Reshaping antibody diversity.

Some species mount a robust antibody response despite having limited genome-encoded combinatorial diversity potential. Cows are unusual in having exceptionally long CDR H3 loops and few V regions, but the mechanism for creating diversity is not understood. Deep sequencing reveals that ultralong CDR H3s contain a remarkable complexity of cysteines, suggesting that disulfide-bonded minidomains may arise during repertoire development. Indeed, crystal structures of two cow antibodies reveal that these CDR H3s form a very unusual architecture composed of a ß strand "stalk" that supports a structurally diverse, disulfide-bonded "knob" domain. Diversity arises from somatic hypermutation of an ultralong DH with a severe codon bias toward mutation to cysteine. These unusual antibodies can be elicited to recognize defined antigens through the knob domain. Thus, the bovine immune system produces an antibody repertoire composed of ultralong CDR H3s that fold into a diversity of minidomains generated through combinations of somatically generated disulfides.

The smallest functional antibody fragment: Ultralong CDR H3 antibody knob regions potently neutralize SARS-CoV-2.

Cows produce antibodies with a disulfide-bonded antigen-binding domain embedded within ultralong heavy chain third complementarity determining regions. This "knob" domain is analogous to natural cysteine-rich peptides such as knottins in that it is small and stable but can accommodate diverse loops and disulfide bonding patterns. We immunized cattle with SARS-CoV-2 spike and found ultralong CDR H3 antibodies that could neutralize several viral variants at picomolar IC50 potencies in vitro and could protect from disease in vivo. The independent CDR H3 peptide knobs were expressed and maintained the properties of the parent antibodies. The knob interaction with SARS-CoV-2 spike was revealed by electron microscopy, X-ray crystallography, NMR spectroscopy, and mass spectrometry and established ultralong CDR H3-derived knobs as the smallest known recombinant independent antigen-binding fragment. Unlike other vertebrate antibody fragments, these knobs are not reliant on the immunoglobulin domain and have potential as a new class of therapeutics.

Germline-Encoded Positional Cysteine Polymorphisms Enhance Diversity in Antibody Ultralong CDR H3 Regions.

Ab "ultralong" third H chain complementarity-determining regions (CDR H3) appear unique to bovine Abs and may enable binding to difficult epitopes that shorter CDR H3 regions cannot easily access. Diversity is concentrated in the "knob" domain of the CDR H3, which is encoded by the DH gene segment and sits atop a ß-ribbon "stalk" that protrudes far from the Ab surface. Knob region cysteine content is quite diverse in terms of total number of cysteines, sequence position, and disulfide bond pattern formation. We investigated the role of germline cysteines in production of a diverse CDR H3 structural repertoire. The relationship between DH polymorphisms and deletions relative to germline at the nucleotide level, as well as diversity in cysteine and disulfide bond content at the structural level, was ascertained. Structural diversity is formed through (1) DH polymorphisms with altered cysteine positions, (2) DH deletions, and (3) new cysteines that arise through somatic hypermutation that form new, unique disulfide bonds to alter the knob structure. Thus, a combination of mechanisms at both the germline and somatic immunogenetic levels results in diversity in knob region cysteine content, contributing to remarkable complexity in knob region disulfide patterns, loops, and Ag binding surface.

Evolution of immunogenetic components encoding ultralong CDR H3.

The genomes of most vertebrates contain many V, D, and J gene segments within their Ig loci to construct highly variable CDR3 sequences through combinatorial diversity. This nucleotide variability translates into an antibody population containing extensive paratope diversity. Cattle have relatively few functional VDJ gene segments, requiring innovative approaches for generating diversity like the use of ultralong-encoding IGHV and IGHD gene segments that yield dramatically elongated CDR H3. Unique knob and stalk microdomains create protracted paratopes, where the antigen-binding knob sits atop a long stalk, allowing the antibody to bind both surface and recessed antigen epitopes. We examined genomes of twelve species of Bovidae to determine when ultralong-encoding IGHV and IGHD gene segments evolved. We located the 8-bp duplication encoding the unique TTVHQ motif in ultralong IGHV segments in six Bovid species (cattle, zebu, wild yak, domestic yak, American bison, and domestic gayal), but we did not find evidence of the duplication in species beyond the Bos and Bison genera. Additionally, we analyzed mRNA from bison spleen and identified a rich repertoire of expressed ultralong CDR H3 antibody mRNA, suggesting that bison use ultralong IGHV transcripts in their host defense. We found ultralong-encoding IGHD gene segments in all the same species except domestic yak, but again not beyond the Bos and Bison clade. Thus, the duplication event leading to this ultralong-encoding IGHV gene segment and the emergence of the ultralong-encoding IGHD gene segment appears to have evolved in a common ancestor of the Bos and Bison genera 5-10 million years ago.

Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows.

No immunogen to date has reliably elicited broadly neutralizing antibodies to HIV in humans or animal models. Advances in the design of immunogens that antigenically mimic the HIV envelope glycoprotein (Env), such as the soluble cleaved trimer BG505 SOSIP, have improved the elicitation of potent isolate-specific antibody responses in rabbits and macaques, but so far failed to induce broadly neutralizing antibodies. One possible reason for this failure is that the relevant antibody repertoires are poorly suited to target the conserved epitope regions on Env, which are somewhat occluded relative to the exposed variable epitopes. Here, to test this hypothesis, we immunized four cows with BG505 SOSIP. The antibody repertoire of cows contains long third heavy chain complementary determining regions (HCDR3) with an ultralong subset that can reach more than 70 amino acids in length. Remarkably, BG505 SOSIP immunization resulted in rapid elicitation of broad and potent serum antibody responses in all four cows. Longitudinal serum analysis for one cow showed the development of neutralization breadth (20%, n = 117 cross-clade isolates) in 42 days and 96% breadth (n = 117) at 381 days. A monoclonal antibody isolated from this cow harboured an ultralong HCDR3 of 60 amino acids and neutralized 72% of cross-clade isolates (n = 117) with a potent median IC50 of 0.028 µg ml-1. Breadth was elicited with a single trimer immunogen and did not require additional envelope diversity. Immunization of cows may provide an avenue to rapidly generate antibody prophylactics and therapeutics to address disease agents that have evolved to avoid human antibody responses.

Diversity of ACE2 and its interaction with SARS-CoV-2 receptor binding domain.

COVID-19, the clinical syndrome caused by the SARS-CoV-2 virus, has rapidly spread globally causing hundreds of millions of infections and over two million deaths. The potential animal reservoirs for SARS-CoV-2 are currently unknown, however sequence analysis has provided plausible potential candidate species. SARS-CoV-2 binds to the angiotensin I converting enzyme 2 (ACE2) to enable its entry into host cells and establish infection. We analyzed the binding surface of ACE2 from several important animal species to begin to understand the parameters for the ACE2 recognition by the SARS-CoV-2 spike protein receptor binding domain (RBD). We employed Shannon entropy analysis to determine the variability of ACE2 across its sequence and particularly in its RBD interacting region, and assessed differences between various species' ACE2 and human ACE2. Recombinant ACE2 from human, hamster, horseshoe bat, cat, ferret, and cow were evaluated for RBD binding. A gradient of binding affinities were seen where human and hamster ACE2 were similarly in the low nanomolar range, followed by cat and cow. Surprisingly, horseshoe bat (Rhinolophus sinicus) and ferret (Mustela putorius) ACE2s had poor binding activity compared with the other species' ACE2. The residue differences and binding properties between the species' variants provide a framework for understanding ACE2-RBD binding and virus tropism.

Formation of ultralong DH regions through genomic rearrangement.

BACKGROUND: Cow antibodies are very unusual in having exceptionally long CDR H3 regions. The genetic basis for this length largely derives from long heavy chain diversity (DH) regions, with a single "ultralong" DH, IGHD8-2, encoding over 50 amino acids. Many bovine IGHD regions have sequence similarity but have several nucleotide repeating units that diversify their lengths. Genomically, most DH regions exist in three clusters that appear to have formed from DNA duplication events. However, the relationship between the genomic arrangement and long CDR lengths is unclear. RESULTS: The DH cluster containing IGHD8-2 underwent a rearrangement and deletion event in relation to the other clusters in the region corresponding to IGHD8-2, with possible fusion of two DH regions and expansion of short repeats to form the ultralong IGHD8-2 gene. CONCLUSIONS: Length heterogeneity within DH regions is a unique evolutionary genomic mechanism to create immune diversity, including formation of ultralong CDR H3 regions.

Somatic hypermutation maintains antibody thermodynamic stability during affinity maturation.

