CD8+ T-Cell Density Imaging with 64Cu-Labeled Cys-Diabody Informs Immunotherapy Protocols.

Purpose: Noninvasive and quantitative tracking of CD8+ T cells by PET has emerged as a potential technique to gauge response to immunotherapy. We apply an anti-CD8 cys-diabody, labeled with 64Cu, to assess the sensitivity of PET imaging of normal and diseased tissue.Experimental Design: Radiolabeling of an anti-CD8 cys-diabody (169cDb) with 64Cu was developed. The accumulation of 64Cu-169cDb was evaluated with PET/CT imaging (0, 5, and 24 hours) and biodistribution (24 hours) in wild-type mouse strains (n = 8/group studied with imaging and IHC or flow cytometry) after intravenous administration. Tumor-infiltrating CD8+ T cells in tumor-bearing mice treated with CpG and αPD-1 were quantified and mapped (n = 6-8/group studied with imaging and IHC or flow cytometry).Results: We demonstrate the ability of immunoPET to detect small differences in CD8+ T-cell distribution between mouse strains and across lymphoid tissues, including the intestinal tract of normal mice. In FVB mice bearing a syngeneic HER2-driven model of mammary adenocarcinoma (NDL), 64Cu-169cDb PET imaging accurately visualized and quantified changes in tumor-infiltrating CD8+ T cells in response to immunotherapy. A reduction in the circulation time of the imaging probe followed the development of treatment-related liver and splenic hypertrophy and provided an indication of off-target effects associated with immunotherapy protocols.Conclusions: 64Cu-169cDb imaging can spatially map the distribution of CD8+ T cells in normal organs and tumors. ImmunoPET imaging of tumor-infiltrating cytotoxic CD8+ T cells detected changes in T-cell density resulting from adjuvant and checkpoint immunotherapy protocols in our preclinical evaluation. Clin Cancer Res; 24(20); 4976-87. ©2018 AACR.

Engineering A11 Minibody-Conjugated, Polypeptide-Based Gold Nanoshells for Prostate Stem Cell Antigen (PSCA)-Targeted Photothermal Therapy.

Currently, there is no curative treatment for advanced metastatic prostate cancer, and options, such as chemotherapy, are often nonspecific, harming healthy cells and resulting in severe side effects. Attaching targeting ligands to agents used in anticancer therapies has been shown to improve efficacy and reduce nonspecific toxicity. Furthermore, the use of triggered therapies can enable spatial and temporal control over the treatment. Here, we combined an engineered prostate cancer-specific targeting ligand, the A11 minibody, with a novel photothermal therapy agent, polypeptide-based gold nanoshells, which generate heat in response to near-infrared light. We show that the A11 minibody strongly binds to the prostate stem cell antigen that is overexpressed on the surface of metastatic prostate cancer cells. Compared to nonconjugated gold nanoshells, our A11 minibody-conjugated gold nanoshell exhibited significant laser-induced, localized killing of prostate cancer cells in vitro. In addition, we improved upon a comprehensive heat transfer mathematical model that was previously developed by our laboratory. By relaxing some of the assumptions of our earlier model, we were able to generate more accurate predictions for this particular study. Our experimental and theoretical results demonstrate the potential of our novel minibody-conjugated gold nanoshells for metastatic prostate cancer therapy.

ImmunoPET Imaging of Murine CD4+ T Cells Using Anti-CD4 Cys-Diabody: Effects of Protein Dose on T Cell Function and Imaging.

PURPOSE: Molecular imaging of CD4+ T cells throughout the body has implications for monitoring autoimmune disease and immunotherapy of cancer. Given the key role of these cells in regulating immunity, it is important to develop a biologically inert probe. GK1.5 cys-diabody (cDb), a previously developed anti-mouse CD4 antibody fragment, was tested at different doses to assess its effects on positron emission tomography (PET) imaging and CD4+ T cell viability, proliferation, CD4 expression, and function. PROCEDURES: The effect of protein dose on image contrast (lymphoid tissue-to-muscle ratio) was assessed by administering different amounts of 89Zr-labeled GK1.5 cDb to mice followed by PET imaging and ex vivo biodistribution analysis. To assess impact of GK1.5 cDb on T cell biology, GK1.5 cDb was incubated with T cells in vitro or administered intravenously to C57BL/6 mice at multiple protein doses. CD4 expression and T cell proliferation were analyzed with flow cytometry and cytokines were assayed. RESULTS: For immunoPET imaging, the lowest protein dose of 2 µg of 89Zr-labeled GK1.5 cDb resulted in significantly higher % injected dose/g in inguinal lymph nodes (ILN) and spleen compared to the 12-µg protein dose. In vivo administration of GK1.5 cDb at the high dose of 40 µg caused a transient decrease in CD4 expression in spleen, blood, lymph nodes, and thymus, which recovered within 3 days postinjection; this effect was reduced, although not abrogated, when 2 µg was administered. Proliferation was inhibited in vivo in ILN but not the spleen by injection of 40 µg GK1.5 cDb. Concentrations of GK1.5 cDb in excess of 25 nM significantly inhibited CD4+ T cell proliferation and interferon-γ production in vitro. Overall, using low-dose GK1.5 cDb minimized biological effects on CD4+ T cells. CONCLUSIONS: Low-dose GK1.5 cDb yields high-contrast immunoPET images with minimal effects on T cell biology in vitro and in vivo and may be a useful tool for investigating CD4+ T cells in the context of preclinical disease models. Future approaches to minimizing biological effects may include the creation of monovalent fragments or selecting anti-CD4 antibodies which target alternative epitopes.

Positive progress in immunoPET--not just a coincidence.

The identification of tumor tissue biomarkers has led to the production, validation, and Food and Drug Administration-approval of a number of antibody-based targeted therapeutics in the past two decades. As a result of the significant role that these immunotherapeutics play in the management of cancer, and the potential utility of complementary imaging agents, immunoPET imaging has generated considerable interest. This update discusses the important factors to consider when designing a PET (positron emission tomography) imaging agent from the molecular target to the biological targeting molecule and radionuclide combination and also reviews recent preclinical and clinical findings in the immunoPET field. Although there are a variety of radionuclides that are currently utilized in PET studies, this update focuses on four of the positron emitters commonly used in labeling proteins: iodine-124, zirconium-89, copper-64, and fluorine-18. Notable advances in the preclinical setting include the continued development of immunoPET probes to predict the biodistribution of related radioimmunotherapeutics, the success of nontraditional radionuclide and antibody fragment combinations, the broader use of zirconium-89, and the recent emergence of (18)F-labeled diabodies for same-day imaging. Antibody-based PET probes constitute a valuable class of molecular imaging agents, and the progress made preclinically should expedite the transition of these targeted diagnostics to clinical applications.