Grabbing the Bull by Both Horns: Bovine Ultralong CDR-H3 Paratopes Enable Engineering of 'Almost Natural' Common Light Chain Bispecific Antibodies Suitable For Effector Cell Redirection

Abstract

A subset of antibodies found in cattle comprises ultralong CDR-H3 regions of up to 70 amino acids. Interestingly, this type of immunoglobulin usually pairs with the single germline VL gene, V30 that is typically very conserved in sequence. In this work, we have engineered ultralong CDR-H3 common light chain bispecific antibodies targeting Epidermal Growth Factor Receptor (EGFR) on tumor cells as well as Natural Cytotoxicity Receptor NKp30 on Natural Killer (NK) cells. Antigen-specific common light chain antibodies were isolated by yeast surface display by means of pairing CDR-H3 diversities following immunization with a single V30 light chain. After selection, EGFR-targeting paratopes as well as NKp30-specific binders were combined into common light chain bispecific antibodies by exploiting the strand-exchange engineered domain (SEED) technology for heavy chain heterodimerization. Biochemical characterization of resulting bispecifics revealed highly specific binding to the respective antigens as well as simultaneous binding to both targets. Most importantly, engineered cattle-derived bispecific common light chain molecules elicited potent NK cell redirection and consequently tumor cell lysis of EGFR-overexpressing cells as well as robust release of proinflammatory cytokine interferon-γ. Taken together, this data is giving clear evidence that bovine bispecific ultralong CDR-H3 common light chain antibodies are versatile for biotechnological applications.

Keywords: NK cell engagers; antibody engineering; bispecific antibodies; bovine ultralong CDR-H3 antibodies; common light chain; effector cell redirection; yeast surface display.

Figure 1

Overview about the generation of cattle-derived ultralong CDR-H3 common light chain bispecific antibodies. After immunization of cattle and library generation antigen-specific paratopes are enriched against both targets (shown in red color and yellow color). To this end, ultralong CDR-H3 regions encoding for stalk/knob architectures are specifically amplified and grafted onto a fixed chimeric Fab scaffold utilizing a single light chain. After selection, common light chain paratopes are reformatted into an IgG-like bispecific format exploiting a heavy chain heterodimerization technique (e.g. the SEED technology). Schemes generated using biorender (www.biorender.com). Model constructed with PYMOL v0.99 based on pdb entries 5dk3 and 5ilt. Individual paratopes based on stalk/knob structures are colored in red and yellow, respectively. Fixed VH region based on IGHV1-7 shown in dark green, utilized VL30 exploited as common light chain shown in light green. Constant regions of the heavy chains colored in dark grey, CLλ shown in light grey. Use of heavy chain heterodimerization technology resulting in two distinct heavy chains indicated by the use of dark blue and light blue segments.

Figure 2

Yeast surface display based selection of NKp30 targeting chimeric bovine x human Fab fragments by yeast surface display as well as sequence analysis after enrichment. (A) Within two sorting rounds a NKp30-binding population was enriched. A two-dimensional sorting strategy was applied to label for functional Fab assembly as well as for NKp30 binding. To this end, library cells were incubated with recombinant human his-tagged NKp30 at a concentration of 1 µM followed by staining using secondary detection reagents directed against the his-tag as well as against the constant region of the human lambda chain. (B) CDR-H3 alignment of sequence unique ultralong CDR-H3 paratopes obtained after library sorting. Sequence of IGHJ2-4 is also shown. Amino acids given in 1-letter code and in different colors. Alignment generated with Geneious Prime^® v2021.1.1.

Figure 3

Biochemical characterization of chimeric ultralong CDR-H3 common light chain bispecific antibodies via Biolayer interferometry. Kinetic measurements against recombinant human EGFR extracellular protein (A) or recombinant human NKp30 ECD (B). Bispecific entities 63D02x60F06 (top) or 63H02x60F06 (bottom) were loaded onto sensor tips. After sensor rinsing, antigen binding was conducted at different concentrations (100 nM, 50 nM, 25 nM and 12.5 nM for EGFR and 50 nM (25 nM for 63H02), 12.5 nM, 6.25 nM and 3.125 nM for NKp30) for 300 s, followed by a dissociation step in kinetics buffer for 300 s. (C) Simultaneous binding of 63D02x60F06 (top) or 63H02x60F06 (bottom) bispecifics against NKp30 ECD and EGFR ECD. Bispecifics were loaded to the sensor tips. After sensor rinsing two consecutive association steps were performed at 100 nM (Nkp30) and 100 nM (EGFR) for 200 s each.

Figure 4

Killing capacities of 26 generated cattle derived common light chain bispecifics (A) and cellular binding (B, left) as well as simultaneous binding to A431 and NKp30 ECD (B, right). Fluorescence-microscopy based killing assay using EGFR-positive A431 target cells and PBMC-purified NK effector cells at an E:T ratio of 5:1 (A) as well as cattle derived bsAbs at a concentration of 50 nM. A monospecific EGFR targeting Fc effector silenced negative control was included (black). Individual bsAbs based on EGFR targeting paratope 60F06 shown in green and entities based on EGFR-specific binder 60H05 given in red. Data was normalized to allow comparison of the independent experiments. Graphs show normalized means ± SEM of n = 3 different healthy donors. (B, left) Cellular binding of selected 60F06 based cattle-derived ultralong CDR-H3 common light chain bsAbs to EGFR expressing A431 cells at 100 nM. B7-H6 competing molecules shown in green, molecules targeting another epitope on NKp30 given in blue. An anti-HEL IgG control was included (grey). Cellular binding properties were detected via a fluorophore conjugated anti-human Fc antibody (B, right) Simultaneous binding properties of generated bispecifics. A431 cells were incubated with engineered common light chain bispecifics at 100 nM (green: B7-H6 competitors, blue: B7-H6 non competitors) followed by incubation with his-tagged NKp30 ECD at 200 nM. Simultaneous binding was detected via a fluorescence-labeled anti-his antibody.

Figure 5

Characterization of selected cattle-derived ultralong CDR-H3 common light chain bsAbs in terms of killing capacities and cytokine release. Fluorescence-microscopy based killing assay using EGFR-positive A431 target cells and PBMC-purified NK effector cells at an E:T ratio of 5:1. Analysis of dose-dependent (A) and maximum (B) target cell killing. B7H6 competitors shown in green, B7-H6 non competitors given in blue. Data was normalized to allow comparison of the independent experiments. Graphs show normalized means ± SEM of n = 8 different healthy donors. (C) Maximum killing capacities at different effector to target (E:T) ratios. Cattle derived common light chain bsAbs were applied at a concentration of 50 nM. Grey: basal killing activities of NK cells i.e. without addition of bsabs. Data was normalized to allow comparison of the independent experiments. Graphs show normalized means ± SEM of n = 3-4 different healthy donors. (D) Donor specific lysis capacities at an E:T ratio of 1:1 and 5:1. BsAbs were added at 50 nM (NKCE). Unstim: Basal killing of NK cells in the absence of bsAbs. Data was normalized to allow comparison of the independent experiments. Graphs show normalized means ± SEM of n = 4 different healthy donors. (E) NK cell-mediated IFN-γ using cytokine HTRF kits for quantification. Purified NK cells were co-cultured with A431 cells for 24 h at an E:T ratio of 5:1 prior to analysis. Graphs show box and whisker plots as superimposition with dot plots of 7 individual experiments. *** p≤0.001, ** p≤0.01. B7-H6 competing molecules shown in green, molecules targeting another epitope on NKp30 given in blue.