Combinatorial Fc modifications for complementary antibody functionality

Abstract

Therapeutic monoclonal antibodies (mAbs) can be functionally enhanced via Fc engineering. To determine whether pairs of mAbs with different Fc modifications can be combined for functional complementarity, we investigated the in vitro activity of two HIV-1 mAb libraries, each equipped with 60 engineered Fc variants. Our findings demonstrate that the impact of Fc engineering on Fc functionality is dependent on the specific Fab clone. Notably, combinations of Fc variants of the same Fab specificity exhibited limited enhancement in functional breadth compared to combinations involving two distinct Fabs. This suggests that the strategic selection of complementary Fc modifications can enhance both functional activity and breadth. Furthermore, while some combinations of Fc variants displayed additive functional effects, others were detrimental, suggesting that the functional outcome of Fc mutations is not easily predicted. Collectively, these results provide preliminary evidence supporting the potential of complementary Fc modifications in mAb combinations. Future studies will be essential to identify the optimal Fc modifications that maximize in vivo efficacy.

Keywords: Broadly functional antibodies; Fc-modifications; complimentary combinations.

Figure 1.

Functional characterization of the Fc-engineered antibodies mAbs RI808 and RI10953. Fc variants of the two clones were evaluated for induction of ADCP, ADNP, ADCD, NK cell degranulation (%CD107a+ of NK cells), NK cell secretion of IFNγ (%IFNγ+ of NK cells), and NK secretion of MIP-1β (MIP-1β+ of NK cells) after immune complex formation with recombinant HIV Envelope glycoprotein. (a) The heatmaps show the Z-Scored values of each individual readout (columns) and Fc variant (rows) for RI808 (left) and RI10953 (right). (b+c) Principal Component Analysis (PCA) of the variant data for RI808 (b) and RI10953 (c). Each point represents one variant (as labeled). Color and spheres show a k-means clustering (k = 4). Loadings plot (small plots within the PCA) indicate the contribution of the different Fc functions to separation in the PCA. (d) Comparison of the respective cluster each variant of each clone was assigned to by the k-means clustering.

Figure 2.

Heterologous combination of engineered mAbs can modulate Fc activity. (a) The flower plots summarize the functional activity of the respective combinations (variants of RI808 in rows with variants of RI10953 in columns; the first column and row represent the combination of the same variant of the same clone). Each petal represents the average of the Z-scored value for the indicated feature. Assay-specific total antibody concentrations for lone Fc variants (first column/row) were the same as the total concentration used for the combinations. (b) Visual representation of expected vs. observed functional breadth. To calculate the expected functional activity, we assumed that both variants would contribute with half of the respective activity that was observed when tested uncombined. Observed is the actual measured data after Z scoring. (c) We calculated the difference of observed and expected value per function. Shown is the Z-Score of this difference matrix (per individual function). Combinations with a difference outside 95% confidence interval (Z-Score >|2|) are indicated (left variant is clone RI808, right variant is clone RI10953).