Efficacy In Preclinical Models Research Articles

Combinations of an acetyl CoA carboxylase inhibitor with hepatic lipid modulating agents do not augment antifibrotic efficacy in preclinical models of NASH and fibrosis.

Dysregulated hepatocyte lipid metabolism is a hallmark of hepatic lipotoxicity and contributes to the pathogenesis of nonalcoholic steatohepatitis (NASH). Acetyl CoA carboxylase (ACC) inhibitors decrease hepatocyte lipotoxicity by inhibiting de novo lipogenesis and concomitantly increasing fatty acid oxidation (FAO), and firsocostat, a liver‐targeted inhibitor of ACC1/2, is under evaluation clinically in patients with NASH. ACC inhibition is associated with improvements in indices of NASH and reduced liver triglyceride (TG) content, but also increased circulating TG in subjects with NASH and preclinical rodent models. Here we evaluated whether enhancing hepatocyte FAO by combining ACC inhibitors with peroxisomal proliferator‐activated receptor (PPAR) or thyroid hormone receptor beta (THRβ) agonists could drive greater liver TG reduction and NASH/antifibrotic efficacy, while ameliorating ACC inhibitor–induced hypertriglyceridemia. In high‐fat diet–fed dyslipidemic rats, the addition of PPAR agonists fenofibrate (Feno), elafibranor (Ela), lanifibranor (Lani), seladelpar (Sela) or saroglitazar (Saro), or the THRb agonist resmetirom (Res), to an analogue of firsocostat (ACCi) prevented ACCi‐induced hypertriglyceridemia. However, only PPARα agonists (Feno and Ela) and Res provided additional liver TG reduction. In the choline‐deficient high‐fat diet rat model of advanced liver fibrosis, neither PPARα (Feno) nor THRβ (Res) agonism augmented the antifibrotic efficacy of ACCi. Conclusion: These data suggest that combination therapies targeting hepatocyte lipid metabolism may have beneficial effects on liver TG reduction; however, they may not be sufficient to drive fibrosis regression.

Abstract 3366: Novel oncolytic adenovirus for systemic treatment of disseminated lung cancer

Abstract The increased cancer incidence and associated mortality call for the development of novel treatment modalities to address needs of cancer patients, which do not respond to existing therapies. Oncolytic viruses emerge as a new modality in cancer therapy due to their natural ability to infect and kill cancer cells, and to stimulate patient's own immune system to eliminate cancer cells throughout the body. For this study, we used novel engineered oncolytic adenovirus, Ad5/35-3M (AVID-317), which demonstrated greatly-improved safety profile after systemic virus administration due to a set of targeted mutations in the adenovirus capsid (Atasheva et al, Science Translational Medicine, 2020, eabc6659). Systemic administration of AVID-317 to NCr/Nude mice with disseminated lung adenocarcinoma slowed tumor progression and significantly extended survival, compared to buffer-treated control tumor-bearing mice. In AVID-317 treated group, about 25% of mice demonstrated complete rejection of disseminated tumors and the rest of mice showed significantly extended survival. To understand the mechanism of AVID-317-mediated tumor suppression and to design more effective combination therapies, we analyzed disseminated tumors from the lungs in NCr/Nude mice using single cell RNA-seq and Mass Cytometry (Cy-TOF) after virotherapy. Our scRNA-seq analysis showed that in a subset of mice responding poorly to AVID-317 therapy, tumor-associated CSF1R-expressing myeloid cells expressed higher levels of MHC-II, Ctsb, and Arg1. In addition, CD3e+ T cells expressed functional exhaustion marker, PD-1, exclusively in mice that responded poorly to virotherapy. Mass Cytometry analysis with 37 metal isotope-labeled antibodies confirmed elevated expression of activation markers on myeloid cells after virotherapy. Next, we analyzed the efficacy of a combination therapy where in addition to AVID-317 administration, tumor-bearing mice were treated with anti PD-1 antibody to block immune-suppressive signaling in T cells, anti-CSF1R antibody to block CSF1R signaling, and anti-Asialo GM1 antibody to deplete anti-viral NK cells. Our analyses showed that combination therapy regimen significantly improved therapeutic outcomes in mice with disseminated lung cancer, by extending median survival to 95 days from 55-56 days observed in mice treated with either virus alone or combination drugs without the virus. Taken together, our study demonstrated feasibility of using AVID-317 oncolytic adenovirus for systemic therapy of disseminated lung cancer. Our study also showed that a combination of virotherapy with a defined select of immunotherapeutic drugs allows for the significant improvement of therapeutic efficacy in pre-clinical model of disseminated lung cancer. Citation Format: Jia Yao, Svetlana Atasheva, Dmitry Shayakhmetov. Novel oncolytic adenovirus for systemic treatment of disseminated lung cancer [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 3366.

Abstract 6218: Oncolytic Seneca Valley virus (SVV-001) overcomes checkpoint inhibitor resistance and demonstrates a systemic anti-tumor immune response in a syngeneic pancreatic cancer murine model

