Development and Evaluation of a Set of Spike and Receptor Binding Domain-Based Enzyme-Linked Immunosorbent Assays for SARS-CoV-2 Serological Testing.

The implementation and validation of anti-SARS-CoV-2 IgG serological assays are reported in this paper. S1 and RBD proteins were used to coat ELISA plates, and several secondary antibodies served as reporters. The assays were initially validated with 50 RT-PCR positive COVID-19 sera, which showed high IgG titers of mainly IgG1 isotype, followed by IgG3. Low or no IgG2 and IgG4 titers were detected. Then, the RBD/IgG assay was further validated with 887 serum samples from RT-PCR positive COVID-19 individuals collected at different times, including 7, 14, 21, and 40 days after the onset of symptoms. Most of the sera were IgG positive at day 40, with seroconversion happening after 14-21 days. A third party conducted an additional performance test of the RBD/IgG assay with 406 sera, including 149 RT-PCR positive COVID-19 samples, 229 RT-PCR negative COVID-19 individuals, and 28 sera from individuals with other viral infections not related to SARS-CoV-2. The sensitivity of the assay was 99.33%, with a specificity of 97.82%. All the sera collected from individuals with infectious diseases other than COVID-19 were negative. Given the robustness of this RBD/IgG assay, it received approval from the sanitary authority in Mexico (COFEPRIS) for production and commercialization under the name UDISTEST-V2G®.

Structural insights into humanization of anti-tissue factor antibody 10H10.

Murine antibody 10H10 raised against human tissue factor is unique in that it blocks the signaling pathway, and thus inhibits angiogenesis and tumor growth without interfering with coagulation. As a potential therapeutic, the antibody was humanized in a two-step procedure. Antigen-binding loops were grafted onto selected human frameworks and the resulting chimeric antibody was subjected to affinity maturation by using phage display libraries. The results of humanization were analyzed from the structural perspective through comparison of the structure of a humanized variant with the parental mouse antibody. This analysis revealed several hot spots in the framework region that appear to affect antigen binding, and therefore should be considered in human germline selection. In addition, some positions in the Vernier zone, e.g., residue 71 in the heavy chain, that are traditionally thought to be crucial appear to tolerate amino acid substitutions without any effect on binding. Several humanized variants were produced using both short and long forms of complementarity-determining region (CDR) H2 following the difference in the Kabat and Martin definitions. Comparison of such pairs indicated consistently higher thermostability of the variants with short CDR H2. Analysis of the binding data in relation to the structures singled out the ImMunoGeneTics information system® germline IGHV1-2*01 as dubious owing to two potentially destabilizing mutations as compared to the other alleles of the same germline and to other human germlines.

Structural diversity in a human antibody germline library.

To support antibody therapeutic development, the crystal structures of a set of 16 germline variants composed of 4 different kappa light chains paired with 4 different heavy chains have been determined. All four heavy chains of the antigen-binding fragments (Fabs) have the same complementarity-determining region (CDR) H3 that was reported in an earlier Fab structure. The structure analyses include comparisons of the overall structures, canonical structures of the CDRs and the VH:VL packing interactions. The CDR conformations for the most part are tightly clustered, especially for the ones with shorter lengths. The longer CDRs with tandem glycines or serines have more conformational diversity than the others. CDR H3, despite having the same amino acid sequence, exhibits the largest conformational diversity. About half of the structures have CDR H3 conformations similar to that of the parent; the others diverge significantly. One conclusion is that the CDR H3 conformations are influenced by both their amino acid sequence and their structural environment determined by the heavy and light chain pairing. The stem regions of 14 of the variant pairs are in the 'kinked' conformation, and only 2 are in the extended conformation. The packing of the VH and VL domains is consistent with our knowledge of antibody structure, and the tilt angles between these domains cover a range of 11 degrees. Two of 16 structures showed particularly large variations in the tilt angles when compared with the other pairings. The structures and their analyses provide a rich foundation for future antibody modeling and engineering efforts.

Antibody engineering and therapeutics, The Annual Meeting of the Antibody Society: December 8-12, 2013, Huntington Beach, CA.

The 24th Antibody Engineering & Therapeutics meeting brought together a broad range of participants who were updated on the latest advances in antibody research and development. Organized by IBC Life Sciences, the gathering is the annual meeting of The Antibody Society, which serves as the scientific sponsor. Preconference workshops on 3D modeling and delineation of clonal lineages were featured, and the conference included sessions on a wide variety of topics relevant to researchers, including systems biology; antibody deep sequencing and repertoires; the effects of antibody gene variation and usage on antibody response; directed evolution; knowledge-based design; antibodies in a complex environment; polyreactive antibodies and polyspecificity; the interface between antibody therapy and cellular immunity in cancer; antibodies in cardiometabolic medicine; antibody pharmacokinetics, distribution and off-target toxicity; optimizing antibody formats for immunotherapy; polyclonals, oligoclonals and bispecifics; antibody discovery platforms; and antibody-drug conjugates.

