Subunit vaccines have been investigated in over 1000 clinical trials of cancer immunotherapy, but have shown limited efficacy. Nanovaccines may improve efficacy but have rarely been clinically translated. By conjugating molecular vaccines with Evans blue (EB) into albumin-binding vaccines (AlbiVax), here we develop clinically promising albumin/AlbiVax nanocomplexes that self-assemble in vivo from AlbiVax and endogenous albumin for efficient vaccine delivery and potent cancer immunotherapy. PET pharmacoimaging, super-resolution microscopies, and flow cytometry reveal almost 100-fold more efficient co-delivery of CpG and antigens (Ags) to lymph nodes (LNs) by albumin/AlbiVax than benchmark incomplete Freund’s adjuvant (IFA). Albumin/AlbiVax elicits ~10 times more frequent peripheral antigen-specific CD8+ cytotoxic T lymphocytes with immune memory than IFA-emulsifying vaccines. Albumin/AlbiVax specifically inhibits progression of established primary or metastatic EG7.OVA, B16F10, and MC38 tumors; combination with anti-PD-1 and/or Abraxane further potentiates immunotherapy and eradicates most MC38 tumors. Albumin/AlbiVax nanocomplexes are thus a robust platform for combination cancer immunotherapy.

A subset of CCR6+, γδ-low (GDL) T cells that express Th17 cytokines in mouse skin participates in IL-23-induced psoriasisform dermatitis. We use CCR6-deficient (KO) and wildtype (WT) mice to analyze skin trafficking patterns of GDL T cells and function-blocking mAbs to determine the role of CCR6 in IL-23-mediated dermatitis. Herein, CCL20 was highly upregulated in IL-23-injected WT mouse ear skin as early as 24 hours after initial treatment, and large numbers of CCR6+ cells were observed in the epidermis of IL-23-injected WT mice. Anti-CCL20 mAbs reduced psoriasiform dermatitis and blocked recruitment of GDL T cells to the epidermis. In CCR6 KO mice, GDL T cells failed to accumulate in the epidermis after IL-23 treatment, but total numbers of GDL T cells in the dermis of WT and CCR6 KO mice were equivalent. There was a ~70% reduction in the proportion of IL-22+ GDL T cells in the dermis of CCR6 KO mice (vs. WT mice), suggesting that effector function as well as epidermal recruitment of GDL T cells are impaired in CCR6-deficient mice. Thus, these data show CCR6 regulates epidermal trafficking of γδ T cell subsets in skin and suggest the potential of CCR6 as a therapeutic target for psoriasis.

Chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) infection is accompanied by inflammation and fibrosis eventually leading to cirrhosis. The chemokine CXCL12 is involved in chronic inflammatory conditions. The role of the CXCL12/CXCR4 pathway in HCV-and HBV-associated liver inflammation and fibrosis was therefore studied. The levels and tissue localization of CXCL12 in liver and plasma of HCV and HBV patients were tested using immunohistochemistry and ELISA. The expression and function of CXCR4 on liver-infiltrating lymphocytes (LIL) were tested by FACS and transwell migration assays. We found that CXCL12 is expressed by bile duct epithelial cells in normal liver tissue. Bile duct proliferation and liver fibrosis in chronic HCV and HBV infection result in the anatomical re-distribution of CXCL12 in the liver. Moreover, CXCL12 is up-regulated in the endothelium of neo-bloodvessels formed in active inflammatory foci and is significantly elevated, compared with controls, in the plasma of patients with advanced liver fibrosis. Complementing these observations were others indicating that over 50% of LIL express CXCR4 and, in response to CXCL12, migrated and adhered to fibronectin. These observations suggest an important role for the CXCL12/CXCR4 pathway in recruitment and retention of immune cells in the liver during chronic HCV and HBV infection.

Expression of the chemokine receptor CXCR4 by cancers has been shown to correlate with tumor aggressiveness and poor prognosis and may also contribute to metastatic seeding of organs that express its ligand SDF-1. However, fully optimized PET agents for determining CXCR4 expression by tumor cells in vivo are not yet available. This study aims to develop a stable, 18F-labeled peptide that enables in vivo quantification of CXCR4 in cancer. Methods 4-F-benzoyl-TN14003 (4-F-T140), a short peptide antagonist of CXCR4 with 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl protecting groups on the ε-amino groups of the lysine residues, was labeled with 18F-fluoride via N-succini-midyl-4-18F-fluorobenzoate conjugation, followed by deprotection to give 4-18F-T140 that was exclusively labeled on the α-amine at the N terminus. Cell binding, migration, biodistribution, and small-animal PET studies of 4-18F-T140 were performed. Results 4-F-T140 was radiolabeled by coupling with N-succinimidyl-4-18F-fluorobenzoate, with an overall decay-corrected radiochemical yield of 15% ± 5% calculated from the start of synthesis. The mean measured specific activity (±SD) was 7 ± 2 GBq/μmol (0.19 ± 0.05 Ci/μmol), and radio-chemical purity was greater than 99%. 4-18F-T140 was found to bind specifically to red blood cells in vitro and in vivo. The binding of 4-18F-T140 to red blood cells was blocked with a small amount of cold 4-F-T140, which led to higher uptake of 4-18F-T140 by Chinese hamster ovarian (CHO)-CXCR4 tumors. Biodistribution experiments at 3 h after injection with the addition of 10 μg of cold 4-F-T140 showed a 3.03 ± 0.31 percentage injected dose per gram uptake in CHO-CXCR4 tumors, with a tumor-to-blood ratio of 27.1 ± 8.7 and a tumor-to-muscle ratio of 21.6 ± 7.1. PET studies demonstrated clear visualization of CXCR4-transfected, but not CXCR4-negative, CHO tumors. Conclusion 4-18F-T140 can be used as a PET tracer to image tumor expression of CXCR4, with a high tumor-to-background ratio. The knowledge of whether tumors express or do not express CXCR4 might be beneficial in determining appropriate treatment and monitoring.

CXCR4 is a chemokine receptor which has been shown to be exploited by various tumors for increased survival, invasion, and homing to target organs. We developed a one step radiosynthesis for labeling the CXCR4-specific antagonist AMD3100 with Cu-64 to produce 64 Cu-AMD3100 with a specific activity of 11.28 Ci/μmol (417 GBq/μmol) at the end of radiosynthesis. Incorporation of Cu(II) ion into AMD3100 did not change its ability to inhibit cellular migration in response to the (only) CXCR4 ligand, SDF-1/CXCL12. 64 Cu-AMD3100 binding affinity to CXCR4 was found to be 62.7 μM. Biodistribution of 64 Cu-AMD3100 showed accumulation in CXCR4-expressing organs and tissues, a renal clearance pathway, and an anomalous specific accumulation in the liver. We conclude that 64 Cu-AMD3100 exhibits promise as a potential PET imaging agent for visualization of CXCR4-positive tumors and metastases and might be used to guide and monitor anti-CXCR4 tumor therapy.