Activity In Patient-derived Xenograft Models Research Articles

Abstract 1880: Ecubectedin and PM54 demonstrate antitumor activity in patient-derived xenograft models of soft tissue sarcoma

Abstract Objective: Soft tissue sarcoma (STS) is a heterogenous group of rare, malignant tumors arising from mesenchymal tissue. Doxorubicin (DOX)-based chemotherapy is the standard for advanced or metastatic STS, despite low response rates and poor disease control. We explored the antitumor activity of two novel compounds, ecubectedin (ECU; PM14) and PM54 (PharmaMar), in two patient-derived xenograft (PDX) models of STS, leiomyosarcoma (LMS) and CIC-rearranged sarcoma (CRS). Mode of action of ECU and PM54 is similar to lurbinectedin (LUR), inhibiting oncogenic transcription by eviction of oncogenic transcription factors from gene promotors’ recognition sequences, while inducing the generation of double strand breaks through DNA adduct formation. Methods: Female NMRI nu/nu mice (n=104) were transplanted bilaterally with STS PDX: UZLX-STS134 (CRS) and UZLX-STS22_2 (LMS). Xenografted mice were randomized to six treatment groups: 1) vehicle (VEH) (5% dextrose) 5 mL/kg once weekly (QW) intravenously (IV); 2) DOX 5 mg/kg QW IV; 3) trabectedin (TRA) 0.15 mg/kg QW IV; 4) LUR 0.18 mg/kg QW IV; 5) ECU 1.2 mg/kg QW IV; 6) PM54 1.2 mg/kg QW IV. Treatment lasted 16 days and antitumor activity was assessed by tumor volume (TV) analysis, histopathology and western blot. The Mann-Whitney U test was used to compare groups, the Wilcoxon test to compare to baseline. Results: ECU and PM54 caused TV shrinkage in UZLX-STS134, while TV in other groups grew significantly on day 16 (Table 1). VEH treatment led to the largest TV increase on day 16, compared to any treatment. PM54 treatment showed TV stabilization in UZLX-STS22_2, and significantly smaller tumors than VEH and TRA treated tumors (p<0.05 and p<0.01, respectively). Histopathological evaluation corroborate the results from the relative TV analysis. Conclusion: Novel drugs exhibit promising results in the PDX models tested, supporting ongoing exploration in additional PDX models of synovial sarcoma and dedifferentiated liposarcoma. Table 1: TV change on day 16 versus baseline, and comparison of proliferative and apoptotic activity Tumor volume change Proliferative activity (pHH3) Apoptotic activity (cPARP) Subtypes UZLX-STS134 UZLX-STS22_2 UZLX-STS134 UZLX-STS22_2 UZLX-STS134 UZLX-STS22_2 VEH(5 ML/kg) 387%*** 349%** n/a n/a n/a n/a DOX (5 mg/kg/QW IV) 177%*** 243%** ↓*** ↓ ↑** ↑ TRA(0.15 mg/kg/QW IV) 230%** 254%*** ↓** ↓** ↑** ↑*** LUR(0.18 mg/kg/QW IV) 202%** 232%** ↓*** ↓*** ↑*** ↑** ECU(1.2 mg/kg/QW IV) 41%*** 181%** ↓*** ↓*** ↑*** ↑*** PM54 (1.2 mg/kg/QW IV) 76%*** 162% ↓*** ↓*** ↑*** ↑ Citation Format: Daniël Gorgels, Agnieszka Wozniak, Chao-Chi Wang, Pablo M. Avilés, Maria J. Guillen, Carmen Cuevas, Patrick Schöffski. Ecubectedin and PM54 demonstrate antitumor activity in patient-derived xenograft models of soft tissue sarcoma [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 1880.

Abstract LB_C09: QTX3034, a potent and selective multi-KRAS inhibitor, synergizes with EGFR inhibitors and enhances anti-tumor activity

Abstract KRAS, one of the most frequently mutated oncogenes in cancer, was considered undruggable until the development of covalent KRASG12C inhibitors. While these KRASG12C-targeted therapies exhibit clinical benefit, they do not address the most common KRAS mutant variants. QTX3034 is a potent, highly selective, orally bioavailable allosteric multi-KRAS inhibitor that is efficacious against a broad spectrum of the more common KRASG12D- and KRASG12V-mutated cancers in vitro and in vivo. In an effort to enhance anti-tumor activity of QTX3034 and to minimize the development of possible resistance mechanisms, we explored the potential synergy of combining QTX3034 with EGFR inhibitors in multiple KRAS-mutated tumor models. Cell viability assays over a 7-day period revealed that QTX3034 synergized with three different EGFR inhibitors (afatinib, cetuximab, erlotinib) to profoundly inhibit proliferation across multiple KRASG12D, KRASG12V, and KRASWT Amp mutant cell lines with strong ZIP synergy scores (>10). Combination of QTX3034 and afatinib exhibited a markedly greater inhibitory effect on KRAS-mutant colony formation compared to the respective single agents. Analysis of RAS downstream signaling revealed that the combination of QTX3034 and afatinib produced a more potent suppression of the MAPK pathway than either single agent alone in KRAS-mutant cells. Moreover, QTX3034 monotherapy induced a dose-dependent increase in EGFR phosphorylation in the KRASG12D-mutated cell line GP2D, while co-administration with afatinib robustly inhibited this EGFR activation. Combining QTX3034 and cetuximab elicited durable tumor regressions in KRASG12D-mutant cell line-derived xenograft models. Furthermore, tumor regressions in these combination studies were obtained using meaningfully lower doses of QTX3034 than needed to achieve tumor regression by monotherapy. Similar combinations improved QTX3034 anti-tumor activity in patient-derived xenograft models that exceeded monotherapy with either agent. Taken together, these data support the rationale to co-administer QTX3034 and EGFR inhibitors to optimize therapeutic benefit for patients with KRAS-mutated cancers. Citation Format: Yang J. Zhang, Jillian M. Silva, Yang W. Zhang, Elizabeth Donohue Vo, John M. Micozzi, Douglas Krauth, Arghyotri Sinha, Ben Reid, Brooke McDonough, Gregory M. Lee, Christopher C. Pan, Audrey Hospital, Juan I. Luengo, Cameron Pitt, Hong Lin. QTX3034, a potent and selective multi-KRAS inhibitor, synergizes with EGFR inhibitors and enhances anti-tumor activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C09.