Abstract Here we evaluated the efficacy of Seneca Valley Virus (SVV), an oncolytic virus in the Picornaviridae family, in combination with checkpoint inhibitors (CPI) using the CPI-resistant murine syngeneic pancreatic tumor model Pan02. SVV, serendipitously identified in 2001, is an extremely tumor selective and potent oncolytic virus for a variety of solid human cancers, primarily due to the expression of the receptor of SVV, TEM8, being selectively expressed on the surface of malignant cancer cells and cancer stem cells within a variety of solid tumors. SVV has been tested in three clinical trials of patients having neuroendocrine neoplasms as a single intravenous dose monotherapy. Despite this, the results were encouraging with patients having evidence of clinical benefit and only one DLT was reported in the 76 patients treated. Recent data have demonstrated that some oncolytic viruses can enhance efficacy in preclinical models and in clinical trials when the oncolytic virus was injected intratumorally in combination with systemic administration of a CPI(s). The mechanism is primarily believed to be due to the oncolytic virus enhancing T cell infiltration and fostering an anti-tumor immune response. SVV was injected intra-tumorally in Pan02 tumors along with aPD1and/or aCTLA4. SVV reversed any resistance to CPIs and enhanced efficacy over CPI(s) alone resulting in complete cures in >83% of mice with primary and abscopal tumors. SVV plus aPD1+aCTLA4 resulted in 5 of 6 mice cured of their primary tumor. The animals were challenged on their contralateral flank with Pan02 to evaluate systemic and abscopal immune effects. All mice (5/5) that cleared the primary Pan02 tumor also eradicated the abscopal secondary tumors. Control treated Pan02 tumor-bearing mice were all sacrificed due to tumor burden by day 70 (median survival <50 days). Control animals treated with aPD1+aCTLA4 showed transient tumor regressions but these animals all grew large tumors, requiring sacrifice. In contrast, the SVV+aPD1+aCTLA4 primary tumors were eliminated within 44 days and these animals remain tumor-free out to >110+ days. Contralateral tumors were eradicated in all 5 animals that rejected the primary tumor, demonstrating potent systemic immunity. In the control aPD1+aCTLA4 group, only one animal with contralateral tumor showed prolonged tumor regression. SVV treatment caused increases in T-cell infiltration; the combination of SVV+aPD1+aCTLA4 showing the highest infiltration. Phenotypic and gene expression analyses of infiltrating cells will be presented. These studies serve as a foundation for translating SVV oncolytic virotherapy combined with anti-PD-1 and anti-CTLA4 antibodies in patients with neuroendocrine neoplasms with a clinical trial anticipated to begin in H1, 2021. Citation Format: Paul L. Hallenbeck, Sunil Chada. Oncolytic Seneca Valley virus (SVV-001) overcomes checkpoint inhibitor resistance and demonstrates a systemic anti-tumor immune response in a syngeneic pancreatic cancer murine model [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 6218.

Abstract CT147: Phase 1 study of recombinant interleukin 15 (rhIL-15) in combination with checkpoint inhibitors nivolumab and ipilimumab in subjects with refractory cancers

Abstract Introduction: Recombinant human interleukin 15 (rhIL-15) has been shown to stimulate the activation and expansion in the number of effector T lymphocytes and natural killer cells in patients. The addition of the immune checkpoint inhibitors (ICI) CTLA-4 and PD-1 has shown increased antitumor efficacy in preclinical models, and the triplet is hypothesized to enhance anti-tumor immune response by augmenting effector cell expansion, differentiation, cytotoxic activity, and immune checkpoint inhibition. Results of the lead-in safety arms of rhIL-15/ipilimumab (Ipi) or rhIL-15/nivolumab (N) doublets were previously reported (O’Sullivan et al. AACR. 2019) and we now report results for the dose escalation phase of the triplet combination (NCT03388632). Methods: This phase 1, open-label, 3+3 dose escalation trial enrolled adult patients (pts) with measurable metastatic or refractory solid tumors and ECOG performance status ≤2. Prior therapy with 2 of the 3 study agents was permitted. Pts were treated on three dose levels (DL 1-3) of rhIL-15 (0.5, 1, or 2 mcg/kg/day) administered subcutaneously on days (D)1-8 and D22-29 of a 6-week cycle for the first 4 cycles only. Nivolumab (240mg IV) was administered on D8, 22, 36 and ipilimumab (1mg/kg IV) on D8 of every cycle. Response was assessed by RECIST 1.1. Upon progression, pts could continue for response assessment utilizing iRECIST if clinically stable and without toxicity. Treatment-induced changes in circulating and tumor T cell activation, signaling, and the PD-L1 checkpoint will be assessed with validated pharmacodynamic biomarker assays. Results: A total of 17 pts were enrolled on the triplet dose escalation phase; 15 pts were evaluable for response and 6 pts had received prior treatment with a PD-1/PD-L1 inhibitor. Median time on treatment (MTT) was 12 weeks (range 3-101). A confirmed partial response (PR) was observed in a pt with intrahepatic cholangiocarcinoma (MSI-H) after 3 cycles (total time on treatment 101 weeks). Across all dose levels, 7 pts (46.7%) had stable disease (SD) that prolonged their time on treatment with MTT of 20.4 weeks; 7 pts (46.7%) experienced progressive disease (PD) with MTT of 6.4 weeks. To better profile toxicity, 5 pts were replaced due to incomplete dosing of C1. Dose limiting toxicities at DL 3 (rhIL-15 at 2 mcg/kg/day) were photosensitivity and rash. Main drug-related adverse events included grade 4 lymphopenia (n=1) and grade 3 colitis, hyperthyroidism, hyponatremia, myocarditis, neutropenia, and photosensitivity (n=1, each). No pts discontinued therapy due to toxicity events. No deaths occurred on study. Conclusions: The recommended phase 2 dose (RP2D) of rhIL-15 is 1 mcg/kg/day when administered in combination with N+Ipi. The triplet dose expansion phase with the RP2D is currently accruing and includes biomarker studies in blood and tumor biopsies to assess the tumor microenvironment. This study was funded in part by NCI Contract HHSN261200800001E. *In memorium: Dr. Thomas Waldmann7. Citation Format: Sarah J. Shin, Geraldine O'Sullivan Coyne, Howard Streicher, Naoko Takebe, Ashley Bruns, Elad Sharon, Richard Piekarz, Lamin Juwara, Larry Rubinstein, Ralph Parchment, Kristin Fino, King L. Fung, Katherine Ferry-Galow, Arjun Mittra, Abdul Rafeh Naqash, Kevin Conlon, James H. Doroshow, Alice P. Chen. Phase 1 study of recombinant interleukin 15 (rhIL-15) in combination with checkpoint inhibitors nivolumab and ipilimumab in subjects with refractory cancers [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 CT147.