Somatic hypermutation and clonal selection lead to B cells expressing high-affinity antibodies. Here we show that somatic mutations not only play a critical role in antigen binding, they also affect the thermodynamic stability of the antibody molecule. Somatic mutations directly involved in antigen recognition by antibody 93F3, which binds a relatively small hapten, reduce the melting temperature compared with its germ-line precursor by up to 9 °C. The destabilizing effects of these mutations are compensated by additional somatic mutations located on surface loops distal to the antigen binding site. Similarly, somatic mutations enhance both the affinity and thermodynamic stability of antibody OKT3, which binds the large protein antigen CD3. Analysis of the crystal structures of 93F3 and OKT3 indicates that these somatic mutations modulate antibody stability primarily through the interface of the heavy and light chain variable domains. The historical view of antibody maturation has been that somatic hypermutation and subsequent clonal selection increase antigen-antibody specificity and binding energy. Our results suggest that this process also optimizes protein stability, and that many peripheral mutations that were considered to be neutral are required to offset deleterious effects of mutations that increase affinity. Thus, the immunological evolution of antibodies recapitulates on a much shorter timescale the natural evolution of enzymes in which function and thermodynamic stability are simultaneously enhanced through mutation and selection.

Bispecific small molecule-antibody conjugate targeting prostate cancer.

Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative of the prostate-specific membrane antigen-binding small molecule DUPA was selectively conjugated to a mutant αCD3 Fab containing the unnatural amino acid, p-acetylphenylalanine, at a defined site. Homogeneous conjugates were generated in excellent yields and had good solubility. The efficacy of the conjugate was optimized by modifying the linker structure, relative binding orientation, and stoichiometry of the ligand. The optimized conjugate showed potent and selective in vitro activity (EC50 ~ 100 pM), good serum half-life, and potent in vivo activity in prophylactic and treatment xenograft mouse models. This semisynthetic approach is likely to be applicable to the generation of additional bispecific agents using drug-like ligands selective for other cell-surface receptors.

Fc-Small Molecule Antibody Mimetics.

Antibody therapeutics are a promising drug class due to their high specificity and favorable pharmacokinetics. While there are many methods for the development of antibodies specific to disease associated antigens, selecting antibodies against functional epitopes with high specificity and affinity can be difficult for certain epitopes. We describe a generalizable method for synthesizing antibody mimetics by site specifically conjugating small molecules (with high affinity and specificity to disease associated antigens) to an Fc fragment to develop drugs with the benefits of an antibody. As a proof of concept, an E269pAcPhe Fc antibody Fc fragment was produced and subsequently site-specifically labeled with a linker-modified folic acid compound to generate an Fc-folic acid antibody-mimetic. This was chosen as the model system because the high-affinity folate receptor FR-α is highly expressed in a number of cancer types including breast and ovarian cancer. The specificity of the Fc-folic acid conjugate was assessed via flowcytometry with the folate-receptor positive breast cancer cell line MDA-MB-231 by measuring Fc-folic acid binding in both the absence and presence of an excess of folic acid. Fc-small molecule conjugates could be developed into a unique class of antibody-like therapeutics.

Antibody microarrays utilizing site-specific antibody-oligonucleotide conjugates.

Protein arrays are typically made by random absorption of proteins to the array surface, potentially limiting the amount of properly oriented and functional molecules. We report the development of a DNA encoded antibody microarray utilizing site-specific antibody-oligonucleotide conjugates that can be used for cell immobilization as well as the detection of genes and proteins. This technology allows for the facile generation of antibody microarrays while circumventing many of the drawbacks of conventionally produced antibody arrays. We demonstrate that this method can be used to capture and detect SK-BR-3 cells (Her2+ breast cancer cells) at concentrations as low as 10(2) cells/mL (which is equivalent to 10 cells per 100 µL array) without the use of microfluidics, which is 100- to 10(5)-fold more sensitive than comparable techniques. Additionally, the method was shown to be able to detect cells in a complex mixture, effectively immobilizing and specifically detecting Her2+ cells at a concentration of 10(2) SK-BR-3 cells/mL in 4 × 10(6) white blood cells/mL. Patients with a variety of cancers can have circulating tumor cell counts of between 1 and 10(3) cells/mL in whole blood, well within the range of this technology.

Antibody conjugates with unnatural amino acids.

Antibody conjugates are important in many areas of medicine and biological research, and antibody-drug conjugates (ADCs) are becoming an important next generation class of therapeutics for cancer treatment. Early conjugation technologies relied upon random conjugation to multiple amino acid side chains, resulting in heterogeneous mixtures of labeled antibody. Recent studies, however, strongly support the notion that site-specific conjugation produces a homogeneous population of antibody conjugates with improved pharmacologic properties over randomly coupled molecules. Genetically incorporated unnatural amino acids (uAAs) allow unique orthogonal coupling strategies compared to those used for the 20 naturally occurring amino acids. Thus, uAAs provide a novel paradigm for creation of next generation ADCs. Additionally, uAA-based site-specific conjugation could also empower creation of additional multifunctional conjugates important as biopharmaceuticals, diagnostics, or reagents.

