Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody V
H
domain library from which we identified a high-affinity V
H
binder ab8. Bivalent V
H
, V
H
-Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. V
H
-Fc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of V
H
-Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic.
Immunoglobulin binding proteins (IBPs) are broadly used as reagents for the purification and detection of antibodies. Among the IBPs, the most widely used are Protein-A and Protein-G. The C2 domain of Protein-G from Streptococcus is a multi-specific protein domain; it possesses a high affinity (KD ~ 10 nM) for the Fc region of the IgG, but a much lower affinity (KD ~ low εM) for the constant domain of the antibody fragment (Fab), which limits some of its applications. Here, we describe the engineering of the Protein-G interface using phage display to create Protein-G-A1, a variant with 8 point mutations and an approximately 100-fold improved affinity over the parent domain for the 4D5 Fab scaffold. Protein-G-A1 is capable of robust binding to Fab fragments for numerous applications. Furthermore, we isolated a variant with pH-dependent affinity, demonstrating a 10,000-fold change in affinity from pH 7 to 4. Additional rational mutagenesis endowed Protein-G with significantly enhanced stability in basic conditions relative to the parent domain while maintaining high affinity to the Fab. This property is particularly useful to regenerate Protein-G affinity columns. Lastly, the affinity-matured Protein-G-A1 variant was tethered together to produce dimers capable of providing multivalent affinity enhancement to a low affinity antibody fragment-antigen interaction. Engineered Protein-G variants should find widespread application in the use of Fab-based affinity reagents.
Reversible, high affinity immobilization tags are critical tools for myriad biological applications. However, inherent issues are associated with a number of the current methods of immobilization. Particularly, a critical element in phage display sorting is functional immobilization of target proteins. To circumvent these problems, we have used a mutant (N5A) of calmodulin binding peptide (CBP) as an immobilization tag in phage display sorting. The immobilization relies on the ultra high affinity of calmodulin to N5A mutant CBP (RWKKNFIAVSAANRFKKIS) in presence of calcium (KD ~ 2pM), which can be reversed by EDTA allowing controlled “capture and release” of the specific binders. To evaluate the capabilities of this system, we chose eight targets, some of which were difficult to overexpress and purify with other tags and some had failed in sorting experiments. In all cases, specific binders were generated using a Fab phage display library with CBP fused constructs. KD of the Fabs were in sub to low nanomolar (nM) ranges and were successfully used to selectively recognize antigens in cell-based experiments. Some of these targets were problematic even without any tag, so the fact that all led to successful selection endpoints means that borderline cases can be worked on with a high probability of positive outcome. Taken together with examples of successful case specific high level applications like generation of conformation, epitope and domain specific Fabs, we feel that the CBP tag embodies all the attributes of covalent immobilization tags, but does not suffer from some of their well documented drawbacks.
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