The rapidly advancing field of cancer immunotherapy is currently limited by the scarcity of noninvasive and quantitative technologies capable of monitoring the presence and abundance of CD8+ T cells and other immune cell subsets. In this study, we describe the generation of 89Zr-desferrioxamine-labeled anti-CD8 cys-diabody (89Zr-malDFO-169 cDb) for noninvasive immuno-positron emission tomography (immuno-PET) tracking of endogenous CD8+ T cells. We demonstrate that anti-CD8 immuno-PET is a sensitive tool for detecting changes in systemic and tumor-infiltrating CD8 expression in preclinical syngeneic tumor immunotherapy models including antigen-specific adoptive T cell transfer, agonistic antibody therapy (anti-CD137/4-1BB), and checkpoint blockade antibody therapy (anti-PD-L1). The ability of anti-CD8 immuno-PET to provide whole body information regarding therapy-induced alterations of this dynamic T cell population provides new opportunities to evaluate antitumor immune responses of immunotherapies currently being evaluated in the clinic.

Significance Anti-CD8 immuno-PET imaging agents provide the potential to monitor the localization, migration, and expansion of CD8-expressing cells noninvasively in vivo. Shown here is the successful generation of functional anti-CD8 imaging agents based on engineered antibodies for use in a variety of preclinical disease and immunotherapeutic models.

The proliferation and trafficking of T lymphocytes in immune responses are crucial events in determining inflammatory responses. To study whole body T lymphocyte dynamics non-invasively in vivo, we have generated anti-CD4 and -CD8 cys-diabodies (cDbs) derived from the parental antibody hybridomas GK1.5 and 2.43, respectively, for 89Zr-immunoPET detection of helper and cytotoxic T cell populations. Methods Anti-CD4 and -CD8 cys-diabodies were engineered, produced via mammalian expression, purified using immobilized metal affinity chromatography, and characterized for T cell binding. The cys-diabodies were site-specifically conjugated to maleimide-desferrioxamine for 89Zr radiolabeling and subsequent microPET/CT acquisition and ex vivo biodistribution in both wild type mice and a model of hematopoietic stem cell (HSC) transplantation. Results ImmunoPET and biodistribution studies demonstrate targeting and visualization of CD4 and CD8 T cell populations in vivo in the spleen and lymph nodes of wild type mice, with specificity confirmed through in vivo blocking and depletion studies. Subsequently, a murine model of HSC transplantation demonstrated successful in vivo detection of T cell repopulation at 2, 4, and 8 weeks post-HSC transplant using the 89Zr-radiolabeled anti-CD4 and -CD8 cDbs. Conclusion These newly developed anti-CD4 and -CD8 immunoPET reagents represent a powerful resource to monitor T cell expansion, localization and novel engraftment protocols. Future potential applications of T cell targeted immunoPET include monitoring immune cell subsets in response to immunotherapy, autoimmunity, and lymphoproliferative disorders, contributing overall to preclinical immune cell monitoring.

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 89Zr-labeled GK1.5 cDb resulted in significantly higher % injected dose/gram 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 post-injection; 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.