SARS-CoV-2S-Protein-Ace2 Binding Analysis Using Surface Plasmon Resonance.

Surface plasmon resonance (SPR) allows for the label-free determination of the binding affinity and rate constants of bimolecular interactions. Here, we describe the method used for the analysis of the Ace2-SARS-CoV2 S-protein interaction using indirect capture of the S-protein onto the SPR surface, and flowing monomeric Ace2. This method will allow for the determination of the rate constants for affinity, with additional analysis that is achievable using S-protein capture levels in conjunction with the sensorgram response for relative activity benchmarking.

Exploring rigid-backbone protein docking in biologics discovery: a test using the DARPin scaffold.

Accurate protein-protein docking remains challenging, especially for artificial biologics not coevolved naturally against their protein targets, like antibodies and other engineered scaffolds. We previously developed ProPOSE, an exhaustive docker with full atomistic details, which delivers cutting-edge performance by allowing side-chain rearrangements upon docking. However, extensive protein backbone flexibility limits its practical applicability as indicated by unbound docking tests. To explore the usefulness of ProPOSE on systems with limited backbone flexibility, here we tested the engineered scaffold DARPin, which is characterized by its relatively rigid protein backbone. A prospective screening campaign was undertaken, in which sequence-diversified DARPins were docked and ranked against a directed epitope on the target protein BCL-W. In this proof-of-concept study, only a relatively small set of 2,213 diverse DARPin interfaces were selected for docking from the huge theoretical library from mutating 18 amino-acid positions. A computational selection protocol was then applied for enrichment of binders based on normalized computed binding scores and frequency of binding modes against the predefined epitope. The top-ranked 18 designed DARPin interfaces were selected for experimental validation. Three designs exhibited binding affinities to BCL-W in the nanomolar range comparable to control interfaces adopted from known DARPin binders. This result is encouraging for future screening and engineering campaigns of DARPins and possibly other similarly rigid scaffolds against targeted protein epitopes. Method limitations are discussed and directions for future refinements are proposed.

Tuning the immune response: sulfated archaeal glycolipid archaeosomes as an effective vaccine adjuvant for induction of humoral and cell-mediated immunity towards the SARS-CoV-2 Omicron variant of concern.

Liposomes composed of sulfated lactosyl archaeol (SLA) have been shown to be a safe and effective vaccine adjuvant with a multitude of antigens in preclinical studies. In particular, SLA-adjuvanted SARS-CoV-2 subunit vaccines based on trimeric spike protein antigens were shown to be immunogenic and efficacious in mice and hamsters. With the continued emergence of SARS-CoV-2 variants, we sought to evaluate next-generation vaccine formulations with an updated antigenic identity. This was of particular interest for the widespread Omicron variant, given the abundance of mutations and structural changes observed within its spike protein compared to other variants. An updated version of our resistin-trimerized SmT1 corresponding to the B.1.1.529 variant was successfully generated in our Chinese Hamster Ovary (CHO) cell-based antigen production platform and characterized, revealing some differences in protein profile and ACE2 binding affinity as compared to reference strain-based SmT1. We next evaluated this Omicron-based spike antigen for its immunogenicity and ability to generate robust antigen-specific immune responses when paired with SLA liposomes or AddaS03 (a mimetic of the AS03 oil-in-water emulsion adjuvant system found in commercialized SARS-CoV-2 protein vaccines). Immunization of mice with vaccine formulations containing this updated antigen with either adjuvant stimulated neutralizing antibody responses favouring Omicron over the reference strain. Cell-mediated responses, which play an important role in the neutralization of intracellular infections, were induced to a much higher degree with the SLA adjuvant relative to the AddaS03-adjuvanted formulations. As such, updated vaccines that are better capable of targeting towards SARS-CoV-2 variants can be generated through an optimized combination of antigen and adjuvant components.

TNX-1500, a crystallizable fragment-modified anti-CD154 antibody, prolongs nonhuman primate renal allograft survival.

