Benjamin Larimer

Granzyme B PET Imaging as a Predictive Biomarker of Immunotherapy Response

While cancer immunotherapy can produce dramatic responses, only a minority of patients respond to treatment. Reliable response biomarkers are needed to identify responders, and conventional imaging modalities have not proved adequate. Here, we provide a preclinical proof of concept for the use of granzyme B, a downstream effector of tumoral cytotoxic T cells, as an early biomarker for tumors responding to immunotherapy. We designed novel PET imaging probes for the murine and human granzyme B isoforms that specifically and quantitatively bind granzyme B. Immunotherapy-treated mice were imaged prior to therapy-induced tumor volume reduction. Imaging distinguished treated responders from nonresponders with excellent predictive ability. To assess the clinical value of a granzyme B imaging paradigm, biopsy specimens from melanoma patients on checkpoint inhibitor therapy were analyzed. A marked differential in granzyme B expression was observed between treated responders and nonresponders. Additionally, our human probe was able to specifically detect granzyme B expression in human samples, providing a clear candidate for clinical application. Overall, our results suggest granzyme B PET imaging can serve as a quantitatively useful predictive biomarker for efficacious responses to cancer immunotherapy. Cancer Res; 77(9); 2318-27. ©2017 AACR.

Differential Receptor Tyrosine Kinase PET Imaging for Therapeutic Guidance.

Inhibitors of the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway hold promise for the treatment of breast cancer, but resistance to these treatments can arise via feedback loops that increase surface expression of the receptor tyrosine kinases (RTK) epidermal growth factor receptor 1 (EGFR) and human epidermal growth factor receptor 3 (HER3), leading to persistent growth pathway signaling. We developed PET probes that provide a method of imaging this response in vivo, determining which tumors may use this escape pathway while avoiding the need for repeated biopsies. Methods: Anti-EGFR-F(ab′)2 and anti-HER3-F(ab′)2 were generated from monoclonal antibodies by enzymatic digestion, conjugated to DOTA, and labeled with 64Cu. A panel of breast cancer cell lines was treated with increasing concentrations of the AKT inhibitor GDC-0068 or the PI3K inhibitor GDC-0941. Pre- and posttreatment expression of EGFR and HER3 was compared using Western blot and correlated to probe accumulation with binding studies. Nude mice xenografts of HCC-70 or MDA-MB-468 were treated with either AKT inhibitor or PI3K inhibitor and imaged with either EGFR or HER3 PET probe. Results: Changes in HER3 and EGFR PET probe accumulation correlate to RTK expression change as assessed by Western blot (R2 of 0.85–0.98). EGFR PET probe PET/CT imaging of HCC70 tumors shows an SUV of 0.32 ± 0.03 for vehicle-, 0.50 ± 0.01 for GDC-0941–, and 0.62 ± 0.01 for GDC-0068–treated tumors, respectively (P < 0.01 for both comparisons to vehicle). HER3 PET probe PET/CT imaging of MDAMB468 tumors shows an SUV of 0.35 ± 0.02 for vehicle- and 0.73 ± 0.05 for GDC-0068–treated tumors (P < 0.01). Conclusion: Our imaging studies, using PET probes specific to EGFR and HER3, show that changes in RTK expression indicative of resistance to PI3K and AKT inhibitors can be seen within days of therapy initiation and are of sufficient magnitude as to allow reliable clinical interpretation. Noninvasive PET monitoring of these RTK feedback loops should help to rapidly assess resistance to PI3K and AKT inhibitors and guide selection of an appropriate combinatorial therapeutic regimen on an individual patient basis.

Identification of a Peptide from In vivo Bacteriophage Display with Homology to EGFL6: A Candidate Tumor Vasculature Ligand in Breast Cancer.

Background A crucial step in tumorigenesis is the recruitment of novel vasculature to the site of neoplasia. Currently, a number of high throughput techniques are employed to identify genes, mRNA and proteins that are aberrantly expressed in tumor vasculature. One drawback of such techniques is the lack of functional in vivo data that they provide. Bacteriophage (phage) display has been demonstrated in vivo to select peptides that home to tumors and tumor vasculature. The peptides can be compared to sequences of putative cancer-related proteins, in order to identify novel proteins essential for tumorigenesis. Objectives It was hypothesized that an in vivo selection for phage which targeted human breast cancer xenografts could identify peptides with homology to cancer-related proteins for in vivo imaging of breast cancer. Methods Following four rounds of in vivo selection in human MDA-MB-435 breast cancer xenografted mice, peptide 3-G03 was discovered with significant homology to a putative secreted protein termed EGFL6. Egfl6 mRNA is upregulated in several transcriptomic analyses of human cancer biopsies, and the protein may play a role in tumor vascularization. Results Egfl6 mRNA expression was demonstrated in MDA-MB-435 cells and EGFL6 protein was secreted from these cells. Based on homology of 3-G03 to EGFL6, an EGFL6 peptide was synthesized and shown to target MDA-MB-435 cells. EGFL6 peptide was radiolabeled with 111In and analyzed for biodistribution and tumor imaging capabilities. Single photon emission computed tomography imaging revealed uptake of the peptide in a manner consistent with other tumor vasculature targeting agents.