Abstract ND13: The discovery and characterization of CFT7455: A potent and selective degrader of IKZF1/3 for the treatment of relapsed/refractory multiple myeloma

Abstract Introduction: Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 inhibits the growth of multiple myeloma (MM) cells, confirming their dependency on IKZF1/3. IMiDs (lenalidomide, pomalidomide) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. However, most patients treated with lenalidomide or pomalidomide eventually develop progressive disease due to acquired resistance, underscoring the unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high binding affinity to cereblon (CRBN), rapid and deep IKZF1/3 degradation, and potent dose-dependent efficacy in vivo. Results: A series of novel benzoimidazolone-based CRBN ligands with potent binding affinity were discovered and their binding modes were informed by CRBN co-crystal structures. Although the benzoimidazolone-based CRBN binders did not exhibit IKZF1/3 degradation activity, structural insights into their unique binding modes and knowledge of the IKZF1/3 degradation pharmacophore were combined to enable identification of a novel benzoisoindolone-based ligand that exhibited a 10-fold potency increase in biochemical CRBN binding and a 30-fold potency increase in H929 MM cell growth inhibition when compared to lenalidomide. Additional rounds of structure-based drug design, degradation and phenotypic profiling led to the discovery of CFT7455, a highly potent, selective and orally bioavailable degrader of IKZF1/3. CFT7455 demonstrated an 800 and 1600-fold improvement in CRBN binding compared to pomalidomide in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced >75% degradation of IKZF1 within 1.5 hrs. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines, as well as H929 cells made resistant to IMiDs. In vivo, CFT7455 catalyzed deep and durable degradation of IKZF3, translating into potent antitumor activity in multiple myeloma xenograft models. CFT7455 also retained its activity in models resistant or insensitive to clinically approved IMiDs as single agent or in combination with standard of care agent dexamethasone. Conclusion: Overall, CFT7455 is a next generation IKZF1/3 degrader, with improved potency and anticancer efficacy in preclinical models compared to existing IMiDs. These features make CFT7455 an exciting drug candidate, as a single agent or for use in combination. CFT7455 is currently being studied in a Ph1 clinical trial. Citation Format: James A. Henderson, Scott J. Eron, Andrew Good, R Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Ashley A. Hart, Christina S. Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Adam S. Crystal, Roy M. Pollock, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. The discovery and characterization of CFT7455: A potent and selective degrader of IKZF1/3 for the treatment of relapsed/refractory multiple myeloma [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 ND13.

Abstract 1894: Click Activated Protodrugs Against Cancer (CAPAC™): A modular platform for tumor directed oncology therapeutics

Abstract The CAPAC platform aims to help patients beat cancer by activating powerful cancer therapies at the tumor site(s). The core click chemistry technology is agnostic to therapeutic agent or indication. The modular nature of the platform allows targeting strategies based on tumor’s location, antigens, or both to be combined with therapeutics utilizing multiple mechanisms of action to deliver highly effective treatments tailored to each patient. The lead clinical candidate SQ3370 consists of a tetrazine-modified sodium hyaluronate biopolymer (SQL70) that is injected at the tumor site and followed by multiple systemic doses of trans-cyclooctene (TCO)-modified protodrug based on doxorubicin (Dox). Efficient covalent reaction between tetrazine and TCO moieties activates the protodrug and releases active Dox. SQ3370 provides enhanced safety and efficacy in preclinical models compared to conventional Dox, and is being evaluated in a Phase I study in advanced solid tumors (NCT04106492). The efficacy of SQ3370 is from biopolymer-induced activation of protodrug at the tumor site. Data will be presented from mice treated with biopolymer injected peritumorally versus distal-to-tumor, prior to protodrug dosing. Results show significant improvement in tumor growth inhibition and survival in peritumorally-activated SQ3370 compared to distally-activated SQ3370 or conventional Dox. Quantification of protodrug activation in a pharmacokinetic model in rats showed at least 10 to 20% of the protodrug dose is activated by the biopolymer, resulting in over 300 times higher exposure of Dox to tumor versus systemic (plasma) exposure. New TCO-modified protodrugs have been synthesized and preclinical results for the candidate based on monomethyl auristatin E (MMAE) paired with SQL70 will be presented. TCO-MMAE was selected for in vivo evaluation because of its high cytotoxicity-attenuation (93-fold) in vitro. In naive mice, the maximum tolerated dose (MTD) of TCO-MMAE, given as 5 daily doses after an injection of SQL70 biopolymer, was established at 20-times higher than the MTD of conventional MMAE. The antitumor efficacy of TCO-MMAE will be evaluated in future studies. In addition, preliminary results on the use of tetrazine-conjugated antibodies as a targeting agent capable of treating disperse disease when paired with an appropriate TCO-protodrug will be presented. In conclusion, the CAPAC platform is a novel approach that enables targeting of injectable and non-injectable tumors. CAPAC expands the therapeutic potential of anticancer drugs by increasing exposure to tumors and minimizing systemic adverse effects. The CAPAC platform can also be combined with other therapies to modulate the immune response and modify the tumor microenvironment. Citation Format: Jesse M. McFarland, Amir Mahmoodi, Michael Zakharian, Sangeetha Srinivasan, Nathan A. Yee, José M. Mejía Oneto. Click Activated Protodrugs Against Cancer (CAPAC™): A modular platform for tumor directed oncology therapeutics [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 1894.

Abstract 6377: Developing combination therapy with SHP2 inhibition for CIN-type gastroesophageal adenocarcinoma with KRAS amplification