The blockade of the CD154-CD40 pathway with anti-CD154 monoclonal antibody has been a promising immunomodulatory approach to prevent allograft rejection. However, clinical trials of immunoglobulin G1 antibodies targeting this pathway revealed thrombogenic properties, which were subsequently shown to be mediated by crystallizable fragment (Fc)-gamma receptor IIa-dependent platelet activation. To prevent thromboembolic complications, an immunoglobulin G4 anti-CD154 monoclonal antibody, TNX-1500, which retains the fragment antigen binding region of ruplizumab (humanized 5c8, BG9588), was modified by protein engineering to decrease Fc binding to Fc-gamma receptor IIa while retaining certain other effector functions and pharmacokinetics comparable with natural antibodies. Here, we report that TNX-1500 treatment is not associated with platelet activation in vitro and consistently inhibits kidney allograft rejection in vivo without clinical or histologic evidence of prothrombotic phenomena. We conclude that TNX-1500 retains efficacy similar to that of 5c8 to prevent kidney allograft rejection while avoiding previously identified pathway-associated thromboembolic complications.

A CHO stable pool production platform for rapid clinical development of trimeric SARS-CoV-2 spike subunit vaccine antigens.

Protein expression from stably transfected Chinese hamster ovary (CHO) clones is an established but time-consuming method for manufacturing therapeutic recombinant proteins. The use of faster, alternative approaches, such as non-clonal stable pools, has been restricted due to lower productivity and longstanding regulatory guidelines. Recently, the performance of stable pools has improved dramatically, making them a viable option for quickly producing drug substance for GLP-toxicology and early-phase clinical trials in scenarios such as pandemics that demand rapid production timelines. Compared to stable CHO clones which can take several months to generate and characterize, stable pool development can be completed in only a few weeks. Here, we compared the productivity and product quality of trimeric SARS-CoV-2 spike protein ectodomains produced from stable CHO pools or clones. Using a set of biophysical and biochemical assays we show that product quality is very similar and that CHO pools demonstrate sufficient productivity to generate vaccine candidates for early clinical trials. Based on these data, we propose that regulatory guidelines should be updated to permit production of early clinical trial material from CHO pools to enable more rapid and cost-effective clinical evaluation of potentially life-saving vaccines.

An anti-HER2 biparatopic antibody that induces unique HER2 clustering and complement-dependent cytotoxicity.

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.

Production of afucosylated antibodies in CHO cells by coexpression of an anti-FUT8 intrabody.

Some effector functions prompted by immunoglobulin G (IgG) antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC), strongly depend on the N-glycans linked to asparagine 297 of the Fc region of the protein. A single α-(1,6)-fucosyltransferase (FUT8) is responsible for catalyzing the addition of an α-1,6-linked fucose residue to the first GlcNAc residue of the N-linked glycans. Antibodies missing this core fucose show a significantly enhanced ADCC and increased antitumor activity, which could help reduce therapeutic dose requirement, potentially translating into reduced safety concerns and manufacturing costs. Several approaches have been developed to modify glycans and improve the biological functions of antibodies. Here, we demonstrate that expression of a membrane-associated anti-FUT8 intrabody engineered to reside in the endoplasmic reticulum and Golgi apparatus can efficiently reduce FUT8 activity and therefore the core-fucosylation of the Fc N-glycan of an antibody. IgG1-producing CHO cells expressing the intrabody secrete antibodies with reduced core fucosylation as demonstrated by lectin blot analysis and UPLC-HILIC glycan analysis. Cells engineered to inhibit directly and specifically alpha-(1,6)-fucosyltransferase activity allows for the production of g/L levels of IgGs with strongly enhanced ADCC effector function, for which the level of fucosylation can be selected. The quick and efficient method described here should have broad practical applicability for the development of next-generation therapeutic antibodies with enhanced effector functions.

Structure-based dual affinity optimization of a SARS-CoV-1/2 cross-reactive single-domain antibody.

