General strategy for the generation of human antibody variable domains with increased aggregation resistance

Abstract

The availability of stable human antibody reagents would be of considerable advantage for research, diagnostic, and therapeutic applications. Unfortunately, antibody variable heavy and light domains (V(H) and V(L)) that mediate the interaction with antigen have the propensity to aggregate. Increasing their aggregation resistance in a general manner has proven to be a difficult and persistent problem, due to the high level of sequence diversity observed in human variable domains and the requirement to maintain antigen binding. Here we outline such an approach. By using phage display we identified specific positions that clustered in the antigen binding site (28, 30-33, 35 in V(H) and 24, 49-53, 56 in V(L)). Introduction of aspartate or glutamate at these positions endowed superior biophysical properties (non-aggregating, well-expressed, and heat-refoldable) onto domains derived from common human germline families (V(H)3 and V(κ)1). The effects of the mutations were highly positional and independent of sequence diversity at other positions. Moreover, crystal structures of mutant V(H) and V(L) domains revealed a surprising degree of structural conservation, indicating compatibility with V(H)/V(L) pairing and antigen binding. This allowed the retrofitting of existing binders, as highlighted by the development of robust high affinity antibody fragments derived from the breast cancer therapeutic Herceptin. Our results provide a general strategy for the generation of human antibody variable domains with increased aggregation resistance.

Fig. 1.

Effect of mutations in human antibody variable domains on aggregation resistance. Surface residues in variable heavy and light domains (human V_H3, human V_κ1) are targeted for substitution with aspartic acid (aspartate). Aggregation resistance of the domains is determined by measuring retained binding to superantigen after heating to 80 °C on phage (20). Mutations are mapped on the variable domain surface structure (blue: 100% retained binding; white: 0%; wild type residue: WT; means, standard deviation (SD) shown, n = 2). Numbering according to Kabat (30). Complementarity determining regions are indicated as H1, H2 for V_H and L1, L2 for V_L. (A) Single mutations in human V_H. (B) Single mutations in human V_L. (C) Double mutations in human V_H (Left) and human V_L (Right). (D) Mutations in human V_H repertoire (Left) and human V_L repertoire (Right). Synthetic repertoires closely mimicking CDR amino acid diversity in the natural antibody repertoire were generated (23). Mutant repertoires carry mutations in H1 (32D/33D) and L2 (52D/53D). Graph shows mean aggregation resistance of repertoires (***, p < 0.001).

Fig. 2.

Aggregation resistance of human variable domains. Representative human variable domains are targeted for substitution with aspartate/glutamate in CDR H1 (V_H) or L2 (V_L), expressed and purified (see SI Text for details). The graphs show sample turbidity after incubation at aggregation-promoting temperatures (as measured by absorbance at 360 nm; means, SD shown for single and double mutations, V_H: n = 3, V_L: n = 4). (A) Human V_H domains at 80 °C. (B) Human V_L domains at 85 °C.

Fig. 3.

Crystal structures of mutant human variable domains: structure of triple mutants of (A) human V_H and (B) human V_L (in tan). CDR regions are shown in yellow/blue/red. Mutant residues are highlighted (sticks). The structures of the mutant domains tightly superpose onto structures of representative human variable domains [with the exception of H3 which is conformationally diverse in antibodies (40); representative structures shown in gray].

Fig. 4.

Retrofitting of human variable domains (I): effects on biological activity of IgG. Variants of Trastuzumab (Herceptin) are generated by introduction of aspartate substitutions in CDR H1 and/or L2. Means, SD shown. (A) Binding to HER2 antigen on cells. The graph shows binding of IgG to SK-BR-3 breast cancer cells (n = 2). (B) Inhibition of cellular proliferation (as determined by incubation of SK-BR-3 cells with IgG; n = 2). (C) Serum concentrations (after intra-peritoneal injection of IgG into C57/BL6 mice at 1 mg/kg, n = 4).

Fig. 5.

Retrofitting of human variable domains (II): effects on antibody fragments. Variants of Trastuzumab (Herceptin) are generated by introduction of aspartate substitutions in CDR H1 and/or L2. (A) Affinity of mutants for recombinant HER2 (as determined by surface plasmon resonance using scFv fragments). (B) Aggregation resistance of mutants. The graph shows sample turbidity of scFv fragments at 85 °C (as measured by absorbance at 360 nm). (C) Aggregation resistance (visual appearance) before (-) and after (+) heating to 85 °C. Panel shows V_H domains (Left), V_L domains (Center), and paired as scFv fragments (Right).