT mice by flow cytometry (Ctrl: n=5, Vx: n=8);
  • FIG. 5F is a graph showing results of measurement of HER2-responsive T cells of NeuT mice by IFN-γ ELISPOT (Ctrl: n=5, Vx: n=5);
  • FIG. 5G is a graph showing results of measurement of neu-responsive T cells of NeuT mice by IFN-γ ELISPOT (Ctrl: n=5, Vx: n=5);
  • FIG. 6A is a graph showing tumor volume in TUBO-bearing vaccinated BALB/c mice (n=11). TUBO cells were inoculated 13 days prior to the start of vaccinations, to allow for variability in tumor volumes at treatment onset. Vaccines were given on days 0 and 14. PET imaging was conducted on day 28;
  • FIG. 6B is a graph showing weekly tumor growth rate, calculated by regression analysis of log tumor growth, versus 89Zr-anti-IFN-γ labeled-antibody conjugate uptake plotted for each mouse and evaluated by Pearson’s correlation;
  • FIG. 6C is a graph showing tumor growth during passive immunotherapy with anti-neu mAb 7.16.4, given as 5 doses at 1.5 mg i.p. every 3-4 days as indicated beginning 15 days after tumor inoculation. 89Zr-anti-IFN-γ (n=5) or 89Zr-rat-IgG control (n=6) PET imaging was conducted 30 days after treatment onset;
  • FIG. 6D is a graph showing tumor VOIs calculated for 89Zr-anti-IFN-γ or 89Zr-rat-IgG labeled-antibody conjugate in 7.16.4 treated TUBO-bearing mice;
  • FIG. 6E is a graph showing results relating to control and 7.16.4-treated tumors (n=4 each) which were dissociated and analyzed for T cell infiltration by flow cytometry by staining for CD45 and CD8;
  • FIG. 6F is a graph showing PD-1 expression analyzed by flow cytometry on CD8+ tumor infiltrates from (FIG. 6F);
  • FIG. 7A shows time course imaging of 89Zr-anti-IFNγ. Mice were injected with 89Zr-anti-IFNy and images of MIP are shown for each time point. L = liver, S = spleen;
  • FIG. 7B is a graph of the volumes-of-interest obtained from select tissues over time from 24-120 h p.i.;
  • FIG. 8A shows time course imaging of 89Zr-anti-CD3. Mice were injected with 89Zr-anti-CD3 and images of MIP are shown for each time point. L = liver, S = spleen;
  • FIG. 8B is a graph of the volumes-of-interest obtained from select tissues over time from 4-72 h p.i.;
  • FIG. 9A shows MIP images of 89Zr-anti-IFNγ detection in all control (left) and vaccinated (right) TUBO bearing mice;
  • FIG. 9B shows MIP images of 89Zr-anti-CD3 detection in all control (left) and vaccinated (right) TUBO bearing mice;
  • FIG. 10A is a plot of flow cytometry results showing T cell detection in TUBO-bearing BALB/c mice. Tumors from untreated TUBO-bearing mice were dissociated and stained with CD45, to detect total leukocyte infiltrates, and the T cell receptor beta chain (TCRβ), to identify the T cell fraction, by flow cytometry;
  • FIG. 10B is a graph showing Spleen VOIs calculated for each TUBO-bearing mouse imaged with either 89Zr-anti-IFNγ or 89Zr-anti-CD3;
  • FIG. 11A shows an image demonstrating immunoblot detection of T cell markers in imaged TUBO-bearing mice. Western blots from lysed decayed tumor tissues post-IFN-y PET were conducted to analyze presence of CD3 and CD8 protein in the Vx vs Ctrl mice. Beta-tubulin is detected as a gel loading control;
  • FIG. 11B shows an image demonstrating immunoblot detection of T cell markers in imaged TUBO-bearing mice. Western blots from lysed decayed tumor tissues post-CD3 PET were conducted to analyze presence of CD3 and CD8 protein in the Vx vs Ctrl mice. Beta-tubulin is detected as a gel loading control;
  • FIG. 12A shows MIP images of 89Zr-anti-IFNγ detection in all control (left) and vaccinated (right) NeuT mice bearing spontaneous salivary tumors;
  • FIG. 12B shows MIP images of 89Zr-anti-CD3 detection in all control (left) and vaccinated (right) NeuT mice bearing spontaneous salivary tumors;
  • FIG. 13 shows 89Zr-IFN-γ PET of vaccinated mice for correlation to tumor growth. The MIP images show 89Zr-anti-IFNγ detection in all vaccinated TUBO mice bearing tumors;
  • FIG. 14A is a graph showing tissue biodistribution of anti-IFN-y PET labeled-antibody conjugate. BALB/c mice were treated with 100 µg CpG intramuscular and co-injected with 89Zr-IFN-γ intravenous (mAb clone AN-18 or XMG1.2). Uptake in blood, heart, stomach, small intestine, large intestine, pancreas, brain, bone, muscle, and lymph node was analyzed by gamma counter 72 hours later;
  • FIG. 14B is a graph showing tissue biodistribution of anti-IFN-y PET labeled-antibody conjugates. BALB/c mice were treated with 100 µg CpG intramuscular and co-injected with 89Zr-IFN-γ intravenous (mAb clone AN-18 or XMG1.2). Uptake in lungs, liver, kidney, and spleen, was analyzed by gamma counter 72 hours later; and
  • FIG. 15 is an image showing results of injection of 100-150 µCi anti-IFN-y PET labeled-diabody conjugate into a TUBO-bearing BALB/c mouse after Vx.
  • DETAILED DESCRIPTION OF THE INVENTION
  • While the following terms are believed to be well understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the presently-disclosed subject matter.
  • The term “label” refers to a chemical moiety that can be covalently attached to an antibody and that functions to provide a detectable signal, such as, e.g., radionuclide tracers.
  • The term “antibody” is used herein in its broadest sense and specifically refers to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity (see e.g., Miller et al. (2003) J Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein that is capable of recognizing and binding to a specific antigen, such as, e.g., IFN-γ. (See e.g., Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen may have numerous binding sites, also called epitopes, recognized by complementarity determining regions of antibodies. In embodiments, each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically-active portion of a full-length immunoglobulin molecule, such as, e.g., a molecule that contains an antigen binding site that immunospecifically recognizes and binds to an antigen of a target of interest or part thereof, such as, e.g., IFN-γ. The immunoglobulin disclosed herein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species. In some embodiments, however, the immunoglobulin is of rodent or human origin. According to embodiments, the antibody is an antibody fragment and in specific embodiments, the antibody fragment is a diabody.
  • The term “specifically recognizes and binds to” refers to antibodies that are capable of binding to an antigen of interest, such as, e.g., IFN-γ, with sufficient binding affinity such that the antibody is useful in targeting the antigen of interest. As an example, the antibodies may have a binding affinity KD of from about 10-4 to about 10-15, or from about 10-6 to about 10-13, or from about 10-7 to about 10-12, or from about 10-9 to about 10-10, for the antigen of interest. As another example, where the antibody is one that binds to IFN-γ, it will preferentially bind to IFN-γ as opposed to other antigens and/or extracellular components. As a further example, where the antibody is one that binds to IFN-γ, it may not significantly cross-react with other extracellular components. In embodiments, the extent of binding of the antibody to non- IFN-γ antigens and/or other extracellular components is less than about 10% (such as, e.g., 0% to about 9%), as determined by standard techniques known to those of ordinary skill in the art, such as, e.g., by flow cytometric analysis.
  • The terms “interferon gamma” and “IFN-γ” are synonyms that refer to a cytokine. IFN-γ is a member of the interferon family, which exhibits similar antiviral and anti-proliferative properties characteristic of interferons-a and -β (IFN-α and IFN-β). IFN-γ was originally discovered as a product induced upon mitogenic activation of lymphocytes. The recombinant production of human IFN-γ was first reported by Gray, Goeddel and co-workers (Gray et al., Nature 295:503-508, 1982), and is subject of U.S. Pat. Nos. 4,762,791, 4,929,544, 4,727,138, 4,925,793, 4,855,238, 5,582,824, 5,096,705, 5,574,137, and 5,595,888, each of which is hereby incorporated by reference in its entirety. The recombinant human IFN-γ of Gray and Goeddel as produced in E. coli consisted of 146 amino acids, the N-terminal position of the molecule commencing with the sequence CysTyrCys. It was later found that the native human IFN-γ (i.e., that arising from mitogen induction of human peripheral blood lymphocytes and subsequent purification) is a polypeptide that lacks the CysTyrCys N-terminus assigned by Gray et al., supra. The crystal structure of E. coli-derived recombinant human IFN-γ (rhIFN-γ) was determined (Ealick et al., Science 252:698-702, 1991), showing that the protein exists as a tightly intertwined non-covalent homodimer, in which the two identical polypeptide chains are oriented in an antiparallel manner. IFN-γ is known to exhibit a broad range of biological activities, including antitumor, antimicrobial and immunoregulatory activities. A particular form of recombinant human IFN-γ is commercially available as an immunomodulatory drug for the treatment of chronic granulomatous disease characterized by severe, recurrent infections of the skin, lymph nodes, liver, lungs, and bones due to phagocyte dysfunction (rhIFN-γ-1b, Actimmune®, Genentech, Inc. South San Francisco, Calif.). IFN-γ was subsequently approved for use in patients with severe, malignant osteopetrosis. IFN-γ may refer to IFN-γ of any species including both human and mouse IFN-γ.
  • The term “bifunctional chelator” refers to a chemical moiety that attaches an antibody to a radionuclide. Bifunctional chelators may include a chelator covalently attached to a first linking group. Bifunctional chelators may function by complexing metal ions with the chelator and by covalently attaching the chelator to an antibody with the first linking group, as is known to those of ordinary skill in the art. For example, bifunctional chelators may be covalently attached to a primary amine group, a hydroxyl group, and/or a cysteine amino acid of an antibody. Examples of suitable chelators include: 1,4,7-triazacyclononane-N,N′,N′-triacetic acid (i.e., NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (i.e., DOTA), ethylenediaminetetraacetic acid (i.e., EDTA), diethylenetriaminepentaacetic acid (i.e., DTPA; CAS Reg. No. 67-43-6), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (i.e., DOPA), N′-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (i.e., deferoxamine or DFO; CAS Reg. No. 70-51-9), 2,2′-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NODA), or a combination of any two or more thereof. Examples of suitable linking groups capable of attaching a chelator to a primary amine group, a hydroxyl group, and/or a cysteine amino acid of an antibody are known to those of skill in the art (see e.g., Denardo et al., 1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug. Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26(8):943-50). Examples of suitable bifunctional chelators include 4-maleimidobutyramidobenzyl-DOTA (which can be prepared following the procedure of Axworthy et al (2000) Proc. Natl. Acad. Sci. USA 97(4):1802-1807), 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (i.e., MX-DTPA, see e.g., International Pub. No. WO 94/11026), p-SCN-Bn-Deferoxamine (i.e., p-SCN-Bn-DFO), or a combination of any two or more thereof. (See also, U.S. Pat. Application Publication No. 2015/0017094, hereby incorporated by reference in its entirety). p-SCN-Bn-DFO has the following structure:
  • Figure US20230338589A1-20231026-C00001
  • With regard to antibodies, the term “isolated” refers to an antibody that has been identified and separated and/or recovered from at least one contaminant component of its natural environment. Contaminant components may be materials that would interfere with diagnostic and/or therapeutic uses for the antibody, such as, e.g., enzymes, hormones, and/or other proteinaceous or nonproteinaceous solutes. In embodiments, the isolated antibody will be purified to greater than about 95% (such as, e.g., about 96% to about 100%) by weight of antibody as determined by the Lowry method, or to greater than about 99% by weight of antibody.
  • The term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, wherein the individual antibodies in the population are identical except for naturally occurring post-translational modifications. Monoclonal antibodies may be highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different antigenic determinants (such as, e.g., epitopes), each monoclonal antibody is directed against a single antigenic determinant on the antigen. Monoclonal antibodies may be synthesized uncontaminated by other antibodies. While monoclonal indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, it is not intended to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be prepared by the Kohler hybridoma method (see e.g., Kohler et al. (1975) Nature 256:495), or they may be prepared by recombinant DNA methods (see e.g., U.S. Pat. No. 4,816,567 and U.S. Pat. No. 5,807,715, each of which is hereby incorporated by reference in its entirety). Further, monoclonal antibodies may be isolated from phage antibody libraries using standard techniques (see e.g., Clackson et al. (1991) Nature, 352:624-628 and Marks et al. (1991) J. Mol. Biol., 222:581-597).
  • The terms “antibody fragment” and “fragment” refer to a portion of a full length antibody that includes a region capable of recognizing and binding to a specific antigen, such as, e.g., the antigen binding, variable, or hypervariable (also known as complementarity determining) region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, F(ab)2, Fv, sFv, and scFv fragments; diabodies; linear antibodies; minibodies (see e.g., Olafsen et al. (2004) Protein Eng. Design & Sel. 17(4):315-323), fragments produced by a Fab expression library, anti-idiotypic (hereinafter, “anti-Id”) antibodies, complementary determining region (hereinafter, “CDR”) and epitope-binding fragments of any of the above that immunospecifically recognize and bind to IFN-γ, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • The term “radiolabel” refers to a radionuclide moiety that can be attached to an antibody and that functions to provide a detectable signal, such as, e.g., radionuclide tracers. Radionuclide tracers include radioisotopes, which by virtue of radioactive decay, are detectable. Radionuclide tracers may be detected by various imaging techniques, such as, e.g., PET and single-photon emission computed tomography (hereinafter, “SPECT”) imaging. In embodiments, radionuclide tracers have an energy of from about 20 to about 4,000 kiloelectronvolts (i.e., keV). Suitable radionuclide tracers include radionuclides that emit gamma radiation, positrons, or a combination of gamma radiation and positrons. In illustrative, non-limiting embodiments, radionuclide tracers have appropriate decay characteristics for optimizing image resolution and quantitative accuracy and/or have appropriate residualization. For example, radionuclide tracers may have a physical half-life (i.e., t½) compatible with the time required for the antibody to achieve optimal specific: non-specific binding ratios. Examples of radionuclide tracers suitable for PET imaging include 11C (t½ ~ 20 min), 13N (t½ ~ 10 min), 15O (t½ ~ 2 min), 18F (t½ ~ 1.83 h), 44Sc (t½ ~ 3.97 h), 45Ti (t½ ~ 3 h), 52Mn (t½ ~ 5.6 days), 64Cu (t½ ~ 12.7 h), 68Ga (t½ ~ 1.13 h), 76Br (t½ ~ 16.2 h), 82Rb (t½ ~ 1.27 min), 86Y (t½ ~ 14.7 h), 89Zr (t½ ~ 78.4 h), 124I (t½ ~ 100.3 h), or a combination of any two or more thereof. Examples of radionuclide tracers suitable for SPECT imaging include 99mTc (t½ ~ 6 h), 111In (t½ ~ 2.80 days), 131I (t½ ~ 8 days), 177Lu (t½ ~ 6.64 days), 186Re (t½ ~ 3.7186 days), or a combination of any two or more thereof.
  • The term “therapeutic radionuclide” refers to a radionuclide moiety that can be attached to an antibody that functions to deliver a cytotoxic dose of radiation, such as, e.g., a radionuclide therapeutic agent, to a target of interest, such as, e.g., a tumor. Suitable radionuclide therapeutic agents include radionuclides that emit beta particle radiation, alpha particle radiation, Auger electron radiation, or a combination of any two or more thereof. Examples of suitable radionuclide therapeutic agents include 125I, 131I, 177Lu, 186Re, 225Ac, 225Ra, or a combination of any two or more thereof.
  • The term “immunoimaging” refers to imaging IFN-γ in vivo in a subject via detection of a labeled anti-IFN-γ antibody conjugate to produce an image indicative of the location and/or level of IFN-γ in the subject, wherein the location and/or level of IFN-γ in the subject is indicative of activated T cells.
  • Embodiments of the present disclosure are directed toward antibody conjugates, including labeled-antibody conjugates and therapeutic radionuclide-antibody conjugates, to methods for imaging, to mouse models, and to methods for assessing the effect of a composition or immune-mediated treatment for cancer, injury or infection, particularly human cancer, human injury or human infection.
