Engineered hapten-binding antibody derivatives for modulation of pharmacokinetic properties of small molecules and targeted payload delivery

Abstract

Hapten-binding antibodies have for more than 50 years played a pivotal role in immunology, paving the way to antibody generation (as haptens are very important and robust immunogens), to antibody characterization (as the first structures generated more than 40 years ago were those of hapten binders), and enabled and expanded antibody engineering technologies. The latter field of engineered antibodies evolved over many years and many steps resulting in recombinant humanized or human-derived antibody derivatives in multiple formats. Today, hapten-binding antibodies are applied not only as reagents and tools (where they still play an important part) but evolved also to engineered targeting and pretargeting vehicles for disease diagnosis and therapy. Here we describe recent applications of hapten-binding antibodies and of engineered mono- and bispecific hapten-binding antibody derivatives. We have designed and applied these molecules for the modulation of the pharmacokinetic properties of small compounds or peptides. They are also integrated as additional binding entities into bispecific antibody formats. Here they serve as non-covalent or covalent coupling modules to haptenylated compounds, to enable targeted payload delivery to disease tissues or cells.

Keywords: biotin; digoxigenin; protein engineering; protein structure; targeted therapy.

Figure 1

The first X‐ray structure of an antibody was that of the hapten‐binding antibody NEW without bound antigen ( PDB 7 FAB ). Shortly thereafter, the structure of the antibody MC/PC603 (shown here, PDB entry 2MCP 1984 by Padlan, Cohen & Davies) bound to its cognate hapten was solved. These structures demonstrated for the first time the molecular basis of antibody–antigen interactions. These and many more structures to come opened the field of rational structure‐based antibody engineering.

Figure 2

Structure of an antibody that specifically binds conjugated biotin (biocytinamide in this figure) but not free biotin ( PDB 4S1D). Note the negatively charged aspartate 31 and aspartate 52 of the VH domain lining the sides of the binding pocket. The negative charges of their side chains generate a charge repulsion which prevents entry of (COOH‐containing) free biotin. In contrast, biotin that is coupled to a payload and thereby is uncharged (in the same manner as biotcytinamide that was used to solve the structure) can enter and stay in the binding pocket without charge repulsion.

Figure 3

Engineered protein formats that have been applied to generate hapten‐binding bispecific antibodies. (A) 2 + 2 ‘Morrison‐Type’ bsAbs which are nowadays in most cases stabilized by interchain disulfides (indicated in yellow) in the attached scFvs. (B) 2 + 1 IgG‐derived bsAbs with knob‐into‐hole Fc regions and disulfide‐stabilized VH and VL, respectively, at the C‐termini of CH3. (C) Fab‐derived bsAb format that has a disulfide‐stabilized Fv attached to the C‐termini of CH1 or CL, respectively. (D) CrossMabs generate the desired H‐chain heterodimers via knob‐into‐hole mutations placed into their constant regions. They contain in addition one designed domain‐exchanged Fab arm to assure correct light chain pairing.

Figure 4

Engineered hapten‐binding antibodies enable covalent payload attachment via hapten‐positioned spontaneous disulfide shuffling. (A) Structure of the Fab fragment of a digoxigenin‐binding antibody (PDB 3RA7). (B) Position of the engineered cysteine in the rim region of hapten‐binding antibodies which is in close proximity to the binding pocket of most hapten binders yet does not interfere with antigen binding. (C) Hapten‐positioned spontaneous disulfide shuffling links thiol‐containing haptenylated payloads to the antibody.

Figure 5

Modulation of the pharmacokinetic properties of a small compound by use of engineered hapten‐binding antibodies. Pharmacokinetic parameters were analyzed in a mouse model. A haptenylated fluorophore (BiotDig‐Cy5) was applied (intravenously) and its presence in serum at different time intervals detected by fluorescence measurements 22. The level of hapten‐binding antibody was determined by ELISA (detecting the Fc region). Due to its small size, the hapten‐fluorophore by itself is rapidly cleared from the serum. (A) Non‐covalent complexing with hapten‐binding antibodies prolongs the serum half‐life significantly. Differences between payload levels (fluorescence) and IgG levels reveal that the non‐covalently linked payload becomes released over time from the PK‐modulating antibody. (B) Covalent attachment via hapten‐mediated disulfide shuffling generates stable antibody conjugates with even further improved serum half‐life.

Figure 6

Hapten‐binding bsAbs for targeted payload delivery. (A) Dig‐coupled flourophore complexed with bsAbs that target tumor‐associated cell surface antigens (e.g. HER2) accumulate the payload at the tumors. (B) Hapten‐coupled nucleic acids (Dig‐payload) can be coupled to bsAbs and delivered to and into target cells where payload (red) and targeting vehicles (green) separate in vesicular compartments.

Figure 7

Cell‐surface targeting and hapten‐binding bispecific antibodies as research tools. (A) Chemical structure of the small molecule DIG‐Cy5. (B) Binding possibilities of a 1 + 1 and 2 + 2 format emphasizing that a 2 + 2 BsAb can also bind monovalently. (C) Concentration‐dependent analysis of BsAb cell surface binding to a growth factor receptor (cMET) in H1993 as well as histogram analysis at preset concentration. Red curve = 1 + 1 IgG, green = 1 + 1 Fab‐scFv, blue = 2 + 2 IgG) (D) Histogram of MESF calibration beads used for generating a calibration curve for quantitation. (E) Visualization of a growth factor receptor (cMET) on tumor cells by confocal microscopy using the indicated antibodies. (F) Analysis of cMET cell surface expression and internalized cMET in H1993. Cells were incubated with antibodies for 60 min at 37°C. Signals for Met2v2 were corrected for the number of Dig‐binding scFv per antibody.