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KRAS is one of the most commonly mutated oncogenes in lung, colorectal, and pancreatic cancers. Recent clinical trials directly targeting KRAS G12C presented encouraging results for a large population of non-small cell lung cancer (NSCLC), but resistance to treatment is a concern. Continued exploration of new inhibitors and preclinical models is needed to address resistance mechanisms and improve duration of patient responses. To further enable the development of KRAS G12C inhibitors, we present a preclinical framework involving translational, non-invasive imaging modalities (CT and PET) and histopathology in a conventional xenograft model and a novel KRAS G12C knock-in mouse model of NSCLC. We utilized an in-house developed KRAS G12C inhibitor (Compound A) as a tool to demonstrate the value of this framework in studying in vivo pharmacokinetic/pharmacodynamic (PK/PD) relationship and anti-tumor efficacy. We characterized the Kras G12C-driven genetically engineered mouse model (GEMM) and identify tumor growth and signaling differences compared to its Kras G12D-driven counterpart. We also find that Compound A has comparable efficacy to sotorasib in the Kras G12C-driven lung tumors arising in the GEMM, but like observations in the clinic, some tumors inevitably progress on treatment. These findings establish a foundation for evaluating future KRAS G12C inhibitors that is not limited to xenograft studies and can be applied in a translationally relevant mouse model that mirrors human disease progression and resistance.

Understanding a drug's whole-body biodistribution and tumor targeting can provide important information regarding efficacy, safety, and dosing parameters. Current methods to evaluate biodistribution include in vivo imaging technologies like positron electron tomography and single-photon emission computed tomography or ex vivo quantitation of drug concentrations in tissues using autoradiography and standard biochemical assays. These methods use radioactive compounds or are cumbersome and do not give whole-body information. Here, for the first time, we show the utility of fluorescence molecular tomography (FMT) imaging to determine the biodistribution and targeting of an antibody–drug conjugate (ADC). An anti–5T4-antibody (5T4-Ab) and 5T4-ADC were conjugated with a near-infrared (NIR) fluorophore VivoTag 680XL (VT680). Both conjugated compounds were stable as determined by SEC-HPLC and plasma stability studies. Flow cytometry and fluorescence microscopy studies showed that VT680-conjugated 5T4-ADC specifically bound 5T4-expressing cells in vitro and also exhibited a similar cytotoxicity profile as the unconjugated 5T4-ADC. In vivo biodistribution and tumor targeting in an H1975 subcutaneous xenograft model demonstrated no significant differences between accumulation of VT680-conjugated 5T4-Ab or 5T4-ADC in either normal tissues or tumor. In addition, quantitation of heart signal from FMT imaging showed good correlation with the plasma pharmacokinetic profile suggesting that it (heart FMT imaging) may be a surrogate for plasma drug clearance. These results demonstrate that conjugation of VT680 to 5T4-Ab or 5T4-ADC does not change the behavior of native biologic, and FMT imaging can be a useful tool to understand biodistribution and tumor-targeting kinetics of antibodies, ADCs, and other biologics. Mol Cancer Ther; 15(10); 2530–40. ©2016 AACR.

Supplemental Figure 1: Standard curve of VT680 showing the 3-D quantitation and linearity by FMT imaging; Supplemental Figure 2: Evaluation of VT680 conjugated 5T4-Ab and 5T4-ADC; Supplemental Figure 3: Determination of antibody binding capacity (ABC) and receptor saturation in H1975 and MDA-MB-468 cells by flow cytometry; Supplemental Figure 4: Ex vivo FMT imaging showing the accumulation of 5T4-Ab-VT680, 5T4-ADC-VT680, Isotype control-Ab-VT680 and VT680 alone 48 h post-injection in tumor (A) and liver (B).

Radiotherapy (RT) has traditionally been seen as a means to induce targeted tumor cell death, and more than 50% of all cancer patients receive RT. RT is also known to induce immune cell activation, and the advent of immunotherapeutic treatments such as checkpoint inhibition has sparked increased interest in using RT to enhance an anti-tumor immune response. However, to take advantage of the immunological effects of RT, a deeper understanding of the effects of RT on tumor-infiltrating leukocytes (TILs) is essential. Therefore, we systematically analyzed the effects of RT on tumor growth and the tumor-infiltrating immune cells in five syngeneic tumor models with diverse immune cell-infiltration profiles. These tumor types show strong differences in the overall immune cell infiltration, as well as in the composition of the infiltrating immune cell populations. After RT, tumors that were characterized as hot (e.g. CT26) showed increased sensitivity and dose-dependent tumor growth inhibition compared to cold tumor models (e.g. B16F10). Immune cell profiling indicated that RT led to strong changes in TILs of some tumor types, such as MC38 and CT26. These changes included reduced fractions of macrophages and increases in NK cells and also CD8 T cells. Single cell RNA sequencing also revealed an increase in CD8 T cells expressing proliferation-related genes. Furthermore, macrophage clusters expressing markers of proliferation were specifically eradicated by RT, while monocytes and neutrophils were less affected. The monocytes and neutrophils in the models that showed little changes in TILs after RT expressed marker genes of type-I interferon response. These findings predict that tumors that are highly infiltrated by neutrophils and monocytes, with little intra-tumoral proliferation and type-I interferon response signature, are likely resistant to an RT-mediated anti-tumor immune response. The present work has laid a strong foundation to develop next-generation combinatorial treatments using RT and immunotherapy. Citation Format: Tristan Wirtz, Catherine Lee, Tao Xie, Lisa Manzuk, Manfred Kraus, Christopher Dillon, Timothy Affolter, Anand Giddabasappa. Effects of targeted radiotherapy on tumor immune landscape in diverse murine tumor models [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P065.