  • 1. Antibody Conjugates
  • In one or more embodiments, the disclosure relates to antibody conjugates. In embodiments, antibody conjugates include both labeled-antibody conjugates and therapeutic radionuclide-antibody conjugates. In embodiments, labeled-antibody conjugates include an antibody that specifically recognizes and binds to IFN-γ, and at least one detection label conjugated to the antibody, wherein the at least one detection label includes a radionuclide tracer.
  • Suitable antibodies that specifically recognize and bind to IFN-γ may be prepared by standard techniques known to those of ordinary skill in the art. In embodiments, the antibody is an isolated antibody. In embodiments, the antibody is selected from a monoclonal antibody, an antibody fragment, or combination thereof. In embodiments, the antibody is a monoclonal antibody expressed by a hybridoma cell line or by CHO cells, NS0 cells, Sp2/0 cells, HEK cells, BHK cells, or PER.C6 cells.
  • In embodiments, the antibody is an antibody fragment. In further embodiments, the antibody is a Fab fragment. Antibody fragments that recognize specific epitopes can be produced by standard techniques known to those of ordinary skill in the art. For example, F(ab′)2 fragments can be produced by pepsin digestion of the antibody molecule. (See e.g., U.S. Pat. Nos. 4,036,945 and 4,331,647 and references contained therein; see also Nisonoff et al., Arch Biochem. Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959), Edelman et al., in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and Coligan et al. Current Protocols in Immunology, Vol. 1, pages 2.8.1-2.8.10 and 2.10.-2.10.4 (John Wiley & Sons 1991)). Alternatively, Fab′ expression libraries can be constructed (see e.g., Huse et al., 1989, Science, 246:1274-1281) to allow rapid and easy identification of monoclonal Fab′ fragments with desired specificity.
  • In embodiments, a single chain Fv molecule (i.e., scFv) includes a VL domain and a VH domain. The VL and VH domains may associate to form a target binding site. In embodiments, VL and VH domains are covalently linked by a peptide linker (i.e., L). In embodiments, a scFv molecule is denoted as either VL-L-VH if the VL domain is the N-terminal part of the scFv molecule, or as VH-L-VL if the VH domain is the N-terminal part of the scFv molecule. scFv molecules can be produced by standard techniques known to those of ordinary skill in the art. (See U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E. Bird and B. W. Walker, Single Chain Antibody Variable Regions, TIBTECH, Vol 9:132-137 (1991)).
  • Other antibody fragments, such as, e.g., single domain antibody fragments, are also known to those of ordinary skill in the art. Single domain antibodies (i.e., VHH) may be obtained, for example, from camels, alpacas or llamas by standard immunization techniques known to those of ordinary skill in the art. (See, e.g., Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). In embodiments, the VHH has potent antigen-binding capacity and can interact with novel epitopes that may be inaccessible to conventional VH-VL pairs. (See e.g., Muyldermans et al., 2001). In embodiments, alpaca serum IgG contains about 50% camel heavy chain only IgG antibodies (i.e., HCAbs) (see e.g., Maass et al., 2007). In embodiments, alpacas may be immunized with known antigens, such as, e.g., TNF-α, and VHHS can be isolated that bind to and neutralize the target antigen (see e.g., Maass et al., 2007). PCR primers that amplify virtually all alpaca VHH coding sequences have been identified and may be used to construct alpaca VHH phage display libraries, which can be used for antibody fragment isolation by standard biopanning techniques well known to those of ordinary skill in the art. (See Maass et al., 2007).
  • In embodiments, an antibody fragment is produced by proteolytic hydrolysis of a full-length antibody and/or by expression in E. coli, CHO cells, or another host of DNA coding for the antibody fragment. In embodiments, antibody fragments can be obtained by pepsin or papain digestion of full-length antibodies by standard techniques known to those of ordinary skill in the art. For example, an antibody fragment can be produced by enzymatic cleavage of antibodies with pepsin to provide an ~100 kDa fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce a ~ 50 kDa Fab′ monovalent fragment. In alternative embodiments, an enzymatic cleavage using papain produces two monovalent Fab fragments and an Fc fragment directly.
  • In embodiments, antibody fragments may include peptides coding for a single complementary determining region (i.e., CDR). In embodiments, a CDR is a segment of the variable region of an antibody that is complementary in structure to the epitope to which the antibody binds and is more variable than the rest of the variable region. Accordingly, in embodiments, a CDR is referred to as a hypervariable region. In embodiments, a variable region includes three CDRs. In embodiments, CDR peptides can be produced by constructing genes encoding the CDR of an antibody of interest. Such genes may be prepared, for example, by using the polymerase chain reaction (i.e., PCR) to synthesize the variable region from RNA of antibody-producing cells. (See e.g., Larrick et al., Methods: A Companion to Methods in Enzymology 2:106 (1991); Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds), pages 166-179 (Cambridge University Press 1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al., (eds.), pages 137-185 (Wiley-Liss, Inc. 1995).
  • According to embodiments, the antibody conjugate is an anti-IFN-γ diabody that specifically recognizes and binds to IFN-γ. A diabody included in a labeled-antibody conjugate is a noncovalent dimer of single-chain Fv (scFv) fragment that has the heavy chain variable (VH) and light chain variable (VL) regions connected by a linker, such as a peptide linker. According to embodiments, the diabody includes heavy chain variable region (VH) having the amino acid sequence disclosed herein as SEQ ID NO:11 and light chain variable region (VL) having the amino acid sequence disclosed herein as SEQ ID NO:12. The linker used is too short to allow pairing between the two domains on the same chain, such that the linked VH - VL domains are forced to pair with complementary domains of another chain, creating two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993). An exemplary linker is SGGGGS (SEQ ID NO:13). According to embodiments, an scFv included in an anti-IFN-γ diabody that specifically recognizes and binds to IFN-γ has SEQ ID NO:14. According to embodiments, an scFv included in an anti-IFN-γ diabody that specifically recognizes and binds to IFN-γ has SEQ ID NO:15.
  • Variants of the scFv of SEQ ID NO:14 and/or SEQ ID NO:15 can also be included in an anti-IFN- γ diabody in labeled-antibody conjugate according to embodiments.
  • A functional variant of the scFv of SEQ ID NO:14 has at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, sequence identity with the amino acid sequence set forth in SEQ ID NO:14 wherein the diabody including the variant or variants specifically recognizes and binds to IFN- γ.
  • A functional variant of the scFv of SEQ ID NO:15 has at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, sequence identity with the amino acid sequence set forth in SEQ ID NO:15 wherein the diabody including the variant or variants specifically recognizes and binds to IFN- γ.
  • The diabody including scFv of SEQ ID NO:14 and/or SEQ ID NO:15 or a variant of either thereof can be isolated or generated recombinantly or synthetically.
  • Mutations can be introduced using standard molecular biology techniques such as site-directed mutagenesis and PCR-mediated mutagenesis to produce a diabody including a variant of the scFv SEQ ID NO: 14 and/or SEQ ID NO:15. One of skill in the art will recognize that one or more amino acid mutations can be introduced without altering the functional properties of the diabody.
  • Variants of the diabody VH and VL domains of SEQ ID NO:11 and SEQ ID NO:12 can also be included in a labeled-antibody conjugate according to embodiments.
  • A functional variant of the diabody VH and VL domains of SEQ ID NO:11 and SEQ ID NO:12 has at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, sequence identity with the amino acid sequences set forth in SEQ ID NO:11 and SEQ ID NO:12 wherein the diabody including the variant or variants specifically recognizes and binds to IFN- γ.
  • The diabody including VH and VL domains of SEQ ID NO:11 and SEQ ID NO:12 or a variant of either thereof can be isolated or generated recombinantly or synthetically.
  • Mutations can be introduced using standard molecular biology techniques such as site-directed mutagenesis and PCR-mediated mutagenesis to produce a diabody including a variant of VH and VL domains of SEQ ID NO:11 and SEQ ID NO:12. One of skill in the art will recognize that one or more amino acid mutations can be introduced without altering the functional properties of the diabody.
  • Conservative amino acid substitutions can be made in the diabody sequences of SEQ ID NO:11, SEQ ID NO:12, and/or SEQ ID NO:14, to produce functional variants. Conservative amino acid substitutions are art recognized substitutions of one amino acid for another amino acid having similar characteristics. For example, each amino acid can be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar, hydrophobic, and hydrophilic. A conservative substitution is a substitution of one amino acid having a specified structural or functional characteristic for another amino acid having the same characteristic. Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine, and valine; aromatic amino acids include phenylalanine, tyrosine, and tryptophan; polar amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, and tryptophan; and conservative substitutions include substitutions among amino acids within each group. Amino acids can also be described in terms of steric effects or relative size, e.g., alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine, and valine are all typically considered to be small.
  • To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in a first amino acid or nucleotide sequence for optimal alignment with a second amino acid or nucleotide sequence using the default parameters of an alignment software program). The amino acids at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid as the corresponding position in the second sequence, then the sequences are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical aligned positions ÷ total number of aligned positions · 100%). In some embodiments, the two sequences have the same length.
  • The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, PNAS USA 87:2264-68, e.g., as modified as in Karlin and Altschul, 1993, PNAS USA 90:5873-77. In calculating percent identity, only exact matches are typically counted.
  • Other methods of cleaving antibodies, such as, e.g., separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the antibody fragments specifically bind to the antigen that is recognized by the intact antibody.
  • The detection label or radionuclide therapeutic agent is conjugated directly, or indirectly, to the antibody to produce an antibody conjugate. Suitable linkers, chelating agents and methods of use for conjugation of a detection label or radionuclide therapeutic agent to an antibody are well-known in the art, see for example, Shan S. Wong et al., Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation, Second Edition, CRC Press, 2011.
  • According to aspects of the present disclosure, an antibody conjugate is an imaging agent which can be used in diagnostic procedures as well as for localization of activated T cells. Imaging can be performed by many procedures well-known to those having ordinary skill in the art, for example, by positron emission tomography (PET), single photon emission computed tomography (SPECT), Cerenkov imaging, photoacoustic imaging, ultrasound imaging, optical coherent tomography, optical imaging, including fluorescence imaging, and/or bioluminescence imaging, or a combination of any two or more thereof.
  • In embodiments, the labeled-antibody conjugates include at least one detection label conjugated to the antibody, wherein the at least one detection label includes a radionuclide tracer. In embodiments, the at least one detection label is a combination of a radionuclide tracer and a fluorophore.
  • In embodiments, the radionuclide tracer is selected from 11C, 13N, 15O, 18F, 44Sc, 45Ti, 52Mn, 64Cu, 68Ga, 76Br, 82Rb, 86Y, 89Zr, 99mTc, 111In, 124I, 131I, 186Re, 43K, 52Fe, 57Co, 67Cu, 67Ga, 77Br, 81Rb, 81mKr, 87mSr, 89Zr, 113mIn, 123I, 125I, 127Cs, 129Cs, 132I, 177Lu, 186Re, 197Hg, 203Pb, 206Bi, 225Ac, 225Ra, or combination of any two or more thereof. In illustrative, non-limiting embodiments, the radionuclide tracer is 89Zr.
  • In embodiments wherein the detection label is a radionuclide tracer, the radionuclide tracer is optionally conjugated to the antibody with a bifunctional chelator. In embodiments wherein the radionuclide tracer is 11C, 13N, 18F, 76Br 123I, 124I, or 125I, a bifunctional chelator is not required for conjugation to the antibody wherein 11C, 13N, 18F, 76Br 123I, 124I, or 125I, can be directly conjugated to the antibody with standard techniques known to those of ordinary skill in the art. In embodiments, the bifunctional chelator includes a chelator attached to a first linking group. Suitable chelators and first linking groups are known to those of ordinary skill in the art. In embodiments, the bifunctional chelator includes a chelator selected from DFO, NOTA, DOTA, EDTA, DOPA, DTPA, NODA, or a combination of any two or more thereof. In illustrative, non-limiting embodiments, the bifunctional chelator is p-SCN-Bn-DFO.
  • In embodiments, the bifunctional chelator is attached to the antibody. In further embodiments, the radionuclide tracer is complexed to the bifunctional chelator, wherein the bifunctional chelator is covalently attached to the antibody. In embodiments, the bifunctional chelator is attached to the antibody by reacting the bifunctional chelator with the antibody by standard techniques known to those of ordinary skill in the art. In embodiments, an ion of the radionuclide tracer is complexed to the bifunctional chelator by standard techniques known to those of ordinary skill in the art. In illustrative, non-limiting embodiments, (1) the bifunctional chelator p-SCN-Bn-DFO is reacted with the anti-IFN-γ antibody at a ratio of about 5:1 in saline solution at a pH ~ 9 for about 1 hour at about 37° C. for covalent attachment thereof; and (2) the radionuclide tracer 89Zr4+ is reacted with the bifunctional chelator p-SCN-Bn-DFO covalently attached to the antibody at a pH ~ 7.0-7.2 for about 1 hour at room temperature to form a radiolabeled-antibody conjugate.
  • In embodiments, the labeled-antibody conjugates include at least one detection label conjugated to the antibody, wherein the at least one detection label includes a radionuclide tracer. In embodiments, the at least one detection label also includes a fluorophore. In embodiments, the fluorophore is a fluorescent dye. In embodiments, the fluorophore emits fluorescence in the visible (i.e., 400-700 nm) or near-infrared (i.e., 700-1400 nm) region.
  • In embodiments, the antibody conjugates include therapeutic radionuclide-antibody conjugates. In embodiments, therapeutic radionuclide-antibody conjugates include an antibody that specifically recognizes and binds IFN-γ, a bifunctional chelator conjugated to the antibody, and a radionuclide therapeutic agent complexed to the bifunctional chelator. In embodiments, the antibody is as described herein with regard to labeled-antibody conjugates. Additionally, in embodiments, the bifunctional chelator is as described herein with regard to labeled-antibody conjugates.
  • In embodiments, the therapeutic radionuclide-antibody conjugate includes a radionuclide therapeutic agent complexed to the bifunctional chelator. In embodiments, the radionuclide therapeutic agent is selected from 125I, 131I, 177Lu, 186Re, 225Ac, 225Ra, or a combination of any two or more thereof. In embodiments, the bifunctional chelator is attached to the antibody. In further embodiments, the radionuclide therapeutic agent is complexed to the bifunctional chelator, wherein the bifunctional chelator is covalently attached to the antibody. In embodiments, the bifunctional chelator is attached to the antibody by reacting the bifunctional chelator with the antibody. Then, in embodiments, an ion of the radionuclide therapeutic agent is complexed to the bifunctional chelator.
  • In embodiments, the therapeutic radionuclide-antibody conjugate includes a labeled-antibody conjugate attached to a therapeutic moiety. The therapeutic moiety can be, without limitation, a small molecule drug, an oligonucleotide or polynucleotide, such as an antisense oligonucleotide or polynucleotide, an siRNA, an mRNA, an miRNA, an shRNA, a peptide or protein. According to embodiments, a therapeutic moiety is attached to, enclosed in or partially enclosed in, a particle.
