CD16A-Specific Tetravalent Bispecific Immune Cell Engagers Potently Induce Antibody-Dependent Cellular Phagocytosis (ADCP) on Macrophages
Authors: Wingert Susanne , Reusch Uwe , Beez Armin , Pahl Jens , Cerwenka Adelheid , Koch Joachim , Treder Martin 
Published: October 2019

Abstract
Introduction
Affimed has developed high affinity tetravalent bispecific immune cell engagers for redirected optimized cell killing (ROCK platform). Using anti-CD16A and anti-tumor target-specific antibody domains, the engagers activate NK cells to efficiently kill target cells. The most advanced ROCK-based immune cell engager, AFM13, targeting CD30 on tumor cells and CD16A on immune effectors, is currently being evaluated in several clinical trials to treat CD30-positive malignancies.
Based on the fact that CD16A is not exclusively expressed on NK cells, but also on macrophages, we hypothesized that CD16A-specific immune cell engagers would also be able to activate CD16A expressing macrophages through antibody-dependent cellular phagocytosis (ADCP) contributing to anti-tumor response.
Macrophages are an essential component of the innate immune system and are a major constituent of normal tissues. They can be broadly classified into different subtypes including M1 (classically activated, generally characterized as pro-inflammatory and immuno-supportive) and M2 (alternatively activated, primarily of an anti-inflammatory profile) subtypes. Those subtypes greatly differ in their phenotype and function and appear to be highly plastic. While M1 macrophages are generally considered to be tumoricidal, M2 macrophages are mostly tumorigenic, depending on their context within the tumor microenvironment. Therapeutic agents focusing on macrophages such as the CD47/SIRPa axis, CSF-1R antibodies and elimination of tumor-associated macrophages (TAMs) have recently come into focus in immuno-oncology.
Methods
Peripheral monocytes derived from primary human hematopoietic cells of healthy donors were used to generate various macrophage subtypes (unpolarized macrophages, M1, M2a, M2c) in vitro using well-defined cytokine cocktails. These subtypes were characterized phenotypically for their CD16A expression and a wide number of additional markers. Subsequently, they were used to investigate the ability of a number of different CD16A-specific immune cell engagers derived from Affimed´s ROCK platform and control antibodies in vitro to activate and induce ADCP of target cells by flow cytometry and microscopy.
Results
We demonstrated that all of the macrophage subtypes generated in this study expressed CD16A and mediated ADCP of tumor cells. In addition, we showed that ADCP of tumor cells by several CD16A-specific engagers was both fast and robust for all investigated macrophage subtypes. Specifically, ADCP was detected as early as 2 hours after co-incubation of tumor cells with M1 or M2 macrophages and CD16A-specific immune cell engagers. Using appropriate control antibodies, it was demonstrated that ADCP mediated by CD16A-specific immune cell engagers was selective and at least as potent as ADCP mediated by classical monoclonal antibodies pan-specific for Fc-gamma receptors.
Summary and conclusion:
We have demonstrated a new mechanism whereby Affimed´s CD16A-specific immune cell engaging antibodies eliminate tumor cells by ADCP, mediated by different subsets of macrophages. Our data suggest that these antibodies may have the potential to boost tumoricidal function within the tumor microenvironment. Future directions of leveraging innate immunity as a therapeutic option in immuno-oncology will be presented.

Disclosures
Wingert: Affimed: Employment. Reusch:Affimed: Employment. Beez:Affimed: Employment. Pahl:Affimed: Research Funding. Cerwenka:Affimed: Research Funding; Dragonfly Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment.