Tumor-Specific Activation of an EGFR-Targeting Probody Enhances Therapeutic Index

Abstract

Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. To redirect the activity of antibodies recognizing widely distributed targets to the site of disease, we have applied a prodrug strategy to create an epidermal growth factor receptor (EGFR)-directed Probody therapeutic-an antibody that remains masked against antigen binding until activated locally by proteases commonly active in the tumor microenvironment. In vitro, the masked Probody showed diminished antigen binding and cell-based activities, but when activated by appropriate proteases, it regained full activity compared to the parental anti-EGFR antibody cetuximab. In vivo, the Probody was largely inert in the systemic circulation of mice, but was activated within tumor tissue and showed antitumor efficacy that was similar to that of cetuximab. The Probody demonstrated markedly improved safety and increased half-life in nonhuman primates, enabling it to be dosed safely at much higher levels than cetuximab. In addition, we found that both Probody-responsive xenograft tumors and primary tumor samples from patients were capable of activating the Probody ex vivo. Probodies may therefore improve the safety profile of therapeutic antibodies without compromising efficacy of the parental antibody and may enable the wider use of empowered antibody formats such as antibody-drug conjugates and bispecifics.

Preclinical studies of a Pro-antibody-drug conjugate designed to selectively target EGFR-overexpressing tumors with improved therapeutic efficacy

“…Activated PanP-DM1 was obtained by incubating PanP-DM1 with recombinant uPA in vitro as described previously and characterized using SDS-PAGE ( Fig S2). 13,25 Activated PanP-DM1 and activated PanP exhibited mostly equivalent ability to bind to EGFR-overexpressing A431 cells at saturating (10 mg/ml) and subsaturating (1 mg/ml) concentrations, suggesting that attachment of DM1 did not affect the uPA-mediated activation (Fig. 2B).…”

“…11,12 Recently, Desnoyers et al designed a pro-antibody based on cetuximab that improved the safety profile without compromising the pre-clinical efficacy. 13 Antibody-drug conjugates (ADCs) are emerging as powerful anti-tumor therapeutics that combine tumor-targeted antibodies with active cytotoxic agents. 14 Generally speaking, the ADC components consist of an antibody that targets internalized cell surface molecule and a highly cytotoxic compound.…”

Generation and characterization of a target-selectively activated antibody against epidermal growth factor receptor with enhanced anti-tumor potency

“…43 In another approach, the Probody TM Platform was developed to improve the safety profile of cetuximab. 24 The novel 'Prodrug' strategy involves tumor-specific activation achieved by 'mask sequence' and 'cleavable protease substrate'.…”

“…24,25 As shown in Figure 3A, the indicated peptide was fused to the light chain amino terminus of Pan. The sequence consists of blocking peptide (IYPPLLRTSQAM), substrate peptide (LSGRSDNH) and serine-glycine linker peptide (GSSGGSGGSGGSG).…”

“…1 It is noteworthy that a recent study has shown that a novel Probody TM technology platform was able to limit cutaneous side effects in preclinical study. 24 As a tumor-specific protease activated EGFR-directed antibody, a Probody TM remains inert in healthy tissues and systemic circulation, but is activated locally at the tumor site. 24,25 To some extent, the technology will be an excellent solution to the problem of potent skin toxicities when developing an ADCC-enhanced mAb from panitumumab.…”