Development of Tetravalent, Bispecific CCR5 Antibodies with Antiviral Activity against CCR5 Monoclonal Antibody-Resistant HIV-1 Strains

Abstract

In this study, we describe novel tetravalent, bispecific antibody derivatives that bind two different epitopes on the HIV coreceptor CCR5. The basic protein formats that we applied were derived from Morrison-type bispecific antibodies: whole IgGs to which we connected single-chain antibodies (scFvs) via (Gly 4 Ser) n sequences at either the C or N terminus of the light chain or heavy chain. By design optimization, including disulfide stabilization of scFvs or introduction of 30-amino-acid linkers, stable molecules could be obtained in amounts that were within the same range as or no less than 4-fold lower than those observed with monoclonal antibodies in transient expression assays. In contrast to monospecific CCR5 antibodies, bispecific antibody derivatives block two alternative docking sites of CCR5-tropic HIV strains on the CCR5 coreceptor. Consequently, these molecules showed 18-to 57-fold increased antiviral activities compared to the parent antibodies. Most importantly, one prototypic tetravalent CCR5 antibody had antiviral activity against virus strains resistant to the single parental antibodies. In summary, physical linkage of two CCR5 antibodies targeting different epitopes on the HIV coreceptor CCR5 resulted in tetravalent, bispecific antibodies with enhanced antiviral potency against wild-type and CCR5 antibodyresistant HIV-1 strains.

CCR5 Antagonist TD-0680 Uses a Novel Mechanism for Enhanced Potency against HIV-1 Entry, Cell-mediated Infection, and a Resistant Variant

“…Continuing efforts will be given to address this issue in future studies. Schanzer et al (48) recently demonstrated that a bi-specific CCR5 antibody displays potent neutralizing activity against resistant viruses by targeting both the Nt and ECL2. Our future studies should investigate the potency of TD-0680 on other R5-tropic non-competitive CCR5 antagonist-resistant viruses, which might be more dependent on the Nt of CCR5 for entry (41,45).…”

Dual targeting strategies with bispecific antibodies

“…ScFv molecules have been fused to the N-terminus but also the C-terminus of the of the heavy or light chain of a mAb, [21][22][23] generally without compromising productivity or antigen-binding activity, although issues regarding stability have recently been addressed. 24 This group of IgG-like bispecific molecules also includes DVD-Igs, where a second V H and V L domain is fused to the heavy and light chain, respectively, of a mAb, 25 two-in-one antibodies, where a second specificity is introduced into the natural binding site of an IgG molecule, 26 and mAb 2 molecules, where a second specificity is build into the C H 3 domain of the Fc region. 27 A characteristic feature of all these molecules is a symmetry caused by dimeric assembly of two identical heavy chains, an intrinsic property of these chains.…”

Nanocell targeting using engineered bispecific antibodies

“…21,22,31 Previous studies have found that inclusion of a number of design modifications, including variations in CH3-scFv linker length and type, and also scFv stability engineering via addition of disulfide bonds, improved construct stability. [70][71][72][73][74][75] A concern with the Fc-linked dimeric design was whether the presence of 2 anti-LPS scFvs would result in EDV TM nanocell aggregation via binding of multiple nanocells rather than high avidity binding of a single nanocell. An alternative Fc-linked BsAb was engineered consisting of 2 EGFR targeting scFvs at the N-terminus (high avidity) and a single anti-LPS scFv at the C-terminus of the final product.…”

“…Cysteine stabilization substitutions were made at Kabat position VH44 and VL100 of the scFvs. 72,73 Variants included either a short (G4S) linker or a longer linker (SSDKTHTSPPSPGGGGSGGGGSGGGGSGGGG), as described in Moore et al, connecting the CH3 domain to the 1H10-scFv. 75 A "knob" was produced by amino acid substitution for a longer side chain (T366'W), whereas a "hole" was produced by amino acid substitutions for shorter side chains (T366'S:L368'A:Y407'V); the sequence positions are as indicated by Kabat et al 80 The tandem scFv included 6xHis and c-myc tags for purification and detection, while the other constructs utilised the Fc domain for purification and detection.…”