Phase 1/2 study of PRO1160, a CD70-directed antibody-drug conjugate, in patients with advanced solid tumors and hematologic malignancies

Abstract Background: PRO1160 is a novel antibody-drug conjugate (ADC) directed to CD70, an antigen mediating immuno-suppression that is overexpressed in multiple solid tumors and hematologic malignancies, with limited distribution in normal tissues. PRO1160 comprises (1) a human monoclonal antibody specific for CD70, (2) a protease-cleavable proprietary hydrophilic linker, and (3) exatecan, a topoisomerase 1 inhibitor. Comprehensive prior work demonstrated that the hydrophilic linker confers excellent physicochemical properties and pharmacokinetics (PK) across a range of payload mechanisms and is superior to conventional linkers on these critical parameters for ADCs. In addition, exatecan is broadly active in many tumor types, is membrane permeable, and is not a substrate of multidrug resistance pumps, thus likely lending strong bystander effects and durable treatment responses. PRO1160 is highly potent in cell-derived xenograft models of renal cell carcinoma (RCC), non-Hodgkin lymphoma (NHL), and nasopharyngeal carcinoma (NPC). PRO1160 also demonstrates marked antitumor activity in patient-derived xenograft models of diverse tumor sites, histologies, molecular subtypes, target expression levels, and Epstein Barr Virus status. PRO1160 is stable in circulation and displays PK characteristics indistinguishable from the parent antibody in rats. In a GLP toxicity study in cynomolgus monkeys, the primary PRO1160-related toxicity resided in the thymus and bone marrow, was consistent with exatecan toxicities, and was reversible. PRO1160-001 is an ongoing, open-label Phase 1/2 study to evaluate the safety, tolerability, PK, and antitumor activity of PRO1160 in patients with metastatic RCC, metastatic or relapsed NPC, or advanced relapsed/refractory NHL. Patients must have measurable disease per the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 or the Lugano Classification for NHL. Patients must also have previously received therapies known to confer clinical benefit unless considered ineligible, refused by the patient, or not available in the region. PRO1160 is given by intravenous infusion on Day 1 of a 21-day cycle and treatment may continue until disease progression, unacceptable toxicity, or other reason for discontinuation. The primary objectives are to evaluate the safety and tolerability of PRO1160 and to identify the maximum tolerated dose, if reached, and recommended phase 2 dose (RP2D). This study consists of 2 parts, Part A: dose-escalation and dose-level expansion, and Part B: 3 tumor-specific expansion cohorts (metastatic RCC, metastatic or relapsed NPC, or advanced relapsed/refractory NHL) treated at the RP2D. PK, immunogenicity, and antitumor activity will also be evaluated. The study is currently enrolling at sites in the US, with future enrollment in China planned (Clinicaltrials.gov: NCT05721222).

Dose escalation results from a first-in-human study of ABBV-400, a novel c-Met–targeting antibody-drug conjugate, in advanced solid tumors.

3015 Background: c-Met overexpression is common in non-small cell lung cancer (NSCLC), gastroesophageal adenocarcinoma (GEA), and colorectal cancer (CRC). There is a need for effective therapies that target c-Met–overexpressing tumors, as there are currently no approved options available. The antibody-drug conjugate (ADC) ABBV-400 consists of the c-Met–targeting antibody telisotuzumab conjugated to a potent topoisomerase 1 inhibitor (Top1i) payload. ABBV-400 exhibited sustained antitumor activity in patient-derived xenograft models of c-Met–expressing NSCLC, GEA, and CRC. Herein, we present data from the dose escalation of the first-in-human study of ABBV-400 monotherapy. Methods: A phase 1 dose escalation/expansion study of ABBV-400 has been initiated in patients with advanced solid tumors (NCT05029882). Primary objectives are to evaluate safety, tolerability, and pharmacokinetics (PK) of ABBV-400, to determine the recommended phase 2 dose, and to assess preliminary efficacy. PK will be characterized for the conjugate, total antibody, and free payload. Adults (≥18 years) with an advanced solid tumor that progressed on standard therapies were enrolled during dose escalation, without restrictions on the basis of c-Met overexpression status. Results: As of January 2023, 47 patients had enrolled in dose escalation with a median follow-up of 6.5 months. Median age was 58 years (range, 34–79), 25 (53%) patients were male, and the most common cancer types were CRC (n=21), NSCLC (n=5), and GEA (n=5). The median number of prior treatment lines was 4 (1–13). Most common hematologic adverse events (AEs) were anemia (66%; grade [G]≥3: 38%), neutropenia (62%; G≥3: 43%), thrombocytopenia (43%; G≥3: 26%), and leukopenia (32%; G≥3: 19%). Nausea (60%, all grade 1–2), fatigue (49%; G≥3: 4%), and vomiting (38%, all grade 1–2) were the most frequent non-hematologic AEs. One patient experienced grade 1 interstitial lung disease. Neutropenia and thrombocytopenia were the dose-limiting toxicities (DLTs) and were dose and exposure dependent. Clinical activity was observed with ABBV-400, with an objective response rate of 24.4% (11/45; 95% CI: 12.9, 39.5). All responses were confirmed partial responses (PR). In addition, 23 patients (50.0%) had stable disease (SD) and 7 (15.2%) had progressive disease. Among patients with CRC, 4/21 (19.0%) had a PR and 13/21 (61.9%) had durable SD, with a disease control rate of 81.0% (95% CI: 58.1, 94.6). Outside of CRC, PRs were observed in patients with NSCLC ( EGFR wildtype and mutant), uterine cancer, and acral melanoma. Conclusions: On the basis of DLTs, a maximum tolerated dose of ABBV-400 was identified. At this dose, safety results appear comparable with other Top1i ADCs. Promising antitumor activity was seen with ABBV-400 across tumor types, justifying further evaluation in the ongoing dose expansion in NSCLC, GEA, and CRC. Clinical trial information: NCT05029882 .

Abstract CT222: Multimodal mechanism of action of the GSK-3 inhibitor 9-ING-41 (elraglusib) includes an immunomodulatory component: preliminary results from the 1801 phase 1/2 trial

Abstract 9-ING-41 (elraglusib) is a potent and selective GSK-3 inhibitor that has shown anti-tumor activity in patient-derived xenograft models and phase1/2 clinical studies in patients (pts) with advanced solid tumors. Preclinical studies have demonstrated that elraglusib downregulates PD-1, TIGIT and LAG-3, upregulates expression of MHC class I proteins in tumor cells from “cold tumors”, and shows synergy when combined with PD-1 blockade in mouse xenografts. Clinical activity has been observed both as a single agent and in combination with standard of care chemotherapies in several advanced cancer histologies. Here we present initial ‘omics data from our phase 1/2 study (NCT03678883; ACT1801) spanning >100 pts with advanced cancer evaluating both single agent and chemotherapy combinations. The patients (pts) included in this study received elraglusib as a ≥3rd line therapy for advanced disease. Most pts with melanoma treated with elraglusib monotherapy (8/9; 89%) stayed on study for >2 cycles. One patient with melanoma refractory to checkpoint inhibitors achieved a confirmed and durable CR. Clinical benefit was also observed among pts with colorectal cancer with 4/12 (33.3%) pts treated with monotherapy and 12/15 (80%) that received elraglusib plus irinotecan rechallenge stayed on study >2 cycles reaching median overall survival of 106 and 211 days, respectively. Based on emerging in vivo and in vitro results demonstrating that elraglusib activates T and NK cells promoting anti-tumor immune responses, we hypothesize immunomodulation by elraglusib may be contributing to anti-tumor immune response in the 1801 trial. We have acquired TCRseq and RNAseq profiles of PBMC samples from seven patients in 1801 during the first two weeks of treatment with elraglusib monotherapy. In these pts, reduced TCR clonality was observed and specific TCR clonotypes expanded after treatment, indicating T cell activation and expansion. These pts also showed changes in PBMC populations during elraglusib therapy as measured by immune deconvolution of PBMC RNAseq. Taken together, these data support a novel, previously unrecognized immunomodulatory mechanism of action for elraglusib and could provide rationale for future clinical development of elraglusib in pts with advanced malignancies. Additional analysis from TCRseq, immune profiling and cytokine analysis from expanded cohort of pts is ongoing. Citation Format: Taylor Weiskittel, Joseph McDermott, Daniel Billadeau, Hu Li, Anwaar Saeed, Devalingam Mahalingam, Solmaz Sahebjam, Pamela Munster, Ludimila Cavalcante, Frank J. Giles, Andrew Mazar, Benedito Carneiro. Multimodal mechanism of action of the GSK-3 inhibitor 9-ING-41 (elraglusib) includes an immunomodulatory component: preliminary results from the 1801 phase 1/2 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT222.

