Intra-Domain Cysteines (IDC), a New Strategy for the Development of Original Antibody Fragment–Drug Conjugates (FDCs)

International audienceAntibody–drug conjugates (ADCs) derived from a full immunoglobulin-G (IgG) are associated with suboptimal solid-tumor penetration and Fc-mediated toxicities. Antibody fragment–drug conjugates (FDCs) could be an alternative. Nevertheless, innovative solutions are needed to implant cysteines as conjugation sites in the single-chain fragment variable (scFv) format, which is the backbone from which many other antibody formats are built. In addition, the bioconjugation site has the utmost importance to optimize the safety and efficacy of bioconjugates. Our previous intra-tag cysteine (ITC) strategy consisted of introducing a bioconjugation motif at the C-terminal position of the 4D5.2 scFv, but this motif was subjected to proteolysis when the scFv was produced in CHO cells. Considering these data, using three intra-domain cysteine (IDC) strategies, several parameters were studied to assess the impact of different locations of a site-specific bioconjugation motif in the variable domains of an anti-HER2 scFv. In comparison to the ITC strategy, our new IDC strategy allowed us to identify new fragment–drug conjugates (FDCs) devoid of proteolysis and exhibiting enhanced stability profiles, better affinity, and better ability to kill selectively HER2-positive SK-BR-3 cells in vitro at picomolar concentrations. Thus, this work represents an important optimization step in the design of more complex and effective conjugates

Internalization of Foldamer-Based DNA Mimics through a Site-Specific Antibody Conjugate to Target HER2-Positive Cancer Cells

International audienceInhibition of protein–DNA interactions represents an attractive strategy to modulate essential cellular functions. We reported the synthesis of unique oligoamide-based foldamers that adopt single helical conformations and mimic the negatively charged phosphate moieties of B-DNA. These mimics alter the activity of DNA interacting enzymes used as targets for cancer treatment, such as DNA topoisomerase I, and they are cytotoxic only in the presence of a transfection agent. The aim of our study was to improve internalization and selective delivery of these highly charged molecules to cancer cells. For this purpose, we synthesized an antibody-drug conjugate (ADC) using a DNA mimic as a payload to specifically target cancer cells overexpressing HER2. We report the bioconjugation of a 16-mer DNA mimic with trastuzumab and its functional validation in breast and ovarian cancer cells expressing various levels of HER2. Binding of the ADC to HER2 increased with the expression of the receptor. The ADC was internalized into cells and was more efficient than trastuzumab at inhibiting their growth in vitro. These results provide proof of concept that it is possible to site-specifically graft high molecular weight payloads such as DNA mimics onto monoclonal antibodies to improve their selective internalization and delivery in cancer cells

Branched pegylated linker-auristatin to control hydrophobicity for the production of homogeneous minibody-drug conjugate against HER2-positive breast cancer

International audienceTrastuzumab emtansine (Kadcyla®) was the first antibody-drug conjugate (ADC) approved by the Food and DrugAdministration in 2013 against a solid tumor, and the first ADC to treat human epidermal growth factor receptor2 positive (HER2+) breast cancer. However, this second generation ADC is burden by several limitationsincluded heterogeneity, limited activity against heterogeneous tumor (regarding antigen expression) and suboptimaltumor penetration. To address this, different development strategies are oriented towards homogeneousconjugation, new drugs, optimized linkers and/or smaller antibody formats. To reach better developed nextgeneration ADCs, a key parameter to consider is the management of the hydrophobicity associated with thelinker-drug, increasing with and limiting the drug-to-antibody ratio (DAR) of the ADC. Here, an innovativebranched pegylated linker was developed, to control the hydrophobicity of the monomethyl auristatin E (MMAE)and its cathepsin B-sensitive trigger. This branched pegylated linker-MMAE was then used for the efficientgeneration of internalizing homogeneous ADC of DAR 8 and minibody-drug conjugate of DAR 4, targeting HER2.Both immunoconjugates were then evaluated in vitro and in vivo on breast cancer models. Interestingly, this studyhighlighted that the minibody-MMAE conjugate of DAR 4 was the best immunoconjugate regarding in vitrocellular internalization and cytotoxicity, gamma imaging, ex vivo biodistribution profile in mice and efficientreduction of tumor size in vivo. These results are very promising and encourage us to explore further fragmentdrugconjugate development

Site-specific conjugation of auristatins onto engineered antibody fragments to target her2-positive breast cancer in vitro

poster + "pitch" de présentation du poster de 90sThe combination of a highly potent cytotoxic agent (drug) with a specific therapeutic monoclonal antibody (mAb) via a suitably constructed spacer arm (linker) (Figure 1) appear to be an ideal embodiment of the “magic bullet” concept leading to the development of a novel therapeutic class named Antibody-Drug-Conjugates (ADC). This armed antibodies can be viewed as a way to improve tumor-cell killing while sparing normal tissues. While ADC have been successfully implemented in clinical strategies for the treatment of hematological cancers, the case of solid tumors suffer from insufficient ADCs activity at the maximum doses that can be tolerated. Currently, almost all ADC in clinical trials are based on canonical IgG molecules associated with limitations including bad tumor penetration as well as Fc-mediated off-target toxicity, due to an increase of normal tissue exposure (due to long half-life via FcRn recycling) and cross reaction with immune cells (due to Fc R interactions). Thereby, the aim of our project is to use antibody fragments to try to circumvent this limitations.Our strategy is based on a site-specific conjugation of an auristatin derivative onto an engineered anti-HER2 antibody fragment including single chain fragment variable (scFv) of the trastuzumab antibody, generating new scFv-drug conjugates (SDC). Cysteines were judiciously incorporated in the scFv aminoacid sequence to allow controlled bioconjugation of a heterobifunctional linker, either cleavable (for monomethyl auristatin E) or noncleavable (for monomethyl auristatin F) (Figure 1)