Abstract Recent studies from the cancer genome atlas (TCGA. Nature. 2014) (TCGA. Nature. 2017) and other groups showed that a molecular subtype with chromosomal instability (CIN) is the most common subtype of gastroesophageal adenocarcinoma (GEA) and is characterized by aneuploidy without somatic hypermutation. The oncogene KRAS is frequently amplified in CIN type gastric cancer (14.3%) and esophageal adenocarcinoma (10.4%). SHP2 is a phosphatase which is a part of a machinery activating KRAS from GDP-bound inactive status to GTP-bound active status, and our group previously reported that KRAS-amplified gastric tumors are relatively resistant to MEK inhibition but sensitive to MEK/SHP2 co-inhibition. While the combination of MEK and SHP2 inhibitors showed efficacy in pre-clinical models, the translation of this combination has been challenging, in part due to on-target toxicity from inhibitors of the MAPK pathway (Auliac, et al. Cancers. 2020). Therefore, we sought better combination strategy with SHP2 inhibition, using genome-wide CRISPR screen. We conducted primary CRISPR screening with two GEA cell lines (KE-39 and HUG1-N) and CRISPR-KO lentivirus library (Brunello CRISPR knockout pooled library, 77,741 sgRNAs/19,114 genes, Broad Institute Genomic Perturbation Platform) and identified candidate targets both within the MAPK pathway and among upstream tyrosine kinases that may enhance the efficacy of a SHP2 inhibitor in KRAS-amplified GEA. Prior to detailed analyses of potential hits from the screen, we performed the secondary screen with additional KRAS-amplified and -mutant cell lines (CAT12, YCC-1, and GSU) using a custom CRISPR-Cas9 sgRNA library targeting 509 genes of interest including top hits in each cell line data set, and confirmed that knockout of KRAS upstream and downstream genes have additive cytotoxicity to SHP2 inhibition. Given our focus on finding targets that could be readily translated into therapeutics, we focused our analysis on those of most ready translational relevance and specifically chose ERBBs, FAK, SRC, RAFs, ERK1/2, and CDK4/6 as candidate targets. Further analyses showed the potent cytotoxicity of SHP2 inhibition with a pan-ERBB kinase inhibitior (Afatinib) and with a CDK4/6 inhibitor (Ribociclib), with greater efficacy of this combination in KRAS-amplified tumors compared to KRAS-mutant tumors, both in vitro and in vivo experiments. Overall, these results suggest co-inhibition of SHP2 and upstream/downstream of KRAS as a promising treatment strategy against KRAS-amplified CIN-type GEA. Citation Format: Osamu Kikuchi, Tianxia Li, Jin Zhou, Yichen Wang, Klavdija Bastl, Prafulla C. Gokhale, Aine Knott, Yanxi Zhang, John G. Doench, Zandra Ho, Daniel V. Catenacci, Adam J. Bass. Developing combination therapy with SHP2 inhibition for CIN-type gastroesophageal adenocarcinoma with KRAS amplification [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 6377.

Abstract 1260: Nonclinical evaluation of rucaparib in tumors with mutations in non-BRCA1/2 homologous recombination repair (HRR) genes

Abstract Background: Rucaparib is a PARP inhibitor approved to treat ovarian cancer (OC) and metastatic castration-resistant prostate cancer (mCRPC). Deficiencies in HRR resulting from genetic or epigenetic alterations in BRCA1/2 sensitize tumors to rucaparib through synthetic lethality. Inactivation of other HRR genes may also confer sensitivity to PARP inhibition. Here, we evaluated rucaparib efficacy in preclinical models with genomic or epigenetic alterations in an HRR gene panel. Methods: BRCA1/2 and 14 additional genes were selected for their role in DNA repair and mutation frequency in OC and mCRPC. Small interfering RNA (siRNA) knockdowns were performed in OC (OVCAR-3, OAW28, SK-OV-3) and mCRPC (DU145, PC-3) cell lines to model their inactivation and assess impact on rucaparib sensitivity (IC50) in cell viability assays. Patient-derived xenografts (PDX) harboring deleterious alterations in BRCA1/2, BARD1, BRIP1, NBN, PALB2, RAD51B, RAD51C, or RAD51D, or BRCA1/RAD51C promoter hypermethylation were treated with up to 150 mg/kg rucaparib BID for 25-42 days and tumor growth was monitored. Mutational status was confirmed by next-generation sequencing. Results: In addition to BRCA1/2 knockdown, depletion of BARD1, PALB2, or RAD51 resulted in potent rucaparib activity in OC and mCRPC cells, where the rucaparib IC50 was reduced by >70% vs control in at least 4 of the 5 cell lines tested. Rucaparib also displayed greater potency in cells with siRNA-mediated decreased expression of FANCA, NBN, RAD51C, or RAD54L, where at least 1 cell line had >50% decrease in IC50. Rucaparib treatment in 19/33 HRR-deficient PDX models resulted in significant tumor growth inhibition (TGI). These included BRCA1/2, PALB2, NBN, RAD51B, RAD51C, or RAD51D-mutated and BRCA1/RAD51C-hypermethylated models. Efficacy was strongly associated with biallelic inactivation of BRCA1/2, PALB2, RAD51C, or RAD51D with 87% vs 29% mean TGI for monoallelic BRCA1/2 alterations (P<0.0001). In addition, a single PDX model each for the exploratory genes NBN and RAD51B had 101% and 80% TGI, respectively. No significant difference was observed between PDX models with BRCA1/2 vs non-BRCA1/2 (PALB2, NBN, RAD51B, RAD51C, or RAD51D) biallelic alterations (98% vs 80% mean TGI, respectively; P=0.21). In support of these nonclinical findings, we highlight a patient with RAD51B-mutant mCRPC treated with rucaparib in the TRITON2 trial (NCT02952534) who had confirmed radiographic and PSA responses. Conclusions: Cell lines and PDX models with biallelic alterations in HRR genes other than BRCA1/2, including RAD51B, RAD51C, RAD51D, PALB2, and NBN, are comparably sensitive to rucaparib. Rucaparib is being evaluated in the LODESTAR trial (NCT04171700) in patients with tumors associated with deleterious alterations in BRCA1/2, PALB2, RAD51C, and RAD51D (cohort A) and BARD1, BRIP1, FANCA, NBN, RAD51, and RAD51B (exploratory cohort B). Citation Format: Liliane Robillard, Kevin K. Lin, Andrea Loehr, Tanya Kwan, Rachel Dusek, Andrew D. Simmons, Thomas C. Harding, Brieuc Sautois, Minh Nguyen. Nonclinical evaluation of rucaparib in tumors with mutations in non-BRCA1/2 homologous recombination repair (HRR) genes [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 1260.