Comprehensive Profiling of Clinical JAK2 Inhibitors in Myeloproliferative Neoplasms

Since the approval of ruxolitinib (JAK1/JAK2) in 2011, two additional inhibitors, fedratinib (JAK2/FLT3) and pacritinib (JAK2/FLT3/IRAK1/CSF1R), have received FDA approval for myelofibrosis, and a new drug application for a fourth inhibitor, momelotinib (JAK1/JAK2/ACVR1/TBK1) has been submitted in June 2022. Although all four inhibitors potently suppress JAK2, the targeting of additional auxiliary targets may contribute to distinct responses observed in the clinic. Here, we take a comprehensive approach to assess the unique proliferative, transcriptional, and signaling pathways altered by these four JAK2 inhibitors in myeloproliferative neoplasms (MPN). In three human JAK2 mutant leukemic cell lines (HEL, UKE-1, SET2) and murine Ba/F3 EpoR-JAK2VF cells, ruxolitinib consistently showed the lowest IC50. Following, CD34+ hematopoietic stem/progenitor cells (HSPCs) from 8 MPN patients and 3 healthy donors were seeded for colony formation assays. Erythroid colonies were highly sensitive to ruxolitinib, fedratinib, and pacritinib inhibition. In contrast, momelotinib demonstrated greater relative sparing of erythroid colonies, consistent with clinical anemia amelioration, albeit potentially via a ACVR1/ALK2 and hepcidin-independent manner. Overall, greatest potency was demonstrated by pacritinib ex vivo, followed by ruxolitinib, fedratinib, and momelotinib. These findings were somewhat distinct from in vitro assays, likely reflecting additional underlying biology of human HSPCs. We then performed high dimensional mass cytometry to assess signaling changes at single cell resolution across different cell populations in 9 MPN patient samples and 3 healthy donors treated ex vivo with JAK inhibitors. We observed potent suppression of pSTAT1, pSTAT3, and pSTAT5 in CD34+ cells, with the 4 JAK2 inhibitors exerting similar degrees of inhibition. In B cells, pacritinib suppressed IκBα, pERK, and pCREB levels, while momelotinib inhibited pTBK1. Similar observations were noted in T cells, which in addition exhibited suppression of pSTAT1 and pSTAT3. These findings indicate distinct suppression responses across specific cell types and further highlight nuances beyond sole suppression of JAK-STAT signaling by these inhibitors. RNA-sequencing of HEL cells treated with the 4 JAK2 inhibitors also identified unique alteration of expression profiles and pathways (Fig. A). Differential gene expression (DEG) analysis revealed pacritinib inducing the greatest transcriptional alterations after 4 hours (3,121 genes) and ruxolitinib (1,969 genes) following 48 hours treatment duration. Only 82 and 103 DEGs relative to control were shared across the 4 JAK2 inhibitor treatment at 4 and 48 hours, respectively. Additional pathway analysis revealed at least 3 out of 4 inhibitors demonstrating capacity to suppress 1) JAK-STAT signaling, 2) inflammatory response, and 3) TNF signaling via NFκB pathways. Upregulation of multiple metabolic pathways was observed such as oxidative phosphorylation by momelotinib and pacritinib, potentially reflecting adaptive transcriptional feedback, as Seahorse Mito Stress assays revealed strongest suppression of oxygen consumption rate of PBMCs mediated by pacritinib and fedratinib. Lastly, we compared the in vivo potency of JAK2 inhibitors using patient-derived xenograft (PDX) models (Fig. B). Engrafted immunodeficient NSGS mice were treated with JAK2 inhibitors for 4 weeks across 2 sets of PDX experiments with CD34+ cells from unique JAK2 and CALR mutant donors. Pacritinib demonstrated greatest potency in reduction of peripheral blood and bone marrow human CD45+ leukemic cells, followed by ruxolitinib, and fedratinib/momelotinib. Vehicle treated mice were moribund by week 3 of treatment and survival was significantly prolonged with all JAK2 inhibitor treatments, with all animals surviving in the pacritinib treatment group after 4 weeks of treatment at end point. Despite all four JAK2 inhibitors demonstrating affinity in suppressing JAK-STAT signaling, our assays reveal varying degrees of alteration of transcriptional, proteomic, and metabolic profiles. In conjunction with comparative assessment of in vivo activity in PDX models, these findings provide insights with potential relevance to distinct responses observed with these inhibitors in the clinic and may help guide the use of specific JAK2 inhibitors in combination studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 5284: M1231 is a bispecific anti-MUC1xEGFR antibody-drug conjugate designed to treat solid tumors with MUC1 and EGFR co-expression

Abstract Mucin 1 (MUC1) and epidermal growth factor receptor (EGFR) are co-expressed at high prevalence in multiple cancer indications such as non-small cell lung cancer (NSCLC), esophageal squamous cell carcinoma (ESCC), head and neck squamous cell carcinoma, triple negative breast cancer and ovarian cancer. Tumor-associated MUC1 is hypoglycosylated and exposes peptide epitopes within its extracellular domain, which are not accessible to antibodies in normal epithelial tissue. MUC1 co-localizes with EGFR as a result of loss of cell polarity in tumor cells. M1231 is a bispecific antibody-drug conjugate (ADC) based on a SEED (strand-exchange engineered domain)-antibody scaffold targeting tumor-associated MUC1 and EGFR. Sutro Biopharma’s cell free expression system Xpress CF+™ enables incorporation of non-natural amino acids at precise positions in the SEED-antibody for site-specific linker-payload conjugation. The cytotoxic payload of M1231 (SC209) is a hemiasterlin-related microtubule inhibitor. The cleavable linker (ValCit-PABA) maintained ADC stability in human plasma in vitro, ensuring low systemic payload release. Pre-clinical activity of M1231 was assessed in vitro in cancer cell lines and in vivo in NSCLC and ESCC patient-derived xenograft (PDX) models. M1231 inhibited tumor cell viability in vitro at sub-nanomolar IC50 values. The bispecific MUC1xEGFR antibody has demonstrated superior internalization and lysosomal trafficking compared to monospecific bivalent antibodies. Efficient ADC uptake resulted in superior antitumor activity of M1231 in PDX models compared to monospecific bivalent ADCs. M1231 demonstrated durable and dose-dependent antitumor activity in PDX models. Strong antitumor activity with complete regression was observed in NSCLC and ESCC PDX models following a single treatment of M1231. M1231 antitumor activity was associated with high target expression in NSCLC PDX models and was observed across a range of target expression levels in ESCC models. In summary, efficient payload delivery to tumor cells and strong antitumor activity of M1231 indicate that M1231 may have the potential to deliver an efficacy benefit to patients with tumors co-expressing MUC1 and EGFR. Citation Format: Christine Knuehl, Lars Toleikis, Julia Dotterweich, Jianguo Ma, Seema Kumar, Edith Ross, Claudia Wilm, Martina Schmitt, Hans Juergen Grote, Christiane Amendt. M1231 is a bispecific anti-MUC1xEGFR antibody-drug conjugate designed to treat solid tumors with MUC1 and EGFR co-expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5284.

