Background:There remains a need for animal models with human translatability in lung cancer (LC) research. Findings in pigs have high impact on humans due to similar anatomy and physiology. We present the characterization of a bronchoscopically-induced LC model in Oncopigs carrying inducible KRASG12D and TP53R167H mutations. Methods:Twelve Oncopigs underwent 29 injections via flexible bronchoscopy. Eighteen Adenovirus-Cre recombinase gene (AdCre) inductions were performed endobronchially (n=6) and transbronchially with a needle (n=12). Eleven control injections were performed without AdCre. Oncopigs underwent serial contrast-enhanced chest CT with clinical follow-up for 29 weeks. Following autopsy, lung and organ tissues underwent histopathology, immunohistochemistry, and RNA-sequencing with comparative analysis with The Cancer Genome Atlas (TCGA) human LC data. Results:All 18 sites of AdCre injections had lung consolidations on CT imaging. Transbronchial injections led to histopathologic invasive cancer and/or carcinoma in situ (CIS) in 11/12 (91.7%), and invasive cancer (excluding CIS) in 8/12 (66.6%). Endobronchial inductions led to invasive cancer in 3/6 (50%). A soft tissue metastasis was observed in one Oncopig. Immunohistochemistry confirmed expression of Pan-CK+/epithelial cancer cells, with macrophages and T cells infiltration in the tumor microenvironment. Transcriptome comparison showed 54.3% overlap with human LC (TCGA), in contrast to 29.88% overlap of KRAS-mutant mouse LC with human LC. Conclusions:The transgenic and immunocompetent Oncopig model has a high rate of LC following bronchoscopic transbronchial induction. Overlap of the Oncopig LC transcriptome with human LC transcriptome was noted. This pig model is expected to have high clinical translatability to the human LC patient.

Pigs are playing an increasingly vital role as translational biomedical models for studying human pathophysiology. The annotation of the pig genome was a huge step forward in translatability of pigs as a biomedical model for various human diseases. Similarities between humans and pigs in terms of anatomy, physiology, genetics, and immunology have allowed pigs to become a comprehensive preclinical model for human diseases. With a diverse range, from craniofacial and ophthalmology to reproduction, wound healing, musculoskeletal, and cancer, pigs have provided a seminal understanding of human pathophysiology. This review focuses on the current research using pigs as preclinical models for cancer research and highlights the strengths and opportunities for studying various human cancers.

Abstract Lung cancer (LC) is the leading cause of cancer-associated death in the United States. Preclinical findings in small animal models do not translate into human clinics. There is an urgent need to develop relevant preclinical large animal models for LC. We describe a novel method to induce LC in transgenic pigs carrying Cre-inducible KrasG12D and TrP53R167H mutations (Oncopigs). The Oncopigs (n=12; 9 females, and 3 males) at 9 weeks of age were anesthetized and adenovirus (1 × 1011 PFU) carrying Cre recombinase gene (Ad-Cre) ± polybrene (1:100) ± IL-8 (5 ng/ml) was injected via flexible bronchoscopy with two techniques: (1) Bronchial lavage (n=6) and (2) Transbronchial needle injection (n=6) into different lung lobes. As controls, a combination of polybrene ± IL-8 was injected without AdCre into the contralateral lobes in the same pigs (n=8). Oncopigs were monitored via clinical monitoring, blood counts, basic metabolic panel, and contrast-enhanced computed tomography (CT) imaging for LC progression and metastasis at 3-, 4-, 7-, 10-, 16-, and 26 weeks post-intervention. All animals tolerated the injections with expected weight gains. Blood counts and metabolic panels remained normal during LC progression. At the site of injection/lavage, lung masses were detected on CT imaging at 3 weeks post-injections. However, a decrease in masses was observed on CT at 7 weeks post-intervention. The decrease in size of the lung masses coincided with increasing attenuation. No distant metastases were observed on CT imaging. Lavage-based lesions appeared to be more heterogeneous with surrounding inflammatory changes. Transbronchial-injection-based lesions appeared more consolidated and coin-shaped without surrounding inflammatory changes. Control injection sites did not show any mass growth on CT imaging. Immunohistochemistry analysis on lung tumors revealed increased pancytokeratin, trichrome, E-cad, Ki-67, IBA, and CD3 expression. Further, immunofluorescence revealed decreased E-Cad, CK-19, and Zo-1 expression, whereas increased expression of N-Cad, FN1, snail, CD44, α-SMA, and γ-catenin, was observed in the LC and metastatic tissue compared to normal. We successfully generated and characterized a novel transgenic Oncopig LC model. There is robust tumor production status post AdCre delivery. One of four Oncopigs had carcinomatous metastatic skin growth. The remaining 7 Oncopigs are monitored longitudinally for tumor growth and development of metastasis. Citation Format: Kirtan Joshi, Nagabhishek Sirpu, Amanda Schmelzle, Ravikanth Poonooru, Benjamin Nelson, Pradeep Subramanyam, Colleen Garrett, Mariana Sponchiado, Sarah Schlink, Samantha Gerb, Jesse Porter, Dae Kim, Jeffrey Kunin, Jeffrey Bryan, Timothy Hoffman, Bhanu Telugu, Jussuf Kaifi, Satyanarayana Rachagani. Generation and characterization of transgenic oncopig model for lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3392.