Abstract 5559: Development of a novel PD-L1xCD40 bispecific antibody with excellent efficacy and safety profile for cancer therapy

Abstract CD40 agonistic antibody is emerging as a front-line immunotherapy for patients with cancer. However, despite its remarkable curative effects, serious immune-related adverse reactions limit the clinical application of CD40 agonists. Crosslinking-dependent activation is one of the promising approaches to diminish the adverse effects of CD40 agonists. Agonistic CD40 stimulation on antigen presenting cells (APCs) not only promotes activation of T cells, but also results in PD-L1 upregulation on APCs, thus creating a negative feedback loop to dampen the anti-tumor immune response. Therefore, we hypothesized that the combination of CD40 agonist and blockade of PD-1/PD-L1 interaction may further induce a synergistic effect on T cell activation and anti-tumor activity. Here, we report a novel PD-L1xCD40 Bispecific Antibody (BsAb) designed to have synergistic curative effects as well as improved safety profiles. The PD-L1/CD40 BsAb was generated using the Harbour HBICE࣪® platform. It exhibited superior binding activities to both PD-L1 and CD40, respectively. CD40 activation was fully relied on the PD-L1 crosslinking. The PD-L1xCD40 BsAb showed potent tumor growth inhibition in preclinical efficacy models. Meanwhile, it showed much improved safety profile compared to a reference anti-CD40 agonistic antibody in the preclinical tox models. Overall, the PD-L1xCD40 BsAb demonstrated remarkable in vivo anti-tumor efficacy and improved safety profiles in preclinical models, which presents it as a promising candidate for the development of next-generation cancer immune therapeutics. Citation Format: Haishan luo, Zihong Meng, Jimmy Rong, Yuandong Wang, Yunxing Yang, Fei Chen, Youhong Wang, Victor Chen, Gang Deng, Yebo He, Xin Gan, Yiping Rong. Development of a novel PD-L1xCD40 bispecific antibody with excellent efficacy and safety profile for cancer therapy [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 5559.

Abstract 1643: B-hIL12A/hIL12B/hIL12RB1/hIL12RB2: A novel animal model for generation of IL12 therapies

Abstract The pro-inflammatory cytokine, interleukin 12 (IL12), is primarily responsible for cell-mediated immunity and is chiefly produced by antigen presenting cells. Prior preclinical studies elucidated a promising role for IL12 mediating anti-tumor effects, however, clinical studies using cancer patients showed disappointing efficacy results, as well as severe adverse effects, with administration of systemic IL12 therapies. Intriguingly, progress in targeted delivery to the tumor microenvironment renewed high expectations on IL12 as a promising anti-tumor drug either alone or in combination due to its ability to inhibit and/or reprogram immunosuppressive cell subsets. Therefore, to evaluate the efficacy of IL12 and IL12 analogs in preclinical studies, Biocytogen has successfully generated a novel hIL12A/hIL12B/hIL12RB1/hIL12RB2 knock-in mouse strain. In this mouse model, the full coding sequences (CDS) of the mouse Il12a or Il12b gene were replaced by their human counterpart IL12A or IL12B gene sequences respectively. In addition, chimeric CDS, composed of human IL12RB1 or IL12RB2 extracellular region plus mouse Il12rb1 or Il12rb2 cytoplasmic region, was inserted into the mouse endogenous gene locus in in these animals, to express an IL12 receptor (IL12R) capable of recognizing human IL12 signaling. Expression analysis by RT-PCR detected human IL12A/IL12B/IL12RB1/IL12RB2 mRNA in humanized mice but not in wild-type mice. Additionally, human IL12 p70 was successfully detected in the serum of humanized mice by ELISA. The immune cell subpopulations in spleen, blood, and lymph node did not differ between humanized mice and wild-type mice suggesting no impact on cell differentiation and maturation. Anti-tumor activity of a modified human IL12 was assessed in humanized mice bearing colon cancer MC38 cells and showed tumor inhibitory effects. Moreover, the tumor growth recovered when the administration of the drug was stopped. Altogether, B-hIL12A/hIL12B/hIL12RB1/hIL12RB2 humanized mice provide a promising preclinical efficacy model for human IL12 therapy alone or in combination. Keywords: IL12, humanized mice, antibody efficacy Citation Format: Linlin Wang, Chang Liu, Veronika Chromikova, Ruili Lyu, Tianquan Jin, Mari Kuraguchi. B-hIL12A/hIL12B/hIL12RB1/hIL12RB2: A novel animal model for generation of IL12 therapies [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 1643.

Effect of Chiral Purity on Adjuvanticity of Archaeol-Based Glycolipids.

Archaeosomes composed of sulfated lactosyl archaeol (SLA) glycolipids from stereoisomerically pure archaeol (1) are vaccine adjuvants that can boost immunogenicity and vaccine efficacy in preclinical models. Herein, we report a new synthesis of 2,3-bis((3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol (3) by treating (±)-3-benzyloxy-1,2-propanediol with a mesylated phytol derivative through a double nucleophilic substitution reaction, followed by reductive debenzylation. Three SLA archaeosomes from archaeols of different chiral purities were prepared, and the effect of stereochemistry on their adjuvanticity toward ovalbumin was investigated. It was found that all SLA archaeosomes induced strong humoral and cell-mediated antigen-specific immune responses following immunization of C57BL/6NCrl mice, with no significant differences, irrespective of the chiral purities. The responses were comparable or better than those obtained using mimetics of approved adjuvants. The performance of SLA archaeosomes during immunization and their lack of dependence on the stereochemistry of archaeol points toward a promising, safe, scalable, and economically viable vaccine adjuvant system.

A phase 1 dose-finding and dose-expansion study evaluating the safety, tolerability, pharmacokinetics, and efficacy of a highly selective WEE1 inhibitor (Debio 0123) in adult patients with advanced solid tumors.

