Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies.

Monoclonal antibodies (mAbs) are one of the most successful and versatile protein-based pharmaceutical products used to treat multiple pathological conditions. The remarkable specificity of mAbs and their affinity for biological targets has led to the implementation of mAbs in the therapeutic regime of oncogenic, chronic inflammatory, cardiovascular, and infectious diseases. Thus, the discovery of novel mAbs with defined functional activities is of crucial importance to expand our ability to address current and future clinical challenges. In vitro, antigen-driven affinity selection employing phage display biopanning is a commonly used technique to isolate mAbs. The success of biopanning is dependent on the quality and the presentation format of the antigen, which is critical when isolating mAbs against membrane protein targets. Here, we provide a comprehensive investigation of two established panning strategies, surface-tethering of a recombinant extracellular domain and cell-based biopanning, to examine the impact of antigen presentation on selection outcomes with regards to the isolation of positive mAbs with functional potential against a proof-of-concept type I cell surface receptor. Based on the higher sequence diversity of the resulting antibody repertoire, presentation of a type I membrane protein in soluble form was more advantageous over presentation in cell-based format. Our results will contribute to inform and guide future antibody discovery campaigns against cell surface proteins.

Landscape of human antibody recognition of the SARS-CoV-2 receptor binding domain.

Potent neutralizing monoclonal antibodies are one of the few agents currently available to treat COVID-19. SARS-CoV-2 variants of concern (VOCs) that carry multiple mutations in the viral spike protein can exhibit neutralization resistance, potentially affecting the effectiveness of some antibody-based therapeutics. Here, the generation of a diverse panel of 91 human, neutralizing monoclonal antibodies provides an in-depth structural and phenotypic definition of receptor binding domain (RBD) antigenic sites on the viral spike. These RBD antibodies ameliorate SARS-CoV-2 infection in mice and hamster models in a dose-dependent manner and in proportion to in vitro, neutralizing potency. Assessing the effect of mutations in the spike protein on antibody recognition and neutralization highlights both potent single antibodies and stereotypic classes of antibodies that are unaffected by currently circulating VOCs, such as B.1.351 and P.1. These neutralizing monoclonal antibodies and others that bind analogous epitopes represent potentially useful future anti-SARS-CoV-2 therapeutics.

Tracking molecular recognition at the atomic level with a new protein scaffold based on the OB-fold.

The OB-fold is a small, versatile single-domain protein binding module that occurs in all forms of life, where it binds protein, carbohydrate, nucleic acid and small-molecule ligands. We have exploited this natural plasticity to engineer a new class of non-immunoglobulin alternatives to antibodies with unique structural and biophysical characteristics. We present here the engineering of the OB-fold anticodon recognition domain from aspartyl tRNA synthetase taken from the thermophile Pyrobaculum aerophilum. For this single-domain scaffold we have coined the term OBody. Starting from a naïve combinatorial library, we engineered an OBody with 3 nM affinity for hen egg-white lysozyme, by optimising the affinity of a naïve OBody 11,700-fold over several affinity maturation steps, using phage display. At each maturation step a crystal structure of the engineered OBody in complex with hen egg-white lysozyme was determined, showing binding elements in atomic detail. These structures have given us an unprecedented insight into the directed evolution of affinity for a single antigen on the molecular scale. The engineered OBodies retain the high thermal stability of the parental OB-fold despite mutation of up to 22% of their residues. They can be expressed in soluble form and also purified from bacteria at high yields. They also lack disulfide bonds. These data demonstrate the potential of OBodies as a new scaffold for the engineering of specific binding reagents and provide a platform for further development of future OBody-based applications.

The retroviral hypermutation specificity of APOBEC3F and APOBEC3G is governed by the C-terminal DNA cytosine deaminase domain.

The human proteins APOBEC3F and APOBEC3G restrict retroviral infection by deaminating cytosine residues in the first cDNA strand of a replicating virus. These proteins have two putative deaminase domains, and it is unclear whether one or both catalyze deamination, unlike their homologs, AID and APOBEC1, which are well characterized single domain deaminases. Here, we show that only the C-terminal cytosine deaminase domain of APOBEC3F and -3G governs retroviral hypermutation. A chimeric protein with the N-terminal cytosine deaminase domain from APOBEC3G and the C-terminal cytosine deaminase domain from APOBEC3F elicited a dinucleotide hypermutation preference nearly indistinguishable from that of APOBEC3F. This 5'-TC-->TT mutational specificity was confirmed in a heterologous Escherichia coli-based mutation assay, in which the 5'-CC-->CT dinucleotide hypermutation preference of APOBEC3G also mapped to the C-terminal deaminase domain. An N-terminal APOBEC3G deletion mutant displayed a preference indistinguishable from that of the full-length protein, and replacing the C-terminal deaminase domain of APOBEC3F with AID resulted in an AID-like mutational signature. Together, these data indicate that only the C-terminal domain of APOBEC3F and -3G dictates the retroviral minus strand 5'-TC and 5'-CC dinucleotide hypermutation preferences, respectively, leaving the N-terminal domain to perform other aspects of retroviral restriction.

Retroviral restriction by APOBEC proteins.

A powerful mechanism of vertebrate innate immunity has been discovered in the past year, in which APOBEC proteins inhibit retroviruses by deaminating cytosine residues in nascent retroviral cDNA. To thwart this cellular defence, HIV encodes Vif, a small protein that mediates APOBEC degradation. Therefore, the balance between APOBECs and Vif might be a crucial determinant of the outcome of retroviral infection. Vertebrates have up to 11 different APOBEC proteins, with primates having the most. APOBEC proteins include AID, a probable DNA mutator that is responsible for immunoglobulin-gene diversification, and APOBEC1, an RNA editor with antiretroviral activities. This APOBEC abundance might help to tip the balance in favour of cellular defences.

APOBEC3F properties and hypermutation preferences indicate activity against HIV-1 in vivo.

APOBEC3G (CEM15 ) deaminates cytosine to uracil in nascent retroviral cDNA. The potency of this cellular defense is evidenced by a dramatic reduction in viral infectivity and the occurrence of high frequencies of retroviral genomic-strand G --> A transition mutations. The overwhelming dinucleotide hypermutation preference of APOBEC3G acting upon a variety of model retroviral substrates is 5'-GG --> -AG. However, a distinct 5'-GA --> -AA bias, which is difficult to attribute to APOBEC3G alone, prevails in HIV-1 sequences derived from infected individuals (e.g., ). Here, we show that APOBEC3F is also a potent retroviral restrictor but that its activity, unlike that of APOBEC3G, is partially resistant to HIV-1 Vif and results in a clear 5'-GA --> -AA retroviral hypermutation preference. This bias is also apparent in a bacterial mutation assay, suggesting that it is an intrinsic APOBEC3F property. Moreover, APOBEC3F and APOBEC3G appear to be coordinately expressed in a wide range of human tissues and are independently able to inhibit retroviral infection. Thus, APOBEC3F and APOBEC3G are likely to function alongside one another in the provision of an innate immune defense, with APOBEC3F functioning as the major contributor to HIV-1 hypermutation in vivo.