PDF file - 77KB, Pharmacodynamic effects of LFA102 in the rat mammary gland

Cell line sensitivity table

PDF file - 59KB, Correlation between serum LFA102 and serum PRL over time in rats

PDF file - 55KB, Pharmacokinetic properties of LFA102 in rodents

PDF file - 113KB, Effect of LFA102 on serum PRL levels in rats

PDF file - 49KB, Cell line PRLR expression by WB

PDF file - 53KB, Effect of LFA102 monotherapy on DMBA tumor growth

Background Casitas B-lineage lymphoma proto-oncogene B (CBL-B) is an E3 ubiquitin ligase expressed in multiple immune cell lineages and is a master regulator of the immune response. NX-1607 is a first-in-class oral small molecule inhibitor of CBL-B that has demonstrated potent anti-tumor activity in animal models by reversing T cell dysfunction and overcoming suppressive signaling in the tumor microenvironment.1 Nurix has identified and characterized novel proximal biomarkers of CBL-B inhibition that correlate with anti-tumor activity in syngeneic tumor models. Assays for these biomarkers characterize the activity of NX-1607 in a first-in-human clinical trial (NCT05107674). Methods Agnostic screening campaigns utilizing flow cytometry protein phosphorylation signals and mass spectrometry-based methods to measure direct ubiquitination substrates identified multiple proprietary proximal biomarkers of CBL-B inhibition in activated T cells. Validation of these biomarkers in mouse and non-human primate (NHP) in vivo models, coupled with allometric scaling of pharmacokinetic (PK) profiles, were used to inform clinical dose selection. PK and pharmacodynamic (PD) data are currently being monitored in a Phase I trial with daily oral dosing in 21-day cycles. Results Phosphorylated hematopoietic lineage cell-specific protein 1 (pHS1), a regulator of T cell-receptor signaling, was identified as a robust and reproducible biologically relevant proximal biomarker for monitoring pharmacologic inhibition of CBL-B in whole blood. Validation of proximal biomarkers in vivo demonstrated target engagement with dose-dependent increases of pHS1 in CD8+ T cells in both mice and NHP dosed orally with NX-1607. As of June 16, 2022, 10 patients have enrolled on study at 4 ascending oral dose levels (5, 15, 25 and 50 mg once daily). Dose-proportional increases of pHS1-expressing T cells were observed in cycle 1. Preliminary PK data suggest a half-life of 4 to 9 hours and dose-proportional exposures with no apparent accumulation. Together these data are consistent with preclinical human dose projections and compare to pHS1 levels associated with anti-tumor activity in mouse models. Conclusions NX-1607 is a first-in-class oral CBL-B inhibitor displaying linear PK, dose-dependent target engagement as measured by the validated proximal biomarker, pHS1, and downstream signals of T cell engagement. PK and PD data derived from non-clinical and clinical studies have shown remarkable concordance with biomarker levels observed in the clinic corresponding to those levels associated with potent anti-tumor activity observed in mouse models. The study continues to enroll, and updated PK and PD data will be presented. Trial Registration NCT05107674 Reference Rountree R, Cohen F, Tenn-McClellan A, et al. Small molecule inhibition of the ubiquitin ligase CBL-B with NX-1607 results in potent T and NK cell mediated anti-tumor response. Cancer Res. 2021;81(Suppl. 13; poster #1595). Ethics Approval The study obtained ethics committee(s) approval, and participants gave informed consent before taking part.

Abstract The cell surface glycoprotein P-cadherin is highly expressed in a number of malignancies, including those arising in the epithelium of the bladder, breast, esophagus, lung, and upper aerodigestive system. PCA062 is a P-cadherin specific antibody–drug conjugate that utilizes the clinically validated SMCC-DM1 linker payload to mediate potent cytotoxicity in cell lines expressing high levels of P-cadherin in vitro, while displaying no specific activity in P-cadherin–negative cell lines. High cell surface P-cadherin is necessary, but not sufficient, to mediate PCA062 cytotoxicity. In vivo, PCA062 demonstrated high serum stability and a potent ability to induce mitotic arrest. In addition, PCA062 was efficacious in clinically relevant models of P-cadherin–expressing cancers, including breast, esophageal, and head and neck. Preclinical non-human primate toxicology studies demonstrated a favorable safety profile that supports clinical development. Genome-wide CRISPR screens reveal that expression of the multidrug-resistant gene ABCC1 and the lysosomal transporter SLC46A3 differentially impact tumor cell sensitivity to PCA062. The preclinical data presented here suggest that PCA062 may have clinical value for treating patients with multiple cancer types including basal-like breast cancer.