  • An included particle can be selected from among a lipid particle; a polymer particle; an inorganic particle; and an inorganic/organic particle. A mixture of particle types can also be included. The particles can be of any shape, size, composition, or physiochemical characteristics compatible with administration to a subject. The particles can be organic or inorganic particles, such as glass or metal and can be particles of a synthetic or naturally occurring polymer, such as polystyrene, polycarbonate, silicon, nylon, cellulose, agarose, dextran, and polyacrylamide.
  • In particular aspects, the particle is a lipid particle including, but not limited to, liposomes, micelles, unilamellar or mulitlamellar vesicles; polymer particles such as hydrogel particles, polyglycolic acid particles or polylactic acid particles; inorganic particles such as calcium phosphate particles such as described in for example U.S. Pat. No. 5,648,097; and inorganic/organic particulate carriers such as described for example in U.S. Pat. No. 6,630,486. Further description of liposomes and methods relating to their preparation and use may be found in Liposomes: A Practical Approach (The Practical Approach Series, 264), V. P. Torchilin and V. Weissig (Eds.), Oxford University Press; 2nd ed., 2003.
  • An included particle is typically formulated such that the particle has a diameter, or longest dimension, in the range of about 1 nm - 10 microns. In particular aspects, an included particle is a nanoparticle, formulated such that the nanoparticle has a diameter, or longest dimension, in the range of about 1 nm — about 1000 nm. Further aspects of nanoparticles are described in S.M. Moghimi et al., FASEB J. 2005, 19, 311-30; Choi, et al., Mol Imaging. 2010 Dec; 9(6): 291-310; and Bogart et al., ACS Nano, 2014, 8 (4), pp 3107-3122.
  • II. Methods for Imaging
  • In one or more embodiments, the disclosure discloses methods for imaging. In embodiments, methods for imaging include: (a) administering a labeled-antibody conjugate to a subject, wherein the labeled-antibody conjugate includes: an antibody that specifically recognizes and binds to IFN-γ, and at least one detection label conjugated to the antibody, wherein the at least one detection label includes a radionuclide tracer; and (b) detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging. In embodiments, the labeled-antibody conjugate is as described herein with regard to antibody conjugates. In further embodiments, the antibody and the at least one detection label are as described herein with regard to antibody conjugates.
  • In embodiments, the methods include administering a labeled-antibody conjugate to a subject. In embodiments, the methods include administering an effective amount of a labeled-antibody conjugate to a subject. The labeled-antibody conjugate may be administered by any suitable route known to those of ordinary skill in the art. In embodiments, administration of the labeled-antibody conjugate is by intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intratumoral, perfusion through a regional catheter, and/or direct intralesional injection. In embodiments wherein the labeled-antibody conjugate is administered by injection, the administration may be by continuous infusion, by single bolus, and/or by multiple boluses. In some embodiments, administering the radiolabeled-antibody conjugate is non-immunogenic to the subject. In embodiments, the subject is a mammal. In some embodiments, the subject is a mammal selected from humans, non-human primates, canines, felines, murines, bovines, equines, ovines, porcines, and lagomorphs. In illustrative, non-limiting embodiments, the subject is a mouse or a human.
  • In embodiments, the labeled-antibody conjugate administered to a subject includes a radionuclide tracer. In some embodiments, the labeled-antibody conjugate administered to a subject includes a radionuclide tracer only, i.e. without a fluorophore.
  • In embodiments, the radionuclide tracer is selected from 11C, 13N, 15O, 18F, 44Sc, 45Ti, 52Mn, 64Cu, 68Ga, 76Br, 82Rb, 86Y, 89Zr, 99mTc, 111In, 124I, 131I, 186Re, 43K, 52Fe, 57Co, 67Cu, 67Ga, 77Br, 81Rb, 81mKr, 87mSr, 89Zr, 113mIn, 123I, 125I, 127Cs, 129Cs, 132I, 177Lu, 186Re, 197Hg, 203Pb. 206Bi, 225Ac, 225Ra, or combination of any two or more thereof.
  • In illustrative, non-limiting embodiments, the radionuclide tracer is 89Zr.
  • In alternative embodiments, the labeled-antibody conjugate administered to a subject includes a fluorophore. In embodiments, the fluorophore is a fluorescent dye.
  • In embodiments, the methods for imaging include detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging. In embodiments, the presence of the labeled-antibody conjugate is detected in real time. In embodiments, the presence of the labeled-antibody conjugate is detected non-invasively and/or minimally-invasively.
  • In embodiments wherein the labeled-antibody conjugate includes a radionuclide tracer, the presence of the labeled-antibody conjugate may be detected in vivo by PET imaging, SPECT imaging, or combination thereof. In embodiments wherein the radionuclide tracer is selected from 11C, 13N, 15O, 18F, 64Cu, 68Ga, 76Br, 82Rb, 86Y, 89Zr, 124I, or a combination of any two or more thereof, the presence of the labeled-antibody conjugate may be detected in vivo by PET imaging. In embodiments wherein the radionuclide tracer is selected from 99mTc, 111In, 131I, 186Re, or a combination of any two or more thereof, the presence of the labeled-antibody conjugate may be detected in vivo by SPECT imaging.
  • In embodiments wherein the labeled antibody conjugate includes a fluorophore, the presence of the labeled-antibody conjugate may be detected in vivo by optical imaging. Various in vivo optical imaging techniques are known to those of ordinary skill in the art. (See e.g., Ntziachristos, Annu. Rev. Biomed. Eng. 2006, 8:1-33; Troyan, S. L. et al., Ann. Surg. Oncol. 16, 2943-2952 (2009); Luker, G. D. & Luker, K. E., J. Nucl. Med. 49, 1-4 (2008); Tromberg, B. J. et al., Med. Phys. 35, 2443-2451 (2008), and their potential applicability to imaging-guided diagnostic and surgical methods has been proposed in several preclinical studies; Kirsch, D. G. et al., Nat. Med. 13, 992-997 (2007); von Burstin, J. et al., Int. J. Cancer 123, 2138-2147 (2008); and U.S. Pat. No. 9,409,923). In embodiments, in vivo optical imaging is selected from confocal microscopy, planar imaging, fluorescence molecular tomography, complete projection tomography, fluorescence tomography direct imaging, two-photon in vivo imaging or a combination of any two or more thereof. In further embodiments, planar imaging is selected from epi-illumination (i.e., photographic) imaging, trans-illumination imaging, tomographic imaging, or a combination of any two or more thereof.
  • In embodiments wherein the presence of the labeled-antibody conjugate is detected by in vivo optical imaging, the fluorophore may be selected such that it emits fluorescence in the visible or near-infrared region. In illustrative, non-limiting embodiments, fluorophores emitting fluorescence in the visible or near-infrared region may be selected from fluorescein, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, Texas Red® (Molecular Probes, Inc., Eugene, OR), AlexaFluor® (Molecular Probes, Inc., Eugene), AlexaFluor 350, AlexaFluor 405, AlexaFluor 430, AlexaFluor 488, AlexaFluor 500, AlexaFluor 532, AlexaFluor 546, AlexaFluor 568, AlexaFluor 594, AlexaFluor 610, AlexaFluor 633, AlexaFluor 647, AlexaFluor 660, AlexaFluor 680, AlexaFluor 700, AlexaFluor 750, BODIPY FL, BODIPY R6G, BODIPY TMR, BOPDIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, Rhodamine Green™-X (Molecular Probes, Inc.), Rhodamine Red™-X (Molecular Probes, Inc.), Rhodamine 6G, TMR, TAMRA™ (Applied Biosystems), or a combination of any two or more thereof.
  • In embodiments the presence of the labeled-antibody conjugate is detected by in vivo photoacoustic imaging. In illustrative, non-limiting embodiments, photoacoustic dyes included in the conjugate may be selected from Methylene blue, Evan’s blue, Trypan blue, Patent blue, Indocyanine Green, IRDye800CW, DiR, Cy7, Cy7.5, and porphyrins or a combination of any two or more thereof.
  • In embodiments wherein the labeled antibody conjugate includes a combination of a radionuclide tracer and a fluorophore, the presence of the labeled-antibody conjugate may be detected in vivo by a combination of PET or SPECT and optical imaging.
  • In embodiments wherein the labeled antibody conjugate includes a combination of a radionuclide tracer and a fluorophore, the presence of the labeled-antibody conjugate may be detected in vivo by a combination of PET or SPECT and photoacoustic imaging.
  • In embodiments the presence of the labeled-antibody conjugate may be detected in vivo by a combination of any two or more of: PET, SPECT, Cerenkov imaging, photoacoustic imaging, ultrasound imaging, optical coherent tomography, optical imaging, including fluorescence imaging, and/or bioluminescence imaging.
  • According to embodiments, a method for imaging a subject includes 1) administering a labeled-antibody conjugate to a subject, wherein the labeled-antibody conjugate includes: an antibody that specifically binds to IFN-γ, and a detection label conjugated to the antibody, wherein the detection label is a radionuclide tracer or fluorophore; and 2) detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging. According to particular embodiments, the subject has cancer. According to particular embodiments, the cancer is bladder cancer, brain tumors, breast cancer, cervical cancer, colorectal cancer, kidney cancer, leukemia, lung cancer, melanoma, myeloma, pancreatic cancer, gastric cancer, intestinal cancer, esophageal cancer, mesothelioma cancer, endometrial cancer, ovarian cancer, head and neck cancer, bone cancer, sarcoma, cholangiocarcinoma, gall bladder cancer, testicular cancer, thyroid cancer, or prostate cancer.
  • According to particular embodiments, the subject has an inflammatory condition or a disease associated with inflammation, such as, but not limited to, infection, tissue injury, Alzheimer’s disease, obesity, stroke, heart attack, Hepatitis C, and Crohn’s disease.
  • Combination Treatments
  • Combinations of a labeled-antibody conjugate and at least one therapeutic agent are administered according to aspects of the present invention. In some embodiments, a labeled-antibody conjugate and at least one additional active agent are administered to a subject to treat cancer in a subject in need thereof. In still further aspects, a labeled-antibody conjugate and at least two additional active agents are administered to a subject to treat cancer in a subject in need thereof.
  • The term “additional active agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide). The additional active agent can be, without limitation, a small molecule drug, an oligonucleotide or polynucleotide, such as an antisense oligonucleotide or polynucleotide, an siRNA, an mRNA, an miRNA, an shRNA, a peptide or protein. An additional active agent can be a therapeutic agent or a diagnostic agent according to embodiments.
  • Additional active agents which are therapeutic agents included in aspects of methods and compositions of the present invention include, but are not limited to, antibiotics, antivirals, antineoplastic agents, analgesics, antipyretics, antidepressants, antipsychotics, anti-cancer agents, antihistamines, anti-osteoporosis agents, anti-osteonecrosis agents, antiinflammatory agents, anxiolytics, chemotherapeutic agents, diuretics, growth factors, hormones, non-steroidal antiinflammatory agents, steroids and vasoactive agents.
  • According to embodiments, combination treatments include: (1) administration of pharmaceutical compositions that include a labeled antibody conjugate in combination with one or more additional active agents; (2) co-administration of a labeled antibody conjugate with one or more additional active agents wherein the labeled antibody conjugate and the one or more additional active agents are formulated in the same composition and (3) co-administration of a labeled antibody conjugate with one or more additional active agents wherein the labeled antibody conjugate and the one or more additional therapeutic agents have not been formulated in the same composition. When using separate formulations, the labeled antibody conjugate and the one or more additional active agents may be administered at the same time or at different times.
  • An adjunct anti-cancer treatment can be administered in combination with a labeled antibody conjugate, such as prior to, simultaneous with or after administration of the labeled antibody conjugate, such as an anti-cancer radiation treatment of a subject or an affected area of a subject’s body.
  • An additional active agent can be an anti-cancer agent according to embodiments, such as a targeted therapy agent or chemotherapeutic.
  • Anti-cancer agents are described, for example, in Goodman et al., Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 8th Ed., Macmillan Publishing Co., 1990.
  • Anti-cancer agents illustratively include acivicin, aclarubicin, acodazole, acronine, adozelesin, aldesleukin, alitretinoin, allopurinol, altretamine, ambomycin, ametantrone, amifostine, aminoglutethimide, amsacrine, anastrozole, anthramycin, arsenic trioxide, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene, bisnafide dimesylate, bizelesin, bleomycin, brequinar, bropirimine, busulfan, cactinomycin, calusterone, capecitabine, caracemide, carbetimer, carboplatin, carmustine, carubicin, carzelesin, cedefingol, celecoxib, chlorambucil, cirolemycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dexormaplatin, dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, droloxifene, dromostanolone, duazomycin, edatrexate, eflornithine, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin, erbulozole, esorubicin, estramustine, etanidazole, etoposide, etoprine, fadrozole, fazarabine, fenretinide, floxuridine, fludarabine, fluorouracil, flurocitabine, fosquidone, fostriecin, fulvestrant, gemcitabine, hydroxyurea, idarubicin, ifosfamide, ilmofosine, interleukin II (IL-2, including recombinant interleukin II or rIL2), interferon alfa-2a, interferon alfa-2b, interferon alfa-n1, interferon alfa-n3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan, lanreotide, letrozole, leuprolide, liarozole, lometrexol, lomustine, losoxantrone, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, metoprine, meturedepa, mitindomide, mitocarcin, mitocromin, mitogillin, mitomalcin, mitomycin, mitosper, mitotane, mitoxantrone, mycophenolic acid, nelarabine, nocodazole, nogalamycin, ormnaplatin, oxisuran, paclitaxel, pegaspargase, peliomycin, pentamustine, peplomycin, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, plomestane, porfimer, porfiromycin, prednimustine, procarbazine, puromycin, pyrazofurin, riboprine, rogletimide, safingol, semustine, simtrazene, sparfosate, sparsomycin, spirogermanium, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, talisomycin, tamoxifen, tecogalan, tegafur, teloxantrone, temoporfin, teniposide, teroxirone, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapazamine, topotecan, toremifene, trestolone, triciribine, trimetrexate, triptorelin, tubulozole, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine, vincristine sulfate, vindesine, vinepidine, vinglycinate, vinleurosine, vinorelbine, vinrosidine, vinzolidine, vorozole, zeniplatin, zinostatin, zoledronate, and zorubicin.
  • According to embodiments, the additional active agent is an anti-cancer immunotherapy agent. According to embodiments, the anti-cancer immunotherapy agent is an immune checkpoint inhibitor, a cytokine, a vaccine, an adoptive cell transfer therapy, or any therapeutic wherein the mechanism of action of the therapeutic is an increased number of tumor-infiltrating lymphocytes in the subject and/or an increased activation state of a tumor-infiltrating lymphocyte population in the subject. According to embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor, PD-L1 inhibitor, or CTLA-4 inhibitor. According to embodiments, the immune checkpoint inhibitor selected from atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab, pembrolizumab, and an antigen-binding fragment of any one of the foregoing. According to embodiments, the subject is human and the antibody specifically binds to human IFN-γ.
