CRB-701: a Second-Generation Nectin-4 Targeted Antibody-Drug Conjugate with Optimized Stability and Pharmacokinetics for the Treatment of Solid Tumors.
Aug 2026· Cancer Research Communications· 0 citations
Medicine
TL;DR
Results suggest that CRB-701 may be a promising alternative therapeutic for the treatment of nectin-4 expressing tumors, providing a more favorable therapeutic window and potentially enabling regimens with higher doses and less frequent dosing than are required for EV, the only currently approved nectin-4 targeting ADC.
Abstract
The adhesion protein nectin-4 is a clinically validated tumor-selective antigen that is overexpressed across multiple cancer types and has been successfully exploited as a target for antibody-drug conjugates (ADCs), most notably enfortumab vedotin (EV), which is approved for treatment of urothelial carcinoma. Here, we report the design and preclinical characterization of a second-generation nectin-4 targeted ADC, CRB-701 (also identified as SYS6002). CRB-701 was synthesized using microbial transglutaminase (mTGase) technology to conjugate two molecules of monomethyl auristatin E (MMAE) via a cleavable linker to a highly selective, high-affinity anti-nectin-4 monoclonal antibody. CRB-701 exhibited potent in vitro cytotoxicity in nectin-4 expressing cell lines and in vivo antitumor activity in cell line-derived and patient-derived murine xenograft models of human cancer (cell line-derived models: PC-3-NECTIN4 prostate cancer, MDA-MB-468 breast cancer, and HT1376 bladder cancer; patient-derived model: BL0597 bladder cancer), exhibiting efficacy similar to or great than to EV across tumors with varying levels of nectin-4 expression. CRB-701 delivered high levels of MMAE upon internalization in tumor cells and was markedly more stable in circulation than EV, with lower systemic MMAE release, an approximately two-fold longer half-life, and at least two-fold higher safety margin in monkeys. These results suggest that CRB-701 may be a promising alternative therapeutic for the treatment of nectin-4 expressing tumors, providing a more favorable therapeutic window and potentially enabling regimens with higher doses and less frequent dosing than are required for EV, the only currently approved nectin-4 targeting ADC.
Antibody-drug conjugates (ADCs) have transformed the treatment of advanced breast cancer, yet resistance to HER2- and TROP2-directed ADCs is an increasing clinical challenge. New ADC targets should be linked to aggressive disease, retained after current ADC exposure, and sufficiently tumor-selective for therapeutic development. CD44 variant isoform 9 (CD44v9) is an attractive next-generation ADC target because it marks stem-like, invasive, and treatment-refractory breast cancer phenotypes while showing limited expression in most normal tissues. In prior work, we identified CD44v9 as a tumor-selective and therapeutically accessible surface antigen in aggressive breast cancer and demonstrated proof-of-concept efficacy with a chimeric anti-CD44v9-MMAF conjugate. To advance translation, we generated fully humanized anti-CD44v9 antibody variants.
CD44v9 expression was assessed in human breast cancer cell lines, patient-derived xenografts, tumor specimens, and normal tissues. Two fully humanized anti-CD44v9 variants, SUM24.2 and SUM24.3, were generated using CUMAb, a structural and energy-focused computational humanization tool, and veneering, a surface humanization approach , respectively. The parental chimeric antibody SUM24.1 and humanized variants were conjugated to monomethyl auristatin F (MMAF). Binding, internalization, and cytotoxicity were evaluated in triple-negative, inflammatory, and ADC-resistant breast cancer models. Antitumor activity was assessed in refractory in vivo models.
Humanized anti-CD44v9 variants SUM24.2 and SUM24.3 retained selective recognition of membrane-bound CD44v9 and supported efficient target-dependent internalization (p<0.01). SUM24.2-MMAF induced antigen-dependent cytotoxicity in CD44v9-positive breast cancer cells, whereas no cell-killing effect was observed in antigen-negative controls or human peripheral blood mononuclear cells. CD44v9 expression persisted in models resistant to FDA-approved ADCs, including trastuzumab deruxtecan- and sacituzumab govitecan-resistant models, and CD44v9-directed ADC therapy remained active in these settings, with retained selectivity, supporting a strategy mechanistically distinct from HER2- and TROP2-directed pathways. These results reproduced the activity observed in earlier chimeric proof-of-concept studies.
CD44v9 is a compelling ADC target for treatment-refractory breast cancer because it is associated with aggressive, stem-like, and therapy-resistant disease biology and remains targetable in models resistant to current breast cancer ADCs. Humanization preserved target recognition, internalization, and ADC activity, representing a key translational advance beyond chimeric proof-of-concept studies. These findings support further preclinical development of a humanized CD44v9-directed ADC for advanced breast cancer after resistance to existing ADCs.