E3 ubiquitin ligases play critical roles in directing cellular protein fate by controlling the specificity of ubiquitin conjugation to substrate proteins and targeting them for cellular relocalization or degradation by the ubiquitin proteasome system. The E3 ubiquitin ligase CBL-B is expressed in immune cell lineages and negatively regulates activity of the T-cell receptor (TCR) by imposing a requirement for a costimulatory signal to mount a productive immune response upon TCR engagement. Mice deficient in Cbl-b, and more specifically in the RING Zn-finger ligase domain of Cbl-b, demonstrate a tumor rejection phenotype mediated by CD8+ T cells (Paolino et al., JI, 2011). We have reproduced these results and demonstrate that Cbl-b deficient mice show enhanced anti-tumor activity. In addition, we show that CD4+ and CD8+ T cells from mice deficient in Cbl-b have 5 to 10-fold enhanced secretion of IL-2 and IFN γ when stimulated ex vivo. These data provide a genetic rationale for the development of a small molecule inhibitor of CBL-B ligase activity for use in patients with tumor-mediated immune suppression of effector T cells. We have identified a series of small molecule inhibitors of CBL-B activity with biochemical potency at low nanomolar concentrations. CBL-B inhibitors increased cytokine secretion in vitro at low nanomolar concentrations, as measured by IL-2 and IFN γ secretion, in primary human and mouse T cells stimulated with CD3/CD28 or CD3 alone. The compounds also stimulated proliferation and elevated levels of the T cell surface activation markers CD25 and CD69. CBL-B inhibitors enhanced an antigen recall response in human PBMCs ex vivo, as measured by approximately 5-fold higher secretion of GM-CSF, TNF-α and RANTES, and demonstrated effects in an ex vivo model of exhausted T cell function. Oral dosing of an optimized CBL-B inhibitor enhanced anti-CD3 stimulated T cell activation in mouse CD4+ and CD8+ T cells, demonstrating a dose proportional pharmacodynamic effect. Oral administration over 28 days in the syngeneic CT-26 tumor model was well tolerated and resulted in single agent tumor growth inhibition. These data support the continued advancement of small molecule oral CBL-B inhibitors for future development in immuno-oncology. Citation Format: Jennifa Gosling, Ryan Rountree, Asad Taherbhoy, Chenbo Wang, Thomas Cummins, Frederick Cohen, Hiroko Tanaka, Dahlia Weiss, Mario Cardozo, Christopher Karim, May Tan, Joseph Juan, Austin Tenn-McClellan, Szerenke Kiss von Soly, Julie Sheung, Kathleen Boyle, Ketki Dhamnaskar, Katherine Kurylo, Jilliane Bruffey, Jennifer McKinnell, Dane Karr, Andria Christianson, Anne-Renee Van Der Vuurst de Vries, Pallavur Sivakumar, Mark Gallop, Paul A. Barsanti, Anjanabha Saha, Neil F. Bence, Christoph W. Zapf. Genetic and pharmacologic evaluation of the ubiquitin ligase CBL-B as a small-molecule, tumor immunotherapy target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2696.

Abstract The cell surface glycoprotein P-cadherin is an attractive target for an antibody-drug conjugate (ADC) therapy as it is known to be highly expressed in a number of malignancies, including those arising in the epithelium of the breast, lung, bladder, esophagus, stomach, endometrium and colon, among others. In breast cancer, P-cadherin is frequently overexpressed in high grade invasive tumors and is a reliable marker of the basal-like breast cancer molecular subtype, a disease with no effective therapeutic treatment options. Based on the expression profile of P-cadherin in human cancer, a highly selective and potent ADC was developed to target cancer types overexpressing this glycoprotein. This ADC consists of a fully human anti-P-cadherin-specific antibody conjugated to the potent maytansine-derived microtubule-disruptor, DM1, via an SMCC non-cleavable thioether linkage (technology licensed from ImmunoGen, Inc.). In vitro, the ADC was demonstrated to selectively bind P-cadherin expressing cell lines, to rapidly internalize and traffic to lysosomes, and to release a sufficient amount of activated payload to potently induce a cytotoxic response in cell viability assays. Profiling of activity in a cell line panel indicated that this ADC can effectively target and kill P-cadherin-positive cancer cells representing breast, head and neck, and bladder carcinomas. In vivo, the ADC was highly efficacious in numerous relevant xenograft models of P-cadherin expressing cancers, including breast, head and neck, bladder and lung. From this promising cellular and in vivo activity, this ADC may be an effective treatment for patients with P-cadherin positive cancers of high unmet medical need. Citation Format: Daniel Menezes, Tinya J. Abrams, Christopher Karim, Yan Tang, Chi Ying, Kathy Miller, Christie Fanton, Majid Ghoddusi, Zhen Wang, Montesa Patawaran, Nancy Pryer, Emma Lees, Jason Damiano. Development and activity of a novel antibody-drug conjugate for the treatment of P-cadherin expressing cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1682. doi:10.1158/1538-7445.AM2015-1682