TPS2702 Background: Debio 0123 is an oral, highly selective inhibitor of the tyrosine kinase WEE1. WEE1 is a key regulator of cell cycle progression that modulates the activity of CDK1 (CDC2), influencing entry into mitosis. WEE1 inhibition results in G2 checkpoint abrogation, triggering mitosis with unrepaired DNA leading to cell death. In vitro and in vivo tumor models have shown Debio 0123 antitumor activity. Continuous exposure seems to be needed to maximize monotherapy efficacy in preclinical models. Preliminary data from an ongoing phase 1 study of intermittent Debio 0123 combined with carboplatin, showed a manageable safety profile, and signals of antitumor activity in patients (pts) with advanced solid tumors. We present the design of a phase 1 study (NCT05109975) of continuous Debio 0123 administered as a single agent in pts with advanced solid tumors. Methods: The study comprises a dose escalation part and a dose expansion part. The primary objective of the dose escalation part of the study is to determine the maximum tolerated dose (MTD) and/or the recommended phase 2 dose (RP2D) of continuous Debio 0123 monotherapy in adults with previously treated advanced solid tumors. Key secondary objectives include characterization of safety and tolerability, pharmacokinetics (PK), and preliminary antitumor activity (overall response rate [ORR]). Key inclusion criteria are histologically or cytologically confirmed locally advanced or metastatic solid tumors, and ECOG-PS 0 or 1. Key exclusion criteria are asymptomatic or unstable brain metastases, history of cardiac disorders, inability to swallow oral medication or abnormalities affecting drug absorption. Approximately 30 dose-limiting toxicity (DLT)-evaluable pts are anticipated to be enrolled based on an escalation with overdose control (EWOC) approach for 21-day treatment cycles. When the pts of each cohort become evaluable, a safety monitoring committee will review safety and tolerability and based on EWOC recommendations will decide the next dose level or will declare the MTD and/or RP2D. Pharmacodynamic biomarkers will be correlated with tumor response and/or PK. The dose-expansion part may start after MTD and/or RP2D determination. The primary objective of this part will be to characterize the safety and tolerability (e.g. percentage of pts with DLTs, serious adverse events or discontinuations) of Debio 0123 monotherapy at the MTD/RP2D, and to evaluate the anti-tumor activity (ORR) in pts with selected recurrent/progressive solid tumors. Key inclusion criteria are measurable disease per RECIST 1.1 and specific tumor types. Based on a Simon 2-stage design, up to 34 pts per tumor type cohort may be enrolled. Currently accrual to the dose escalation is ongoing in the United States and Switzerland. Clinical trial information: NCT05109975.

Phase 1 results of a phase 1/2 trial of CYT-0851, a first-in-class inhibitor of RAD51-mediated homologous recombination, in patients with advanced solid and hematologic cancers.

3084 Background: Homologous recombination (HR) is an essential, high-fidelity mechanism to repair DNA double strand breaks (DSBs) in cancer cells. CYT-0851 inhibits HR leading to an accumulation of unrepaired DSBs and tumor cell death. We are reporting the completed Phase 1 dose-escalation results and RP2D selection to support ongoing development of CYT-0851. Methods: Patients (pts) with advanced hematologic and solid tumors were treated with continuous 28-day cycles of increasing doses of CYT-0851 following a 3+3 design. The primary objective was determining the maximum tolerated dose. Secondary objectives included safety, pharmacokinetics, and anti-tumor activity. Results: As of a 15 Nov 2021 data cutoff (DCO), 73 pts with advanced cancers (NHL n = 18; Sarcoma n = 16; Pancreas n = 11; Breast n = 8; HNSCC n = 6; Ovarian n = 4; SCLC n = 4; Other n = 4; Myeloma n = 2) were treated in 12 cohorts (total daily doses: 30 mg to 1200 mg). One pt experienced a dose-limiting toxicity (DLT) of reversible metabolic acidosis at 1200 mg. Two of 3 pts at 800 mg experienced reversible DLT-like events in Cycle 2 of Gr3 dry skin and Gr3 myalgia and polyarthritis, respectively. Three of 10 pts treated at 600 mg experienced DLT-like events in Cycle 1: 1 pt experienced an SAE of Gr3 anorexia with Gr3 stomatitis, vomiting, and dehydration and 2 pts had Gr3 fatigue. No DLTs occurred at 400 mg daily which was selected as the RP2D. 42 pts (57.5%) experienced a CYT-0851-related adverse event (AE), including 12 (16.4%) with a Gr3/4 AE. There were no treatment-related deaths. AE leading to CYT-0851 withdrawal were reported in 2 pts (2.7%) treated with 600 and 800 mg. The most common CYT-0851-related AEs were primarily Gr1/2 and included fatigue (20.5%), hyperuricemia (11%), nausea (11%), alopecia (9.6%), constipation (8.2%) and headache (8.2%). CYT-0851 exposure was approximately dose-proportional across the evaluated doses with an effective half-life of ̃3 days. Exposure at 400 mg daily was consistent with efficacy in preclinical models. 46 pts were evaluable for response at the DCO. 12 pts with NHL were evaluable by Lugano and included 1 CR in FL and 1 PR in DLBCL treated for 393+ and 244 days respectively. 34 pts with solid tumors were evaluable by RECIST v1.1 with 1 PR in a pt with myxofibrosarcoma treated for 313 days and 16 pts with stable disease. Fifteen pts were treated for 100+ days and 5 for 180+ days. Conclusions: CYT-0851 has demonstrated promising and broad clinical activity in a Ph 1 population of pts with advanced cancers. The safety profile is favorable as characterized by events that were infrequent, primarily Gr1/2, and reversible. Six expansion cohorts (DLBCL, Follicular Lymphoma, Myeloma, Pancreatic, Ovarian and Sarcoma) are enrolling to characterize activity at the RP2D. Ph 1 evaluation of CYT-0851 in combination with 3 chemotherapy backbones is also ongoing. Clinical trial information: NCT03997968.

A microfluidic system that replicates pharmacokinetic (PK) profiles in vitro improves prediction of in vivo efficacy in preclinical models.