Site-specific DNA-antibody conjugates for specific and sensitive immuno-PCR.

Antibody conjugates are widely used as diagnostics and imaging reagents. However, many such conjugates suffer losses in sensitivity and specificity due to nonspecific labeling techniques. We have developed methodology to site-specifically conjugate oligonucleotides to antibodies containing a genetically encoded unnatural amino acid with orthogonal chemical reactivity. These oligobody molecules were used in immuno-PCR assays to detect Her2(+) cells with greater sensitivity and specificity than nonspecifically coupled fragments, and can detect extremely rare Her2(+) cells in a complex cellular environment. Such designed antibody-oligonucleotide conjugates should provide sensitive and specific reagents for diagnostics, as well as enable other unique applications based on oligobody building blocks.

Synthesis of site-specific antibody-drug conjugates using unnatural amino acids.

Antibody-drug conjugates (ADCs) allow selective targeting of cytotoxic drugs to cancer cells presenting tumor-associated surface markers, thereby minimizing systemic toxicity. Traditionally, the drug is conjugated nonselectively to cysteine or lysine residues in the antibody. However, these strategies often lead to heterogeneous products, which make optimization of the biological, physical, and pharmacological properties of an ADC challenging. Here we demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical reactivity to synthesize homogeneous ADCs with precise control of conjugation site and stoichiometry. p-Acetylphenylalanine was site-specifically incorporated into an anti-Her2 antibody Fab fragment and full-length IgG in Escherichia coli and mammalian cells, respectively. The mutant protein was selectively and efficiently conjugated to an auristatin derivative through a stable oxime linkage. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro cytotoxic activity against Her2(+) cancer cells, and complete tumor regression in rodent xenograft treatment models. The synthesis and characterization of homogeneous ADCs with medicinal chemistry-like control over macromolecular structure should facilitate the optimization of ADCs for a host of therapeutic uses.

A genetically encoded aza-Michael acceptor for covalent cross-linking of protein-receptor complexes.

Selective covalent bond formation at a protein-protein interface potentially can be achieved by genetically introducing into a protein an appropriately "tuned" electrophilic unnatural amino acid that reacts with a native nucleophilic residue in its cognate receptor upon complex formation. We have evolved orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs that genetically encode three aza-Michael acceptor amino acids, N(ε)-acryloyl-(S)-lysine (AcrK, 1), p-acrylamido-(S)-phenylalanine (AcrF, 2), and p-vinylsulfonamido-(S)-phenylalanine (VSF, 3), in response to the amber stop codon in Escherichia coli. Using an αErbB2 Fab-ErbB2 antibody-receptor pair as an example, we demonstrate covalent bond formation between an αErbB2-VSF mutant and a specific surface lysine ε-amino group of ErbB2, leading to near quantitative cross-linking to either purified ErbB2 in vitro or to native cellular ErbB2 at physiological pH. This efficient biocompatible reaction may be useful for creating novel cell biological probes, diagnostics, or therapeutics that selectively and irreversibly bind a target protein in vitro or in living cells.

Arrayed antibody library technology for therapeutic biologic discovery.

Traditional immunization and display antibody discovery methods rely on competitive selection amongst a pool of antibodies to identify a lead. While this approach has led to many successful therapeutic antibodies, targets have been limited to proteins which are easily purified. In addition, selection driven discovery has produced a narrow range of antibody functionalities focused on high affinity antagonism. We review the current progress in developing arrayed protein libraries for screening-based, rather than selection-based, discovery. These single molecule per microtiter well libraries have been screened in multiplex formats against both purified antigens and directly against targets expressed on the cell surface. This facilitates the discovery of antibodies against therapeutically interesting targets (GPCRs, ion channels, and other multispanning membrane proteins) and epitopes that have been considered poorly accessible to conventional discovery methods.

HIV envelope trimers and gp120 as immunogens to induce broadly neutralizing antibodies in cows.

