Abstract 677: Effective depletion of M2 macrophages by CD206-NAMPT-ADCs
Authors: Berndt Sandra , Filarsky Katharina , Smith Patrick , Boehnke Niels , Berger Markus , Eser Fionnuala McAleese , Lerchen Hans-Georg , Ellinger Phillip , Gehrmann Mathias , Wu Jim , Hildebrand Dominic , Kreft Bertolt , Gritzan Uwe 
Published: September 2020

Abstract
Introduction/ Purpose of study Targeting M2 macrophages as an immunosuppressive cell population within the tumor-microenvironment has become an important effort within preclinical research to achieve and enhance anti-tumor efficacy. In contrast to reprogramming approaches, we focused on effective and specific depletion of M2 macrophages (Mphs) by a new antibody-drug-conjugate (ADC) comprising a CD206-binding antibody conjugated to a nicotinamide phosphoribosyl transferase (NAMPT) toxophore which was proved to be very potent in cell killing.
Description of procedures Human (hu) and mouse (ms) cross-reactive CD206 antibodies were identified by phage display using recombinant hu/ms CD206-extracellular domains. Selected clones showing strong CD206 binding by ELISA were reformatted into huIgG1 format and cellular targeting confirmed by FACS binding and internalization using CD206-transfected HEK cells. Antibodies were conjugated to a NAMPT toxophore by Cys-conjugation using two different non-cleavable linkers. Murine peritoneal Mphs collected after thioglycolate stimulation were polarized to M1 (LPS + IFNγ) or M2 (IL4 + IL13 + PGE2) within 24h and characterized for cytokines and typical markers via FACS (CD206, CD163, CD80/MHCII) and qPCR (iNOS, TGFβ, ARG1). After 96h treatment with either CD206- or isotype-NAMPT-ADCs, viability of Mphs was determined. In vivo pharmacokinetics (PK) of unconjugated antibodies were analyzed in plasma of female BALB/c mice for up to 72h.
Summary of data Of the resulting 57 hu/ms cross-reactive CD206-binding antibodies, TPP-17829 and TPP-17836 were identified with single-digit nanomolar binding affinity and strong internalization in CD206-transfected cells as a prerequisite for an ADC approach. Conjugation of the antibodies to NAMPT toxophores yielded drug-antibody-ratios of 4-7 as determined by SEC-UV without significant aggregation. These two CD206-NAMPT-ADCs successfully depleted polarized M2-Mphs characterized by high CD206 expression, whereas isotype-NAMPT-ADC controls had no effect. While the unconjugated NAMPT toxophore was potent in depleting primary low-proliferating M1/M2 Mphs, by comparison, the anti-proliferative toxophore KSP (kinesin-spindle-protein inhibitor) was completely ineffective. PK analysis of TPP-17829 and TPP-17836, however, revealed a rapid plasma clearance within the first hours, most likely target-mediated via the liver, rendering them unacceptable for in vivo efficacy testing as NAMPT-ADCs.
Conclusions CD206-NAMPT-ADCs demonstrated proof of concept for specific depletion of M2-Mphs in vitro. However, specific in vivo depletion of M2 Mphs from tumors will be challenging with respect to fast plasma clearance observed for anti-CD206 antibodies.
Citation Format: Sandra Berndt, Katharina Filarsky, Patrick Smith, Niels Boehnke, Markus Berger, Fionnuala McAleese Eser, Hans-Georg Lerchen, Phillip Ellinger, Mathias Gehrmann, Jim Wu, Dominic Hildebrand, Bertolt Kreft, Uwe Gritzan. Effective depletion of M2 macrophages by CD206-NAMPT-ADCs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 677.