Preclinical Antitumor Efficacy of BAY 1129980-a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody-Drug Conjugate for the Treatment of Non-Small Cell Lung Cancer.

C4.4A (LYPD3) has been identified as a cancer- and metastasis-associated internalizing cell surface protein that is expressed in non-small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With the exception of skin keratinocytes and esophageal endothelial cells, C4.4A expression is scarce in normal tissues, presenting an opportunity to selectively treat cancers with a C4.4A-directed antibody-drug conjugate (ADC). We have generated BAY 1129980 (C4.4A-ADC), an ADC consisting of a fully human C4.4A-targeting mAb conjugated to a novel, highly potent derivative of the microtubule-disrupting cytotoxic drug auristatin via a noncleavable alkyl hydrazide linker. In vitro, C4.4A-ADC demonstrated potent antiproliferative efficacy in cell lines endogenously expressing C4.4A and inhibited proliferation of C4.4A-transfected A549 lung cancer cells showing selectivity compared with a nontargeted control ADC. In vivo, C4.4A-ADC was efficacious in human NSCLC cell line (NCI-H292 and NCI-H322) and patient-derived xenograft (PDX) models (Lu7064, Lu7126, Lu7433, and Lu7466). C4.4A expression level correlated with in vivo efficacy, the most responsive being the models with C4.4A expression in over 50% of the cells. In the NCI-H292 NSCLC model, C4.4A-ADC demonstrated equal or superior efficacy compared to cisplatin, paclitaxel, and vinorelbine. Furthermore, an additive antitumor efficacy in combination with cisplatin was observed. Finally, a repeated dosing with C4.4A-ADC was well tolerated without changing the sensitivity to the treatment. Taken together, C4.4A-ADC is a promising therapeutic candidate for the treatment of NSCLC and other cancers expressing C4.4A. A phase I study (NCT02134197) with the C4.4A-ADC BAY 1129980 is currently ongoing. Mol Cancer Ther; 16(5); 893-904. ©2017 AACR.

Evaluation of human pancreatic RNase as effector molecule in a therapeutic antibody platform.

Human antibody-ribonuclease (RNase) fusion proteins, referred to as immunoRNases, have been proposed as an alternative to heterologous immunotoxins, without their immunogenicity and unspecific toxicity issues. In this study, we investigated if human pancreatic RNase will be suitable as effector component in a therapeutic antibody development platform. We generated several fusion proteins consisting of tumor-specific human immunoglobulins (IgGs) and human pancreatic RNase. Transient mammalian cell production was efficient and IgG-RNases were purified to homogeneity. Antigen binding was comparable to the parental antibodies and RNase catalytic activity was retained even in the presence of 50-fold molar excess of human cytosolic RNase inhibitor (RI). Serum stability, cell binding and internalization of IgG-RNases were comparable to the parental IgGs. Despite these promising properties, none of the IgG-RNases revealed significant inhibition of tumor cell growth in vitro even when targeting different antigens putatively employing different endocytotic pathways. The introduction of different linkers containing endosomal protease cleavage sites into the IgG-RNase did not enhance cytotoxicity. Similarly, RI evasive human pancreatic RNase variants mediated only small inhibiting effects on tumor cell growth at high concentrations, potentially reflecting inefficient cytosolic translocation. Taken together, human pancreatic RNase and variants did not prove to be generally suitable as effector component for a therapeutic antibody drug development platform.

A recombinant immunotoxin derived from a humanized epithelial cell adhesion molecule-specific single-chain antibody fragment has potent and selective antitumor activity.

PURPOSE: Epithelial cell adhesion molecule (Ep-CAM) is a tumor-associated antigen overexpressed in many solid tumors but shows limited expression in normal epithelial tissues. To exploit this favorable expression pattern for targeted cancer therapy, an Ep-CAM-specific recombinant immunotoxin was developed and its antitumor activity investigated. EXPERIMENTAL DESIGN: The immunotoxin 4D5MOCB-ETA was developed by genetically fusing a truncated form of Pseudomonas aeruginosa exotoxin A (ETA) (ETA(252-608)KDEL) to the highly stable humanized single-chain antibody fragment (scFv) 4D5MOCB. Cytotoxicity of 4D5MOCB-ETA was measured in cell growth and leucine incorporation assays in vitro. Tumor localization and antitumor activity were assessed in athymic mice bearing established human tumor xenografts. RESULTS: Fusion of the toxin moiety to the scFv did neither affect its thermal stability nor its antigen-binding affinity. In vitro, 4D5MOCB-ETA potently and specifically inhibited protein synthesis and reduced the viability of Ep-CAM-positive carcinoma cells of diverse histological origins with IC(50)s ranging from 0.005 to 0.2 pM. Upon systemic administration in mice, 4D5MOCB-ETA showed similar organ distribution as the scFv 4D5MOCB and preferentially localized to Ep-CAM-positive tumor xenografts with a tumor:blood ratio of 5.4. The potent antitumor activity of 4D5MOCB-ETA was demonstrated by its ability to strongly inhibit the growth and induce regression of relatively large tumor xenografts derived from lung, colon, or squamous cell carcinomas. CONCLUSIONS: We describe for the first time the development of a fully recombinant Ep-CAM-specific immunotoxin and demonstrate its potent activity against solid tumors of various histological origins. 4D5MOCB-ETA is currently being evaluated in a Phase I study in patients with refractory squamous cell carcinoma of the head and neck.