Cancer remains a leading cause of morbidity and mortality, and a paradigm shift is needed to fundamentally revisit drug development efforts. Pigs share close similarities to humans and may serve as an alternative model. Recently, a transgenic ‘Oncopig’ line has been generated to induce solid tumors with organ specificity, opening the potential of Oncopigs as a platform for developing novel therapeutic regimens.

Trithiol chelates are suitable for labeling radioarsenic (72As: 2.49 MeV β+, 26 h; 77As: 0.683 MeV β-, 38.8 h) to form potential theranostic radiopharmaceuticals for positron emission tomography (PET) imaging and therapy. A trithiol(b)-(Ser)2-RM2 bioconjugate and its arsenic complex were synthesized and characterized. The trithiol(b)-(Ser)2-RM2 bioconjugate was radiolabeled with no-carrier-added 77As in over 95% radiochemical yield and was stable for over 48 h, and in vitro IC50 cell binding studies of [77As]As-trithiol(b)-(Ser)2-RM2 in PC-3 cells demonstrated high affinity for the gastrin-releasing peptide (GRP) receptor (low nanomolar range). Limited biodistribution studies in normal mice were performed with HPLC purified 77As-trithiol(b)-(Ser)2-RM2 demonstrating both pancreatic uptake and hepatobiliary clearance.

Introduction: Lead-212 (Pb-212)-RM2 is under evaluation as a potential BB2r targeted alpha therapy (TAT) agent for the treatment of BB2r positive prostate cancers. The purpose of this study was to define maximum tolerated dose (MTD) levels for conducting initial therapeutic efficacy studies using human prostate cancer xenograft models and to identify targeted organs and tissues for assessing potential radiotoxicity effects. Methods: The RM2 peptide (DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) was commercially synthesized and radiolabeled with Pb-212 eluted from a Ra-224/Pb-212 generator. Radiosynthesis was accomplished using an Eckert & Ziegler PharmTracer. HPLC QC confirming >93% product purity was performed prior to and post administration of Pb-212 RM2. CF1 mice were administered a single intravenous bolus of either saline (non-treated controls / NTC), 50uCi, 100uCi, or 200uCi of 212Pb-RM2. Weekly complete blood counts (CBC), body weight (BW), and body condition score (BCS) were obtained up to 30 weeks post administration. Subsets of 3-5 mice/group were harvested at 30, 50, 70, 90 days, and 30 weeks for histopathology analysis and pooled serum analysis. An additional set of mice (n=3/group/time point) were administered saline (NTC), 50uCi or 200uCi of Pb-212 RM2 and tissues/serum were obtained at 2, 5, and 7 days to evaluate ALT, AST, BUN, lipase, amylase, and creatinine levels at early timepoints. Results: We observed BW loss in all treatment groups compared to the NTC. We further observed that only the high dose (200uCi) treatment group experienced weight loss that was significantly (P=0.0168) below that of the NTC at 30 weeks. No animals were removed from the study due to BW loss. WBC counts were suppressed at Week1(100uCi and 200uCi groups) and fully recovered by Week 2. WBC counts for all treatment groups were not significantly different at 30 weeks. Platelet counts were slightly suppressed at Week1 (all groups) and fully recovered by Week 2. Week 30 platelet counts were consistent between all treatment groups. Significantly elevated amylase/lipase levels were observed (Day 2) in the 200uCi treatment group with recovery to normal by Day 5. Liver (ALT and AST) and kidney (BUN and creatinine) markers showed nominal changes between groups. Marked changes in pancreas and kidney histopathology were observed only at 30 weeks (100uCi and 200uCi group). Conclusions: Based on BW, CBC, histopathology, and serum analysis, the 50uCi and 100uCi dose levels of Pb-212 RM2 appear to be well tolerated. The high dose Pb-212 RM2 group (200uCi) presented with initially elevated pancreatic enzymes (Day 2) and altered histopathology of the pancreas and kidney appearing only at 30 weeks post treatment. These data suggest that preclinical single dose administration of Pb-212 RM2 TAT at 100uCi or lower should be tolerable. This work was supported by USVA IK6BX004856, USVA IO1BX001699, and NCI RO1CA222293. Citation Format: Tammy L. Rold, Elisabeth A. Devanny, Nkemakonam C. Okoye, Thomas P. Quinn, Timothy J. Hoffman. Preclinical evaluation of maximum tolerated dose (MTD) following single dose administration of 212Pb-RM2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5346.