Antibody modeling assessment II. Structures and models.

To assess the state-of-the-art in antibody structure modeling, a blinded study was conducted. Eleven unpublished Fab crystal structures were used as a benchmark to compare Fv models generated by seven structure prediction methodologies. In the first round, each participant submitted three non-ranked complete Fv models for each target. In the second round, CDR-H3 modeling was performed in the context of the correct environment provided by the crystal structures with CDR-H3 removed. In this report we describe the reference structures and present our assessment of the models. Some of the essential sources of errors in the predictions were traced to the selection of the structure template, both in terms of the CDR canonical structures and VL/VH packing. On top of this, the errors present in the Protein Data Bank structures were sometimes propagated in the current models, which emphasized the need for the curated structural database devoid of errors. Modeling non-canonical structures, including CDR-H3, remains the biggest challenge for antibody structure prediction.

Antigen-binding site anatomy and somatic mutations in antibodies that recognize different types of antigens.

The number of antibody structures co-crystallized with their respective antigens has increased rapidly in the last few years, thus offering a formidable source of information to gain insight into the structure-function relationships of this family of proteins. We have analyzed here 140 unique middle-resolution to high-resolution (<3 Å) antibody structures, including 55 in complex with proteins, 39 with peptides, and 46 with haptens. We determined (i) length variations of the hypervariable loops, (ii) number of contacts with antigen, (iii) solvent accessible area buried upon binding, (iv) location and frequency of antigen contacting residues, (v) type of residues interacting with antigens, and (vi) putative somatic mutations. Except for somatic mutations, distinctive profiles were identified for all the variables analyzed. Compared with contacts, somatic mutations occurred with less abundance at any given position and extended beyond the regions in contact, with no clear difference among antibodies that recognize different types of antigens. This observation is consistent with the fact that although antigen recognition accomplished by shape and physicochemical complementarity is selective in nature, the somatic mutation process is stochastic and selection for mutations leading to improved affinity is not directly related to contact residues. Thus, the knowledge emerging from this study enhances our understanding of the structure-function relationship in antibodies while providing valuable guidance to design libraries for antibody discovery and optimization.

Characterization of a high-affinity human antibody with a disulfide bridge in the third complementarity-determining region of the heavy chain.

Disulfide bridges are common in the antigen-binding site from sharks (new antigen receptor) and camels (single variable heavy-chain domain, VHH), in which they confer both structural diversity and domain stability. In human antibodies, cysteine residues in the third complementarity-determining region of the heavy chain (CDR-H3) are rare but naturally encoded in the IGHD germline genes. Here, by panning a phage display library designed based on human germline genes and synthetic CDR-H3 regions against a human cytokine, we identified an antibody (M3) containing two cysteine residues in the CDR-H3. It binds the cytokine with high affinity (0.4 nM), recognizes a unique epitope on the antigen, and has a distinct neutralization profile as compared with all other antibodies selected from the library. The two cysteine residues form a disulfide bridge as determined by mass spectrometric peptide mapping. Replacing the cysteines with alanines did not change the solubility and stability of the monoclonal antibody, but binding to the antigen was significantly impaired. Three-dimensional modeling and dynamic simulations were employed to explore how the disulfide bridge influences the conformation of CDR-H3 and binding to the antigen. On the basis of these results, we envision that designing human combinatorial antibody libraries to contain intra-CDR or inter-CDR disulfide bridges could lead to identification of human antibodies with unique binding profiles.

Leveraging SBDD in protein therapeutic development: antibody engineering.

Antibodies make up the largest, growing segment of protein therapeutics in the pharmaceutical and biotechnology industries. The development or engineering of therapeutic antibodies is based to a large extent on our knowledge of antibody structure and requires sophisticated methods that continue to evolve. In this chapter, after a review of what is known about the structure and functional properties of antibodies, the current, state-of-the-art antibody engineering methods are described. These methods include antibody humanization, antigen-affinity optimization, Fc engineering for modulated effector function and extended half-life, and engineering for improved stability and biophysical properties. X-ray crystallographic structures of antibody fragments and their complexes can play a critical role in guiding and, in some cases, accelerating these processes. These approaches represent guidelines for developing antibody therapeutics with the desired affinity, effector function, and biophysical properties.

Crystal structure of human germline antibody 3-23/B3.

The human antibody repertoire is dominated by a few combinations of germline sequences, with the genes 3-23 for the heavy chain and B3 for the light chain being among the most frequently used. Despite this fact, there was no experimental structure of the antibody composed of 3-23 and B3. The crystal structure of the Fab fragment of the synthetic antibody composed of the 3-23 and B3 germline sequences was determined to provide a template for antibody modeling. The antigen-binding loops were found in the canonical conformations. Comparison to the other structures where either 3-23 or B3 is paired with a different chain reveals a significant deviation in the orientation of the variable domains.