A phase 1/2 study of BLU-945 in patients with common activating EGFR-mutant non–small cell lung cancer (NSCLC): SYMPHONY trial in progress.

TPS9156 Background: Although EGFR-targeted therapies have improved outcomes in patients with EGFR-mutant ( EGFRm) NSCLC, such as EGFR ex19del and L858R, resistance to these drugs is inevitable. BLU-945 is an investigational next-generation EGFR tyrosine kinase inhibitor (TKI) designed to suppress common activating and on-target resistance EGFR mutations, such as C797S and T790M. Preclinically it has shown activity as monotherapy in osimertinib-resistant patient-derived xenograft (PDX) models. In addition, BLU-945 has > 450-fold selectivity for C797S and T790M mutants over wildtype, advantageous for combinations with complementary EGFR-targeting agents, such as osimertinib. These combinations have shown enhanced activity in PDX models. The SYMPHONY trial (BLU-945-1101; NCT04862780) is an international, open-label, first-in-human, phase 1/2 study designed to evaluate safety, tolerability, and antitumor activity of BLU-945 as monotherapy and in combination with osimertinib in patients with EGFRm NSCLC. Methods: Key eligibility criteria include adults with pathologically confirmed metastatic NSCLC with an activating EGFR mutation, Eastern Cooperative Oncology Group performance status 0–1, and previous treatment with ≥1 EGFR-targeted TKI. Patients with asymptomatic brain metastases who are on stable doses of corticosteroids are eligible. Tumors with additional known driver alterations, including EGFR exon 20 insertions and other kinase drivers, are excluded. Primary endpoints are maximum tolerated dose, recommended phase 2 dose (RP2D) and safety (phase 1); and overall response rate (ORR) by RECIST 1.1 (phase 2). Key secondary endpoints include ORR, duration of response (DOR), pharmacokinetics (PK), and pharmacodynamics (PD; phase 1); and DOR, progression-free survival, overall survival, CNS efficacy, PK, and safety (phase 2). Phase 1 dose escalation will be done using Bayesian optimal interval design; ̃85 patients will receive BLU-945 monotherapy and ̃18 will receive combination BLU-945 and osimertinib. In the phase 2 dose expansion, patients will receive BLU-945 at the RP2D of the monotherapy in 3 groups based on EGFR mutational profile: T790M and C797S (n≈37), T790M, but not C797S (n≈12), and C797S, but not T790M (n≈12). In a combination group, BLU-945 plus osimertinib will be administered at RP2D of the combination to n≈24 patients, ≥12 with both T790M and C797S mutations. Patients may receive treatment until disease progression or unacceptable toxicity. Recruitment has started and approximately 30 sites will be open for enrollment across North America, Europe, and Asia. Clinical trial information: NCT04862780.

Abstract P058: Anti-tumor activity of ATR inhibitor BAY 1895344 in patient-derived xenograft (PDX) models with DNA damage response (DDR) pathway alterations

Abstract Introduction: The ATR (ataxia-telangiectasia and Rad3 related protein) kinase inhibitor BAY 1895344 is currently in clinical development for the treatment of advanced solid tumors and has demonstrated promising antitumor activity in heavily pretreated patients with various advanced solid tumors, particularly those with ATM deleterious mutations and/or loss of ATM protein. There is further need to identify robust predictive biomarkers to optimize patient selection for ATR inhibitors. For that purpose, we tested the antitumor activity of BAY 1895344 in patient-derived xenograft (PDX) models that are characterized by a variety of DDR alterations. Methods: PDX models were characterized by genomic sequencing and ATM loss by immunohistochemistry (IHC). PDX models with deleterious ATM, BRCA1 or BRCA2 mutations or loss derived from a variety of histologies were tested. BAY1895344 treatment was tested with two monotherapy regimens 20 mg/kg and 40 mg/kg PO BID both 3 days on/4 days off. For in vivo studies, the percent tumor volume change per time point was calculated as a relative level of tumor growth change from baseline: , where is the tumor volume at time and is the tumor volume at baseline. T/C ratio was defined as the ratio of tumor volume change in treated vs control group. An event in each animal was defined as a doubling of tumor volume from initial tumor volume. Event free survival was analyzed by Kaplan-Meier survival analysis. Results: Seventeen PDX models from sixteen patients were treated with BAY 1895344. The PDX models spanned multiple tumor types: breast, colon, pancreas, and cholangiocarcinoma. BAY 1895344 has shown potent and dose-dependent antitumor activity. Strongest activity was observed with BAY 1895344 at 40 mg/kg PO BID applied for 3 days on and 4 days off treatment, achieving a regression or T/C ratio <0.4 in 6 of 17 models. Eleven models showed statistically significant prolongation of event-free survival. BAY 1895344 had anti-tumor activity in PDX models with ATM loss as well as BRCA alterations. Notably BAY 1895344 had antitumor activity in an ATM-deleted PDX model with acquired PARP inhibitor resistance generated in the lab as well as a PDX model generated from a BRCA-mutant patient with clinically acquired PARP resistance. Conclusion: ATR inhibition via treatment with BAY 1895344 shows potent antitumor activity as monotherapy in selected models that are characterized by certain DDR alterations and even those that have developed resistance to PARP inhibition. Further analyses are ongoing to define predictors of sensitivity to BAY 1895344 as well as pharmacodynamic markers of efficacy/response as a single agent or in rational combinations Citation Format: Christian X. Cruz Pico, Dali Li, Christopher D. Lanier, Kurt Evans, Maria G. Raso, Timothy DiPeri, Yasmeen Rizvi, Min Jin Ha, Huiqin Chen, Ming Zhao, Argun Akcakanat, Xiaofeng Zheng, Gokce Toruner, Erkan Yuca, Stephen Scott, Antje M. Wengner, Timothy A. Yap, Funda Meric-Bernstam. Anti-tumor activity of ATR inhibitor BAY 1895344 in patient-derived xenograft (PDX) models with DNA damage response (DDR) pathway alterations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P058.