Abstract Introduction: Receptor targeted alpha therapy (TAT) has garnered attention with isotopes such as Pb-212, Bi-213, and Ac-225 due to the receptor specific and potentially lethal damage caused by the in vivo alpha decay of these isotopes when linked to small peptides. We are investigating Pb-212-RM2, a BB2 receptor (BB2r) antagonist as a potential TAT agent for use in BB2r positive prostate cancer. This in vitro study evaluates Pb-212-RM2 at 3 dose levels in a panel of human prostate cancer cell lines assessing cell viability (MTT), DNA damage (53BP1), and clonogenic potential. Methods: Pb-212-RM2 was evaluated in the PC3, LNCAP, C42, C42B, DU-145, VCAP, and 22Rv1 human prostate cancer cell lines. Cells were plated the day before treatment. Pb-212-RM2 was prepared using an automated radiosynthesizer and QC was performed by HPLC. Cells were exposed to 3, 6, 13, or 25uCi/mL of Pb-212 RM2. Cell viability was evaluated at 24, 48, 72, 96, 120, and 144hrs after treatment. DNA damage was evaluated at 24hrs post treatment using the 53BP1 marker. Media in clonogenic plates was refreshed every 5-7 days until colonies of ≥50 cells were present. All assays were conducted in triplicate on different days. Results: LNCAP, C42, 22Rv1, and C42B cell lines were ≤50% viable at all dose levels by 72hrs. PC3 and DU-145 did not show a decrease to 50% viability until 96-144hrs post administration. At 144hrs post treatment, only the 25uCi/mL treated VCAP cells showed less than 50% viability (43.9%±4.1%). Observed DNA damage was highest in the PC3 and LNCAP cell lines with a range of 5.4±0.7 to 26.6±1.7 and 2.4±0.5 to 19.7±1.2 avg 53BP1 foci/cell at 3 and 25uCi/mL, respectively. Clonogenic potential was lowest in PC3 and LNCAP lines with <20% surviving fraction (SF) at 3uCi/mL and decreasing to <5% SF at 6uCi/mL. DU-145, C42B, 22Rv1, and C42 showed a SF of 71.4±11.8%, 57.1±8.8%, 55.5±2.8%, and 41.5±6.5% at 3uCi/mL. Conclusions: Targeted alpha therapy (TAT), in the form of 212Pb-RM2, was evaluated in the PC3, LNCAP, C42, C42B, DU-145, VCAP, and 22Rv1 human prostate cancer cell lines representing the spectrum of human prostate cancer in relation to androgen dependence and sensitivity. At dose levels of 3, 6, 13, or 25uCi/mL, treatment response does not appear to be related to androgen dependence or sensitivity. Cell viability decreased with increasing time and dose level in all cell lines treated with 212Pb-RM2. The VCAP cell line demonstrated the most resistance to Pb-212-TAT. DNA damage, using the 53BP1 marker, was highest in the PC3 and LNCAP cell lines. As expected, this sensitivity was also demonstrated in clonogenic response with the PC3 and LNCAP cell lines exhibiting the lowest surviving fraction. These studies provide the foundation for further exploration of the therapeutic efficacy of 212Pb-RM2 for the treatment of BB2r positive prostate cancer. This work was supported by USVA IK6BX004856, USVA IO1BX001699, and NCI RO1CA222293. Citation Format: Tammy L. Rold, Nkemakonam C. Okoye, Thomas P. Quinn, Timothy J. Hoffman. Treatment efficacy of 212Pb-RM2 targeted alpha therapy in human prostate cancer cell lines: An in vitro investigation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5344.