  • According to embodiments, an anti-cancer immunotherapy agent is administered to a subject and a labeled antibody conjugate is administered simultaneously or subsequently followed by detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging to image IFN-γ and thereby monitor the effectiveness of the treatment with the anti-cancer immunotherapy agent. According to embodiments, the subject received an anti-cancer immunotherapy prior to administering and detecting the presence of the labeled-antibody conjugate, and wherein the mechanism of action of the immunotherapy results in an increased number of tumor-infiltrating lymphocytes in the subject and/or an increased activation state of a tumor-infiltrating lymphocyte population in the subject; or wherein administering and detecting the presence of the labeled-antibody conjugate is performed prior to administration of a therapy to determine the state of active immunity in the subject. Thus, efficacy of the anti-cancer immunotherapy is monitored non-invasively and in real-time in the subject.
  • In embodiments, significant binding of the labeled-antibody conjugate to the IFN-γ antigen indicates the presence of activated lymphocytes and/or human cancer cells. In embodiments, significant binding of the labeled-antibody conjugate to the IFN-γ antigen indicates the presence of a localized immune response in the subject. In embodiments, PET imaging is quantitative wherein a correlation is determined between uptake of the radionuclide tracer quantified by radioactivity measurements of excised tissues and uptake estimated noninvasively by PET. Thus, efficacy of the anti-cancer immunotherapy is monitored non-invasively and in real-time in the subject.
  • According to embodiments, an anti-inflammation immunotherapy agent is administered to a subject and a labeled antibody conjugate is administered simultaneously or subsequently followed by detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging to image IFN-γ and thereby monitor the effectiveness of the treatment with the anti-inflammation immunotherapy agent. According to embodiments, the subject received an anti-inflammation immunotherapy prior to administering and detecting the presence of the labeled-antibody conjugate, and wherein the mechanism of action of the immunotherapy results in an increased number of activated lymphocytes in the subject and/or an increased activation state of an activated lymphocyte population in the subject; or wherein administering and detecting the presence of the labeled-antibody conjugate is performed prior to administration of a therapy to determine the state of active immunity in the subject. Thus, efficacy of the anti-inflammation immunotherapy is monitored non-invasively and in real-time in the subject.
  • In embodiments, significant binding of the labeled-antibody conjugate to the IFN-γ antigen indicates the presence of activated lymphocytes. In embodiments, significant binding of the labeled-antibody conjugate to the IFN-γ antigen indicates the presence of a localized immune response in the subject. In embodiments, PET imaging is quantitative wherein a correlation is determined between uptake of the radionuclide tracer quantified by radioactivity measurements of excised tissues and uptake estimated noninvasively by PET. In embodiments, PET imaging is quantitative wherein uptake of the radionuclide tracer is quantified by analysis of acquired images. Thus, efficacy of the anti-inflammation immunotherapy is monitored non-invasively and in real-time in the subject.
  • According to embodiments, methods for imaging a subject include administering a labeled-antibody conjugate to a subject, wherein the labeled-antibody conjugate comprises: an antibody that specifically binds to IFN-γ, and a detection label conjugated to the antibody, wherein the detection label is a radionuclide tracer or fluorophore; and detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging.
  • According to embodiments, a labeled-antibody conjugate is administered to a subject prior to immunotherapy treatment, wherein the labeled-antibody conjugate includes an antibody that specifically binds to IFN-γ, and a detection label conjugated to the antibody, wherein the detection label is a radionuclide tracer or fluorophore; and the presence of the labeled-antibody conjugate in the subject is detected in vivo by imaging to establish a baseline reading indicative of a baseline level of IFN-γ to determine the state of active immunity in the subject. An immunotherapy is then administered wherein the mechanism of action of the immunotherapy results in an increased number of activated lymphocytes in the subject and/or an increased activation state of an activated lymphocyte population in the subject. The labeled-antibody conjugate is then administered to a subject again after the administration of the immunotherapy to establish a post-treatment reading indicative of a subsequent level of IFN-γ to determine the state of active immunity in the subject. The steps of administration of the immunotherapy and administration/detection of the labeled-antibody conjugate may be performed 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times, over a period of treatment time in the range of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, 1, 2, 3, 4, 5, 6, 7, or more days, 1, 2, 3, 4, 5, 6, 7, 8, or more weeks, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more months, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more years, depending on the course of the disease and the course of treatment.
  • According to embodiments, a labeled-antibody conjugate is administered to a subject prior to immunotherapy treatment of cancer in a subject having or suspected of having cancer, wherein the labeled-antibody conjugate includes an antibody that specifically binds to IFN-γ, and a detection label conjugated to the antibody, wherein the detection label is a radionuclide tracer or fluorophore; and the presence of the labeled-antibody conjugate in the subject is detected in vivo by imaging to establish a baseline reading indicative of a baseline level of IFN-γ to determine the state of active immunity in the subject. An anti-cancer immunotherapy is then administered wherein the mechanism of action of the anti-cancer immunotherapy results in an increased number of tumor-infiltrating lymphocytes in the subject and/or an increased activation state of a tumor-infiltrating lymphocyte population in the subject. The labeled-antibody conjugate is then administered to a subject again after the administration of the anti-cancer immunotherapy to establish a post-treatment reading indicative of a subsequent level of IFN-γ to determine the state of active immunity in the subject. The steps of administration of the anti-cancer immunotherapy and administration/detection of the labeled-antibody conjugate may be performed 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times, over a period of treatment time in the range of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more hours, 1, 2, 3, 4, 5, 6, 7, or more days, 1, 2, 3, 4, 5, 6, 7, 8, or more weeks, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more months, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more years, depending on the course of the disease and the course of treatment.
  • III. Mouse Models
  • In one or more embodiments, the disclosure discloses mouse models.
  • In embodiments, the mouse models include: a mouse having a human cancer xenograft and a labeled-antibody conjugate, wherein the labeled-antibody conjugate includes: an antibody that specifically recognizes and binds to IFN-γ, and at least one detection label conjugated to the antibody, wherein the at least one detection label is selected from a radionuclide tracer or a fluorophore. In embodiments, the labeled-antibody conjugate is as described herein with regard to antibody conjugates and methods of imaging.
  • In embodiments, the mouse model has a human cancer xenograft. In embodiments, the human cancer xenograft is transplanted into the mouse. In embodiments, the human cancer xenograft is established from human cancer cells, human cancer tissues, or combination thereof. In embodiments, the human cancer xenograft includes human cancer cells and/or human cancer tissues of primary cancer and/or metastatic cancer. In embodiments, the human cancer xenograft includes human cancer cells and/or human cancer tissues of primary cancer and/or metastatic cancer selected from brain cancer, melanoma, colon cancer, kidney cancer, prostate cancer, breast cancer, gastric cancer, pancreatic cancer, ovarian cancer and testicular cancer.
  • In embodiments, the mouse model having a human cancer xenograft is immunodeficient and further includes engrafted human hematopoetic stem cells such that the mouse has a “humanized” immune system which produces human IFN-γ.
  • In embodiments, the methods include determining the effect of the composition or treatment on the growth of a human cancer xenograft in the mouse. In embodiments, the effect of the composition or treatment on the growth of the human cancer xenograft is determined by imaging. In embodiments, suitable imaging techniques include those described herein with regard to imaging methods. In embodiments, the effect of the composition or treatment is determined by comparing the growth of the human cancer xenograft in the mouse administered the composition or treatment to growth of a human cancer xenograft in a mouse not administered the composition or treatment.
  • In embodiments, the mouse models include: a mouse having an injury or infection and a labeled-antibody conjugate, wherein the labeled-antibody conjugate includes: an antibody that specifically recognizes and binds to IFN-γ, and at least one detection label conjugated to the antibody, wherein the at least one detection label is selected from a radionuclide tracer or a fluorophore. In embodiments, the labeled-antibody conjugate is as described herein with regard to antibody conjugates and methods of imaging immune-mediated treatment for injury or infection.
  • In embodiments, the mouse models include: an immune competent mouse having a murine cancer, such as an allograft or induced murine tumor, and a labeled-antibody conjugate, wherein the labeled-antibody conjugate includes: an antibody that specifically recognizes and binds to murine IFN-γ, and at least one detection label conjugated to the antibody, wherein the at least one detection label is selected from a radionuclide tracer or a fluorophore. In embodiments, the labeled-antibody conjugate is as described herein with regard to antibody conjugates and methods of imaging.
  • EXAMPLES
  • The following non-limiting examples illustrate the methods and compositions of the present disclosure.
  • Mice
  • Female BALB/c mice (6-8 week old) were purchased from Charles River Laboratories (Wilmington, MA). Heterozygous BALB/NeuT (NeuT) mice were in-house bred. NeuT male mice, which express a transforming rat neu, develop atypical ductal hyperplasia in 1-2 parotid glands by 6 weeks of age which progresses to multifocal acinic cell adenocarcinoma in situ at about 19 weeks of age.
  • Radiochemistry
  • All antibodies were obtained from eBioscience (San Diego, CA) whereas the non-specific IgG isotype controls were purchased from Jackson ImmunoResearch (Westgrove, PA) and Thermo Fisher (Waltham, MA) unless otherwise stated. Chelators were obtained from Macrocyclics (Plano, TX). Radioisotopes were obtained from 3D Imaging, LLC (Little Rock, AR). Synthesis of 89Zr-anti-CD3, 89Zr-anti-IFN-y, and non-specific 89Zr-anti-IgG was achieved as described herein. For this synthesis, p-SCN-Bn-Desferrioxamine (DFO, Macrocyclics, Plano, TX) was incubated with 145-2C11 (anti-CD3ε, Armenian hamster, IgG, eBioscience, San Diego, CA), or AN-18 (anti-IFNy, rat IgG1, kappa, eBioscience, San Diego, CA) at a ratio of 1:5 (Ab:DFO) in phosphate buffered saline at pH ~9 for 1 h at 37° C. and purified via centrifugation with molecular weight column filter (MWCO: 30 kDa, GE Vivaspin 500). Facile 89Zr-radiolabeling (3D Imaging, Little Rock, AR) of the 145-2C11- or AN-18- conjugates proceeded in a neutral pH environment in saline at room temperature after 1 h of incubation. Unbound 89Zr (t½ = 3.27 d) was removed via centrifugation with molecular weight column filters (MWCO: 30 kDa, GE Vivaspin 500). Labeling yields of >85% were achieved as monitored through radio-instant thin layer chromatography (iTLC, MiniScan, Eckert and Ziegler, Berlin, Germany) within 1 h. A radiochemical purity of >98% was obtained after filtration based on iTLC. Specific activities of 4.20 ± 0.12 mCi/mg for 89Zr-anti-IFN-γ and 3.98 ± 0.08 mCi/mg for 89Zr-anti-CD3 were achieved.
  • PET Imaging
  • Tumor-bearing animals were injected intravenously (i.v.). with the radiolabeled antibody (~180-240 µCi/mouse, 42.8 - 57.1 µg in 150 µL sterile saline) in the lateral tail vein. Imaging was acquired from 4-120 h post-injection (p.i.). The animals were anesthetized with 3-5% isoflurane (Baxter Healthcare, Deerfield, IL) in air for induction, then lowered to 1.5-2% for maintenance. Images were acquired with a microPET R4 camera (Siemens Medical Solutions, USA formerly Concorde Microsystems). Manually drawn 3-dimensional volumes-of-interest (VOI) were selected to determine maximum and mean percent injected dose per gram (%ID/g) in various tissues. Images were decay-corrected to the time of injection and analyzed for labeled-antibody conjugate uptake using ASIpro VMTM software v. 6.3.3.0.
  • Detecting Elevated IFN-γ in Mice Treated With the TLR9 Agonist CpG-ODN
  • IFN-γ was systemically induced in naïve BALB/c mice by intramuscular (i.m.) injection of 100 µg CpG-ODN (CpG-oligodeoxynucleotides, Integrated DNA Technologies, Coralville, IA). Within an hour of treatment, the mice were injected with 89Zr-anti-IFN-γ in the lateral tail vein for imaging (180-240 µCi/mouse, 49.9 ± 7.2 µg). PET imaging was conducted at 72 h p.i. Labeled-antibody conjugate uptake within the spleen and peripheral immune tissues were analyzed and compared against a control, untreated group.
  • Biodistribution and Competitive Inhibition Blocking Assay
  • Mice were injected i.v. in the lateral tail vein with 89Zr-anti-IFN-γ (20-30 µCi, 4.7-7.1 µg) in 100-150 µL saline followed by euthanasia via CO2 asphyxiation after 72 h p.i., the time point identified at which tumor-to-background is sufficient. For blocking studies, 80 µg of cold AN-18 was co-injected with the probe in a separate cohort of mice. Select organs were harvested post-sacrifice, weighed and measured for bound radioactivity with a gamma counter (Perkin Elmer 2480 Wizard 2). The %ID/g was calculated as the % of activity bound to the tissue normalized against total administered activity per gram of tissue weight.
  • In Vitro 89Zr-Anti-IFN-γ Internalization Assay
  • TUBO cells (4.5 × 105) were plated into 6 well plates in triplicate and allowed to adhere overnight. In separate wells, cells were either left untreated or incubated with recombinant IFN-γ (10 ng/mL) for 15 minutes before being washed 3 times with PBS. 89Zr-anti-IFN-γ (150 ng, 0.63 nCi) was added and cells were incubated for 1 h at 37° C. Following the incubation period, the media was collected and the cells were rinsed with 1× phosphate buffered saline (PBS) twice. Surface-bound activity was removed by washing the cells in 100 mM acetic acid + 100 mM glycine (1:1, pH 3.5) at 4° C. The cells were then lysed with 1 M NaOH. All washes (media plus PBS, acid and alkaline) were collected in separate tubes and measured for bound activity using a gamma counter (Perkin Elmer). The %-internalized activity was calculated as the ratio of the activity of the lysate and the total activity collected from the media, PBS, acid and base washes, normalized to 450,000 cells counted using a Countess II Automated Cell Counter (Thermo Fisher).
  • Tumor Inoculation
  • The neu+ TUBO line cloned from a spontaneous mammary tumor in a female NeuT mouse was obtained through Dr. Guido Forni (U. Torino, Torino, Italy). Mice were inoculated in the #4 mammary fat pad with 2.5 × 105 tumor cells. Tumor growth was monitored by palpation and caliper measurement. Tumor volume was calculated as v = (l x w2)/2.
  • DNA Vaccination
  • The HER2/neu DNA vaccine consists of 20 µg of pGM-CSF (encoding murine GM-CSF) and 50 µg pE2TM (encoding the extracellular and transmembrane regions of human HER2) in 50 µL PBS, which is injected i.m. into each gastrocnemius followed immediately by application of electrode gel and square wave electroporation using a BTX830 (BTX Harvard Apparatus, Holliston, MA). For NeuT vaccination, mice were depleted of Tregs by intraperitoneal (i.p.) injection of 500 µg anti-CD25 mAb PC61 10 days prior to the first vaccination.
  • Quantitative Real-Time PCR
  • Tumor tissue was snap frozen in liquid nitrogen. Total tumor RNA was collected by Trizol preparation (Thermo Fisher, Waltham, MA) after homogenization. cDNA was synthesized with ProtoScript II reverse transcriptase (New England Biolabs, MA). Real-time qPCR was conducted with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA) using 10 ng cDNA/well and 500 nM primers specific to the indicated gene (Life Tech, Carlsbad, CA). Primer sequences used are: CD3 forward primer: CACTCTGGGCTTGCTGATGG, SEQ ID NO:1; CD3 reverse primer: TCATAGTCTGGGTTGGAACAGG, SEQ ID NO:2; CD8 forward primer: GCTGGTAGTCTGCATCCTGCTTC, SEQ ID NO:3; CD8 reverse primer: TTGCTAGCAGGCTATCAGTGTTGTG, SEQ ID NO:4; IFN-γ forward primer: GAGCTCATTGAATGCTTGGC, SEQ ID NO:5; IFN-γ reverse primer: GCGTCATTGAATCACACCTG, SEQ ID NO:6; PD-1 forward primer: CGTCCCTCAGTCAAGAGGAG, SEQ ID NO:7; PD-1 reverse primer: GTCCCTAGAAGTGCCCAACA, SEQ ID NO:8; GAPDH forward primer: AAGCTCACTGGCATGGCCTTC, SEQ ID NO:9; and GAPDH reverse primer: TGCTTCACCACCTTCTTGATGTC, SEQ ID NO:10.
