As SARS-CoV-2 variants grow in type and frequency, Covid-19 researchers are on the hunt for parts of the virus that remain consistent across variants in order to create Covid-19 treatments that work for multiple strains of the virus. This is the third in a series discussing these potential Achilles’ heels for Covid-19. Read more from this series in part one and part two.

Antibodies have been at the forefront in the search for drugs to prevent and treat Covid-19. Combination antibodies effectively reduce infection length if given early enough and prevent infection for those exposed. However, there are some inherent difficulties with antibodies as drugs. They are relatively large molecules that are expensive to produce. Additionally,  SARS-CoV-2 mutates to resist single and, in some cases, to double-antibody cocktails.  Here we describe a new approach to the same end using antibodies derived from camelids, a biological family which includes mammals such as camels, llamas, and alpacas. 

Camelid-derived antibodies lack a light chain and are composed of two identical heavy chains. These unique heavy-chain antibodies are also known as nanobodies. Camelid nanobodies are less than a quarter of the size of human antibodies. They are more stable, more simply modified, and less inexpensive than traditional monoclonal antibody drugs. These unique qualities have prompted researchers to develop camelid-derived nanobody drugs for Covid-19.

In a study by Koenig et al., a llama and an alpaca were immunized with a SARS-CoV-2 spike protein from an inactivated virus. They then identified four nanobodies that resulted from immunization that potently neutralized SARS-CoV-2: E, U, V, and W. Three of these—U, V, and W—bind to the Covid-19 spike protein as indicated in the figure below. In contrast, E is bound to an extended loop overlapping the receptor-binding domain. 

Each of the four nanobodies effectively neutralized SARS-CoV-2 by roughly 50% in a plaque-reduction assay on their own. To improve the activity, combinations of two individual nanobodies were joined by a flexible linker to create drug candidates that might be more effective by binding to two different sites simultaneously.

The receptor-binding domain that binds to ACE2 resembles a spring-loaded “up-down” device. When the receptor-binding domain is in the “down” position, it is not primed to bind to ACE2. However, when in the “up” position, the receptor-binding domain is open to ACE2 binding. 

A series of experiments were done to link the nanobodies in sets of two or three to determine which combinations were the most potent neutralizers. The ones which were selected were V and E.

V+E neutralized at a dilution 62 times greater and had an affinity at least 22 times greater than singular nanobodies, indicating a highly neutralizing combination nanobody. The E nanobody seems to bind to the “up” position in the stead of ACE2, releasing the fusion activity of the trimeric protein prematurely and inactivating the structure. On the other hand, the V nanobody improves the ability of V+E to bind by stabilizing the receptor-binding domain in the two-up position, allowing the E nanobody a better opportunity to bind and prematurely activate the trimer.

The researchers also aimed to find a multivalent nanobody that neutralizes rapidly spreading variants found to resist neutralizing antibodies from convalescent sera and vaccines. The V nanobody avoids the high-escape amino acids of 417, 484, and 501, suggesting it could potentially neutralize variants like B.1.1.7 and B.1.351, which contain mutations at those positions. However, the E nanobody contains binding at both 484 and 501, indicating it would be less tolerant of changes at these positions. Therefore, the V+E combination nanobody may not have the neutralizing prowess against variants as it does against the wild type. 

The other nanobodies—U and W—were overcome by spike mutations at positions 371 and 378. While mutations at these positions are less common than mutations at positions 417, 484, and 501, they still denote a shortcoming in these nanobodies. While they may be potently neutralizing, it seems they may not overcome rapidly spreading variants of concern.

The V+E combination nanobody is a potent neutralizer and could be used as a new approach to prophylaxis, therapeutics, and improving upon existing antibodies. However, this is not a universal solution, as mutations in the spike protein decrease the binding of individual camelid-derived nanobodies. This can be partially overcome by combining nanobodies, but not entirely because mutations may arise in both domains, which reduce potency.

It may be possible to use the camelid approach to find conserved epitopes which do not affect antibody neutralization, such as those described by Starr et al. in part two of this series. Combination nanobodies could potentially target these conserved regions, in addition to the binding regions, to produce a highly potent and versatile neutralizing bispecific nanobody. With further research, combination nanobodies to prevent and treat highly infectious and immune-evasive SARS-CoV-2 variants may well be on their way.