Abstract Introduction: Targeted alpha therapy (TAT) employing radiolabeled receptor targeted peptides holds the potential to be clinically effective for the treatment of advanced prostate cancer. We are evaluating the use of the TAT agent, Pb-212-RM2, for targeting the BB2r receptor shown to be expressed in prostate cancer. The current study is the initial therapeutic efficacy evaluation of Pb-212-RM2 TAT administered as a single bolus injection using the androgen independent/insensitive PC3 human prostate cancer xenograft model. Methods: SCID mice (4-5 weeks of age) were injected subcutaneously with 5 million cultured PC3 cells in both rear flanks as a suspension in RPMI 1640 media. The RM2 peptide was obtained commercially. Pb-212 was eluted from a Ra-224/Pb-212 generator and synthesis of Pb-212-RM2 performed using an Eckert & Ziegler PharmTracer. QC was conducted using HPLC. PC3 xenografted SCID mice were administered a single intravenous bolus of saline (NTC), 0.3ug Pb-RM2 (Cold RM2), 50uCi or 100uCi of 212Pb-RM2 in approximately 100uL via the tail vein. Weekly caliper tumor measurements, complete blood counts (CBC), body weight (BW), and body condition score (BCS) were taken predose and up to 17 weeks post injection (pi). Results: At 18 days pi, tumor regression was observed in the 100uCi group with a maximum percent change of -49.3%, while the 50uCi dose group revealed slight tumor growth (+17.2%). As expected, the NTC and Cold RM2 groups demonstrated rapid tumor growth (+325.3% and +304.5% change, respectively). By day 40 pi, tumor regrowth was observed in the 100uCi group (+91.6% change from predose with a +221.1% change by day 47). Body weights of the NTC, Cold RM2, 50uCi and 100uCi groups at 7 days pi were 101.5%, 101.5%, 98.5%, and 95.4%, respectively. Platelet count comparisons between the NTC group and the 50uCi (P=0.0544) and 100uCi (P=0.0624) groups were comparable. Conclusions: Both Pb-212-RM2 TAT treatment groups (50uCi and 100uCi) demonstrated initial tumor control for 4-5 weeks post treatment. While tumor growth suppression was evident in only the 100uCi dose group, this suppression was transient with tumor volume increasing steadily beginning at 32 days pi. As expected, the non-radioactive Pb-RM2 had no effect on tumor growth. Body weights for all dose groups remained >95% of predose weights until disease progression required subject removal from the study. These results warrant further evaluation of multi-dose administration of Pb-212-RM2 with and without standard of care chemotherapy to evaluate optimal tumor control properties of BB2r TAT. This work was supported by USVA IK6BX004856, USVA IO1BX001699, and NCI RO1CA222293. Citation Format: Tammy L. Rold, Elisabeth A. Devanny, Nkemakonam C. Okoye, Thomas P. Quinn, Timothy J. Hoffman. Preliminary evaluation of BB2r TAT using 212Pb-RM2 in a PC3 human prostate cancer xenograft model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5347.