Abstract 925: Introduction to the preclinical profile of SYD1875, a novel site-specifically conjugated duocarmycin-based 5T4-targeting antibody-drug conjugate

Abstract 5T4 is an oncofetal antigen with low expression in healthy tissues which is frequently overexpressed in different tumor types. Despite its overexpression in various malignancies and its association with poor prognosis, there are currently no marketed drugs that target this tumor antigen. We developed a novel antibody-drug conjugate (ADC), named SYD1875, comprising a humanized, HC41-cysteine-engineered IgG1 monoclonal antibody directed against 5T4, site-specifically conjugated to a proprietary cleavable synthetic duocarmycin-based linker-drug (vc-seco-DUBA). SYD1875 showed sub-nanomolar binding affinity to human and cynomolgus 5T4, rapid internalization, and induction of both target- and bystander-mediated cell killing. SYD1875 showed superior in vitro cytotoxicity and in vivo activity compared to A1-mcMMAF -also known as PF-6263507-, a 5T4-targeting ADC that is no longer in clinical development. A single dose of SYD1875 induced strong anti-tumor activity in patient-derived xenograft models of multiple tumor types with low, moderate and high 5T4 expression. The GLP toxicity study in cynomolgus monkey showed an acceptable toxicity and PK profile. A first-in-human dose-finding trial was initiated to evaluate safety and explore efficacy (NCT04202705). Citation Format: Patrick Groothuis, Danielle Jacobs, Kim Berentsen, Monique van der Vleuten, Ruud Coumans, Ronald Elgersma, Marion Blomenrohr, Diels van den Dobbelsteen, Patrick Beusker, Ruud Ubink, Miranda van der Lee, Wim H.A. Dokter. Introduction to the preclinical profile of SYD1875, a novel site-specifically conjugated duocarmycin-based 5T4-targeting antibody-drug conjugate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 925.

Abstract 3090: The MAT2A inhibitor, AG-270, combines with both taxanes and gemcitabine to yield enhanced anti-tumor activity in patient-derived xenograft models

Abstract MAT2A (methionine adenosyltransferase 2 alpha) is a critical enzyme within the methionine salvage pathway responsible for generating the universal methyl group donor, S-adenosyl methionine (SAM). We have developed a first-in-class small molecule inhibitor of MAT2A, AG-270, currently in a phase 1 clinical study (ClinicalTrials.gov NCT03435250) for the treatment of patients with solid tumors or lymphomas with MTAP (methylthioadenosine phosphorylase) deletion. The MTAP gene is deleted in approximately 15% of all human cancers, including non-small cell lung cancer (NSCLC; ~15-25%), pancreatic (~25%) and esophageal (~30%) cancer, and glioblastoma (~50%). To prioritize candidate combination partners for AG-270, a cell-based in vitro screening approach was employed using MTAP-null cell lines, in which AG-270 was combined with standard-of-care (SOC) agents as well as agents targeting pathways with hypothesized mechanistic links to MAT2A. Some of the best performing enhancers from this screen included paclitaxel (and docetaxel, using orthogonal screens) and gemcitabine. To assess the robustness of these combination findings in clinically relevant in vivo models, a series of patient-derived xenograft (PDX) experiments was undertaken to evaluate tolerability and efficacy in mice. Results demonstrated that AG-270, when combined with taxanes (paclitaxel/docetaxel) or gemcitabine, was well tolerated using SOC plasma exposures less than or equal to those achieved in patients. Importantly, combining AG-270 with taxanes and gemcitabine yielded additive-to-synergistic anti-tumor activity, with the docetaxel combination yielding 50% complete tumor regressions (CRs) in 2-3 PDX models. To study the mechanism of action, MAT2A was inhibited in vitro within HCT-116 MTAP −/− and wild-type cells, and we observed RNA splicing changes (via detained introns) altering genes involved in cell cycle regulation and DNA damage response, with a more pronounced effect found in the MTAP −/− genetic setting. Moreover, detained introns involving these same two pathways were modulated in MTAP −/− NSCLC PDX models treated with AG-270. Taken together, these data suggest AG-270 complements the known mechanism of action of taxanes and gemcitabine, and leads to enhanced DNA damage and inhibition of cellular proliferation. This work has helped identify a therapeutic strategy of combining AG-270 with taxanes and gemcitabine, which is currently being explored in an ongoing phase 1 clinical trial (NCT03435250). Page 1 of 1 Citation Format: Marc L. Hyer, Peter Kalev, Mark Fletcher, Chi-Chao Chen, Elia Aguado-Fraile, Everton Mandley, Sheila Newhouse, Max Lein, Raj Nagaraja, Yesim Tuncay, Josh Murtie, Kevin M. Marks, Katya Marjon. The MAT2A inhibitor, AG-270, combines with both taxanes and gemcitabine to yield enhanced anti-tumor activity in patient-derived xenograft models [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 3090.

Plocabulin, a novel tubulin inhibitor, has potent antitumor activity in patient-derived xenograft models of gastrointestinal stromal tumors

The majority of patients with gastrointestinal stromal tumors (GIST) eventually become resistant with time due to secondary mutations in the driver receptor tyrosine kinase. Novel treatments that do not target these receptors may therefore be preferable. For the first time, we evaluated a tubulin inhibitor, plocabulin, in patient-derived xenograft (PDX) models of GIST, a disease generally considered to be resistant to cytotoxic agents. Three PDX models of GIST with different KIT genotype were generated by implanting tumor fragments from patients directly into nude mice. We then used these well characterized models with distinct sensitivity to imatinib to evaluate the efficacy of the novel tubulin inhibitor. The efficacy of the drug was assessed by volumetric analysis of the tumors, histopathology, immunohistochemistry and Western blotting. Plocabulin treatment led to extensive necrosis in all three models and significant tumor shrinkage in two models. This histological response can be explained by the drug's vascular-disruptive properties, which resulted in a shutdown of tumor vasculature, reflected by a decreased total vascular area in the tumor tissue. Our results demonstrated the in vivo efficacy of the novel tubulin inhibitor plocabulin in PDX models of GIST and challenge the established view that GIST are resistant to cytotoxic agents in general and to tubulin inhibitors in particular. Our findings provide a convincing rationale for early clinical exploration of plocabulin in GIST and warrant further exploration of this class of drugs in the management of this common sarcoma subtype.