  • Relative mRNA quantities are calculated by 2-ΔCT compared to GAPDH.
  • ELISA
  • Tumor tissue was homogenized in standard RIPA buffer with protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was measured by BCA assay (ThermoFisher). High protein binding plates (ThermoFisher) were coated with 3 µg/mL anti-mouse-IFN-y mAb clone AN-18 (eBioscience) in coating buffer (0.1 M Na2HPO4, pH to 9.0) and washed prior to addition of samples or standard curve using recombinant mouse IFN-γ (Peprotech, Rocky Hill, NJ) in duplicate. IFN-γ was detected with biotin-conjugated anti-mouse IFN-γ clone R4-6A2 (eBioscience), avidin-HRP (ThermoFisher), and TMB substrate (ThermoFisher).
  • Serum IgG Measurement
  • Serum HER2- and neu-specific IgG were quantified by flow cytometry with a BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ), using HER2 over-expressing SKOV3 cells or neu transfected 3T3/NKB cells. Serum was incubated with the appropriate cell line at a 1:100 dilution, followed by washes and detection with PE-conjugated anti-mouse IgG secondary antibody and additional washes prior to detection by flow cytometry. Standard curves for HER2 and neu were run using antibody clones TA-1 and 7.16.4, respectively. Regression analysis was conducted using standard curves of anti-HER2 mAb TA-1 (Calbiochem, Burlington, MA) or anti-neu mAb 7.16.4 (Calbiochem, Burlington, MA).
  • Ifn-γ Elispot
  • HER2- and neu-specific IFN-γ production was measured by ELISPOT assay. For this, recombinant HER2 or neu (10 µg/mL, Sino Biologicals, Beijing, China) were incubated with splenocytes for 48 h in round-bottom wells, followed by transfer to anti-IFN-y coated (clone AN-18, eBioscience) ELISPOT plates (Millipore Sigma, Burlington, MA) for an additional 48 h. Spots were detected by biotinylated anti-IFN-y (clone R4-6A2, eBioscience) and avidin-HRP (Becton Dickinson, Franklin Lakes, NJ), followed by enumeration with an ImmunoSpot analyzer (Cellular Technology Limited, Cleveland, OH). Results are expressed as spot forming units (SFU) per 106 cells.
  • Treatment With Anti-Neu mAb 7.16.4
  • Mice bearing TUBO tumors were injected i.p. 5 times every 3-4 days with sterile-filtered ascites containing 1 mg anti-neu mAb 7.16.4 diluted in PBS to a final volume of 300 µL.
  • Immunohistochemistry (IHC) and H&E Staining
  • IHC was performed using anti-CD8 (clone D4W2Z, Cell Signaling) antibody. After euthanasia, tumors were harvested and fixed in formalin before being embedded in paraffin. Blocks were sectioned into 4 µm sections using a Sakura Accu-Cut SRM microtome (Catalog#: SRM- 200 CV) and adhered onto positively charged slides (Histomax Plus, VWR). Slides were then incubated for 12 minutes at 65° C. and deparrafinized in washes of xylene and graded alcohols. Antigen retrieval was performed in PT module buffer (TA-250-PM4X, Fisher) for CD8 (1:200). Primary antibody incubations were performed for 1 h at room temperature in a humidified chamber. Secondary antibody incubations and DAB were performed following manufacturers protocols. CD8 T cell enumeration was conducted by a blinded board certified pathologist. Each tumor sample was screened for hotspots of CD8 lymphocytes using a Nikon Eclipse Ci microscope at 100x magnification. The number of CD8+ T lymphocytes were counted in the three regions of highest infiltration at 400x magnification with a 0.55 mm field diameter, and an average was calculated. For H&E staining, tissue sections were subjected to xylenes, graduated alcohol and distilled water washes. They were then stained with hematoxylin (TA-125-MH, Fisher) for 5 minutes, rinsed with an acid wash for 1 minute and a bluing agent for 15 seconds. Eosin staining was applied to slides for 1 minute and slides were rinsed in 95% ethanol three times. Lastly, sections went through a series of graded alcohol and xylenes steps to dehydrate sections in preparation for mounting with Permount (UN1294, Fisher). Pictures were taken with a Spot Idea camera using Spot 5.2 software (Spot, Sterling Heights, MI).
  • Tumor Dissociation and Flow Cytometry
  • TUBO tumors from untreated BALB/c mice were dissociated using the GentleMACs Dissociator and mouse tumor dissociation kit (Miltenyi, Germany) following the manufacturer protocol. Cells were stained with a combination of either: FITC-conjugated CD45 mAb (clone 30-F11), APC-conjugated TCRβ mAb (clone H57-597), and eFluor780-conjugated viability dye or FITC-conjugated CD45 mAb (clone 30-F11), PE-Cy7-conjugated CD8 mAb (clone 53-6.7), APC-conjugated PD-1 mAb (J43), and eFluor780-conjugated viability dye. All antibodies/dyes were purchased from eBIoscience (San Diego, CA). Samples were analyzed on a BD FACSCantoII flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and samples were gated on the viable fraction.
  • Statistical Analyses
  • Statistical analyses were conducted using GraphPad Prism 7. Data shown are presented as mean ± standard deviation. All tests use one-way ANOVA (estimating a different variance for each group) with Tukey’s post-test unless otherwise noted. Weekly tumor growth rate for each mouse was calculated by regression analysis of log tumor growth from day 14, when T cells become active after the initial vaccination, until the final measurement on day 38, versus time in weeks. The relationship between the estimated growth rate per week (slope from the regression analysis) for each mouse and the 89Zr-anti-IFN-γ labeled-antibody conjugate uptake measured at day 28 is assessed using Pearson’s correlation. *p<0.05, **p<0.01, ***p<0.001.
  • Results 89Zr-Anti-IFN-γ PET Labeled-Antibody Conjugate Identifies Localized IFN-γ Production
  • The rat mAb AN-18 to murine IFN-γ was labeled with 89Zr using desferrioxamine as the chelate to produce 89Zr-anti-IFN-γ in good yields and purities. In mice treated with CpG-ODN to stimulate IFN-γ, whole-body PET images were acquired 72 h p.i., a time point identified to exhibit reliable labeled-antibody conjugate uptake in the tumor, with low liver and blood pool background (FIG. 7A, FIG. 7B, FIG. 8A, FIG. 8B). VOIs drawn on splenic tissues demonstrated higher labeled-antibody conjugate accumulation (3.50 ± 0.61 %ID/g, n=3) in CpG-ODN-treated groups compared to untreated controls (Ctrl: 0.83 ± 0.12 %ID/g, n=3) (FIG. 1A).
  • Tissue distribution of 89Zr-anti-IFN-γ at 72 h p.i. demonstrated 20.04 ± 12.2 %ID/g uptake in the spleen (FIG. 1B, Table 1).
  • TABLE 1
    Control 10x mAb block
    Mean ± S.D. Mean ± S.D. p-value
    Spleen 20.04 ± 12.20 1.88 ± 2.74 0.0061
    Blood 0.67 ± 0.69 19.46 ± 12.69 0.0043
    Heart 0.30 ± 0.04 10.57 ± 8.91 0.2956
    Liver 9.77 ± 9.12 11.69 ± 9.82 0.9999
    Kidneys 3.93 ± 0.60 10.64 ± 7.18 0.7755
    Bone 4.19 ± 1.18 1.01 ± 0.94 0.9951
    Muscle 0.73 ± 0.65 1.08 ± 1.23 >0.9999
  • Uptake within the blood circulation (0.67 ± 0.69 %ID/g), as well as tissues responsible for excretion, liver (9.77 ± 9.12 %ID/g), and kidneys (3.93 ± 0.6 %ID/g) were low. There was also low uptake in the bone and muscle. Specificity was further confirmed through competitive binding experiment where a decrease in spleen uptake was observed (20.04 ± 12.20 vs. 1.88 ± 2.74 %ID/g, n=4, p=0.0061) with 10X cold mAb blockade, consequently increasing non-specific tissue accumulation in the blood (19.46 ± 12.69 %ID/g, p=0.0043), heart (10.57 ± 8.91, p=0.30), and liver (11.69 ± 9.82 %ID/g, p=0.99). Notable differences in splenic uptake in the imaging and tissue distribution (10-fold lower mass) are due to “mass effects”, wherein a greater mass of protein administered potentially saturated receptor binding sites and rendered slower pharmacokinetics.
  • Since IFN-γ is a soluble protein, the mechanism of localized IFN-γ imaging was investigated. Plated TUBO tumor cells were exposed to IFN-γ and/or 89Zr-anti-IFN-γ labeled-antibody conjugate in quintuplicate followed by analysis of membrane binding and internalization (FIG. 1C). TUBO cells incubated with 89Zr-anti-IFN-γ alone show limited labeled-antibody conjugate surface binding (1.13 ± 0.28%) and internalization (0.29 ± 0.13%). When TUBO is preincubated with IFN-γ, enhanced 89Zr-anti-IFN-γ surface binding (13.62 ± 2.60%) and internalization (3.93 ± 1.07%) is observed (membrane: p=0.00039, internalized: p=0.0015). Detection of labeled-antibody conjugate binding to TUBO cells after IFN-γ exposure suggests localized imaging may be due to sequestration of IFN-γ on its receptor in vivo.
  • As further verification of labeled-antibody conjugate uptake, the spleen, liver, heart and spleen tissues were removed and scanned ex vivo (FIG. 1D).
  • IFN-γ PET Detects Active Anti-Tumor Immunity in Situ in a Syngeneic Tumor Model
  • To test the capacity of 89Zr-anti-IFN-γ as a non-invasive measure of anti-tumor immune response, neu+ TUBO tumor bearing BALB/c mice were imaged after receiving two rounds of HER2/neu DNA vaccination as detailed in FIG. 2A and FIG. 2B. This vaccine induces HER2-specific humoral and T cell responses and ~10% equivalent of cross-reactive anti-neu T cells without cross-reactive neu-specific antibody. Tumor volumes began to stabilize or regress within 1 week after the second vaccination compared to untreated TUBO-bearing mice (FIG. 2A and FIG. 2B). Mice were injected with 89Zr-anti-IFN-γ labeled-antibody conjugate for PET imaging (FIG. 2C, FIG. 2D and FIG. 9A) at 72 h p.i. A nearly two-fold increase in tumor uptake was observed in vaccinated (Vx: 10.07 ± 1.50 %ID/g, n=6) versus control mice (Ctrl: 5.97 ± 0.61 %ID/g, n=6, p=0.0001). A 89Zr-labeled rat IgG isotype control labeled-antibody conjugate demonstrated similar tumor accumulation (72 h p.i.) in both untreated (5.27 ± 0.79 %ID/g) and vaccinated (5.93 ± 0.85 %ID/g) mice. This suggests baseline intratumoral IFN-γ levels are low without treatment. The notable low accumulation of the isotype control labeled-antibody conjugate after vaccination supports the specificity of the IFN-γ labeled-antibody conjugate and suggests increased 89Zr-anti-IFN-γ uptake is not simply due vascular permeability and retention effects post-ITx.
  • Detection of Tumor Infiltrating Lymphocytes Via CD3 ImmunoPET
  • Total T cell presence in the tumor microenvironment was assessed in separate groups of mice via immunoPET imaging of CD3+ tumor infiltrating lymphocytes using 89Zr-anti-CD3 (FIG. 2E, FIG. 2F, and FIG. 9B). Vaccinated tumors exhibited a modest, insignificant increase of CD3 labeled-antibody conjugate binding compared to control (6.25 ± 0.37 %ID/g, n=6 vs. 4.58 ± 0.83 %ID/g, n=5, p=0.16). Both cohorts failed to demonstrate a significant change in uptake compared to Armenian hamster isotype control IgG (5.90 ± 1.26 %ID/g, n=3, p=0.87 (Ctrl), p=0.49 (Vx)). Untreated TUBO tumors have endogenous T cell infiltrates as detected by flow cytometry upon dissociation (FIG. 10A). However, CD3 immunoPET suboptimally detected these TILs in both untreated and vaccinated mice with measured VOIs similar to the non-specific IgG tumor accumulation. This may be due to excessive uptake by the spleen, a T cell-homing secondary lymphoid tissue (Ctrl: 17.06 ± 3.56 %ID/g, Vx: 18.36 ± 1.49 %ID/g, FIG. 10B), which can act as a labeled-antibody conjugate “sink.” In contrast, limited splenic accumulation was observed with the IFN-γ PET probe (Ctrl: 3.58 ± 0.81 %ID/g, p<0.0001, Vx: 4.97 ± 0.97 %ID/g, p<0.0001).
  • Ex Vivo Validation Via IHC, qPCR, and ELISA
  • Upon completion of imaging, tissues were collected for ex vivo validation. Tumor tissue was assessed to verify CD3+ and CD8+ T cell presence, as well as expression of IFN-γ. Transcripts levels of CD3, CD8 and IFN-γ were increased in tumor tissue after vaccination (FIG. 3A, FIG. 3B, FIG. 3C, respectively, Ctrl: n=11, Vx: n=13), in concordance with the PET imaging data. Cultured TUBO cell cDNA is included as a negative control. CD3 and CD8 proteins were increased after treatment (FIG. 11A and FIG. 11B) and intratumoral IFN-γ protein was also confirmed and quantitated by ELISA (FIG. 3D). ELISA results showed higher total IFN-γ in Vx (n=11) versus Ctrl (n=10) TUBO tumors (85.37 ± 65.89 vs. 41.69 ± 20.12 pg/mg tissue, p=0.043).
  • Peripheral vaccine-induced immunity was measured by HER2/neu-specific serum IgG (FIG. 3E and FIG. 3F) and splenic T cell responses (FIG. 3G and FIG. 3H). HER2-specific IgG was only detected in vaccinated mice (18.68 ± 7.40 µg/mL, n=14, p<0.0001). TUBO tumors constitutively express the cell surface oncogene neu, which is foreign in wild-type BALB/c mice. Neu-specific IgG is detected in unvaccinated control TUBO-bearing mice (1.58 ± 1.60 µg/mL, n=13), which is further increased in vaccinated animals (6.18 ± 7.34 µg/mL, n=14, p=0.0019). While the HER2 DNA vaccine itself does not induce anti-neu IgG, tumor cell killing likely enhances immune activity to this foreign antigen. Detection of HER2-specific IFN-γ-producing T cells was restricted to vaccinated mice, similar to anti-HER2 IgG (119.40 ± 95.18/106 splenocytes (SC), n=7, vs. 0.83 ± 2.04/106 SC in untreated controls, n=6, p=0.0012). Peripheral anti-neu T cells were detected in all vaccinated animals (8.33 ± 7.75/106 SC) while only 1 of 4 untreated controls showed T cell responsiveness to neu (0.83 ± 2.04/106 SC, p=0.033). The absolute quantities of HER2 and neu-specific IgG and T cells were ~10-fold lower than similarly vaccinated non-tumor-bearing mice. This may be due to tumor-associated immune suppression by myeloid-derived suppressor cells or regulatory T cells (Tregs), which are reportedly increased in TUBO-bearing mice.
  • Detection of ITx Response in a Spontaneous Tumor Model
  • In this example, the capacity of IFN-γ PET imaging to detect anti-tumor immune activity in a spontaneous tumor setting was tested. Neu transgenic (NeuT) mice are engineered to express a transforming rat neu under direction of the mouse mammary tumor virus promoter, allowing immune system recognition of neu as a self-antigen. Male NeuT mice, which develop 1-2 spontaneous neu+ salivary tumors between 30-40 weeks of age, were used. Once tumors were palpable, Tregs were depleted using anti-CD25 mAb clone PC61 to enhance ITx response given NeuT mice are immune tolerant to rat neu, followed by two HER2/neu DNA vaccinations as detailed in FIGS. 4A and 4B. Vaccination of NeuT mice (n=7) controlled tumor growth rate compared to untreated (n=6) tumor-bearing NeuT mice (FIG. 4A and FIG. 4B, p=0.032). IFN-γ PET of vaccinated tumors displayed a nearly two-fold higher uptake of 89Zr-anti-IFN-γ (8.37 ± 0.35 %ID/g, n=4) vs. control (4.63 ± 0.47 %ID/g, n=3, p=0.001), indicating infiltration of functional anti-tumor T cells (FIG. 4C, FIG. 4D, and FIG. 12A). An examination of tumor infiltrates via CD3 PET (FIG. 4E, FIG. 4F, and FIG. 12B) revealed a similar trend (8.05 ± 1.47 %ID/g vs. 4.43 ± 0.72 %ID/g, n=3 per group, p=0.012).
  • Validation of T cell infiltration and IFN-γ production was conducted by qPCR in tumor tissue samples (FIG. 5A, FIG. 5B, and FIG. 5C). CD3 and CD8 detection showed a variable modest, insignificant increase after vaccination, while IFN-γ mRNA increased (Ctrl: n=3, Vx: n=5, p=0.036). Peripheral immune response to the vaccine was evaluated by measuring serum anti-HER2 and anti-neu IgG as well as spleen-resident HER2- and neu-responsive IFN-γ-producing T cells. Tolerance to HER2/neu in NeuT mice was apparent with a comparatively lower ITx response vs. wild-type BALB/c mice bearing TUBO tumors in FIGS. 2 . HER2-specific IgG was detected in vaccinated animals (FIG. 5D, FIG. 5E, 8.7 ± 4.9 µg/mL, n=8, p=0.0016 vs. Ctrl, n=5), while anti-neu IgG was negligible or absent in all samples tested. Despite increased intratumoral detection of IFN-γ in vaccinated NeuT mice by PET, peripheral T cell response to neu was low (FIG. 5F, FIG. 5G, 15.80 ± 8.84/106 SC, n=5) with HER2 vaccination, and was not significantly increased relative to untreated control (n=5, p=0.27). T cell response to the cognate vaccine antigen was evident in anti-HER2 T cells (15.80 ± 8.84/106 SC, n=5, p=0.0079). The ratio of anti-neu to anti-HER2 T cells detected was ~5.1% in NeuT mice, which is two-fold lower relative to that detected in non-immune tolerant BALB/c mice bearing TUBO (10.0%, FIG. 3G and FIG. 3H). These results support the hypothesis that peripheral immune monitoring may be an inadequate measure of anti-tumor immunity with tumor-responsive T cells preferentially localizing within the tumor, demonstrating the utility of in situ analysis methods using PET imaging of anti-IFN-γ.
  • IFN-γ PET Imaging is an Indicator of Immune Activation Status In Situ