Purpose: Lung cancer screening of high-risk subjects with low-dose CT (LDCT) significantly reduces mortality. Integrating liquid biomarkers circulating tumor cells (CTCs) screening could significantly improve the accuracy of findings on LDCT. Experimental Design: In a prospective, observational clinical trial (NCT02838836; NCT03551951), high-risk screening subjects (≥30 pack-years smoking history, age 55-80) undergoing screening LDCT were enrolled. Peripheral blood (7.5ml) was collected, CTCs were enriched by microfilter isolation and immunofluorescence staining was performed for cytokeratin, EpCAM, CD14/45, PD-L1, vimentin, N-cadherin. Lung cancer tissues were similarly analyzed. Results: In total, 136 subjects were enrolled prospectively. CTCs/7.5ml of blood were significantly higher in 14 subjects with proven lung cancer identified by screening LDCT (mean 23.57, SEM: ±3.73), in comparison to 37 high-risk subjects with benign-appearing lung nodules on LDCT (3.92 (±0.62)) (p<0.0001). A threshold of ≥12 CTCs in screening subjects with a lung nodule had an accuracy of 96.1% to detect lung cancer. CTCs were absent in 15 healthy controls, and rare in 12 subjects with no lung nodules on LDCT (1.08 (±0.47)). Distinct, large, polymorphonuclear CD14/45+CTCs were at significantly increased ratios in stage III/IV lung cancer patients (p=0.005). PD-L1+, vimentin+, and N-cadherin+CTCs were present in all patients, and expression was found at higher rates in CTCs than in patient-matched lung cancer tissues. Conclusions: This seminal study suggests that CTC detection can accurately identify lung cancers in high-risk subjects with a nodule on screening LDCT. CTC phenotyping in lung cancer patients allows real-time insights that are potentially highly relevant for personalized treatment strategies. Table 1.Subjects’ characteristics and analysis for total CTCsCTCs presentCTC mean (±SEM); median (range)p valueSubjects included (total)136High-risk LDCT screening subjects6348 (76.2%)7.73 (±1.4); 3 (0-56)No lung nodules125 (41.7%)1.08 (±0.5); 0 (0-4)Benign lung nodules3729 (78.4%)3.92 (±0.6); 3 (0-14)n.s.*(vs. no lung nodule)Lung cancers (NSCLC)1414 (100%)23.57 (±3.7); 19 (13-56)<0.0001*(vs. benign lung nodules)Lung cancer patients (NSCLC)7272 (100%)25.01 (±1.5); 22 (9-80)Not screened58 (81%)58 (100%)25.36 (±1.6); 23 (9-80)0.32† (vs. lung cancers diagnosed by screening)NSCLC stage (AJCC 8th ed.)•I•II•III•IV26 (36%)14 (20%)19 (26%)13 (18%)22 (±2.3); 18 (13-48)21.4 (±2.1); 20 (9-35)26.4 (±2.2); 27 (10-45)32.9 (±5.3); 24 (12-80)Healthy controls1500AJCC: American Joint Committee on Cancer; NSCLC: non-small cell lung cancer Citation Format: YARISWAMY MANJUNATH, Sathisha Upparahalli Venkateshaiah, Eric T. Kimchi, Kevin F. Staveley-O'Carroll, Jared Coberly, Diego M. Avella, Timothy J. Hoffman, Chelsea Deroche, Klaus Pantel, Guangfu Li, Jussuf T. Kaifi. Circulating tumor cells accurately detect and characterize lung cancers in high-risk subjects undergoing low-dose CT screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 917.

Abstract Background: Talazoparib (TAL), an oral poly ADP-ribose polymerase inhibitor, is under investigation in multiple oncologic clinical trials and has been submitted to the US FDA for use in patients (pts) with germline BRCA-mutated, HER2-negative advanced breast cancer. International Conference on Harmonisation guidance recommends all new drugs be evaluated for effects on cardiac repolarization in a well-controlled clinical study. For drugs for which such evaluation cannot be conducted in healthy volunteers (eg, most anticancer agents), collection of robust corrected QT (QTc) interval data from a dedicated QTc study (hybrid thorough QT/QTc study) in pts is required in the registration dossier. The effect of steady-state (ss) TAL (1 mg once daily) on cardiac repolarization in pts with advanced solid tumors was evaluated in an open-label phase 1 study (NCT03042910). Methods: Continuous 12-lead electrocardiogram (ECG) recordings were collected at baseline (Day -1); time-matched pharmacokinetic (PK) samples and continuous ECG recordings were obtained on Days 1, 2, and 22 (when TAL concentrations achieved ss). On Day -1, pts had continuous 12-lead ECG recording starting at Time 0 (Day 1 dosing time) for 6 hrs. On Days 1 and 22, ECG recording started 45 min before TAL administration and continued for 6 hrs post dose and blood samples for PK were collected before dose and at 1, 2, 4, and 6 hrs post dose. On Day 2, a 30-min ECG recording and a PK sample were obtained before dose at Time 0. Continuous ECG recordings were submitted to a central laboratory; triplicate 10-sec ECGs were extracted from a 5-min extraction window beginning 15 min before each PK collection time. ECG measurements were reported via