IgG2m4, an engineered antibody isotype with reduced Fc function

Abstract

The Fc region of an antibody mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and plays a key role in the in vivo half-life of an antibody. In designing antibody therapeutics, it is sometimes desirable that the antibody has altered Fc-mediated properties. In the case of a "benign blocker" antibody, it is often desirable to diminish or abolish the ADCC and CDC functions while retaining its PK profile. Here, we report a novel engineered IgG isotype, IgG2m4, with reduced Fc functionality. IgG2m4 is based on the IgG2 isotype with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S and P331S). An IgG2m4 antibody has an overall reduction in complement and Fc gamma receptor binding in in vitro binding analyses while maintaining the normal in vivo serum half-life in rhesus.

Figure 1

IgG2m4 constant region amino acid sequence and its alignment with those of IgG1, IgG2 and IgG4. The amino acid positions are indicated on top of each row of alignments according to the EU numbering system. The CH domains and hinge regions are indicated under the alignments. The positions for the four amino acid changes (H268Q, V309L, A330S and P331S) are presented in bold and underlined.

Figure 2

IgG2m4 expression. Both IgG1 and IgG2m4 antibodies with identical variable regions were expressed in stable CHO cells. An aliquot of each antibody was loaded onto SDS-PAGE, and run under reduced and non-reduced conditions. Lane A1, IgG1 antibody (non-reduced conditions); Lane B1, IgG2m4 antibody, (non-reduced conditions); Lane A2, IgG1 antibody (reduced conditions); Lane B2, IgG2m4 antibody (reduced conditions); Lane M, protein molecular weight markers in kilodaltons.

Figure 3

Fcγ receptor binding. Purified IgG1 and IgG2m4 antibodies (Fig. 2), along with controls were serially diluted for an ELISA Fcγ receptor binding assay. Experimental details were described in the Materials and Methods section. (A) FcγRI; (B) FcγRIIa H131; (C) FcγRIIb/c; (D) FcγRIII V158 and (E) FcγRIII F158. Closed circle, IgG1; closed square, IgG2m4 and closed triangle, negative control.

Figure 4

C1q binding. Purified IgG1 and IgG2m4 antibodies were serially diluted for an ELISA based C1q binding assay. Experimental details were described in the Materials and Methods section. Closed circle: IgG2m4 antibody, closed square: IgG1.

Figure 5

Rhesus monkey in vivo PK study. The serum concentration-time data for monkey 1 (filled circles) and monkey 2 (open circles). The two-compartment model fits are shown for monkey 1 (solid line) and monkey 2 (dotted line).