Engineering tetravalent IgGs with enhanced agglutination potencies for trapping vigorously motile sperm in mucin matrix

Abstract

Multivalent antibodies such as sIgA can crosslink motile entities such as sperm and bacteria, creating agglomerates that are too large to permeate the dense mucin matrix in mucus, a process commonly referred to as immune exclusion. Unfortunately, sIgA remains challenging to produce in large quantities, and easily aggregates, which prevented their use in clinical applications. To develop sIgA-like tetravalent antibodies that are stable and can be easily produced in large quantities, we designed two IgGs possessing 4 identical Fab domains, with the Fabs arranged either in serial or in the diametrically opposite orientation. As a proof-of-concept, we engineered these tetravalent IgG constructs to bind a ubiquitous sperm antigen using a Fab previously isolated from an immune infertile woman. Both constructs possess at least 4-fold greater agglutination potency and induced much more rapid sperm agglutination than the parent IgG, while exhibiting comparable production yields and identical thermostability as the parent IgG. These tetravalent IgGs offer promise for non-hormonal contraception and underscores the multimerization of IgG as a promising strategy to enhance antibody effector functions based on immune exclusion.

Keywords: Agglutination; Antibodies; Antibody engineering; Contraception; Sperm; Tetravalent IgG.

Fig. 1.. Production and characterization of tetravalent anti-sperm IgG Abs.

(a) Schematic diagrams of anti-sperm IgG, Fab-IgG and IgG-Fab. (b) Non-reducing and reducing SDS-PAGE analysis of the indicated Abs (1 μg). (c) Demonstration of the purity and homogeneity of the indicated Abs (50–100 μg) using SEC-MALS analysis. Y-axis indicates the total percentage of Abs representing their theoretical molecular weights.

Fig. 2.. Multimerization markedly enhances the agglutination potency and kinetics of anti-sperm IgG Abs.

(a) Sperm agglutination potency of the parent IgG, Fab-IgC and IgG-Fab measured by the CASA-based quantification of the percentage of sperm that remains PM after Ab-treatment compared to pre-treatment condition. (b) The sperm agglutination potency of the Abs normalized to media control. (c) The sperm agglutination kinetics of the indicated Abs measured by the quantification of time required to achieve 90% agglutination of PM sperm compared to the media control. (d) The rate of sperm agglutination determined by the reduction in the percentage of PM sperm count at three timepoints after Ab-treatment compared to the media control. Purified sperm at the final concentration of 5 × 10⁶ PM sperm/mL was used for both agglutination potency and kinetics studies. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Lines indicate arithmetic mean values and standard deviation.

Fig. 3.. Scanning electron microscopy images of the agglutinated sperm.

20 million washed sperm were treated with IgG, Fab-IgG and IgG-Fab for 5 min and fixed using 4% paraformaldehyde.Images were obtained at 2500X magnification. Scale bar, 10 μm.

Fig. 4.. Tetravalent sperm-binding IgG constructs conserve the trapping potency of the parent IgG.

The trapping potency of the indicated Abs measured by quantifying the percentage of fluorescently labeled PM sperm in Ab-treated CVM using neural network tracker analysis software. 25 μg/mL of Abs and purified sperm at the final concentration of 5.8 × 10⁴ PM sperm/mL were used. Motavizumab (anti-RSV IgG) was used as the isotype control. Data were obtained from n = 6 independent experiments with 6 unique combinations of semen and CVM specimens. P values were calculated using a one-tailed t-test between control Ab- and anti-sperm Ab- treated sperm samples. * P < 0.05. Lines indicate arithmetic mean values and standard deviation.