blinded manual adjudication process and included PR interval, QT interval, RR interval, and QRS complex. The QT interval was corrected for effect of heart rate using Fridericia's correction (QTcF) and Bazett's correction (QTcB). The estimate of change from time-matched baseline and its 2-sided 90% confidence interval (CI) was calculated for each nominal time point using PROC MEANS. Additionally, a prespecified PK/pharmacodynamic (PD) model was used to describe the relationship between plasma TAL concentrations ([TAL]) and QTc. The prespecified linear mixed-effects model included [TAL], time (categorical), and treatment with random pt effects on [TAL] and the intercept. If the upper bounds (UB) of 1-sided 95% CIs of time-matched ΔQTc for all ECG time points were <20 msec and the UB of 1-sided 95% CIs of the predicted ΔQTc at the mean ss maximum [TAL] was <20 msec, the effect of TAL on QTc was not of clinical relevance. Results: 37 of 38 pts enrolled received TAL and were included in the ECG and PK/PD analyses. No pts had a postbaseline absolute maximum QTcF or QTcB ≥500 msec or ΔQTc ≥60 msec. The UB of the 1-sided 95% CI for the time-matched ΔQTcF and ΔQTcB were <12 msec at all nominal ECG time points. In the PK/PD analysis, the slopes (95% CI) of QTcF-[TAL] and QTcB-[TAL] relationships were -0.14 (-0.78 to 0.50) msec/ng/mL and -0.24 (-0.88 to 0.41) msec/ng/mL, respectively, indicating that TAL did not have a concentration-dependent effect on QTcF or QTcB. Conclusion: TAL does not have a clinically relevant effect on QTc. Funding: Medivation LLC, acquired by Pfizer. Citation Format: Hoffman J, Chakrabarti J, Wainberg ZA, Plotka A, Babu S, Milillo Naraine A, Kanamori D, Moroose R, Nguyen L, Wang D. Evaluation of the effects of talazoparib on QT interval prolongation [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-14-07.

Abstract Introduction: Although recent studies employing 177Lu-PSMA beta emitting targeted radiotherapy (TRT) have demonstrated efficacy in treating patients with CRPC, there remains significant opportunities for improvement. Since the TRT mode of action involves DNA damage, inhibiting DNA damage repair with a PARP inhibitor (PARPi) may lead to increased cell death. This pilot in vitro study aims to evaluate the potential synergy of two FDA approved PARPi's, Olaparib and Rucaparib, when used in combination with 177Lu-RM2 TRT to prevent repair of DNA damage caused by TRT in PC3 cells. 177Lu-RM2 TRT, a beta emitting TRT agent targeting the BB2 receptor frequently expressed in CRPC. Methods: 177Lu-RM2 was prepared via automated radiosynthesis with HPLC QC validation of product purity >90%. PC3 cells cultured overnight were treated with media or 177Lu-RM2 TRT (5uCi to 40uCi/160uL). Cells were incubated with TRT for 4 h prior to addition of fresh media with or without the PARP inhibitors, Olaparib (0.25, 0.5, 1.0, 1.5uM) or Rucaparib (0.5, 1.0, 1.5, 2.0uM). DNA damage was assessed at 24 hrs after TRT addition by enumeration of 53BP1 and gamma H2AX foci. Clonogenic survival was evaluated in cells treated with TRT and Olaparib at 0.25, 0.5, 1.0, 1.5uM as single and combination treatments with media refreshed every 4-7 days. CalcuSyn software (Biosoft™) was used to evaluate synergism. Results: At 24 hrs after TRT, the addition of either Olaparib or Rucaparib to the cell culture media showed significant increases in DNA damage markers. Combined with TRT dose levels of 10-20uCi/160uL, the addition of Olaparib significantly increased the number of both markers at 0.5-1.25uM as compared to TRT alone. Treatment with Rucaparib resulted in extreme significance (P < 0.0001) being observed in the number of 53BP1 foci at 13/16 drug combinations as compared to TRT alone. In 7/16 drug combinations gamma H2AX foci were noted as extremely significant (P < 0.0001), and the single combination of 40uCi + 1.5uM was noted as very significant (P < 0.005) as compared to TRT alone. Synergy assessment of TRT plus Olaparib showed increasing levels of synergy (synergism to very strong synergism) with increasing dose levels of TRT. Conclusions: This in vitro investigation supports the use of either Olaparib or Rucaparib in combination with beta emitting TRT to increase PC cell death resulting from beta particle induced DNA damage. Additional in vitro and in vivo preclinical therapeutic evaluation is warranted to support the clinical translation of TRT/PARPi combination therapy. Acknowledgements: US Veterans Administration (VA) BX001699 and USVA Research Career Scientist (TJH) Citation Format: Tammy Rold, Reneise White, Nkemakonam C. Okoye, Timothy J. Hoffman. Combination BB2r targeted radiotherapy with PARP inhibitors in human prostate cancer cells: An in vitro evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3215.