Gedatolisib, a PI3K/mTOR dual inhibitor delivered intravenously, is in early clinical trials in women with metastatic ER+ breast cancer. We reasoned that the therapeutic index could be further maximized by encapsulation of gedatolisib in nanoparticles (NP), thereby providing longer half-life, sustained release, blunting of Cmax and associated toxicity, asymmetric distribution to leaky tumor vasculature and the possibility of less frequent dosing. PF-07034663 is a polymeric NP encapsulating gedatolisib, targeted to prostate-specific membrane antigen (PSMA), a clinically validated tumor antigen expressed on prostate cancer cells and the neovasculature of some nonprostate solid tumors. Studies in mice, rats, and cynomolgus monkeys demonstrated that the terminal half-life of PF-07034663 following intravenous administration increased by ~1.5 fold and the Cmax of released gedatolisib from PF-07034663 decreased by ~75 fold compared to gedatolisib delivered by the same route. Gedatolisib released from PF-07034663 exhibited markedly enhanced intratumoral accumulation compared to gedatolisib (~40 fold), indicating enhanced tissue delivery and/or prolonged retention. In exploratory toxicology studies, PF-07034663 caused no hyper-insulinemic spike in rats, in contrast to gedatolisib. To evaluate the antitumor activity of PF-07034663, we performed tumor growth inhibition (TGI) studies in PSMA-positive C4-2 prostate and in PSMA-negative MDAMB361 ER+ breast and A549 lung xenograft tumor models. In the C4-2 model, PF-07034663 showed dose-dependent antitumor efficacy as a single agent, and, in combination with enzalutamide, exhibited enhanced activity leading to tumor regressions. PF 07034663 also exhibited prolonged, dose-dependent inhibition of phosphorylation of S6 (pS6) in the C4-2 model compared to gedatolisib. Interestingly, increased pharmacodynamics (PD) responses compared to gedatolisib were not restricted to PSMA-positive tumors. Moreover, TGI responses were increased in the PSMA-negative A549 lung cancer model, relative to those observed with gedatolisib. PF 07034663 treatment in spontaneous prostate tumors in PTENnullp53mut GEM mice and in subcutaneous allografts of these spontaneous tumors in wild-type mice showed that treatment of PF-07034663 caused similar pS6 inhibition in both models, suggesting that PD modulation is independent of tumor origin or milieu and is less likely due to altered enhanced permeability and retention (EPR) effect. Taken together, PF-07034663 exhibits potential for improved tolerability, sustained tumor PK, prolonged PD modulation, and less frequent dosing schedule compared to gedatolisib. These results support further exploration of gedatolisib in tumor-targeted or non-tumor-targeted nanoparticles in clinical trials as a promising vehicle for delivery of PI3K/mTOR inhibitors to advanced cancer patients. Citation Format: Lianglin Zhang, Gabriel Troche, Ravi Visswanathan, Wenhu Huang, Young-Ho Song, Mary E. Spilker, Zhenxiong Wang, Hui Wang, Anand Giddabasappa, Louise Cadzow, Susan Low, Greg Troiano, Jennifer Lafontaine, Valeria Fantin, Robert T. Abraham, Shubha Bagrodia. Development of a nanoparticle containing the PI3K/mTOR dual Inhibitor, gedatolisib, for cancer therapy [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B25.