Specific epitopes of domains II and III of Bacillus thuringiensis Cry1Ab toxin involved in the sequential interaction with cadherin and aminopeptidase-N receptors in Manduca sexta.

The Bacillus thuringiensis Cry toxins are specific to different insects. In Manduca sexta cadherin (Bt-R1) and aminopeptidase-N (APN) proteins are recognized as Cry1A receptors. Previous work showed that Cry1Ab binds to Bt-R1 promoting the formation of a pre-pore oligomer that binds to APN leading to membrane insertion. In this work we characterized the binding epitopes involved in the sequential interaction of Cry1Ab with Bt-R1 and APN. A Cry1Ab immune M13 phage repertoire was constructed using antibody gene transcripts of bone marrow or spleen from a rabbit immunized with Cry1Ab. We identified antibodies that recognize domain II loop 3 (scFvL3-3) or beta16-beta22 (scFvM22) in domain III. Enzyme-linked immunosorbent assay and toxin overlay binding competition assays in the presence of scFvL3-3, scFvM22, or synthetic peptides showed that domain II loop 3 is an important epitope for interaction with Bt-R1 receptor, whereas domain III beta16 is involved in the interaction with APN. Both scFvL3-3 and scFvM22 lowered the toxicity of Cry1Ab to M. sexta larvae indicating that interaction with both receptors is important for in vivo toxicity. scFvL3-3 and anti-loop2 scFv (scFv73) promoted the formation of the pre-pore oligomer in contrast to scFvM22. In addition, scFvL3-3 and scFv73 preferentially recognized the monomeric toxin rather than the pre-pore suggesting a conformational change in domain II loops upon oligomerization. These results indicate for the first time that both receptor molecules participate in Cry1Ab toxin action in vivo: first the monomeric toxin binds to Bt-R1 through loops 2 and 3 of domain II promoting the formation of the pre-pore inducing some structural changes, then the pre-pore interacts with APN through beta-16 of domain III promoting membrane insertion and cell death.

Design and validation of a synthetic VH repertoire with tailored diversity for protein recognition.

Previous studies have indicated differences in the specificity-determining residues (SDRs) of antibodies that recognize haptens, peptides, or proteins. Here, we designed a V(H) repertoire based on the human scaffold 3-23/J(H)4 and diversification of high and medium-usage SDRs of anti-protein and anti-peptide antibodies. The repertoire was synthesized by overlapping polymerase chain reaction (PCR) and combined with the V(L) chain of the anti-hen egg-white lysozyme (HEL) antibody D1.3. The resulting chimeric single-chain Fv fragments (scFvs) phage-displayed library was panned in HEL-coated immunotubes. After two rounds of selection under non-stringent conditions, that is, trypsinization after 2 h of incubation at room temperature, 63 of 167 clones analyzed (38%) were found to express scFvs specific to HEL. Twenty clones were characterized by DNA sequencing resulting in 10 unique scFvs. Interestingly, the panel of unique scFvs was highly diverse, with V(H) sequences differing in 16 of the 17 positions variegated in the repertoire. Thus, diverse chemico-physical and structural solutions were selected from the library, even when the V(H) repertoire was constrained by the V(L) chain of D1.3 to yield binders against a definite region of HEL surface. The more often selected scFvs, namely H6-1 and B7-1, which differed in eight SDRs, showed levels of expression in E. coli TG1 strain, 6 and 10 times higher than the parental D1.3 Fv fragment, respectively. Dissociation constants (K(Ds)) measured in the BIAcore were 11 and 6.6 nM for H6-1 and B7-1, respectively. These values compared well to the K(D) of 4.7 nM measured for D1.3, indicating that the V(H) repertoire here designed is a valuable source of diverse, well-expressed and high affinity V(H) domains.

Use of human germline genes in a CDR homology-based approach to antibody humanization.

We report a new method of humanizing antibodies by complementarity determining region (CDR) grafting. Our method differs from others in that we choose human framework sequences from the set of human germline genes based on the structural similarity of the human CDRs to those of the mouse antibody to be humanized. The structural similarity is evaluated by scoring residue-to-residue homology of the mouse CDRs to human candidates with the same Chothia canonical structures. The method is illustrated with the humanization of the anti-lysozyme antibody D1.3.

Phage antibody fragments library combining a single human light chain variable region with immune mouse heavy chain variable regions.

We describe the construction of a phage antibody fragments library which combines, in a single cloning step, a synthetic human light chain variable region (V(L)) with a diverse set of heavy chain variable regions, from a mouse immunized with the prostate specific antigen (PSA). Despite V(L) restriction, selection from this library rendered two different single chain Fv antibody fragments, specifically recognizing PSA. The human V(L), used as a general partner for mouse heavy chains, was constructed by linking the germline A27 gene and the J(K)1 minigene segment, both of which are prominently involved in human antibody responses. Our approach offers a fast and simple way to produce half-human molecules, while keeping the advantage of immunizing animals for high affinity antibodies.