Positron-emitting 72As is the PET imaging counterpart for beta-emitting 77As. Its parent, no carrier added (n.c.a.) 72Se, was produced for a 72Se/72As generator by irradiating an enriched 7°Ge metal-graphite target via the 70Ge(α, 2 n)72Se reaction. Target dissolution used a fast, environmentally friendly method with 93% radioactivity recovery. Chromatographic parameters of the 72Se/72As generator were evaluated, the eluted n.c.a. 72As was characterized with a phantom imaging study, and the previously reported trithiol and aryl-dithiol ligand systems were radiolabeled with the separated n.c.a. 72As in high yield.

INTRODUCTION:Trithiol chelates are suitable for labeling radioarsenic (72As: 2.49 MeV β+, 26 h; 77As: 0.683 MeV β-, 38.8 h) to form potential theranostic radiopharmaceuticals for PET imaging and therapy. To investigate the in vivo stability of trithiol chelates complexed with no carrier added (nca) radioarsenic, a bifunctional trithiol chelate was developed, and conjugated to bombesin(7-14)NH2 as a model peptide. METHODS:A trithiol-BBN(7-14)NH2 bioconjugate and its arsenic complex were synthesized and characterized. The trithiol-BBN(7-14)NH2 conjugate was radiolabeled with 77As, its in vitro stability assessed, and biodistribution studies were performed in CF-1 normal mice of free [77As]arsenate and 77As-trithiol- BBN(7-14)NH2. RESULTS:The trithiol-BBN(7-14)NH2 conjugate, its precursors and its As-trithiol-BBN(7-14)NH2 complex were fully characterized. Radiolabeling studies with nca 77As resulted in over 90% radiochemical yield of 77As-trithiol-BBN, which was stable for over 48 h. Biodistribution studies were performed with both free [77As]arsenate and Sep-Pak® purified 77As-trithiol-BBN(7-14)NH2. Compared to the fast renal clearance of free [77As]arsenate, 77As-trithiol-BBN(7-14)NH2 demonstrated increased retention with clearance mainly through the hepatobiliary system, consistent with the lipophilicity of the 77As-trithiol-BBN(714)NH2 complex. CONCLUSION:The combined in vitro stability of 77As-trithiol-BBN(7-14)NH2 and the biodistribution results demonstrate its high in vivo stability, making the trithiol a promising platform for developing radioarsenic-based theranostic radiopharmaceuticals.

Introduction: Treating castration resistant prostate cancer (CRPC) continues to be a challenge for clinicians. We have recently shown that gamma emitting 203Pb-RM2 (DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) efficiently targets the BB2r expressed on CRPC cells in vivo. We are now exploring the potential of using the alpha-emitting radioisotope 212Pb to generate the radiotherapeutic 212Pb-RM2. Using 212Pb-RM2, we have performed in vitro cell studies to examine treatment effects in terms of DNA damage, clonogenic potential, and cell viability and preliminary in vivo xenograft targeting proof of principle studies. Methods: 212Pb-RM2 was prepared using an automated radiosynthesizer using 212Pb eluted from a 212Ra/212Pb generator. DNA damage, colony formation, and cell viability were evaluated in PC-3 cells following treatment with 212Pb-RM2 at 0.51, 1, 2, and 4uCi/160uL. DNA damage was assessed using 53BP1 foci following a 4hr incubation with 212Pb-RM2 (T0) and at 24hrs and 48hrs. Colony formation assays were performed on cells treated with TRT for 4hrs, and analyzed at 12-14 days. The MTT assay was conducted up to 144hrs. All cell experiments were performed in triplicate on different occurrences. PC-3 xenografted SCID mice were administered 212Pb-RM2 and organs/tissues were harvested and counted at 15 min, 1hr, 4hr, and 24hrs post injection. Results: DNA damage was found to be sustained over time with 53BP1 foci/cell at 48hrs of 14.8+1.6, 33.8+2. 6, 29.6+1.1, and 40.6+1.6 (avg+SEM) at 0.51, 1, 2, and 4uCi of 212Pb-RM2/160uL, respectively. Colony formation followed a dose dependent response with surviving fraction decreasing as TRT increased with 17.7+3.2, 4.4+2.9, 0.5+0.5, and 0.0+0.0 (avg+SEM) at 0.51, 1, 2, and 4uCi of 212Pb-RM2/160uL, respectively. 212Pb-RM2 tumor uptake in PC-3 xenografts was found to be 5.2+0.7, 5.0+0.9, 3.9+0.7, and 2.6+0.3 %ID/g at 15min, 1hr, 4hr, and 24hrs respectively. In vivo BB2r blocking studies confirmed 212Pb-RM2 targeting of the BB2r with > 72% blocking of normal pancreas uptake measured at 4hr post injection. Conclusions: Treatment with alpha emitting 212Pb-RM2 TRT showed sustained levels of DNA damage which was confirmed by impaired colony formation at all dose levels of 212Pb-RM2 TRT studied. Preliminary in vivo tumor targeting and retention of 212Pb-RM2 was observed out to 24hrs post injection warranting further evaluation of 212Pb-RM2 in multiple CRPC cell line models assessing potential therapeutic efficacy. Acknowledgements: US Veterans Administration (VA) BX001699 and USVA Research Career Scientist (TJH) Citation Format: Tammy L. Rold, Nkemakonam C. Okoye, Hoffman Timothy. Preliminary evaluation of 212Pb BB2r targeted radiotherapy in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3220.

