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Figure S8 from Improving Intracellular Delivery of an Antibody–Drug Conjugate Targeting Carcinoembryonic Antigen Increases Efficacy at Clinically Relevant Doses In Vivo

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posted on 2024-03-04, 21:06 authored by Ian Nessler, Baron Rubahamya, Anna Kopp, Scott Hofsess, Thomas M. Cardillo, Nalini Sathyanarayan, Jennifer Donnell, Serengulam V. Govindan, Greg M. Thurber

Binding to CEACAM5 and CEACAM6. Binding of XAb and hMN14 were tested against CEACAM5 and CEACAM6. Mouse and human Fc binding beads (Bangs Laboratories) were incubated with XAb and hMN14, respectively. Then varying concentrations of biotinylated CEACAM5 or CEACAM6 were added, followed by streptavidin AF647 to quantify CEACAM binding (therefore, the affinity measured is monovalent). Fluorescence is normalized to adjust for different numbers of binding sites on mouse and human beads. The XAb binds both CEACAM5 and CEACAM6 (KD = 1.03 and 1.04 nM, respectively). hMN-14 only binds CEACAM5 (KD = 2.2 nM). However, XAb does not bind HT-1080 cells but binds HT-1080 cells transfected with CEACAM5 at a higher level than other CEACAM5 antibodies (Fig. S2, (1)). The results are consistent with XAb binding multiple epitopes on CEACAM5 to crosslink the receptor and induce rapid internalization. In addition, bivalent binding of two different antigens would not be anticipated to drive clustering-based internalization.

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ImmunoMedics (Immunomedics Inc)

National Institutes of Health (NIH)

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ARTICLE ABSTRACT

Solid tumor antibody–drug conjugates (ADC) have experienced more clinical success in the last 5 years than the previous 18-year span since the first ADC approval in 2000. While recent advances in protein engineering, linker design, and payload variations have played a role in this success, high expression and readily internalized targets have also been crucial to solid tumor therapy. However, these factors are also paradoxically connected to poor tissue penetration and lower efficacy. Previous work shows that potent ADCs can benefit from slower internalization under subsaturating doses to improve tissue penetration and increase tumor response. In contrast, faster internalization is predicted to increase efficacy under higher, tumor saturating doses. In this work, the intracellular delivery of SN-38 conjugated to an anti-carcinoembryonic antigen (anti-CEA) antibody (Ab) is increased by coadministering a noncompeting (cross-linking) anti-CEA Ab to improve efficacy in a colorectal carcinoma animal model. The SN-38 payload enables broad tumor saturation with clinically-tolerable doses, and under these saturating conditions, using a second CEA receptor cross-linking Ab yields faster internalization, which increases tumor killing efficacy. Our spheroid results show indirect bystander killing can also occur, but the more efficient direct cell killing from targeted intracellular payload release drives a greater tumor response. These results provide a strategy to increase therapeutic effectiveness with improved intracellular delivery under tumor saturating doses with the potential to expand the ADC target repertoire.

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