Nonclinical immunogenicity risk assessment for knobs-into-holes bispecific IgG1 antibodies

Abstract

Bispecific antibodies, including bispecific IgG, are emerging as an important new class of antibody therapeutics. As a result, we, as well as others, have developed engineering strategies designed to facilitate the efficient production of bispecific IgG for clinical development. For example, we have extensively used knobs-into-holes (KIH) mutations to facilitate the heterodimerization of antibody heavy chains and more recently Fab mutations to promote cognate heavy/light chain pairing for efficient in vivo assembly of bispecific IgG in single host cells. A panel of related monospecific and bispecific IgG₁ antibodies was constructed and assessed for immunogenicity risk by comparison with benchmark antibodies with known low (Avastin and Herceptin) or high (bococizumab and ATR-107) clinical incidence of anti-drug antibodies. Assay methods used include dendritic cell internalization, T cell proliferation, and T cell epitope identification by in silico prediction and MHC-associated peptide proteomics. Data from each method were considered independently and then together for an overall integrated immunogenicity risk assessment. In toto, these data suggest that the KIH mutations and in vitro assembly of half antibodies do not represent a major risk for immunogenicity of bispecific IgG₁, nor do the Fab mutations used for efficient in vivo assembly of bispecifics in single host cells. Comparable or slightly higher immunogenicity risk assessment data were obtained for research-grade preparations of trastuzumab and bevacizumab versus Herceptin and Avastin, respectively. These data provide experimental support for the common practice of using research-grade preparations of IgG₁ as surrogates for immunogenicity risk assessment of their corresponding pharmaceutical counterparts.

Keywords: Anti-drug antibodies; T cell epitope; T cell proliferation; bispecific antibody; ex vivo T cell assay; immunogenicity; in silico prediction; knobs-into-holes.

Figure 1.

Overview of the key steps in the generation of a T cell-dependent ADA response to a protein therapeutic such as a bispecific antibody (shown). Methods used in this study to evaluate the immunogenicity risk of monospecific and bispecific antibodies are highlighted: in silico prediction of peptides presented by MHC-II, DC internalization, T cell proliferation and MAPPs. This figure was adapted from “Anti-drug antibodies (ADA) Immunogenicity Assessment” by BioRender.com (2024). Retrieved from https://app.biorender.com/biorender-templates.

Figure 2.

DC internalization assay for immunogenicity risk assessment. The DC internalization index was calculated for each sample: median fluorescence intensity (MFI) total stain – MFI external stain. Samples analyzed include (a) monospecific and bispecific Tras/Bev variants, along with their corresponding research-grade or pharmaceutical-grade parental antibodies (10 human donors), (b) bispecific antibodies containing ATR-107 and bococizumab arms paired with bevacizumab (10 human donors), and (c) the clinical-stage one-armed antibody, onartuzumab (11 human donors). Different donors were used in each panel. The median and interquartile range are overlaid on the donor data points (black bars). The P-values (Wilcoxon matched-pairs signed rank test) from pairwise comparisons are shown: ns: not significant, p > 0.05; ** : p ≤0.01; and *** : p ≤0.001.

Figure 3.

Ex vivo BrdU-based T cell proliferation assay for immunogenicity risk assessment. Human donor-derived PBMCs (n = 72) were co-cultured with (a, b) monospecific and bispecific Tras/Bev antibodies, research-grade or pharmaceutical-grade parental antibodies, (c, d) high ADA monospecific antibodies, ATR-107 and bococizumab, compared to their respective bispecific antibodies with bevacizumab, and (e, f) clinical-grade onartuzumab. (a, c, e) Stimulation index (SI) is defined here as the ratio of the percentage of treated BrdU⁺CD4⁺CD3⁺ T cells to untreated BrdU⁺CD4⁺CD3⁺ T cells. Blue and open circles represent donors that were positive or negative, respectively, in response to KLH, and all donors were used for the following calculations. The median and interquartile range are overlaid on the donor data points (black bars). The P-values from pairwise comparisons (Wilcoxon matched-pairs signed rank test) are shown: ns: not significant, p > 0.05; ** : p ≤0.01; *** : p ≤0.001; and **** : p ≤0.0001. (b, d, f) Shown is the percentage of positive donors out of total number of donors for each sample. A positive response is defined here by using a cut-point value corresponding to the 95% quantile of the SI measurements of Avastin (reference antibody with low incidence of clinical ADAs). Donors with a SI greater than this cut-point are considered positive donors. The P-values (Fisher’s exact test) are shown: ns: not significant and **** : p ≤0.0001.

Figure 4.

MAPPs peptide spectrum matches (PSMs) and in silico immunogenic predictions for (a) the variable domains of pharmaceutical-grade and research-grade bevacizumab, plus the bevacizumab arm of bispecifics with one-cell mutations, (b) the variable domains of pharmaceutical-grade and research-grade trastuzumab, plus the trastuzumab arm of bispecifics with one-cell mutations, (c) C_H3 domain of antibodies including trastuzumab with and without the knob mutation; bevacizumab with and without hole mutations, and onartuzumab with the hole mutations, (d) C_H1 domain of trastuzumab and bevacizumab compared to their respective arms with one-cell mutations, and (e) C_L domain of trastuzumab and bevacizumab compared to their respective arms with one-cell mutations. Peptide clusters detected across 10 human donors for each antibody by MAPPs were collapsed into a single composite heatmap. The heatmap is scaled by area, which is normalized by dividing it by the maximum area among all antibody therapeutics within the corresponding panel. Solid blue bars represent regions of each molecule identified by NetMHCIIpan 4.0 EL to be immunogenic. The immunogenic regions contain one or more nine-mer peptides that are predicted to bind two or more common HLA-DR alleles (percentile rank ≤ 10%), with no matches in the human proteome, and are observed in < 93% of individuals in the OAS. CDR boundaries are labeled with Kabat numbering. the following point mutations are highlighted (red) V_L Q38E and Q38K, C_L V133E and V133K, V_H Q39E and Q39K, C_H1 S183E and S183K, and C_H3 T366W (knob); plus T366S, L368A and Y407V (hole).

Figure 4.

(Continued).