A recent study posted to the bioRxiv* preprint server presented topologically engineered fragment crystallizable (Fc)-fusion proteins and antibodies as therapeutic options for diseases like human coronaviruses (HCoVs) infections and cancers.
Peptide bonding has been used exclusively to link numerous binding domains in multifunctional antibodies that cooperatively bind with targets. Nevertheless, the topology of antibodies limits their capacity to engage with many targets cooperatively. The resultant multipartite fusion proteins are frequently plagued by steric hindrance. This will preclude the required domains from concurrently engaging their respective substrates.
The dimeric character of the antibody molecule, which is a byproduct of the Fc domain, is a critical property that defines its topology. Even though the Fc domain has been exploited to construct dimeric fusion proteins offering various therapeutic benefits such as prolonged plasma half-lives, Fc-fusion proteins are constrained by their topology similar to antibodies.
For instance, due to poor adherence by the viral spike (S) protein, angiotensin-converting enzyme 2 (ACE2) Fc-fusion proteins have limited capacity to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Moreover, affinity-enhanced ACE2 mutants are not the best answer to this problem since they increase the likelihood of viral escape.
About the study
In the current study, the scientists’ utilized non-covalent assembly to circumvent the basic restrictions associated with the topology of antibodies and build therapeutics exhibiting topology superior to antibodies. The team reported a new method for creating antibody-like entities with unique topologies that engage substrates cooperatively by employing cross-dimerization facilitated by the ACE2 collectrin-like domain (CLD) in conjunction with the Fc domain.
The authors evaluated the ability of ACE2 Fc-fusion proteins to cross dimerize. New superdimeric antibodies and Fc-fusion proteins consisting of plasma half-lives similar to antibodies were topologically designed using cross-dimerization to tackle infectious disease and cancer therapies. Structures were validated using specific cleavage by multi-angle light scattering combined with size-exclusion chromatography (SEC-MALS) and IdeZ protease.
Findings and discussions
The results demonstrated that the first (ACE2-615 homodimer) and second (ACE2-740 heterodimer) constructs were generated as separate species. The third construct was a hybrid of two topologically diverse forms. The smaller form was the expected ACE2-740 homodimer. The bigger form was an ACE2-740 superhomodimer, with a unique topology representing cross-dimerization of its two CLD dimerizing polypeptide pairs and two Fc dimerizing polypeptide pairs. The structure of the superdimer reveals that its two Fc dimers and two ACE2 peptidase dimers protrude outward from a central nucleus, forming a tetrahedral-like arrangement.
Stoichiometric competition binding investigations indicated the diverse range of activities of superdimer. The authors named the superheterodimers as Gemini dimers (GEM-DIMERs) due to their twin-like structures. GEM-DIMERs could comprise any mixtures of Fc-fusion proteins and antibodies. Quantitative solution binding testing corroborated the qualitative data from the current competition binding assays. It was observed that ACE2 superdimers have a 51- to 126-time binding advantage over ACE2 dimers.
The ACE2-740/615 superheterodimer and ACE2-740 superhomodimer demonstrated greater potency against SARS-CoV-2 and HCoV NL63 than the ACE2-615 homodimer. ACE2-740/B13A superheterodimer exhibited higher potency against SARS-CoV-2 than B13A antibody and ACE2-740 heterodimer. ACE2-740/615 superheterodimer and ACE2-740 superhomodimer neutralized S adhesion more efficiently than ACE2-740 homodimer. ACE2-740/B13A superheterodimer neutralized S adhesion more efficiently than B13A antibody and ACE2-740 homodimer. In Syrian hamsters, ACE2-740/B13A superheterodimer demonstrated similar potency in precluding weight loss versus REGN10987 and REGN10933 antibody cocktails, further, better efficacy than REGN10933 separately.
Antibody superheterodimers CT1-CT1 and RG2-RG2 neutralized the SARS-CoV-2 B.1.531 variant nearly 100-time superior to the Ct-P59 and REGN10933 parent antibodies. The fact that ACE2 and antibody superdimers attach to individual S trimers more firmly implies that the topology of the superdimer permits it to adopt a configuration that allows it to engage at least two binding domains on the S trimer at the same time. The authors propose that the superdimer arrangement might offer a coherent solution for the Fc-fusion proteins and antibodies’ incapacity to bind numerous multispecific or monospecific targets exhibiting resistance to the synchronous binding.
Employing the human neonatal Fc receptor (FcRn) Tg32 homozygous transgenic mouse model, the researchers validated that antibody superheterodimers demonstrate plasma half-lives similar to antibodies. ACE2-740/B13A and ACE2-740/615 superheterodimers effectively neutralized Omicron B.1.1.529 variant and 11 other major SARS-CoV-2 variants than eight antibodies (CT-P59, VIR-7831, AZD8895, AZD1061, LY-CoV016, LY-CoV555, REGN10933, and REGN10987).
HB1701, a bispecific Fab1/Fab2 superheterodimer comprising REGN10933 and REGN10987 Fabs neutralized HCoVs N439K and SARS-CoV-2 B.1.351 variant 25-time better than REGN10933 /REGN10987 cocktails. The neutralizing activity of HB1701 was equivalent to that of the highly effective ACE2-740/615 and the ACE2-740/B13A superheterodimers, suggesting that bispecific antibody superheterodimers could efficiently neutralize SARS-CoV-2 strains that are immune to their parent antibodies cocktails.
Bispecific Fab1/Fab2 superheterodimers were created using FMC63 (anti-CD19 antibody used in refractory/relapsed B-cell lymphoma) named HB1906 and Rituxan (anti-CD20 antibody used in B-cell lymphoma) called HB1905. Surface plasmon resonance binding investigations revealed that HB1906 and HB1905 bind monovalent CD19 and CD20, complement component C1q and Fc gamma receptors at similar affinity to FMC63 and Rituxan. The antibody-dependent cell-mediated cytotoxicity (ADCC) of the HB1906 and HB1905 was superior versus the FMC63 and Rituxan antibody cocktails.
The study findings showed that ACE2 Fc-fusion proteins instantaneously cross dimerize, establishing topologically unique superdimers. These superdimers exhibited exceptional SARS-CoV-2 intra-S collaborative binding and perniciously neutralize Omicron B.1.1.529 at least 100 times more effectively than eight clinically approved antibodies.
Bispecific superdimers of ACE2-antibody efficiently neutralized all main SARS-CoV-2 variants. In addition, bispecific superdimers of anti-viral and anti-cancer antibodies were more potent than combining two antibodies. These findings imply that superdimers may be created efficiently from single cells, opening up a new treatment avenue for a variety of diseases, including HCoV infections and cancers.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.