The SARS coronavirus 2 (SARS-CoV-2) spike (S) protein binding to the human ACE2 receptor is the molecular event that initiates viral entry into host cells and leads to infection and virus replication. There is a need for agents blocking viral entry into host cells that are cross-reactive with emerging virus variants. VHH-72 is an anti-SARS-CoV-1 single-domain antibody that also exhibits cross-specificity with SARS-CoV-2 but with decreased binding affinity. Here we applied a structure-based approach to affinity-mature VHH-72 for the SARS-CoV-2 spike protein while retaining the original affinity for SARS-CoV-1. This was achieved by employing the computational platform ADAPT in a constrained dual-affinity optimization mode as a means of broadening specificity. Select mutants designed by ADAPT were formatted as fusions with a human IgG1-Fc fragment. These mutants demonstrated improved binding to the SARS-CoV-2 spike protein due to decreased dissociation rates. Functional testing for virus neutralization revealed improvements relative to the parental VHH72-Fc up to 10-fold using a SARS-CoV-2 pseudotyped lentivirus and 20-fold against the SARS-CoV-2 authentic live virus (Wuhan variant). Binding and neutralization improvements were maintained for some other SARS-CoV-2 variants currently in circulation. These improved VHH-72 mutants are predicted to establish novel interactions with the S antigen. They will be useful, alone or as fusions with other functional modules, in the global quest for treatments of COVID-19 infections.

Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX.

The architectural complexity and heterogeneity of the tumor microenvironment (TME) remains a substantial obstacle in the successful treatment of cancer. Hypoxia, caused by insufficient oxygen supply, and acidosis, resulting from the expulsion of acidic metabolites, are prominent features of the TME. To mitigate the consequences of the hostile TME, cancer cells metabolically rewire themselves and express a series of specific transporters and enzymes instrumental to this adaptation. One of these proteins is carbonic anhydrase (CA)IX, a zinc-containing extracellular membrane bound enzyme that has been shown to play a critical role in the maintenance of a neutral intracellular pH (pHi), allowing tumor cells to survive and thrive in these harsh conditions. Although CAIX has been considered a promising cancer target, only two antibody-based therapeutics have been clinically tested so far. To fill this gap, we generated a series of novel monoclonal antibodies (mAbs) that specifically recognize the extracellular domain (ECD) of human CAIX. Here we describe the biophysical and functional properties of a set of antibodies against the CAIX ECD domain and their applicability as: 1) suitable for development as an antibody-drug-conjugate, 2) an inhibitor of CAIX enzyme activity, or 3) an imaging/detection antibody. The results presented here demonstrate the potential of these specific hCAIX mAbs for further development as novel cancer therapeutic and/or diagnostic tools.

Hydrogen-deuterium exchange mass spectrometry reveals three unique binding responses of mAbs directed to the catalytic domain of hCAIX.

Human carbonic anhydrase (hCAIX), an extracellular enzyme that catalyzes the reversible hydration of CO2, is often overexpressed in solid tumors. This enzyme is instrumental in maintaining the survival of cancer cells in a hypoxic and acidic tumor microenvironment. Absent in most normal tissues, hCAIX is a promising therapeutic target for detection and treatment of solid tumors. Screening of a library of anti-hCAIX monoclonal antibodies (mAbs) previously identified three therapeutic candidates (mAb c2C7, m4A2 and m9B6) with distinct biophysical and functional characteristics. Selective binding to the catalytic domain was confirmed by yeast surface display and isothermal calorimetry, and deeper insight into the dynamic binding profiles of these mAbs upon binding were highlighted by bottom-up hydrogen-deuterium exchange mass spectrometry (HDX-MS). Here, a conformational and allosterically silent epitope was identified for the antibody-drug conjugate candidate c2C7. Unique binding profiles are described for both inhibitory antibodies, m4A2 and m9B6. M4A2 reduces the ability of the enzyme to hydrate CO2 by steric gating at the entrance of the catalytic cavity. Conversely, m9B6 disrupts the secondary structure that is necessary for substrate binding and hydration. The synergy of these two inhibitory mechanisms is demonstrated in in vitro activity assays and HDX-MS. Finally, the ability of m4A2 to modulate extracellular pH and intracellular metabolism is reported. By highlighting three unique modes by which hCAIX can be targeted, this study demonstrates both the utility of HDX-MS as an important tool in the characterization of anti-cancer biotherapeutics, and the underlying value of CAIX as a therapeutic target.

Redesigning an antibody H3 loop by virtual screening of a small library of human germline-derived sequences.