TR1801-ADC: a highly potent cMet antibody-drug conjugate with high activity in patient-derived xenograft models of solid tumors.

cMet is a well‐characterized oncogene that is the target of many drugs including small molecule and biologic pathway inhibitors, and, more recently, antibody–drug conjugates (ADCs). However, the clinical benefit from cMet‐targeted therapy has been limited. We developed a novel cMet‐targeted ‘third‐generation’ ADC, TR1801‐ADC, that was optimized at different levels including specificity, stability, toxin–linker, conjugation site, and in vivo efficacy. Our nonagonistic cMet antibody was site‐specifically conjugated to the pyrrolobenzodiazepine (PBD) toxin–linker tesirine and has picomolar activity in cancer cell lines derived from different solid tumors including lung, colorectal, and gastric cancers. The potency of our cMet ADC is independent of MET gene copy number, and its antitumor activity was high not only in high cMet‐expressing cell lines but also in medium‐to‐low cMet cell lines (40 000–90 000 cMet/cell) in which a cMet ADC with tubulin inhibitor payload was considerably less potent. In vivo xenografts with low–medium cMet expression were also very responsive to TR1801‐ADC at a single dose, while a cMet ADC using a tubulin inhibitor showed a substantially reduced efficacy. Furthermore, TR1801‐ADC had excellent efficacy with significant antitumor activity in 90% of tested patient‐derived xenograft models of gastric, colorectal, and head and neck cancers: 7 of 10 gastric models, 4 of 10 colorectal cancer models, and 3 of 10 head and neck cancer models showed complete tumor regression after a single‐dose administration. Altogether, TR1801‐ADC is a new generation cMet ADC with best‐in‐class preclinical efficacy and good tolerability in rats.

Abstract NT-113: SC-003, AN ANTIBODY-DRUG CONJUGATE TARGETING DIPEPTIDASE 3, EXHIBITS POTENT ANTI-TUMOR ACTIVITY IN PATIENT-DERIVED XENOGRAFT MODELS OF HIGH GRADE SEROUS OVARIAN CANCER

Abstract Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Ovarian cancer describes a group of malignancies that remain a serious threat to women's health and claim more than 14,000 deaths in the US each year. Among these, high-grade serous ovarian cancers (HGSC) represent the most common and aggressive form, and tumor recurrence is near-universal following initial response to carboplatin and paclitaxel. While multiple treatment options are available for platinum-sensitive recurrent HGSC, such as continued use of carboplatin/paclitaxel with or without bevacizumab, as well as several FDA-approved PARP inhibitors, there are few effective treatment options for platinum-resistant HGSC. Its inherent heterogeneity, characterized by genomic instability and numerous DNA copy number aberrations, poses challenges to the discovery of novel therapeutic targets. Patient-derived xenograft (PDX) models are an important platform for target identification and efficacy testing of therapeutic agents, as they sustain the intratumoral heterogeneity observed in advanced human tumors more faithfully than established cell lines. The isolation of distinct tumor cell populations by flow cytometry and subsequent testing of their tumorigenic potential can identify tumor-initiating cells (TICs), which have the potential to propagate the tumor over multiple generations in immune-deficient NOD/SCID mice. Thorough characterization of these TIC populations by transcriptome profiling and flow cytometry has identified TIC-enriched cell surface proteins that are targetable by antibody drug conjugates (ADCs), including EFNA4 in mixed Müllerian and ovarian cancers and PTK7 in triple-negative breast and ovarian cancers. ADCs consist of a monoclonal antibody directed against a cell surface epitope linked to a cytotoxic agent, such as auristatin, maytansinoid, or pyrrolobenzodiazepine (PBD). Here, we describe the generation and characterization of a PDX bank for ovarian cancers that led to the identification of dipeptidase 3 (DPEP3) as a TIC-associated target in HGSC. We show that DPEP3 is enriched in the TIC fraction of platinum-sensitive and platinum-resistant HGSC PDX models, where it localizes to the plasma membrane and is detected by flow cytometry and immunohistochemistry (IHC). In contrast, DPEP3 expression is low or absent in most normal adult tissues, thus providing a therapeutic window for an antibody-based therapeutic agent. In order to target DPEP3-expressing ovarian cancer cells, we developed SC-003, an ADC consisting of a humanized monoclonal antibody linked to a PBD dimer via a cleavable linker. We show that SC-003 specifically binds to DPEP3-expressing cells and, upon internalization, elicits cytotoxicity via release of its PBD warhead following lysosomal degradation of the antibody component. A single dose of SC-003 induced tumor regression in DPEP3-positive HGSC PDX models, including platinum-resistant PDX models. Mechanistically, we show that the anti-tumor effect of SC-003 is mediated by a significant reduction in TIC frequency. Moreover, combination with an anti-PD1 antibody potentiated SC-003 efficacy in a syngeneic mouse model engineered to overexpress human DPEP3. In summary, these findings support the clinical development of SC-003 as a novel therapeutic agent for HGSC. Citation Format: Wolf R. Wiedemeyer, Sheila Bheddah, Christine Saechao, Dorothy French, Erik Huntzicker, Aaron Kempema, Julia Gavrilyuk, David Liu, Vikram Sisodiya, Alina He, Zhaomei Zhang, Monette A. Aujay, Kristyn Hayashi, Sandro Vivona, Xi Zhao, Kimberly Walter, Laura R. Saunders, Johannes Hampl, Shravanthi Madhavan, Marybeth Pysz, Alexander J. Bankovich, Holger Karsunky, Scott J. Dylla. SC-003, AN ANTIBODY-DRUG CONJUGATE TARGETING DIPEPTIDASE 3, EXHIBITS POTENT ANTI-TUMOR ACTIVITY IN PATIENT-DERIVED XENOGRAFT MODELS OF HIGH GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-113.

Poor-Risk AML with Complex, Monosomal Karyotype: In Vivo Therapeutic Efficacy of Decitabine (DAC) in Patient-Derived Xenograft (PDX) Mouse Models