Test compounds used on in vitro model systems are conventionally delivered to cell culture wells as fixed concentration bolus doses; however, this poorly replicates the pharmacokinetic (PK) concentration changes seen in vivo and reduces the predictive value of the data. Herein, proof-of-concept experiments were performed using a novel microfluidic device, the Microformulator, which allows in vivo like PK profiles to be applied to cells cultured in microtiter plates and facilitates the investigation of the impact of PK on biological responses. We demonstrate the utility of the device in its ability to reproduce in vivo PK profiles of different oncology compounds over multiweek experiments, both as monotherapy and drug combinations, comparing the effects on tumour cell efficacy in vitro with efficacy seen in in vivo xenograft models. In the first example, an ERK1/2 inhibitor was tested using fixed bolus dosing and Microformulator-replicated PK profiles, in 2 cell lines with different in vivo sensitivities. The Microformulator-replicated PK profiles were able to discriminate between cell line sensitivities, unlike the conventional fixed bolus dosing. In a second study, murine in vivo PK profiles of multiple Poly(ADP-Ribose) Polymerase 1/2 (PARP) and DNA-dependent protein kinase (DNA-PK) inhibitor combinations were replicated in a FaDu cell line resulting in a reduction in cell growth in vitro with similar rank ordering to the in vivo xenograft model. Additional PK/efficacy insight into theoretical changes to drug exposure profiles was gained by using the Microformulator to expose FaDu cells to the DNA-PK inhibitor for different target coverage levels and periods of time. We demonstrate that the Microformulator enables incorporating PK exposures into cellular assays to improve in vitro–in vivo translation understanding for early therapeutic insight.

High-potency synthetic STING agonists rewire myeloid stroma in the tumour microenvironment to amplify immune checkpoint blockade efficacy in refractory pancreatic cancer.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is clinically unresponsive to immune checkpoint blockade (ICB) immunotherapy. Dense immunosuppressive myeloid stroma (MS) and consequent T cell exclusion from the tumour microenvironment renders PDAC resistant to immune-based therapies. Innate immune activation of the MS via cyclic dinucleotide (CDN) agonists of the STING (Stimulator of Interferon Genes) pathway can trigger T cell infiltration into cold tumours leading to robust anti-tumour immunity. Despite proven therapeutic efficacy in preclinical models, the cellular mechanisms of how CDNs reprogram the suppressive MS to sensitise tumours to ICB is poorly understood. Using multi-omic profiling of MDSCs and M2 Macrophages of human and murine origin, we show that the synthetic STING agonist, IACS-8803, rewires these populations from immunosuppressive to immune-permissive phenotypes in part through inhibition of c-Myc signaling, energy metabolism modulation, and antagonism of cell cycle. Furthermore, dimensionality reduction analyses of multiparameter flow cytometry data and survival studies in a KPC-derived model of PDAC show a proinflammatory remodeling of the MS and an increased T/NK cell infiltration and function by synthetic CDN STING agonists into the tumour that underlies an amplified therapeutic response to ICB. We are the first to report that synthetic CDN STING agonists affect MDSC and M2 macrophage repolarization through altering energy metabolism, cell cycle dynamics and c-Myc signalling. This study uncovers molecular and cellular mechanisms by which high-potency STING agonists drive a proinflammatory conversion of the tumour MS to overcome resistance to ICB in an aggressive orthotopic tumour model of PDAC. Supported by grants from PanCan (206100-90-100300-54-00006617-11111)

PDI inhibitor LTI6426 enhances panobinostat efficacy in preclinical models of multiple myeloma

The histone deacetylase inhibitor (HDACi), panobinostat (Pano), is approved by the United States Food and Drug Administration (FDA) and European Medicines Agency (EMA) for treatment of relapsed/refractory multiple myeloma (MM). Despite regulatory approvals, Pano is used on a limited basis in MM due largely to an unfavorable toxicity profile. The MM treatment landscape continues to evolve, and for Pano to maintain a place in that paradigm it will be necessary to identify treatment regimens that optimize its effectiveness, particularly those that permit dose reductions to eliminate unwanted toxicity. Here, we propose such a regimen by combining Pano with LTI6426, a first-in-class orally bioavailable protein disulfide isomerase (PDI) inhibitor. We show that LTI6426 dramatically enhances the anti-MM activity of Pano in vitro and in vivo using a proteasome inhibitor resistant mouse model of MM and a low dose of Pano that exhibited no signs of toxicity. We go on to characterize a transcriptional program that is induced by the LTI6426/Pano combination, demonstrating a convergence of the two drugs on endoplasmic reticulum (ER) stress pathway effectors ATF3 (Activating Transcription Factor 3), DDIT3/CHOP (DNA Damage Inducible Transcript 3, a.k.a. C/EBP Homologous Protein), and DNAJB1 (DnaJ homolog subfamily B member 1, a.k.a. HSP40). We conclude that LTI6426 may safely enhance low-dose Pano regimens and that ATF3, DDIT3/CHOP, and DNAJB1 are candidate pharmacodynamic biomarkers of response to this novel treatment regimen.

IBI379, a novel B cell maturation antigen/CD3 bispecific T-cell engager, displays high antitumor efficacy in preclinical models of multiple myeloma

Multiple myeloma (MM) is a hematological malignancy that results from the malignant proliferation of plasma cells in the bone marrow. B cell maturation antigen (BCMA) is highly selectively expressed in malignant plasma cells and is a novel therapeutic target for MM. Here, we developed a bispecific T cell engager, IBI379, that targets BCMA and CD3, and investigated its antitumor efficacy against MM. IBI379 showed strong binding affinity with both BCMA and CD3, which triggered T cell activation, proliferation, and cytokine release. An in vitro study demonstrated that IBI379 induced the lysis of MM cells expressing differing levels of BCMA on the cell surface. Administration of IBI379 in H929 or Daudi-BCMA cell xenograft mouse models significantly inhibited tumor growth without inducing body weight loss. The mechanism of action study revealed the accumulation of CD4+CD8+ T cells and granzyme B-positive T cells in tumors that were treated with IBI379. Moreover, administration of low dose of IBI379 in cynomolgus monkeys was well-tolerated and induced the depletion of BCMA+ B cells and a mild transient increase of cytokines. Collectively, these results demonstrate that IBI379 is a highly potent therapeutic strategy for depleting BCMA-positive B cells and is a promising approach for the treatment of MM.