Naoto T. Ueno, Dileep R. Reddy, McKenna E. Flynn, Yasuaki Anami, Zhaoxuan Yang, Minji Seo, Kyoji Tsuchikama, Jennifer A. Maynard, Jangsoon Lee. Preclinical Development of a Humanized CD44v9-Directed Antibody-Drug Conjugate for Treatment-Refractory Breast Cancer [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr A013.
N. Ueno, Dileep R. Reddy, McKenna E. Flynn et al.· Clinical Cancer Research· 0 citations
Integrins represent a large family of cell surface receptors that exist as heterodimers with essential roles in key biological processes such as cellular adhesion, motility, and cytokinesis. Among them, integrin beta-6 (IB6), which exclusively dimerizes with integrin alpha-v, has emerged as a clinically relevant target due to its restricted expression in normal adult epithelial tissues and elevated levels in solid tumors. High expression of IB6 is correlated with poor prognosis across multiple solid tumor types. PF-08046876 is an investigational antibody-drug conjugate (ADC) consisting of the anti-IB6 monoclonal antibody conjugated to a camptothecin-class topoisomerase I (TOP1) inhibitor, AMDCPT, using a traceless enzyme-cleavable glucuronide linker. The AMDCPT payload has been optimized for differentiation from other TOP1 inhibitors with improved potency, enhanced bystander activity, and reduced susceptibility to multidrug resistance (MDR) efflux mechanisms. PF-08046876 leverages the same antibody backbone from sigvotatug vedotin (SV) and binds IB6 without cross-reactivity to other alpha-v integrin complexes. In preclinical studies, PF-08046876 has demonstrated significant antitumor activity both in vitro and in vivo across multiple tumor models with IB6 expression, including non-small cell lung (NSCLC), head and neck squamous cell (HNSCC), urothelial (UC), pancreatic (PDAC), and esophageal (ESCA) carcinomas. These findings support further clinical development of PF-08046876 as a promising therapeutic candidate for the treatment of IB6-expressing solid tumors.
R. Mazahreh, C. Lim, Nicolas H Garcia et al.· Molecular Cancer Therapeutic...· 0 citations
Aim Although B-cell acute lymphoblastic leukemia (B-ALL) is highly responsive to antigen-directed immunotherapies, treatment resistance remains a major barrier to achieving durable responses in patients. We recently developed a novel VpreB1 (CD179a)-directed antibody-drug conjugate with calicheamicin (VpreB1-ADC) that exploits the restricted expression of VpreB1 within the surrogate light chain in early B cells, including B-ALL. In the present work, we investigated mechanisms of resistance to the VpreB1-ADC. Methods Mechanisms of resistance were evaluated using a TCF3::HLF B-ALL model, assessing target engagement parameters including VpreB1 surface expression and antibody internalization. The role of the multidrug resistance transporter ABCB1 (P-glycoprotein) was evaluated via pharmacologic inhibition, using tariquidar and zosuquidar, and enforced overexpression across multiple B-ALL cell lines. Sensitivity to alternative non-ABCB1 substrate payloads exatecan and PNU-159682 was also assessed. Results Resistant TCF3: HLF cells retained VpreB1 expression and efficient antibody internalization. Instead, resistance was driven by elevated ABCB1 expression and activity. ABCB1 inhibition with tariquidar or zosuquidar restored VpreB1-ADC sensitivity. Conversely, enforced ABCB1 overexpression conferred ADC resistance, which was reversed by ABCB1 inhibition. Cells with high ABCB1 activity remained fully sensitive to alternative payloads, including exatecan and PNU-159682, which are not ABCB1 substrates. Discussion ABCB1-mediated drug efflux drives intrinsic resistance to calicheamicin-conjugated ADCs in B-ALL. Combining ADCs with ABCB1 inhibitors or selecting payloads non-susceptible to ABCB1 efflux offer viable strategies to overcome resistance and optimize future ADC therapies.
Robin L. Williams, Xiaohong Wang, Jason Ostergaard et al.· bioRxiv· 0 citations
Pancreatic cancer remains a lethal malignancy with limited treatment options. Trophoblast cell surface antigen 2 (TROP2), which is frequently overexpressed in pancreatic tumors and closely associated with poor prognosis, represents a promising therapeutic target. hIMB1636-LDM is a novel antibody-drug conjugate (ADC) developed by our laboratory, comprising the anti-TROP2 antibody hIMB1636 covalently linked to cytotoxic payload lidamycin (LDM) via an uncleavable linker. In this study, we systematically evaluated the antitumor efficacy and underlying mechanism of action of hIMB1636-LDM in pancreatic cancer. The results showed that hIMB1636-LDM exhibited TROP2-dependent binding and internalization, as well as potent cytotoxicity (Half maximal inhibitory concentration (IC50): 0.27-0.5 nM) against pancreatic cancer cells. Mechanistically, beyond inducing cell cycle arrest and apoptosis, it was for the first time found to possess the ability to trigger immunogenic cell death (ICD). In vivo, hIMB1636-LDM at a dose of 0.8 mg/kg significantly suppressed tumor growth in both T3M4 and BxPC3 models, with no obvious toxic reactions observed. hIMB1636-LDM exerts potent antitumor activity through a dual mechanism combining targeted cytotoxicity and ICD induction. Its favorable safety profile, potent efficacy, and unprecedented ICD-inducing property highlight its potential as a promising therapeutic candidate for TROP2-positive pancreatic cancer.
APG157DS is a multi-component investigational drug which is currently the subject of multiple cancer trials. It has shown promising efficacy in patients with head and neck cancer. We report its immuno-modulatory effect in a syngeneic mouse model of glioblastoma (GBM). Long-term treatment with APG157DS led to durable tumor remission in 50% of mice, while all vehicle-treated mice reached humane endpoints within 42 days. Mechanistically, the drug induced selective repolarization of tumor-associated macrophages (TAMs) from an immunosuppressive Arg1high/iNOSlow phenotype to a tumoricidal Arg1low/iNOShigh state, accompanied by increased intratumoral recruitment of activated (NKp46+) natural killer cells and CD8+ cytotoxic T-cells. APG157DS also suppressed vascular endothelial growth factor (VEGF) and Hypoxia-inducible factor 1-alpha (HIF-1α) expression in tumors, further impairing tumor growth. Notably, APG157DS did not elicit off-target macrophage activation in peripheral tissues such as the spleen, highlighting its selective immune targeting. These findings provide a mechanistic rationale for further clinical development of APG157DS in GBM and potentially other immune-evasive cancers.
Shubhasmita Mohapatra, Adrian Guerrero, Neha Rahman et al.· International Journal of Mol...· 0 citations
Preclinical models play a critical role in the development of PSMA-targeted radiopharmaceuticals for prostate cancer. However, many existing models have practical limitations, including slow tumor growth, low engraftment rates, and restricted availability, and all human PSMA-positive lines are confined to immunodeficient hosts. We developed and characterized a novel PSMA-expressing transgenic cell line, CT26-PSMA, as a practical tool for preclinical screening of PSMA-targeting agents. The CT26-PSMA cell line was established by stable transfection of the murine colon carcinoma CT26 cell line with human PSMA using the Sleeping Beauty transposon system. PSMA expression was confirmed by RT-qPCR (reverse transcription quantitative polymerase chain reaction), flow cytometry, and radioligand saturation binding on intact cells. Two [68Ga]Ga-labeled radioconjugates—the well-established PSMA-617 and a newly synthesized conjugate (Conjugate-1)—were used to validate the functionality of the model through in vitro binding, uptake and internalization studies, and through ex vivo biodistribution in CT26-PSMA tumor-bearing athymic male nu/nu mice. The CT26-PSMA cell line demonstrated high and stable PSMA expression, with approximately 95% of cells expressing the biomarker and no measurable loss over 16 passages in antibiotic-free medium. Saturation binding gave a receptor density of ∼3.5 × 106 sites per cell, approximately four-fold higher than that of LNCaP cells (∼0.8 × 106), with dissociation constants that were indistinguishable between the two radioconjugates and between the two cell lines (Kd 9.0–11.6 nM). Subcutaneous tumors reached ~300 mm3 within 8–10 days of inoculation, with a take rate of 10/10 versus 1/10 for LNCaP (Fisher’s exact test, p = 1.2 × 10−4). Both radiotracers showed saturable, 2-PMPA-blockable binding and uptake in CT26-PSMA cells, confirming the functional activity of the recombinant receptor. Biodistribution studies revealed accumulation of both conjugates in CT26-PSMA tumors, with generally comparable tumor-to-background profiles. The CT26-PSMA cell line represents a robust, rapid, and reproducible platform for preclinical evaluation of PSMA-targeting radiopharmaceuticals, and its murine BALB/c origin permits engraftment in immunocompetent or minimally immunosuppressed hosts, whereas existing human PSMA-positive lines do not. It is intended as a screening platform rather than as a model of prostate cancer biology. The validation data obtained with [68Ga]Ga-labelled conjugates confirm the suitability of this cell line for future studies of PSMA-directed compounds.
A. S. Lunev, K. A. Petrosova, M. Rakhimov et al.· International Journal of Mol...· 0 citations
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