No abstract

We developed a method for deep mutational scanning of antibody complementarity-determining regions (CDRs) that can determine in parallel the effect of every possible single amino acid CDR substitution on antigen binding. The method uses libraries of full length IgGs containing more than 1000 CDR point mutations displayed on mammalian cells, sorted by flow cytometry into subpopulations based on antigen affinity and analyzed by massively parallel pyrosequencing. Higher, lower and neutral affinity mutations are identified by their enrichment or depletion in the FACS subpopulations. We applied this method to a humanized version of the anti-epidermal growth factor receptor antibody cetuximab, generated a near comprehensive data set for 1060 point mutations that recapitulates previously determined structural and mutational data for these CDRs and identified 67 point mutations that increase affinity. The large-scale, comprehensive sequence-function data sets generated by this method should have broad utility for engineering properties such as antibody affinity and specificity and may advance theoretical understanding of antibody-antigen recognition.

Synopsis: Checkpoint inhibitor immunotherapy can be associated with severe immune-related adverse events that can limit therapeutic efficacy. The authors show that Probody therapeutics effectively localize checkpoint inhibition to sites of tumor growth, thereby reducing toxicities and maintaining therapeutic efficacy.

Immune checkpoint blockade therapies have been shown to induce potent and durable anti-tumor immunity in many cancer types. Nevertheless, not all patients benefit from immunotherapy, and immune-related adverse events remain a problem. Recently, it has been demonstrated that Antibody Drug Conjugates (ADCs) are not only capable of killing cancer cells but also can act to induce the immunogenic cell death of tumor cells as well as directly activate dendritic cells. These results provided a rationale to combine ADCs with immunotherapy to enhance the potential of immune checkpoint blockade therapies in a broader population of patients. CytomX Therapeutics has developed a new class of antibodies called Probody™ therapeutics, designed to widen the therapeutic window by minimizing binding to target in healthy tissue while being specifically activated in the tumor microenvironment (TME) by tumor-associated proteases. Probody technology has been evaluated in preclinical studies in several antibody formats, with efficacy and increased safety windows observed for Probody therapeutics targeting the PD-1 pathway, Probody drug conjugates (PDCs) targeting highly expressed tumor antigens, and T-cell engaging bispecific Probody therapeutics. Here we extend our evaluation of the Probody platform to the combination of CX-2009, an investigational PDC targeting human CD166, with an investigational Probody therapeutic targeting PD-1. To evaluate the anti-tumor activity of PDC CX-2009 in a syngeneic mouse model, human CD166 was overexpressed on the surface of the CT-26 murine colon carcinoma cell line. The combination treatment of CX-2009 with a surrogate mouse anti-PD-1 Probody molecule significantly inhibited tumor growth in human CD166 positive CT-26 tumor-bearing mice as compared to CX-2009 or anti-PD-1 Probody molecule alone. Tumor rejection is partially dependent on CD8+ T cells as illustrated by the evidence of a CD8+ memory T cell response in a re-challenge assay, and a reduced activity of CX-2009 alone or in combination with a mouse anti-PD-1 Probody molecule after CD8+ T cell depletion. The immunogenic potential of CX-2009 was further evaluated in multiple in vitro assays using human cancer cells and human PBMCs. In contrast to its cytotoxic activity towards CD166+ tumor cells, CX-2009 spares T cells and may enhance T cell priming. These preclinical data demonstrate the potential utility of a combination of PDC CX-2009 with a Probody therapeutic targeting the PD-1 pathway. Generally, these data highlight the potential to combine ADCs or PDCs with immune checkpoint blockade therapies. PROBODY is a trademark of CytomX Therapeutics, Inc. Citation Format: Erwan Le Scolan, Tiffany Tse, Michael Krimm, Will Garner, Hikmat Assi, Jennifer Razo, Laurie Wong, Kenneth Wong, Victoria Singson, Jennifer Leong, Linnea Diep, Jennifer Richardson, Siew Schleyer, Dylan Daniel, Marcia Belvin, Michael Kavanaugh. A probody drug conjugate targeting CD166 (ALCAM) enhances preclinical antitumor activity of a probody therapeutic targeting PD-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3202.