Polyamine Immunometabolism: Central Regulators of Inflammation, Cancer and Autoimmunity.

Polyamines are ubiquitous, amine-rich molecules with diverse processes in biology. Recent work has highlighted that polyamines exert profound roles on the mammalian immune system, particularly inflammation and cancer. The mechanisms by which they control immunity are still being described. In the context of inflammation and autoimmunity, polyamine levels inversely correlate to autoimmune phenotypes, with lower polyamine levels associated with higher inflammatory responses. Conversely, in the context of cancer, polyamines and polyamine biosynthetic genes positively correlate with the severity of malignancy. Blockade of polyamine metabolism in cancer results in reduced tumor growth, and the effects appear to be mediated by an increase in T-cell infiltration and a pro-inflammatory phenotype of macrophages. These studies suggest that polyamine depletion leads to inflammation and that polyamine enrichment potentiates myeloid cell immune suppression. Indeed, combinatorial treatment with polyamine blockade and immunotherapy has shown efficacy in pre-clinical models of cancer. Considering the efficacy of immunotherapies is linked to autoimmune sequelae in humans, termed immune-adverse related events (iAREs), this suggests that polyamine levels may govern the inflammatory response to immunotherapies. This review proposes that polyamine metabolism acts to balance autoimmune inflammation and anti-tumor immunity and that polyamine levels can be used to monitor immune responses and responsiveness to immunotherapy.

An Fc-Competent Anti-Human TIGIT Blocking Antibody Ociperlimab (BGB-A1217) Elicits Strong Immune Responses and Potent Anti-Tumor Efficacy in Pre-Clinical Models.

TIGIT (T-cell immunoglobulin and ITIM domain) has emerged as a promising target in cancer immunotherapy. It is an immune “checkpoint” inhibitor primarily expressed on activated T cells, NK cells and Tregs. Engagement of TIGIT to its ligands PVR and PVR-L2 leads to inhibitory signaling in T cells, promoting functional exhaustion of tumor-infiltrating T lymphocytes. Here, we described the pre-clinical characterization of Ociperlimab (BGB-A1217), a novel humanized IgG1 anti-TIGIT antibody (mAb), and systemically evaluated the contribution of Fc functions in the TIGIT mAb-mediated anti-tumor activities. BGB-A1217 binds to the extracellular domain of human TIGIT with high affinity (KD = 0.135 nM) and specificity, and efficiently blocks the interaction between TIGIT and its ligands PVR or PVR-L2. Cell-based assays show that BGB-A1217 significantly enhances T-cell functions. In addition, BGB-A1217 induces antibody dependent cellular cytotoxicity (ADCC) against Treg cells, activates NK cells and monocytes, and removes TIGIT from T cell surfaces in an Fc-dependent manner, In vivo, BGB-A1217, either alone or in combination with an anti-PD-1 mAb elicits strong immune responses and potent anti-tumor efficacy in pre-clinical models. Moreover, the Fc effector function is critical for the anti-tumor activity of BGB-A1217 in a syngeneic human TIGIT-knock-in mouse model. The observed anti-tumor efficacy is associated with a pharmacodynamic change of TIGIT down-regulation and Treg reduction. These data support the selection of BGB-A1217 with an effector function competent Fc region for clinical development for the treatment of human cancers.

A phase II study of sEphB4-HSA in metastatic castration-resistant prostate cancer.

84 Background: Ephrin receptors and their membrane-localized ligands induce bidirectional signaling and facilitate tumor-stroma interactions. Expression of EphB4 is increased in prostate cancer tissue and cell lines and retained in castration resistant states, and can promote cell migration, invasion, and metastases. Blocking the EphB4-EphrinB2 pathway, which can be accomplished by soluble EphB4 conjugated to human serum albumin (sEphB4-HSA), has efficacy in preclinical models of aggressive prostate cancer. A phase I clinical trial of sEphB4-HSA led to response or stable disease in 56% of patients, with no grade 4 or 5 related adverse events, and combination pembrolizumab sEphB4-HSA led to a 52% response rate in EphrinB2 expressing urothelial cancer. We hypothesized that targeting the EphB4-EphrinB2 pathway may serve as a therapeutic target in the treatment of metastatic castration resistant prostate cancer (mCRPC). Methods: We conducted a single arm, phase II trial in patients with progressive mCRPC and treatment with at least one second generation androgen receptor (AR)-targeted therapy but no more than three prior therapies for mCRPC. On Day 1 of each cycle patients received sEphB4-HSA 1000 mg IV, with cycle length 14 days cycles 1-6 and cycle length 21 days for cycle 7 and beyond. The primary endpoint was confirmed prostate specific antigen (PSA) response rate (confirmed decrease in PSA by > 50%). We employed a Simon two stage Minimax design, requiring two or more responses among the first 15 patients to enroll an additional ten patients. Results: Fourteen eligible patients enrolled in the study. Median age was 73.5 years (range 52-83), patients had a median baseline PSA value of 65.11 ng/mL (range 7.77-2850 ng/mL) and received a median of three prior therapies (range 1-3) for mCRPC. Ten patients received prior taxane for mCRPC or hormone sensitive prostate cancer. The median length of treatment with sEphB4-HSA was 6.5 weeks (range 2-35 weeks). The potentially treatment-related adverse events (AEs) that occurred in more than 25% of patients were hypertension (10 patients) and fatigue (7 patients). Three patients experienced a serious adverse event potentially related to therapy, including one patient with a grade 5 event (cerebral vascular accident) possibly related to study drug. No patient had a confirmed PSA response, and the study was stopped for futility. Thirteen patients had PSA progression ( > 25% increase in PSA), and one patient withdrew due to toxicity prior to having an evaluable PSA response. The median time to PSA progression was 28 days (95% CI 28-64 days), and median time to radiologic progression was 55 days (95% CI 55 days-NR). Of three patients with measurable disease, two had stable disease and one had progressive disease. Conclusions: In patients with mCRPC who progressed on prior second generation AR-targeted therapy, sEphB4-HSA monotherapy had no discernable anti-tumor activity. Clinical trial information: NCT04033432.