Abstract Numerous lines of evidence suggest that the polypeptide hormone prolactin (PRL) may contribute to breast and prostate tumorigenesis through its interactions with the prolactin receptor (PRLR). Here, we describe the biologic properties of LFA102, a humanized neutralizing monoclonal antibody directed against the extracellular domain of PRLR. This antibody was found to effectively antagonize PRL-induced signaling in breast cancer cells in vitro and in vivo and to block PRL-induced proliferation in numerous cell line models, including examples of autocrine/paracrine PRL activity. A single administration of LFA102 resulted in regression of PRL-dependent Nb2-11 tumor xenografts and significantly prolonged time to progression. Finally, LFA102 treatment significantly inhibited PRLR signaling as well as tumor growth in a carcinogen-induced, estrogen receptor-positive rat mammary cancer model as a monotherapy and enhanced the efficacy of the aromatase inhibitor letrozole when administered in combination. The biologic properties of LFA102, elucidated by the preclinical studies presented here, suggest that this antibody has the potential to be a first-in-class, effective therapeutic for the treatment of PRL-dependent cancers. Mol Cancer Ther; 12(3); 295–305. ©2012 AACR.

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Numerous lines of evidence suggest that the polypeptide hormone prolactin (PRL) may contribute to breast and prostate tumorigenesis through its interactions with the prolactin receptor (PRLR). Here we describe the biological properties of LFA102, a humanized neutralizing monoclonal antibody directed against the extracellular domain of PRLR. This antibody was found to effectively antagonize PRL-induced signaling in breast cancer cells in vitro and in vivo and to block PRL-induced proliferation in numerous cell line models, including examples of autocrine/paracrine PRL activity. A single administration of LFA102 resulted in regression of PRL-dependent Nb2-11 tumor xenografts and significantly prolonged time to progression. Finally, LFA102 treatment significantly inhibited PRLR signaling as well as tumor growth in a carcinogen-induced, estrogen receptor (ER)-positive rat mammary cancer model as a monotherapy and enhanced the efficacy of the aromatase inhibitor letrozole when administered in combination. The biological properties of LFA102, elucidated by the preclinical studies presented here, suggest that this antibody has the potential to be a first in class, effective therapeutic for the treatment of PRL-dependent cancers.

Abstract The prolactin receptor (PRLR) is a class I cytokine receptor frequently expressed in breast and prostate cancer. The polypeptide hormone prolactin (PRL) has been demonstrated to induce PRLR signaling through the Jak/Stat, PI3-kinase/AKT and MAPK pathways, leading to cell proliferation and survival. Breast- and prostate-specific overexpression of PRL in transgenic mice leads to a higher incidence of mammary and prostate tumors, respectively. In addition, the PRLR locus is the site of frequent viral integrations in MMTV-derived mammary tumors. Elevated serum PRL levels in humans have been correlated with an increased risk for breast cancer, and an analysis of more than 3000 breast tumor specimens indicates that PRLR is expressed with high prevalence (60-70% of tumors) across all breast cancer subtypes. In prostate cancer specimens, the presence of prolactin and phosphorylated Stat5 have been reported to be associated with high-grade tumors and poor clinical outcomes, suggesting a role of the PRL/PRLR signaling pathway in the pathology of this disease as well. All of these lines of evidence support the hypothesis that targeting the PRL/PRLR axis may be a new approach for addressing unmet medical need in these tumor types. LFA102 is a Human Engineered™ anti-PRLR antibody of the IgG1 isotype that neutralizes the function of PRLR through a nonligand competitive binding interaction. LFA102 blocks PRL-induced signaling and proliferation in T47D and MCF7 human breast cancer cells in vitro, and abolishes PRL-induced phospho-Stat5 signaling in T47D xenograft tumors in vivo. This antibody also cross-reacts with and neutralizes rat PRLR and is capable of potently regressing PRL-dependent Nb2-C11 pre-T cell lymphoma tumors in vivo. In vitro studies have shown that LFA102 can mediate antibody-dependent cellular cytotoxicity (ADCC) and inhibit the PRL-dependent release of the proangiogenic factor VEGF from breast cancer cells. Thus, there are multiple potential mechanisms through which LFA102 could show antitumor activity in vivo. Preclinical toxicological studies of LFA102 indicate that this therapeutic is well tolerated and exhibits a normal pharmacokinetic profile in relevant animal species. The safety and pharmacokinetics of LFA102 in humans are currently being evaluated in a phase I healthy volunteer trial. A phase 1b trial in breast and prostate cancer is planned to evaluate the efficacy of this antibody in patient populations predicted to have the highest probability of benefiting from an anti-PRLR therapeutic. This presentation will provide a summary of the preclinical data supporting the clinical development of LFA102. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr DDT02-02. doi:10.1158/1538-7445.AM2011-DDT02-02