Background: DNA-hypomethylating agents (HMAs) show an encouraging but not yet well-understood activity in AML/MDS patients (pts) with adverse cytogenetics, such as -7/del(7q) embedded in a complex, monosomal karyotype (MK). We have noted that MK pts with multiple monosomies responded better to DAC treatment than pts with only one monosomal autosome (Blum, Greve and Lübbert, Curr Opin Hematol. 2017). This important characteristic of HMAs differentiates them from cytarabine (AraC, lacking DNMT-inhibitory activity) as shown in randomized studies with DAC (Wierzbowska et al., Am. J. Hematology 2018) and AZA (Döhner et al., Leukemia 2018). The mechanism of action is currently under investigation and may involve preferential gene reactivation on monosomic chromosomes by DAC (Greve et al., ASH Annual Meeting 2017, #2612). In order to address this important difference between DAC and AraC, we compared their antileukemic activity in PDX models representing cytogenetically normal (CN) AML and adverse cytogenetics AML (complex, monosomal karyotypes including del(7q) and 1 or more monosomies). Materials and Methods: PDX models were generated for 5 CN and 3 MK AML pts with a median age of 68 years (range 55-80), a median WBC of 3.0 x 103/µl (range 1.4-139.7) and a median of 42% bone marrow (BM) blasts (range 5-97). 7/8 pts were sampled at diagnosis, i.e. prior to first-line treatment (3 pts received DAC, 3 pts induction and 1 pt upfront allografting); 1/8 pt had had multiple relapses. T-cell depleted peripheral blood or bone marrow cells (3x106 cells/mouse) were injected into NSG (NOD/Shi-scid/IL-2Rγnull) mice. Leukemic cell engraftment was determined by flow cytometry (FC) in BM, peripheral blood (PB) and spleen during the course of the experiment and at the end of a study. Leukemic cells were transplanted serially at least 4-5 times to propagate and ensure full leukemic potential of the cells. Mice were treated with low-toxic concentrations of DAC (1 mg/kg/day given intraperitoneally), AraC (15 mg/kg/day given intravenously) or vehicle (PBS) for 5 consecutive days (one cycle). Drug doses were titrated to be equitoxic and as potent as possible without causing side effects. Treatment commenced upon disease onset, as evaluated by FC positivity for human CD33, increased WBC in PB and weight loss. Overall survival served as the read-out. The study was carried out in accordance with the recommendations by the Society of Laboratory Animal Science in an AAALCA accredited animal facility. The animal experiments were approved by the regional council (Regierungspräsidium Freiburg, ref. 35, permit no. G-12/86). Results: We successfully established well-characterized PDX from pts with CN (n = 5), and with MK (n = 3) with a median of 2 autosomal monosomies (range 1-6; of chromosomes 4, 6, 7 [2x], 15, 17 [2x], 18, 21). TP53 (mutational status available for 6/8 pts) was wildtype in 4 CN pts and 1 MK pt, and mutated in 1 MK pt. All models were treated with either DAC, AraC or vehicle. Across both cytogenetic cohorts, DAC treatment significantly prolonged median survival by 36 days when compared to vehicle administration (p = 0.0002, see Fig 1A). Specifically, DAC increased median survival of CN PDX by 33 days (p = 0.0104, range 61-105 days with DAC, compared to 43-65 days with vehicle, Fig 1B), and in MK PDX by 40 days (p = 0.0197; range 72-105 days with DAC, compared to 39-50 days with vehicle, Fig 1C). Notably, disease progression in two DAC-treated PDX (one CN, one MK with 6 monosomies) was delayed to 105 days (Fig 1, marked with ⋆). AraC treatment also resulted in longer median survival across both cohorts compared to vehicle, albeit not as pronounced as with DAC, by an average of 20 days (p = 0.0066). In more detail, AraC increased median survival of CN PDX by 19 days (p = 0.0025, range 53-82 days), and, though not significantly, of MK PDX by 15 days (p = 0.2048, range 46-92 days). Conclusion: In this PDX study recapitulating two different cytogenetic risk groups of AML, DAC showed encouraging activity also in models with complex, monosomal karyotype, even in the presence of 6 autosomal monosomies, and was superior to AraC in extending survival. These data lend further support to the treatment of such adverse genetics AML patients with HMAs. Disclosures No relevant conflicts of interest to declare.

Abstract 1052: Identification of signal transduction pathway activity in patient-derived xenograft models in comparison with their originating clinical samples of a variety of human cancer types

Abstract Background. Targeted drugs are directed against cellular signal transduction pathways, such as the PI3K pathway, and efficacy depends on the active pathway in the tumor. Development and clinical application of targeted drugs for personalized cancer treatment require tests which identify pathway activity in cancer cells/tissue. Patient-derived xenograft (PDX) models are becoming the cornerstone of preclinical drug development. Characterization of PDX models and clinical samples for pathway activity is needed for successful preclinical drug testing and patient drug response prediction. Inference of signal transduction pathway activity from cancer genomics information is often unreliable. A novel analysis method has been developed which enables identification and quantification of activity of signal transduction pathways, based on Bayesian reasoning models which infer an activity score from mRNA levels of pathway target genes (Verhaegh et al, Cancer Res 2014:2936-45). For the AR, ER, PI3K, Hedgehog, TGFbeta, Wnt, and NFkB pathways, models have been biologically validated on a number of different cell/tissue types. Methods. For a number of different cancer types (among which colon, breast, lung, ovarian, bladder, osteosarcoma, melanoma, esophagus), AR, ER, PI3K, Hedgehog, TGFbeta, Wnt, and NFkB pathway activity was inferred from Affymetrix HG-U133Plus2.0 microarray data, available from clinical studies (GEO database) and from PDX models (Charles River). Clinical and xenograft pathway activity scores were compared and where possible correlated to genomic datato identify associated pathway driving alterations. Results. Different cancer types were analyzed and compared. Pathway activity patterns for different cancer types in PDX models resemble pathway activity in clinical patient cohorts. Furthermore, known associated cancer mutations are reproduced in PDX models, such as APC mutations in colon PDX and ovary PDX, and an activating beta-catenin mutation in breast PDX, that show an activated Wnt pathway. Conclusion. Signal transduction pathway activity in PDX models resembles pathway activity in patient cohorts with corresponding cancer types and corresponds with known driving mutations. Functional pathway activity testing in PDX models is expected to increase xenograft model utility for (1) further validation of pathway tests for clinical diagnostic use and for (2) preclinical testing of targeted drug efficacy. Citation Format: Wim Verhaegh, Anja van de Stolpe, Nevisa Caushaj, Manuel Landesfeind, Angelika Zaremba. Identification of signal transduction pathway activity in patient-derived xenograft models in comparison with their originating clinical samples of a variety of human cancer types [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 1052.

Sleeping Beauty Modified CAR+ Lymphocytes Engraft and Exhibit Anti-Tumor Activity in Patient-Derived Xenograft Models of Acute Lymphoblastic Leukemia