In Vitro and In Vivo Evaluation of [99mTc(CO)3]-Radiolabeled ErbB-2-Targeting Peptides for Breast Carcinoma Imaging

ErbB-2 is a type 1 receptor tyrosine kinase over-expressed on ~30% of breast cancers and is an attractive target for the development of new diagnostic and therapeutic agents. In this study, an ErbB-2-targeting peptide, KCCYSL, previously isolated using bacteriophage display, was radiolabeled with [99mTc(H2O)3(CO)3]+ and examined for breast cancer in vitro cell binding and in vivo biodistribution and breast cancer imaging propensities. KCCYSL peptide was synthesized with the chelates diaminopropionc acid (DAP), Nα-histidinyl acetic acid [(NαHis)Ac], and 4-Ala-1,2-3-Triazol-1-acetic acid [(Ala-Triazol)Ac] at its amino-terminus via a gly-ser-gly (GSG) spacer and radiolabeled with [99mTc(H2O)3(CO)3]+. Radiolabeled peptide binding to cultured human MDA-MB-435 breast carcinoma cells was examined. Biodistribution and single photon emission computed tomography (SPECT)/CT imaging of the radiolabeled peptides were evaluated in female SCID mice bearing human MDA-MB-435 breast tumors. Results demonstrated that 99mTc(CO)3-DAP-GSG-KCCYSL, 99mTc(CO)3-(NαHis)Ac-GSG-KCCYSL and 99mTc(CO)3- (Ala-Triazol)Ac-GSG-KCCYSL were stable and bound to MDA-MB-435 cells. In vivo biodistribution studies revealed that tumor uptake of 99mTc(CO)3-DAP-GSG-KCCYSL was 1.67 ±0.16, 1.25 ±0.61, 0.88 ±0.12, 0.30 ±0.06 % ID/g at 1, 2, 4, and 24 h post injection, respectively. Tumor uptake of 99mTc(CO)3-(NαHis)Ac-GSG-KCCYSL was 0.76 ±0.13, 0.75 ±0.40, 0.33 ±0.08, 0.16 ±0.02 % ID/g at 1, 2, 4, and 24 h post injection, respectively. Tumor uptake of 99mTc(CO)3-(Ala- Triazol)Ac-GSG-KCCYSL was 1.15 ±0.12, 0.63 ±0.09, 0.30 ±0.02, 0.09 ±0.02 % ID/g at 1, 2, 4, and 24 h post injection, respectively. SPECT/CT studies showed tumor selective uptake of the peptides in the tumor-bearing mice. Specific uptake was confirmed by competitive receptor blocking studies. 99mTc(CO)3-DAP-GSG-KCCYSL and 99mTc(CO)3-(Ala-Triazol)Ac-GSG-KCCYSL may be better as imaging agents due to their higher tumor to non-target uptake ratios than 99mTc(CO)3-(NαHis)Ac-GSG-KCCYSL.

Specific 18F-FDHT Accumulation in Human Prostate Cancer Xenograft Murine Models Is Facilitated by Prebinding to Sex Hormone–Binding Globulin

Tremendous efforts are currently dedicated to the development of novel therapies targeting the androgen receptor (AR), the major driver of prostate cancer disease and its progression to castration resistance. The ability to noninvasively interrogate AR expression over time in murine models of prostate cancer would permit longitudinal preclinical analysis of novel compounds that could not otherwise be accomplished ex vivo. Although PET imaging with 16β-18F-fluoro-5α-dihydrotestosterone (18F-FDHT) has successfully quantified AR levels clinically, no rodent model of 18F-FDHT imaging has been reported so far. One difference between humans and rodents is the absence in the latter of the sex hormone–binding globulin (SHBG), a glycoprotein that binds to testosterone in the bloodstream, Here, we explore the role of SHBG in developing a working model of rodent AR imaging. Methods: Three human prostate cancer cell lines and xenografts (LNCaP, 22Rv1, and PC3) were used to examine the uptake of free 18F-FDHT and SHBG-bound 18F-FDHT. Both ligands were examined for stability and competitive binding to AR over time in vitro before in vivo studies. PET/CT imaging was used to dynamically measure the uptake of both tracers over 4 h, whereas specificity was determined by competitive binding with the AR antagonist enzalutamide. Results: AR levels correlated with the uptake of both 18F-FDHT and SHBG-18F-FDHT in prostate cancer cell lines. Interestingly, whereas both free and SHBG-bound 18F-FDHT had a similar cellular accumulation at 1 and 2.5 h, SHBG-18F-FDHT accumulated at significantly higher levels after 4 h—evidence that receptor-mediated uptake of SHBG accounted for later time-point differences. This observation was also seen in 22Rv1 tumor–bearing mice, in which SHBG-18F-FDHT exhibited a significantly increased uptake (average tumor-to-background ratio [TBR], 1.62 ± 0.62) in comparison to unbound 18F-FDHT (TBR, 0.81 ± 0.08) at 4 h. Furthermore, the specificity of the SHBG-18F-FDHT accumulation at 4 h was demonstrated by a reduced tumor uptake after AR blockade with enzalutamide (TBR, 1.07 ± 0.13). Conclusion: Prebinding of 18F-FDHT to SHBG allows accurate and quantitative PET imaging of AR levels in murine models of prostate cancer. This procedure may permit the use of PET imaging to study the longitudinal effects of AR-targeting therapies, accelerating novel-drug development.