Introduction: Treating castration resistant prostate cancer (CRPC) continues to be a challenge for clinicians. We have recently shown that gamma emitting 203Pb-RM2 (DOTA-4-amino-1-carboxymethyl-piperidine-D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) efficiently targets the BB2r expressed on CRPC cells in vivo. We are now exploring the potential of using the alpha-emitting radioisotope 212Pb to generate the radiotherapeutic 212Pb-RM2. Using 212Pb-RM2, we have performed in vitro cell studies to examine treatment effects in terms of DNA damage, clonogenic potential, and cell viability and preliminary in vivo xenograft targeting proof of principle studies. Methods: 212Pb-RM2 was prepared using an automated radiosynthesizer using 212Pb eluted from a 212Ra/212Pb generator. DNA damage, colony formation, and cell viability were evaluated in PC-3 cells following treatment with 212Pb-RM2 at 0.51, 1, 2, and 4uCi/160uL. DNA damage was assessed using 53BP1 foci following a 4hr incubation with 212Pb-RM2 (T0) and at 24hrs and 48hrs. Colony formation assays were performed on cells treated with TRT for 4hrs, and analyzed at 12-14 days. The MTT assay was conducted up to 144hrs. All cell experiments were performed in triplicate on different occurrences. PC-3 xenografted SCID mice were administered 212Pb-RM2 and organs/tissues were harvested and counted at 15 min, 1hr, 4hr, and 24hrs post injection. Results: DNA damage was found to be sustained over time with 53BP1 foci/cell at 48hrs of 14.8+1.6, 33.8+2. 6, 29.6+1.1, and 40.6+1.6 (avg+SEM) at 0.51, 1, 2, and 4uCi of 212Pb-RM2/160uL, respectively. Colony formation followed a dose dependent response with surviving fraction decreasing as TRT increased with 17.7+3.2, 4.4+2.9, 0.5+0.5, and 0.0+0.0 (avg+SEM) at 0.51, 1, 2, and 4uCi of 212Pb-RM2/160uL, respectively. 212Pb-RM2 tumor uptake in PC-3 xenografts was found to be 5.2+0.7, 5.0+0.9, 3.9+0.7, and 2.6+0.3 %ID/g at 15min, 1hr, 4hr, and 24hrs respectively. In vivo BB2r blocking studies confirmed 212Pb-RM2 targeting of the BB2r with > 72% blocking of normal pancreas uptake measured at 4hr post injection. Conclusions: Treatment with alpha emitting 212Pb-RM2 TRT showed sustained levels of DNA damage which was confirmed by impaired colony formation at all dose levels of 212Pb-RM2 TRT studied. Preliminary in vivo tumor targeting and retention of 212Pb-RM2 was observed out to 24hrs post injection warranting further evaluation of 212Pb-RM2 in multiple CRPC cell line models assessing potential therapeutic efficacy. Acknowledgements: US Veterans Administration (VA) BX001699 and USVA Research Career Scientist (TJH) Citation Format: Tammy L. Rold, Nkemakonam C. Okoye, Hoffman Timothy. Preliminary evaluation of 212Pb BB2r targeted radiotherapy in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3220.