Background:Adoptive infusion of T lymphocytes modified with anti-CD19 chimeric antigen receptor (CAR) by viral vectors is a therapeutic option proven effective in the treatment of hematological malignancies. In the context of B-lineage neoplasms, CD19-specific CART cells demonstrated unexpected positive results, achieving complete remission and durable response. To overcome high manufacturing costs, regulatory hurdles and scale-up complexities, we recently established a platform for non-viral gene manipulation of Cytokine-Induced Killer (CIK) cells, an effector T cell population characterized by enrichment in highly cytotoxic CD3+CD56+ cells and reduced risk of GvHD, in compliance with Good manufacturing practices (GMP). Therefore, we investigate whether donor-derived CD19CAR redirected CIK cells can significantly increase anti-leukemic responses in Acute Lymphoblastic Leukemia (ALL) by performing a pre-clinical validation of the non-viral platform.Methods:Large-scale production will be performed by nucleofection in presence of two GMP-grade Sleeping Beauty transposon DNA plasmids, coding for the anti-CD19CD28OX40 CAR transgene and for the SB11 transposase. T cells were differentiated according to a procedure derived from the Eudract n 2008-003185-26 study with the addition of a single stimulation with gamma-irradiated PBMCs to rescue the impaired T-cell expansion induced by electroporation.Results:The feasibility of large scale manufacturing process was verified starting from 30-60X10^6 PBMC, reaching efficient expansion with an average of 69.3±15.2-fold increase (Figure 1A) and stable expression of CD19CAR (average 65%, Figure 1B). Immunophenotypic analysis showed the typical enrichment of the CD3+/CD56+ cell subset, and maintenance of naive and central memory CD8+ and CD4+ T cells. Modified cells displayed a specific and effective cytotoxicity and proliferation towards CD19+ cell lines and primary tumors. In order to determine the biological effect of CARCIK-CD19 cells on disease and the correct dose for optimal activity, we performed a CARCIK-cell dose titration in a xenograft mouse model of common BCP-ALL, bearing the feature of a Ph-like gene rearrangement (PAX5/AUTS2). Mice were treated with 5x106, 10x106, 15x106 CAR+ cells or with 10x106 not transduced (NT) cells. CARCIK-CD19 cells showed a dose-dependent antitumor response and persisted in tumor-bearing animals, in peripheral blood (PB), bone marrow (BM) and spleen (Figure 2). Furthermore, to evaluate whether cryopreservation affects the activity of the cell product, we performed functional experiments with both fresh and frozen/thawed CAR CIK cells in vitro and in an established MLL/ENL patient-derived xenograft model. CARCIK.CD19 cells remained functional after freezing and thawing as evidenced by killing activity and decreased level of leukemic engraftment in the spleen of treated mice. Finally, we evaluated CARCIK-CD19 cell bio-distribution, safety and acute toxicity in NOD-SCID-gamma chain-/- (NSG) mice. The infusion of CARCIK-CD19 cells proved to be safe and well tolerated in all mice tested. The infused cells persisted in time in the hematopoietic and post-injection perfused organs with a follow-up of 3 months.Conclusions:Our findings describe a novel donor-derived non-viral CAR approach characterized by efficient cell transfection, expansion and functionality that could be used as valid and sustainable alternative to patient-derived viral approach with the aim to increase the cure rate of children and adults with leukemia. This study provides the proof-of-concept for designing phase I/II study for relapsing and refractory ALL post Hematopoietic Stem Cell Transplantation. [Display omitted] [Display omitted] DisclosuresCooper:Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership; City of Hope: Patents & Royalties; Targazyme, Inc.,: Equity Ownership; Immatics: Equity Ownership; Sangamo BioSciences: Patents & Royalties; MD Anderson Cancer Center: Employment; Miltenyi Biotec: Honoraria. Biondi:Cellgene: Other: Advisory Board; Novartis: Membership on an entity’s Board of Directors or advisory committees, Other: Advisory Board; BMS: Membership on an entity’s Board of Directors or advisory committees.

Mirvetuximab Soravtansine (IMGN853), a Folate Receptor Alpha–Targeting Antibody-Drug Conjugate, Potentiates the Activity of Standard of Care Therapeutics in Ovarian Cancer Models

Elevated folate receptor alpha (FRα) expression is characteristic of epithelial ovarian cancer (EOC), thus establishing this receptor as a candidate target for the development of novel therapeutics to treat this disease. Mirvetuximab soravtansine (IMGN853) is an antibody-drug conjugate (ADC) that targets FRα for tumor-directed delivery of the maytansinoid DM4, a potent agent that induces mitotic arrest by suppressing microtubule dynamics. Here, combinations of IMGN853 with approved therapeutics were evaluated in preclinical models of EOC. Combinations of IMGN853 with carboplatin or doxorubicin resulted in synergistic antiproliferative effects in the IGROV-1 ovarian cancer cell line in vitro. IMGN853 potentiated the cytotoxic activity of carboplatin via growth arrest and augmented DNA damage; cell cycle perturbations were also observed in cells treated with the IMGN853/doxorubicin combination. These benefits translated into improved antitumor activity in patient-derived xenograft models in vivo in both the platinum-sensitive (IMGN853/carboplatin) and platinum-resistant (IMGN853/pegylated liposomal doxorubicin) settings. IMGN853 co-treatment also improved the in vivo efficacy of bevacizumab in platinum-resistant EOC models, with combination regimens causing significant regressions and complete responses in the majority of tumor-bearing mice. Histological analysis of OV-90 ovarian xenograft tumors revealed that concurrent administration of IMGN853 and bevacizumab caused rapid disruption of tumor microvasculature and extensive necrosis, underscoring the superior bioactivity profile of the combination regimen. Overall, these demonstrations of combinatorial benefit conferred by the addition of the first FRα-targeting ADC to established therapies provide a compelling framework for the potential application of IMGN853 in the treatment of patients with advanced ovarian cancer.

Association of EGFR Expression Level and Cetuximab Activity in Patient-Derived Xenograft Models of Human Non–Small Cell Lung Cancer

To explore in a panel of patient-derived xenograft models of human non-small cell lung cancer (NSCLC) whether high EGFR expression, was associated with cetuximab activity. NSCLC patient-derived xenograft models (n=45) were implanted subcutaneously into panels of nude mice and randomization cohorts were treated with either cetuximab, cisplatin, cisplatin plus cetuximab, vehicle control, or else were left untreated. Responses according to treatment were assessed at week 3 by analyzing the relative change in tumor volume and an experimental analogue of the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines. An EGFR IHC score was calculated for each patient-derived xenograft model and response was assessed according to EGFR expression level. When tumors were stratified into high and low EGFR expression groups (IHC score threshold 200; scale 0-300), a stronger antitumor activity was seen in the high EGFR expression group compared with the low EGFR expression group in both the cetuximab monotherapy and cisplatin plus cetuximab combination therapy settings. For tumors treated with cisplatin plus cetuximab, the objective response rate was significantly higher in the high EGFR expression group compared with the low EGFR expression group (68% vs. 29%). Objective response rates were similar in high and low expression groups for tumors treated with cisplatin alone (27% vs. 24%, respectively). Cetuximab activity in NSCLC patient-derived xenograft models was demonstrated clearly only in tumors that expressed high levels of EGFR, as defined by an IHC score of ≥200.

Abstract 2590: Preclinical evaluation of ASG-5ME, a novel antibody-drug conjugate for pancreatic cancer

Abstract Antibody-drug conjugates (ADCs) have demonstrated promising activity and tolerability profiles in oncology clinical trials, including for Hodgkin lymphoma (brentuzumab vedotin) and breast cancer (trastuzumab-DM1), motivating the development of new ADCs targeting antigens in other prevalent cancers. An immunohistochemical survey of expression of the novel tumor antigen AGS-5 revealed strong staining in 90% of pancreatic cancer specimens tested. Importantly, this staining was largely uniform, with essentially all transformed cells demonstrating membranous staining. This observation along with the observation of equally abundant expression of AGS-5 in prostate and other cancers motivated the development of ASG-5ME, an ADC targeting AGS-5. The ADC is comprised of a fully human IgG2 monoclonal antibody conjugated to the potent tubulin-binding drug monomethylauristatin E, conjugated with an average of 3.7 drug molecules per antibody. Conjugation and analysis of this IgG2 ADC will be discussed. The resulting conjugate demonstrated antigen-dependent internalization and in vitro cytotoxicity at sub-saturating ADC concentrations, as well as potent in vivo antitumor activity in patient-derived xenograft models of pancreatic cancer. ASG-5ME has a long (12 day) T1/2 in mice. Taken together, these results support the clinical evaluation of ASG-5ME for treatment of pancreatic cancer Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2590.