The study of immunogens capable of eliciting broadly neutralizing antibodies (bnAbs) is crucial for the development of an HIV vaccine. To date, only cows, making use of their ultralong CDRH3 loops, have reliably elicited bnAbs following immunization with HIV Envelope trimers. Antibody responses to the CD4 binding site have been readily elicited by immunization of cows with a stabilized Env trimer of the BG505 strain and, with more difficulty, to the V2-apex region of Env with a cocktail of trimers. Here, we sought to determine whether the BG505 Env trimer could be engineered to generate new bnAb specificities in cows. Since the cow CD4 binding site bnAbs bind to monomeric BG505 gp120, we also sought to determine whether gp120 immunization alone might be sufficient to induce bnAbs. We found that engineering the CD4 binding site by mutation of a key binding residue of BG505 HIV Env resulted in a reduced bnAb response that took more immunizations to develop. Monoclonal antibodies isolated from one animal were directed to the V2-apex, suggesting a re-focusing of the bnAb response. Immunization with monomeric BG505 g120 generated no serum bnAb responses, indicating that the ultralong CDRH3 bnAbs are only elicited in the context of the trimer in the absence of many other less restrictive epitopes presented on monomeric gp120. The results support the notion of a hierarchy of epitopes on HIV Env and suggest that, even with the presence in the cow repertoire of ultralong CDRH3s, bnAb epitopes are relatively disfavored.

Immunization of cows with HIV envelope trimers generates broadly neutralizing antibodies to the V2-apex from the ultralong CDRH3 repertoire.

The generation of broadly neutralizing antibodies (bnAbs) to specific HIV epitopes of the HIV Envelope (Env) is one of the cornerstones of HIV vaccine research. The current animal models we use have been unable to reliable produce a broadly neutralizing antibody response, with the exception of cows. Cows have rapidly and reliably produced a CD4 binding site response by homologous prime and boosting with a native-like Env trimer. In small animal models other engineered immunogens previously have been able to focus antibody responses to the bnAb V2-apex region of Env. Here, we immunized two groups of cows (n=4) with two regiments of V2-apex focusing immunogens to investigate whether antibody responses could be directed to the V2-apex on Env. Group 1 were immunized with chimpanzee simian immunodeficiency virus (SIV)-Env trimer that shares its V2-apex with HIV, followed by immunization with C108, a V2-apex focusing immunogen, and finally boosted with a cross-clade native-like trimer cocktail. Group 2 were immunized with HIV C108 Env trimer followed by the same HIV trimer cocktail as Group 1. Longitudinal serum analysis showed that one cow in each group developed serum neutralizing antibody responses to the V2-apex. Eight and 11 bnAbs were isolated from Group 1 and Group 2 cows respectively. The best bnAbs had both medium breadth and potency. Potent and broad responses developed later than previous CD4bs cow bnAbs and required several different immunogens. All isolated bnAbs were derived from the ultralong CDRH3 repertoire. The finding that cow antibodies can target multiple broadly neutralizing epitopes on the HIV surface reveals important insight into the generation of immunogens and testing in the cow animal model. The exclusive isolation of ultralong CDRH3 bnAbs, despite only comprising a small percent of the cow repertoire, suggests these antibodies outcompete the long and short CDRH3 antibodies during the bnAb response.

Self-assembled antibody multimers through peptide nucleic acid conjugation.

With the recent clinical success of bispecific antibodies, a strategy to rapidly synthesize and evaluate bispecific or higher order multispecific molecules could facilitate the discovery of new therapeutic agents. Here, we show that unnatural amino acids (UAAs) with orthogonal chemical reactivity can be used to generate site-specific antibody-oligonucleotide conjugates. These constructs can then be self-assembled into multimeric complexes with defined composition, valency, and geometry. With this approach, we generated potent bispecific antibodies that recruit cytotoxic T lymphocytes to Her2 and CD20 positive cancer cells, as well as multimeric antibody fragments with enhanced activity. This strategy should accelerate the synthesis and in vitro characterization of antibody constructs with unique specificities and molecular architectures.

Identification of metal ion binding peptides containing unnatural amino acids by phage display.

The bidentate metal binding amino acid bipyridylalanine (BpyAla) was incorporated into a disulfide linked cyclic peptide phage displayed library to identify metal ion binding peptides. Selection against Ni(2+)-nitrilotriacetic acid (NTA) enriched for sequences containing histidine and BpyAla. BpyAla predominated when selections were carried out at lower pH, consistent with the differential pKa's of histidine and BpyAla. Two peptides containing BpyAla were synthesized and found to bind Ni(2+) with low micromolar dissociation constants. Incorporation of BpyAla and other metal binding amino acids into peptide and protein libraries should enable the evolution of novel binding and catalytic activities.