  • To test the capacity of 89Zr-anti-IFN-γ to predict treatment outcomes, BALB/c mice (n=11) bearing variably-sized TUBO tumors were treated with HER2 vaccine, resulting in a range of growth slopes (FIG. 6A). 89Zr-anti-IFN-γ PET imaging was conducted two weeks after the final vaccination and tumor volume was monitored for an additional ten days. Tumor-localized 89Zr-anti-IFN-γ labeled-antibody conjugate uptake inversely correlated with tumor growth rate (FIG. 6B and FIG. 13 , r=-0.64, 95% CI: (-0.90,-0.06); p=0.034), suggesting IFN-γ PET is an indicator of the effects of ITx on these tumors.
  • IFN-γ PET was then evaluated in a setting where tumor-infiltrating T cells are present but have become exhausted. TUBO-bearing mice were treated with passive ITx, mAb 7.16.4 to rat neu. This mAb has been shown to inhibit neu signaling in addition to initiating host anti-tumor immunity. Once tumors were established at ~50 mm3, 1 mg doses of 7.16.4 were given i.p. at 3-4 day intervals for a total of 5 treatments, which reduced and stabilized tumor growth (FIG. 6C). 89Zr-anti-IFN-γ (n=5) or control IgG (n=6) PET imaging was conducted on day 30 after treatment onset, at which time tumor growth had resumed. Tumor uptake of IFN-γ labeled-antibody conjugate was indistinguishable from IgG control, suggesting a lack of immune activity (FIG. 6D). CD8 T cell infiltration was evaluated by IHC, see Table 2 below.
  • TABLE 2
    Treatment Avg # CD8 Std. Dev.
    Ctrl 3 1
    mAb 7.16.4 36 19
    Vx 74 25
  • Blinded pathologist enumeration of the three regions with highest infiltration were calculated, showing a 12-fold increase in CD8 T cells after 7.16.4 treatment versus control (Ctrl: 3 ± 1, 7.16.4: 36 ± 19). Vaccinated TUBO tumor had the largest detected CD8 infiltration (74 ± 25). Overall, CD8+ tumor infiltration was intermittent, with high-density regions scattered among areas with no detectable CD8+ TILs.
  • CD8 T cell infiltration after 7.16.4 therapy was validated by flow cytometry in a parallel cohort of treated and control mice (FIG. 6E, n=4 each). An overall increase in CD45+ infiltrates (Ctrl: 6.84 ± 1.85%, 7.16.4: 16.95 ± 5.88%, p=0.036) and CD8+ T cells (Ctrl: 0.41 ± 0.19%, 7.16.4: 4.96 ± 1.96%, p=0.018) was detected after mAb treatment. The majority of CD8+ TILs expressed the T cell exhaustion marker PD-1 (FIG. 6F, 79.7 ± 10.3%) compared to control tumors (20.5 ± 9.3%, p=0.0001). Collectively these results suggest this treatment model promotes an inactive and exhausted CD8 T cell status despite tumor infiltration, leading to reduced IFN-y production which can be detected by 89Zr-anti-IFN-γ PET imaging.
  • To date, a variety of monoclonal antibodies have been commercially developed against mouse and human IFN-γ. For the studies presented here, anti-mouse IFN-γ clone AN-18 was utilized, and clone XMG1.2 was also tested in a biodistribution study. BALB/c mice were treated with a single intramuscular injection of 100 µg CpG-ODN and tissue biodistribution was measured after 72 h. From these results, it is not anticipated that the clone of IFN-γ antibody would significantly impact its efficacy as a PET labeled-antibody conjugate.
  • IFN-γ Diabody Production
  • From the derived sequence of the anti-IFNγ (clone: AN18), homodimers of the single chain Fv were produced using CHO cells as expression system (Neoclone, LLC). Octet analysis of the binding affinity (Kd) for the diabody was 37.9 ± 5.5 nM vs. 28.2 ± 0.66 nM for the full-length AN18.
  • Synthesis and Quality Control of ImmunoPET Probes
  • NOTA-PEG4-Tz was made (57 ± 12% yield) by attaching NOTA-Bz-NCS to tetrazine (Tz)-PEG4-NH2 using equimolar ratios in DMSO/TEA solvent for 1 h at room temperature (RT). Purification was done through C18 RP-HPLC. 18F-labeling proceeded using 18F-AlF-NOTA method. Briefly, non-carrier added 18F[F-] will be incubated with 40 nmol AlCl3 in 0.4 M KHCO3 at pH~4 for 10 min. Equimolar NOTA-PEG4-Tz in 3:1 acetonitrile:H2O was added and the solution was incubated for 15 minutes at 90° C. Analytical radio-C18 HPLC monitored the reaction. Purification of 18F-AlF-NOTA-Tz was made by eluting the product through a C18-Solid Phase Extraction cartridge with ethanol. Radiochemical Yield: 14-16% decay-corrected.
  • Diabody (Db) Conjugation to 18F-AlF-NOTA-Tz
  • Labeling of Dbs (MW: ~ 50 kDa, NeoClone, WI) is accomplished via inverse electron demand Diels-Alder bioorthogonal click reaction using Tz and transcyclooctene (TCO), which affords a rapid, mild method of labeling a protein, conserving its configuration. The Db fragment of anti-IFNγ clone AN18 was attached to TCO-NHS, which was then conjugated to 18F-AlF-NOTA-Tz at RT in PBS, pH ~ 7 for 15 min. The labeled product was purified through a spin column (MWCO: 10 kDa). Radio-instant thin layer chromatography and size exclusion chromatography (SEC) indicated >95% purities with an overall decay-corrected radiochemical yield of 6-10%. The specific activity was 3.8-4 mCi/mg. 100-150 µCi Db tracer was injected into a TUBO-bearing BALB/c mouse after Vx (FIG. 15 ). Tumor uptake is detected with an ROI of ~8.0 %ID/g at 3 h p.i., similar to results obtained with full-length mAb (FIG. 2C). The clearance appears to be through the gall bladder and intestines, a clear shift from the hepatic sink typically observed in full mAbs. This validated labeling method will be maintained throughout the proposed studies. A cold 19F-IFN-γ Db compound will be prepared as reference standard for SEC quality control. Retention of affinity post-conjugation is evaluated through binding affinity via surface plasmon resonance.
  • IFN-γ PET methods of the present invention measure active anti-tumor immunity, providing a predictive tool for non-invasive in situ tumor evaluation. This approach is highly specific to the tumor compared to total T cell imaging due to the fact that IFN-γ is secreted by CTLs within the tumor. Imaging CD3, on the other hand, targets the general T cell population that are not only localized in the tumor but also in other lymphoid tissues.
  • These results show that 89Zr-anti-IFN-γ labeled-antibody conjugate uptake can be indicative of response to therapy in both cancer vaccination and TAA-specific mAb models. These results further demonstrate that IFN-γ PET may be more sensitive for determining response to ITx when compared to peripheral immune evaluation. IFN-γ PET has the potential to serve as a universal non-invasive measurement of immune activity in situ for a variety of cancers with virtually any ITx modality with no need for knowledge of specific antigens or cumbersome ex vivo antigen recall assays. Additionally, the utility of IFN-γ PET allows for cancer immune monitoring, and examination of localized inflammatory conditions such as injury, infection, or autoimmune disease.
  • Collectively, these results suggest it may be optimal to detect activity of immune cell populations instead of mere presence. PET imaging of released, soluble IFNγ displayed higher sensitivity of active anti-tumor immunity detection versus total CD3 imaging. A major drawback to the use of general T cell surface markers for PET imaging is labeled-antibody conjugate uptake in normal secondary lymphoid tissues with dense T cell content, such as the spleen, thymus and lymph nodes, which may limit tumor detection. Importantly, these results indicate non-invasive in situ tumor evaluation by PET more clearly depicts responses to immunotherapy when compared to peripheral immune evaluation by ELISPOT. Unlike peripheral T cell assays, IFN-γ PET also does not require knowledge of specific tumor antigens or cumbersome ex vivo antigen recall assays to determine the extent of immunotherapy response. In addition, a mAb-based labeled-antibody conjugate may locally neutralize IFN-γ activity, which could benefit an anti-tumor response by reducing PD-L1 upregulation. Taken together, these results support the development of IFN-γ PET labeled-antibody conjugates for clinical evaluation of tumor immunotherapy.
  • Items
  • Item 1. A method for in vivo immunoimaging IFN-γ as a marker of active T cells in a subject, comprising: administering a labeled-antibody conjugate to a subject, wherein the labeled-antibody conjugate comprises: an antibody that specifically binds to IFN-γ, and a detection label conjugated to the antibody, wherein the detection label is a radionuclide tracer or fluorophore; and detecting the presence of the labeled-antibody conjugate in the subject in vivo by imaging.
  • Item 2. The method of item 1, wherein the subject has cancer.
  • Item 3. The method of item 2, wherein the subject received an immunotherapy prior to administering and detecting the presence of the labeled-antibody conjugate, and wherein the mechanism of action of the immunotherapy results in an increased number of tumor-infiltrating lymphocytes in the subject and/or an increased activation state of a tumor-infiltrating lymphocyte population in the subject; or wherein administering and detecting the presence of the labeled-antibody conjugate is performed prior to administration of a therapy to determine the state of active immunity in the subject.
  • Item 4. The method of item 3, wherein the immunotherapy is an immune checkpoint inhibitor, a cytokine, a vaccine, or an adoptive cell transfer therapy.
  • Item 5. The method of any one of the preceding items, wherein the subject is human and the antibody specifically binds to human IFN-γ.
  • Item 6. The method of any one of the preceding items, wherein the antibody is selected from a monoclonal antibody, an antibody fragment, or combination thereof.
  • Item 7. The method of item 6, wherein the antibody fragment is a Fab fragment.
  • Item 8. The method of item 6, wherein the antibody fragment is a diabody.
  • Item 9. The method of any one of the preceding items, wherein the radionuclide tracer is conjugated to the antibody or antibody fragment with a bifunctional chelator or linker.
  • Item 10. The method of item 9, wherein the bifunctional chelator comprises a chelator selected from N′-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), 1,4,7-triazacyclononane-N,N′,N′-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (i.e., DOPA), diethylenetriaminepentaacetic acid (DTPA), 2,2′-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NODA), or a combination of any two or more thereof.
  • Item 11. The method of item 9 or item 10, wherein the bifunctional chelator is p-SCN-Bn-DFO.
  • Item 12. The method of any one of the preceding items, wherein the radionuclide tracer is selected from the group consisting of: 11C, 13N, 15O, 18F, 64Cu, 68Ga, 76Br, 82Rb, 86Y, 89Zr, 99mTc, 111IN 124I, 131I, 186Re, 43K, 52Fe, 57Co, 67Cu, 67Ga, 77Br, 81Rb, 81mKr, 87mSr, 113mIn, 123I, 125I, 127Cs, 129Cs, 132I, 197Hg, 203Pb, 206Bi, and combination of any two or more thereof.
  • Item 13. The method of item 12, wherein the radionuclide tracer is 89Zr or 18F.
  • Item 14. The method of any one of the preceding items, wherein the imaging comprises positron emission tomography (PET) imaging, single photon emission computed tomography (SPECT) imaging, or a both PET and SPECT.
  • Item 15. The method of item 14, wherein the imaging comprises PET.
  • Item 16. The method of any one of the preceding items, wherein the labeled-antibody conjugate further comprises a fluorophore.
  • Item 17. The method of any one of the preceding items, wherein specific binding of the labeled-antibody conjugate to IFN-γ indicates the presence of activated T cells and/or human cancer cells.
  • Item 18. The method of any one of the preceding items, wherein the presence of the labeled-antibody conjugate is detected in real time.
  • Item 19. A labeled-antibody conjugate comprising: an antibody that specifically binds to IFN-γ, and a detection label conjugated to the antibody, wherein the detection label is a radionuclide tracer.
  • Item 20. The labeled-antibody conjugate of item 19, wherein the antibody is isolated.
  • Item 21. The labeled-antibody conjugate of item 19 or item 20, wherein the antibody is selected from a monoclonal antibody, an antibody fragment, or combination thereof.
  • Item 22. The labeled-antibody conjugate of item 21, wherein the antibody fragment is a Fab fragment.
  • Item 23. The labeled-antibody conjugate of item 21, wherein the antibody fragment is a diabody.
  • Item 24. The labeled-antibody conjugate of any one of items 19 to 23, wherein the radionuclide tracer is conjugated to the antibody with a bifunctional chelator or linker.
  • Item 25. The labeled-antibody conjugate of item 24, wherein the bifunctional chelator comprises a chelator selected from N′-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl} amino)pentyl]-N-hydroxysuccinamide (DFO), 1,4,7-triazacyclononane-N,N′,N′-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (i.e., DOPA), diethylenetriaminepentaacetic acid (DTPA), 2,2′-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NODA), or a combination of any two or more thereof.
  • Item 26. The labeled-antibody conjugate of item 24 or item 25, wherein the bifunctional chelator is p-SCN-Bn-DFO or NOTA.
  • Item 27. The labeled-antibody conjugate of any one of items 19 to 26, wherein the radionuclide tracer is selected from 11C, 13N, 15O, 18F, 64Cu, 68Ga, 76Br, 82Rb, 86Y, 89Zr, 99mTc, 111In, 124I, 131I, 186Re, 43K, 52Fe, 57Co, 67Cu, 67Ga, 77Br, 81Rb, 81mKr, 87mSr, 113mIn, 123I, 125I, 127Cs, 129Cs, 132I, 197Hg, 203Pb, 206Bi, or a combination of any two or more thereof.
  • Item 28. The labeled-antibody conjugate of any one of items 19 to 27, wherein the radionuclide tracer is 89Zr or 18F.
  • Item 29. The labeled-antibody conjugate of any one of items 19 to 28, wherein the antibody specifically binds to human IFN-γ.
  • Sequences
  • Diabody heavy chain variable region (VH) SEQ ID NO: 11
  • LSQVQLKESGPGLVQPSQTLSLTCTVSGFSLDSYNVHWVRQPPGKGLEWM
    GRMKYNGDTSYNSALKSRLRISRDTSKNQVFLKMNSLQTDDTGTYYCTRD
    WHYGYPSPYFEFWGQGVMVTVSSAETTAPSVYPLAPGTALKSNSMVTLGC
    LVKGYFPEPVTVTWNSGALSSGVHTFPAVLQSGLYTLTSSVTVPSSTWPS
    QTVTCNVAHPASSTKVDKKIVPRNCGGDCKPCICTGSEVSSVFIFPPKPK
    DVLTITLTPKVTCVVVDISQDDPEVHFSWFVDDVEVHTAQTRPPEEQFNS
    TFRSVSELPILHQDWLNGRTFRCKVTSAAFPSPIEKTISKPEGRTQVPHV
    YTMSPTKEEMLQNEVSITCMVKGFYPPDIYVEWQMNGQPQENYKNTPPTM
    DTDGSYFLYSKLNVKKEKWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK
  • Diabody light chain variable region (VL) SEQ ID NO: 12
  • DVQMTQSPSYLAASPGESVSISCKASKNINTYLAWYQEKPGKTNKLLIYS
    GSTLQSGTPSRFSGSGSGTDFTLTIRSLEPEDFAVYYCQQHNEYPLTFGS
    GTKLEVKRADAAPTVSIFPPSTEQLATGGASVVCLMNNFYPRDISVQWKI
    DGTERRDGVLDSVTDQDSKDSTYSMSSTLSLTKADYESHNLYTCEVVHKT
    SSSPVVKSFNRNEC
  • Exemplary linker SGGGGS (SEQ ID NO: 13). scFv SEQ ID NO: 14 which can be included in an anti-IFN-g diabody
  • LSQVQLKESGPGLVQPSQTLSLTCTVSGFSLDSYNVHWVRQPPGKGLEWM
    GRMKYNGDTSYNSALKSRLRISRDTSKNQVFLKMNSLQTDDTGTYYCTRD
    WHYGYPSPYFEFWGQGVMVTVSSAETTAPSVYPLAPGTALKSNSMVTLGC
    LVKGYFPEPVTVTWNSGALSSGVHTFPAVLQSGLYTLTSSVTVPSSTWPS
    QTVTCNVAHPASSTKVDKKIVPRNCGGDCKPCICTGSEVSSVFIFPPKPK
    DVLTITLTPKVTCVVVDISQDDPEVHFSWFVDDVEVHTAQTRPPEEQFNS
    TFRSVSELPILHQDWLNGRTFRCKVTSAAFPSPIEKTISKPEGRTQVPHV
    YTMSPTKEEMLQNEVSITCMVKGFYPPDIYVEWQMNGQPQENYKNTPPTM
    DTDGSYFLYSKLNVKKEKWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGKS
    GGGGSDVQMTQSPSYLAASPGESVSISCKASKNINTYLAWYQEKPGKTNK
    LLIYSGSTLQSGTPSRFSGSGSGTDFTLTIRSLEPEDFAVYYCQQHNEYP
    LTFGSGTKLEVKRADAAPTVSIFPPSTEQLATGGASVVCLMNNFYPRDIS
    VQWKIDGTERRDGVLDSVTDQDSKDSTYSMSSTLSLTKADYESHNLYTCE
    VVHKTSSSPVVKSFNRNEC
  • scFv SEQ ID NO:15 which can be included in an anti-IFN-g diabody
  • DVQMTQSPSYLAASPGESVSISCKASKNINTYLAWYQEKPGKTNKLLIYS
    GSTLQSGTPSRFSGSGSGTDFTLTIRSLEPEDFAVYYCQQHNEYPLTFGS
    GTKLEVKRADAAPTVSIFPPSTEQLATGGASVVCLMNNFYPRDISVQWKI
    DGTERRDGVLDSVTDQDSKDSTYSMSSTLSLTKADYESHNLYTCEVVHKT
    SSSPVVKSFNRNECSGGGGSLSQVQLKESGPGLVQPSQTLSLTCTVSGFS
    LDSYNVHWVRQPPGKGLEWMGRMKYNGDTSYNSALKSRLRISRDTSKNQV
    FLKMNSLQTDDTGTYYCTRDWHYGYPSPYFEFWGQGVMVTVSSAETTAPS
    VYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSGVHTFPAVL
    QSGLYTLTSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRNCGGDCK
    PCICTGSEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISQDDPEVHFSWF
    VDDVEVHTAQTRPPEEQFNSTFRSVSELPILHQDWLNGRTFRCKVTSAAF
    PSPIEKTISKPEGRTQVPHVYTMSPTKEEMLQNEVSITCMVKGFYPPDIY
    VEWQMNGQPQENYKNTPPTMDTDGSYFLYSKLNVKKEKWQQGNTFTCSVL
    HEGLHNHHTEKSLSHSPGK
  • All documents cited are incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present disclosure.
  • It is to be further understood that where descriptions of various embodiments use the term “comprising,” and/or “including” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”
  • While particular embodiments of the present disclosure have been illustrated and described, it would be obvious to one skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the disclosure. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this disclosure.
  • It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
  • Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting. As used in the specification and appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Claims (11)