Background: NOTCH3, a cell surface receptor involved in cell-cell communications, is over-expressed or amplified in certain human tumors. NOTCH3 is known to regulate proliferation, differentiation and survival of cancer cells or cancer stem cells and thus an important therapeutic target. NOTCH3 antibody drug conjugate (ADC) is comprised of humanized anti-NOTCH3 antibody conjugated to an auristatin based cytotoxic payload. NOTCH3-ADC has shown promising results in pre-clinical tumor models. In this study we evaluated the kinetics, dose-dependent tissue distribution, tumor accumulation and targeting specificity of the NOTCH3-ADC in OVCAR3 xenograft model using fluorescence molecular tomography (FMT) imaging. The NOTCH3-ADC is mouse cross-reactive thus providing an accurate assessment of biodistribution. Methods: NOTCH3-ADC was conjugated to the near-infrared dye, AlexaFluor680 (AF680). The in vitro cellular binding was evaluated by cell-based ELISA. In vivo biodistribution was evaluated using OVCAR3 subcutaneous xenograft model. NOTCH3-ADC-AF680 (1mg/kg; 3mg/kg and 10mg/kg) was injected when the tumors were ~300mm3 and imaged 5 min, 24, 48, 96, 168 and 240 h post-injection. Ex vivo FMT imaging, pharmacokinetic analysis and immunohistochemistry (IHC) was performed at 48 and 240 h after whole-body perfusion. An in vivo receptor competition FMT study was performed by injecting excess of unconjugated anti-NOTCH3 antibody (Ab) or a non-targeted control Ab. Results: In vitro cell binding studies showed that conjugation of AF680 to NOTCH3-ADC did not change its binding ability. A dose-dependent tumor regression was also observed after a single injection of NOTCH3-ADC-AF680. In vivo FMT imaging showed dose-dependent whole-body clearance kinetics of NOTCH3-ADC. Dose-dependent accumulation in the tumors was observed with peak accumulation at 24-48 h post-injection and a slow decline at later time-points. A maximum accumulation of ~10 %ID/g was observed which was independent of the dose of NOTCH3-ADC-AF680. Ex vivo FMT quantitation of tumor was consistent with the IHC for antibody and LC/MS analysis of released payload. Pharmacological competition with excess unlabeled control Ab did not block tumor accumulation of NOTCH3-ADC-AF680, whereas excess unlabeled NOTCH3-Ab blocked ~47% of NOTCH3-ADC-AF680 accumulation. There was no significant specific accumulation of NOTCH3-ADC in other organs as observed by FMT imaging or IHC. Conclusions: These imaging studies provided understanding of the kinetics, tumor accumulation and biodistribution of NOTCH3-ADC. Further this work showcase the utility of non-invasive FMT imaging in better understanding of pharmacology and behavior of biologic drugs. Citation Format: Anand Giddabasappa, Parul Gupta, Mauricio Leal, Jonathan Golas, Fengping Li, Bing Yang, Antonio Esparza, Christopher Winkelmann, Kenneth Geles. Dose-dependent tissue distribution and tumor targeting of Notch3-ADC using fluorescence molecular tomography imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2861. doi:10.1158/1538-7445.AM2017-2861

Introduction: Previously we have shown the utility of Fluorescent Molecular Tomography (FMT) imaging in evaluating bio-distribution of biologics. P-cadherin LP-DART is a bi-specific Dual Affinity Re-Targeting (DART®) molecule targeting CD3 expressed on T-cells and P-cadherin expressed on tumors. In this study we evaluated the bio-distribution and tumor targeting of P-cadherin LP-DART using FMT imaging in a colorectal xenograft model. Methods: NSG or athymic nude mice with subcutaneous HCT-116 xenografts were used. Studies that included engraftment of T-cells received either PBMNCs or T-cells isolated from healthy human volunteers. Bio-distribution studies were initiated when the tumors reached 300-500 mm3. P-cadherin LP-DART or a negative control-DART (non-targeted domain x CD3 binding domain) was conjugated with a near-infrared fluorophore VivoTag680XL (VT680), and the labeling efficiency was determined by spectrophotometer. T-cells used in trafficking studies were labeled with CellVue815. Cell surface P-cadherin expression and P-cadherin LP-DART binding was determined by flow cytometry. T-cell activity was measured with cytotoxic T-lymphocyte (CTL) assays. FMT imaging was performed longitudinally post injection of labeled bi-specifics. Data was analyzed using TrueQuant software. Plasma and tissues were collected for PK analysis by ELISA or histology. Results: VT680 conjugation to P-cadherin LP-DART did not significantly affect the binding to P-cadherin, whereas CD3 binding was decreased. In vivo FMT imaging revealed high levels of P-cadherin LP-DART accumulation in the tumors. The in vivo kinetics revealed that the peak accumulation in tumors was 96hrs post-injection. At 240hrs post-injection, there was still measurable P-cadherin LP-DART detected in tumors. Ex vivo imaging showed 20-25 fold increase in accumulation of P-cadherin LP-DART compared to negative control DART. Comparison of P-cadherin LP-DART accumulation between PBMNC engrafted and non-engrafted model showed no significant difference in quantity or kinetics. There was no significant difference in the kinetics of elimination in the whole-body, heart or liver between P-cadherin LP-DART or negative control. Ex vivo comparison of accumulation in various organs showed no difference between P-cadherin LP-DART or negative control. Cell trafficking studies with CellVue labeled T-cells showed the co-localization of T-cells and P-cadherin LP-DART in tumors. Conclusion: FMT imaging showed that P-cadherin LP-DART specifically targeted HCT-116 tumors. Cell trafficking studies showed that engrafted T-cells accumulated in tumors. This study shows the utility of FMT in bio-distribution studies of biologics and in vivo cell trafficking. Citation Format: Anand Giddabasappa, Vijay Gupta, Timothy S. Fisher, John David, Norberg Rand, Allison Rohner, Justin Cohen, Tracey Clark, Nahor Haddish-Berhane, Adam Root, Chad May. Bio-distribution and tumor targeting of a P-cadherin x CD3 bi-specific redirected T-cell molecule using fluorescence molecular tomography imaging. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5111. doi:10.1158/1538-7445.AM2015-5111