Fc-Small Molecule Antibody Mimetics

Abstract

Antibody therapeutics are a promising drug class due to their high specificity and favorable pharmacokinetics. While there are many methods for the development of antibodies specific to disease associated antigens, selecting antibodies against functional epitopes with high specificity and affinity can be difficult for certain epitopes. We describe a generalizable method for synthesizing antibody mimetics by site specifically conjugating small molecules (with high affinity and specificity to disease associated antigens) to an Fc fragment to develop drugs with the benefits of an antibody. As a proof of concept, an E269pAcPhe Fc antibody Fc fragment was produced and subsequently site-specifically labeled with a linker-modified folic acid compound to generate an Fc-folic acid antibody-mimetic. This was chosen as the model system because the high-affinity folate receptor FR-α is highly expressed in a number of cancer types including breast and ovarian cancer. The specificity of the Fc-folic acid conjugate was assessed via flowcytometry with the folate-receptor positive breast cancer cell line MDA-MB-231 by measuring Fc-folic acid binding in both the absence and presence of an excess of folic acid. Fc-small molecule conjugates could be developed into a unique class of antibody-like therapeutics.

Figure 1.

A) SDS-PAGE gel of the Fc-Folate conjugate and the E269pAcPhe Fc starting material. B) ESI-MS data of the Fc-Folate conjugate and the E269pAcPhe Fc starting material.

Figure 2.

Specificity of Folate-Fc binding to human breast cancer cells. Flow cytometry analysis of Folate-Fc binding to FR-α+ MDA-MB-231 cells detected with an allophycocyanin (APC) conjugated anti-human Fc mAb secondary antibody. Orange is secondary alone, blue is Folate-Fc and secondary and red is Folate-Fc, secondary, and 10eq of free folic acid. Conjugate binding was detected with an allophycocyanin (APC) conjugated anti-human Fc mAb secondary antibody.

Scheme 1.

Synthesis of the Fc-Folate antibody mimetic. E269pAcPhe Fc (10 mg/mL) was treated with 30 equivalents of aminoxy-modified folic acid in 100 mM sodium acetate (pH 4.5) with 10% DMSO for 48 hrs at 37 °C. Reaction resulted in quantitative yield verified by SDS-PAGE and ESI-MS.