1. A labeled-antibody conjugate comprising:
an antibody that specifically binds to IFN-γ, and
a detection label conjugated to the antibody, wherein the detection label is a radionuclide tracer.
2. The labeled-antibody conjugate of claim 1, wherein the antibody is isolated.
3. The labeled-antibody conjugate of claim 1, wherein the antibody is selected from a monoclonal antibody, an antibody fragment, or combination thereof.
4. The labeled-antibody conjugate of claim 3, wherein the antibody fragment is a Fab fragment.
5. The labeled-antibody conjugate of claim 3, wherein the antibody fragment is a diabody.
6. The labeled-antibody conjugate of claim 1, wherein the radionuclide tracer is conjugated to the antibody with a bifunctional chelator or linker.
7. The labeled-antibody conjugate of claim 6, wherein the bifunctional chelator comprises a chelator selected from N′-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-aminopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), 1,4,7-triazacyclononane-N,N′,N′-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (i.e., DOPA), diethylenetriaminepentaacetic acid (DTPA), 2,2′-(7-(1-carboxy-4-((2,5-dioxopyrrolidin-1-yl)oxy)-4-oxobutyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NODA), or a combination of any two or more thereof.
8. The labeled-antibody conjugate of claim 6, wherein the bifunctional chelator is p-SCN-Bn-DFO or NOTA.
9. The labeled-antibody conjugate of claim 1, wherein the radionuclide tracer is selected from 11C, 13N, 15O, 18F, 64Cu, 68Ga, 76Br, 82Rb, 86Y, 89Zr, 99mTc, 111In, 124I, 131I, 186Re, 43K, 52Fe, 57Co, 67CU, 67Ga, 77Br, 81Rb, 81mKr, 87mSr, 113mIn, 1231, 125I, 127Cs, 129Cs, 132I, 197Hg, 203Pb, 206Bi, or a combination of any two or more thereof.
10. The labeled-antibody conjugate of claim 1, wherein the radionuclide tracer is 89Zr or 18F.
11. The labeled-antibody conjugate of claim 1, wherein the antibody specifically binds to human IFN-γ.
US18/144,382 2017-08-23 2023-05-08 In vivo immunoimaging of interferon-gamma Pending US20230338589A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/144,382 US20230338589A1 (en) 2017-08-23 2023-05-08 In vivo immunoimaging of interferon-gamma

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201762549231P 2017-08-23 2017-08-23
PCT/US2018/047742 WO2019040740A1 (en) 2017-08-23 2018-08-23 In vivo immunoimaging of interferon-gamma
US202016640456A 2020-02-20 2020-02-20
US18/144,382 US20230338589A1 (en) 2017-08-23 2023-05-08 In vivo immunoimaging of interferon-gamma

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
US16/640,456 Division US11672877B2 (en) 2017-08-23 2018-08-23 In vivo immunoimaging of interferon-gamma
PCT/US2018/047742 Division WO2019040740A1 (en) 2017-08-23 2018-08-23 In vivo immunoimaging of interferon-gamma

Publications (1)

Publication Number Publication Date
US20230338589A1 true US20230338589A1 (en) 2023-10-26

Family

ID=65439628

Family Applications (2)

Application Number Title Priority Date Filing Date
US16/640,456 Active 2039-08-19 US11672877B2 (en) 2017-08-23 2018-08-23 In vivo immunoimaging of interferon-gamma
US18/144,382 Pending US20230338589A1 (en) 2017-08-23 2023-05-08 In vivo immunoimaging of interferon-gamma

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US16/640,456 Active 2039-08-19 US11672877B2 (en) 2017-08-23 2018-08-23 In vivo immunoimaging of interferon-gamma

Country Status (4)

Country Link
US (2) US11672877B2 (en)
EP (1) EP3672488A4 (en)
AU (1) AU2018321893A1 (en)
WO (1) WO2019040740A1 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115960230A (en) 2015-08-07 2023-04-14 伊麦吉纳博公司 Antigen binding constructs for targeting molecules
WO2021034943A1 (en) * 2019-08-19 2021-02-25 Wayne State University In vivo immunoimaging of interleukin-12
WO2021113450A2 (en) * 2019-12-05 2021-06-10 Imaginab, Inc. Methods of imaging using multiple imaging agents

Family Cites Families (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4036945A (en) 1976-05-03 1977-07-19 The Massachusetts General Hospital Composition and method for determining the size and location of myocardial infarcts
US4331647A (en) 1980-03-03 1982-05-25 Goldenberg Milton David Tumor localization and therapy with labeled antibody fragments specific to tumor-associated markers
US5582824A (en) 1981-10-19 1996-12-10 Genentech, Inc. Recombinant DES-CYS-TYR-CYS human immune interferon
AU561343B2 (en) 1981-10-19 1987-05-07 Genentech Inc. Human immune interferon by recombinant dna
US5096705A (en) 1981-10-19 1992-03-17 Genentech, Inc. Human immune interferon
US4929544A (en) 1982-04-23 1990-05-29 Sri International Reagents, methods, and test kit for diagnosing/monitoring cancer in humans
US4855238A (en) 1983-12-16 1989-08-08 Genentech, Inc. Recombinant gamma interferons having enhanced stability and methods therefor
US5807715A (en) 1984-08-27 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods and transformed mammalian lymphocyte cells for producing functional antigen-binding protein including chimeric immunoglobulin
US4704692A (en) 1986-09-02 1987-11-03 Ladner Robert C Computer based system and method for determining and displaying possible chemical structures for converting double- or multiple-chain polypeptides to single-chain polypeptides
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5720937A (en) 1988-01-12 1998-02-24 Genentech, Inc. In vivo tumor detection assay
DE3920358A1 (en) 1989-06-22 1991-01-17 Behringwerke Ag BISPECIFIC AND OLIGO-SPECIFIC, MONO- AND OLIGOVALENT ANTI-BODY CONSTRUCTS, THEIR PRODUCTION AND USE
EP0752248B1 (en) 1992-11-13 2000-09-27 Idec Pharmaceuticals Corporation Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma
US5648097A (en) 1995-10-04 1997-07-15 Biotek, Inc. Calcium mineral-based microparticles and method for the production thereof
US6391336B1 (en) 1997-09-22 2002-05-21 Royer Biomedical, Inc. Inorganic-polymer complexes for the controlled release of compounds including medicinals
US20100111856A1 (en) 2004-09-23 2010-05-06 Herman Gill Zirconium-radiolabeled, cysteine engineered antibody conjugates
CN101151277B (en) * 2005-01-27 2013-11-13 诺维莫尼公司 Anti-interferon gamma antibodies and methods of use thereof
KR20140115498A (en) 2013-03-20 2014-10-01 한국과학기술연구원 Complex of drug-fluorophore for specific detection of cancer cell
EP3242691A1 (en) * 2015-01-09 2017-11-15 Immunomedics, Inc. Radiosensitivity of fluorophores and use of radioprotective agents for dual-modality imaging

Also Published As

Publication number Publication date
EP3672488A4 (en) 2021-07-14
US20200206371A1 (en) 2020-07-02
EP3672488A1 (en) 2020-07-01
US11672877B2 (en) 2023-06-13
AU2018321893A1 (en) 2020-03-19
WO2019040740A1 (en) 2019-02-28

Similar Documents

Publication Publication Date Title
US20230338589A1 (en) In vivo immunoimaging of interferon-gamma
JP7105235B2 (en) Radiolabeled anti-PD-L1 antibody for immunoPET imaging
CN1675245B (en) Monoclonal antibody hPAM4
ES2405618T3 (en) Monoclonal antibodies directed to tumors for FZD10 and uses thereof
JP2019054819A (en) Antigen binding constructs to cd8
ES2880709T3 (en) Nucleic acids encoding human antibodies against Sialyl-Lewis A
US7183384B2 (en) Monoclonal antibody 7H11 reactive with human cancer
IL278493B2 (en) Humanised anti kallikrein-2 antibody
JP2018505681A (en) Multispecific antibody having affinity for human A33 antigen and DOTA metal complex and use thereof
TWI822822B (en) Use of antibody-drug conjugate
JP7167041B2 (en) Radiolabeled anti-LAG3 antibody for immunoPET imaging
SG188795A1 (en) Chimeric and humanized anti-cd44 antibodies that mediate cancer cell cytotoxicity
US11427648B2 (en) Anti-CD146 antibodies and uses thereof
AU2022286407A1 (en) Dll3 targeting trispecific proteins and methods of use
US7560095B2 (en) Cancer specific monoclonal antibodies
US20220287658A1 (en) In vivo immunoimaging of interleukin-12
KR20200103751A (en) Antibodies to Centrin-1, methods for preparing the same, and uses
JPWO2019087994A1 (en) Antibodies useful for the treatment of solid tumors and their antibody-drug conjugates and anticancer agents containing them
JP2009529010A (en) Cancerous disease modifying antibodies
EP1996623A1 (en) Cancerous disease modifying antibodies for diagnostic and therapeutic use in breast and prostate cancer

Legal Events

Date Code Title Description
STPP Information on status: patent application and granting procedure in general

Free format text: APPLICATION UNDERGOING PREEXAM PROCESSING

AS Assignment

Owner name: WAYNE STATE UNIVERSITY, MICHIGAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:VIOLA, NERISSA;GIBSON, HEATHER;REEL/FRAME:064554/0542

Effective date: 20200213

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION