While chemotherapy delivery by nanocarriers has modestly improved the survival prospects of pancreatic ductal adenocarcinoma (PDAC), additional engagement of the immune response could be game changing. We demonstrate a nano-enabled approach for accomplishing robust anti-PDAC immunity in syngeneic mice through the induction of immunogenic cell death (ICD) as well as interfering in the immunosuppressive indoleamine 2,3-dioxygenase (IDO) pathway. This is accomplished by conjugating the IDO inhibitor, indoximod (IND), to a phospholipid that allows prodrug self-assembly into nanovesicles or incorporation into a lipid bilayer that encapsulates mesoporous silica nanoparticles (MSNP). The porous MSNP interior allows contemporaneous delivery of the ICD-inducing chemotherapeutic agent, oxaliplatin (OX). The nanovesicles plus free OX or OX/IND-MSNP induce effective innate and adaptive anti-PDAC immunity when used in a vaccination approach, direct tumor injection or intravenous biodistribution to an orthotopic PDAC site. Significant tumor reduction or eradication is accomplishable by recruiting cytotoxic T lymphocytes, concomitant with downregulation of Foxp3+ T cells.

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.

Inflammatory bowel diseases (IBDs) in humans are characterized in part by aberrant CD4-positive (CD4+) T-cell responses. Currently, identification of foci of inflammation within the gut requires invasive procedures such as colonoscopy and biopsy. Molecular imaging with antibody fragment probes could be used to noninvasively monitor cell subsets causing intestinal inflammation. Here, GK1.5 cys-diabody (cDb), an antimouse CD4 antibody fragment derived from the GK1.5 hybridoma, was used as a PET probe for CD4+ T cells in the dextran sulfate sodium (DSS) mouse model of IBD. The DSS mouse model of IBD was validated by assessing changes in CD4+ T cells in the spleen and mesenteric lymph nodes (MLNs) using flow cytometry. Furthermore, CD4+ T cell infiltration in the colons of colitic mice was evaluated using immunohistochemistry.Zr-labeled GK1.5 cDb was used to image distribution of CD4+ T cells in the abdominal region and lymphoid organs of mice with DSS-induced colitis. Region-of-interest analysis was performed on specific regions of the gut to quantify probe uptake. Colons, ceca, and MLNs were removed and imaged ex vivo by PET. Imaging results were confirmed by ex vivo biodistribution analysis. An increased number of CD4+ T cells in the colons of colitic mice was confirmed by anti-CD4 immunohistochemistry. Increased uptake ofZr-maleimide-deferoxamine (malDFO)-GK1.5 cDb in the distal colon of colitic mice was visible in vivo in PET scans, and region-of-interest analysis of the distal colon confirmed increased activity in DSS mice. MLNs from colitic mice were enlarged and visible in PET images. Ex vivo scans and biodistribution confirmed higher uptake in DSS-treated colons (DSS, 1.8 ± 0.40; control, 0.45 ± 0.12 percentage injected dose [%ID] per organ, respectively), ceca (DSS, 1.1 ± 0.38; control, 0.35 ± 0.09 %ID per organ), and MLNs (DSS, 1.1 ± 0.58; control, 0.37 ± 0.25 %ID per organ). Zr-malDFO-GK1.5 cDb detected CD4+ T cells in the colons, ceca, and MLNs of colitic mice and may prove useful for further investigations of CD4+ T cells in preclinical models of IBD, with potential to guide development of antibody-based imaging in human IBD.

Prostate stem cell antigen (PSCA) is expressed on the cell surface in 83%–100% of local prostate cancers and 87%–100% of prostate cancer bone metastases. In this study, we sought to develop immunoPET agents using 124I- and 89Zr-labeled anti-PSCA A11 minibodies (scFv-CH3 dimer, 80 kDa) and evaluate their use for quantitative immunoPET imaging of prostate cancer. Methods A11 anti-PSCA minibody was alternatively labeled with 124I- or 89Zr-desferrioxamine and injected into mice bearing either matched 22Rv1 and 22Rv1× PSCA or LAPC-9 xenografts. Small-animal PET data were obtained and quantitated with and without recovery coefficient–based partial-volume correction, and the results were compared with ex vivo biodistribution. Results Rapid and specific localization to PSCA-positive tumors and high-contrast imaging were observed with both 124I-and 89Zr-labeled A11 anti-PSCA minibody. However, the differences in tumor uptake and background uptake of the radiotracers resulted in different levels of imaging contrast. The nonresidualizing 124I-labeled minibody had lower tumor uptake (3.62 ± 1.18 percentage injected dose per gram [%ID/g] 22Rv1×PSCA, 3.63 ± 0.59 %ID/g LAPC-9) than the residualizing 89Zr-labeled minibody (7.87 ± 0.52 %ID/g22Rv1×PSCA, 9.33 ±0.87 %ID/gLAPC-9, P <0.0001 for each), but the 124I-labeled minibody achieved higher imaging contrast because of lower nonspecific uptake and better tumor–to–soft-tissue ratios (22Rv1×PSCA:22Rv1 positive-to-negative tumor, 13.31 ± 5.59 124I-A11 and 4.87 ± 0.52 89Zr-A11, P = 0.02). Partial-volume correction was found to greatly improve the correspondence between small-animal PET and ex vivo quantification of tumor uptake for immunoPET imaging with both radionuclides. Conclusion Both 124I-and 89Zr-labeled A11 anti-PSCA minibody showed high-contrast imaging of PSCA expression in vivo. However, the 124I-labeled A11 minibody was found to be the superior imaging agent because of lower nonspecific uptake and higher tumor–to–soft-tissue contrast. Partial-volume correction was found to be essential for robust quantification of immunoPET imaging with both 124I- and 89Zr-labeled A11 minibody.

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 Cu, to assess the sensitivity of PET imaging of normal and diseased tissue. Radiolabeling of an anti-CD8 cys-diabody (169cDb) with Cu was developed. The accumulation ofCu-169cDb was evaluated with PET/CT imaging (0, 5, and 24 hours) and biodistribution (24 hours) in wild-type mouse strains ( = 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 ( = 6-8/group studied with imaging and IHC or flow cytometry). 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 -driven model of mammary adenocarcinoma (NDL),Cu-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. Cu-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. .