Characterization of the bispecific VHH antibody tarperprumig (ALXN1820) specific for properdin and designed for low-volume administration

Abstract

The bispecific antibody tarperprumig (ALXN1820) was developed as a treatment option for diseases involving dysregulated complement alternative pathway (AP) activity that could be administered in small volumes, either subcutaneously or intravenously. Tarperprumig incorporates a C-terminal variable domain of a heavy chain only antibody (VHH) that binds properdin (FP) connected via a flexible linker to an N-terminal VHH that binds human serum albumin (HSA). The purified bispecific VHH antibody exhibits an experimental molecular weight average of 27.4 kDa and can be formulated at > 100 mg/mL. Tarperprumig binds tightly to FP and HSA with sub-nanomolar affinity at pH 7.4 and can associate simultaneously with FP and HSA to form a ternary complex. Tarperprumig potently and dose-dependently inhibits to completion in vitro AP-dependent complement C5b-9 formation, AP-dependent hemolysis, and the AP deposition of C3, FP and C9. X-ray crystallography revealed that the isolated FP-binding VHH recognizes the thrombospondin repeat 5 domain of FP, thereby preventing FP from binding to the AP convertase owing to severe steric hindrance. Tarperprumig cross-reacts with cynomolgus monkey FP and serum albumin. In summary, tarperprumig exhibits properties tailored for subcutaneous administration and is currently in clinical development for the treatment of complement AP-related disorders.

Keywords: Antibody analytics; Bispecific VHH antibody; antibody engineering; antibody structure; bispecific; characterization; complement alternative pathway; complement inhibition; preclinical study; properdin; tarperprumig.

Figure 1.

Sequence and physical properties of tarperprumig. (a) Amino acid sequence of tarperprumig. The final purified protein is recovered with an N-terminal pyroglutamate from cyclization of the N-terminal glutamine. The three CDRs within each VHH domain are boxed. The HSAbinding VHH domain is followed by the (Gly4-Glu)2-Gly4 linker which is highlighted in black and is in white font. This is followed by the fp-binding VHH domain. (b) Tarperprumig is smaller than an IgG, migrating at the expected size of around 28 kDa gauged against size markers in a non-reducing, denaturing 4–12% NuPAGE BisTris gel (10 µg applied). (c) Non-reducing CE-SDS electropherogram profile of tarperprumig formulated at >100 mg/mL.

Figure 2.

Tarperprumig can bind hFP and HSA simultaneously. (a) A mixture of tarperprumig, FP and HSA (black trace) formed a larger earlier eluting peak from SEC than individual protein components or all binary protein combinations. (b) HSA and FP do not interact in the absence of tarperprumig.

Figure 3.

Tarperprumig potently and selectively inhibits AP activation of both C3 and C5. Wieslab® assay of the inhibition by tarperprumig (green traces) of membrane attack complex formed by AP (a), CP (b) and LP (c) pathways of human serum, in comparison with the IgG based C5-blocking mAb N19-8 (red traces). Inhibition by tarperprumig of APdependent hemolysis in 20% human serum (d), and in 40% (red trace), 60% (green trace) and 80% (black trace) human serum (e). Inhibition of deposition of C3 (f), FP (g), and C9 (h). Red lines in panels (d), (f), (g) and (h) are fits. IC50 values are located on the figure.

Figure 4.

The fp-binding VHH of tarperprumig recognizes an epitope in TSR5. (a) Outline of the FP domain structure with two FB interacting regions indicated. (b, c) examples of the 2mF_o-DF_c electron density contoured at 1.75 σ with the final atomic model. (d) Cartoon representation of the complex revealing that the VHH epitope is located exclusively in FP TSR5. (e) Open book view of the intermolecular interface. The footprint of the VHH on FP is indicated by the orange area (left) whereas the footprint of FP on TPP3077 is outlined in light blue (right). The positions of selected residues in the interface are indicated. (f, g) magnified views of the interface with atoms colored as in panels b and c. Dotted lines indicate hydrogen bonds and salt bridges connecting FP and TPP-3077. Water molecules are shown as pink spheres.

Figure 5.

Structural basis for tarperprumig inhibition of the AP. (a) The sequence of the epitope explains the selective specificity of TPP-3077 for hFP and cFP. The triangles indicate the epitope on FP that is bound by TPP-3077. (b) The crystal structure of the fp-stabilized proconvertase from PDB entry 7NOZ used for the comparison. (c) The superposition of FPΔ23 from the TPP-3077 complex (domains colored as in Figure 4a) with FP from the proconvertase complex in panel b (colored gray) illustrates that interaction with TPP-3077 does not induce significant conformational changes in the convertase-binding TSR5 and the TSR6 index finger loop. A small difference at the N-terminal end of TSR4 is due to crystal packing interactions and flexibility in this end of TSR4 is a common feature. (d) Model illustrating the major steric clash that would occur if the proconvertase attempted to interact with TPP-3077 bound FP. In panel d, carbon atoms in TPP-3077 are shown in orange, whereas carbon atoms in FP are colored as in panel b.