The design of superior biologic therapeutics, including antibodies and engineered proteins, involves optimizing their specific ability to bind to disease-related molecular targets. Previously, we developed and applied the Assisted Design of Antibody and Protein Therapeutics (ADAPT) platform for virtual affinity maturation of antibodies (Vivcharuk et al. in PLoS One 12(7):e0181490, https://doi.org/10.1371/journal.pone.0181490 , 2017). However, ADAPT is limited to point mutations of hot-spot residues in existing CDR loops. In this study, we explore the possibility of wholesale replacement of the entire H3 loop with no restriction to maintain the parental loop length. This complements other currently published studies that sample replacements for the CDR loops L1, L2, L3, H1 and H2. Given the immense sequence space theoretically available to H3, we focused on the virtual grafting of over 5000 human germline-derived H3 sequences from the IGMT/LIGM database increasing the diversity of the sequence space when compared to using crystalized H3 loop sequences. H3 loop conformations are generated and scored to identify optimized H3 sequences. Experimental testing of high-ranking H3 sequences grafted into the framework of the bH1 antibody against human VEGF-A led to the discovery of multiple hits, some of which had similar or better affinities relative to the parental antibody. In over 75% of the tested designs, the re-designed H3 loop contributed favorably to overall binding affinity. The hits also demonstrated good developability attributes such as high thermal stability and no aggregation. Crystal structures of select re-designed H3 variants were solved and indicated that although some deviations from predicted structures were seen in the more solvent accessible regions of the H3 loop, they did not significantly affect predicted affinity scores.

New soluble angiopoietin analog of Hepta-ANG1 prevents pathological vascular leakage.

Vascular leak is a key driver of organ injury in diseases, and strategies that reduce enhanced permeability and vascular inflammation are promising therapeutic targets. Activation of the angiopoietin-1 (ANG1)-Tie2 tyrosine kinase signaling pathway is an important regulator of vascular quiescence. Here we describe the design and construction of a new soluble ANG1 mimetic that is a potent activator of endothelial Tie2 in vitro and in vivo. Using a chimeric fusion strategy, we replaced the extracellular matrix (ECM) binding and oligomerization domain of ANG1 with a heptameric scaffold derived from the C-terminus of serum complement protein C4-binding protein α. We refer to this new fusion protein biologic as Hepta-ANG1, which forms a stable heptamer and induces Tie2 phosphorylation in cultured cells, and in the lung following intravenous injection of mice. Injection of Hepta-ANG1 ameliorates vascular endothelial growth factor- and lipopolysaccharide-induced vascular leakage, in keeping with the known functions of Angpt1-Tie2 in maintaining quiescent vascular stability. The new Hepta-ANG1 fusion is easy to produce and displays remarkable stability with high multimericity that can potently activate Tie2. It could be a new candidate ANG1 mimetic therapy for treatments of inflammatory vascular leak, such as acute respiratory distress syndrome and sepsis.

AlbuCORE: an albumin-based molecular scaffold for multivalent biologics design.

As biologics have become a mainstay in the development of novel therapies, protein engineering tools to expand on their structural advantages, namely specificity, affinity, and valency are of interest. Antibodies have dominated this field as the preferred scaffold for biologics development while there has been limited exploration into the use of albumin with its unique physiological characteristics as a platform for biologics design. There has been a great deal of interest to create bispecific and more complex multivalent molecules to build on the advantages offered by protein-based therapeutics relative to small molecules. Here, we explore the use of human serum albumin (HSA) as a scaffold for the design of multispecific biologics. In particular, we describe a structure-guided approach to the design of split HSA molecules we refer to as AlbuCORE, that effectively and spontaneously forms a native albumin-like molecule, but in a heterodimeric state upon co-expression. We show that the split AlbuCORE designs allow the creation of novel fusion entities with unique alternate geometries. We also show that, apart from these AlbuCORE fusion entities, there is an opportunity to explore their albumin-like small hydrophobic molecule carrying capacity as a drug conjugate in these designs.

Structure-based engineering of pH-dependent antibody binding for selective targeting of solid-tumor microenvironment.

Recent development of monoclonal antibodies as mainstream anticancer agents demands further optimization of their safety for use in humans. Potent targeting and/or effector activities on normal tissues is an obvious toxicity concern. Optimization of specific tumor targeting could be achieved by taking advantage of the extracellular acidity of solid tumors relative to normal tissues. Here, we applied a structure-based computational approach to engineer anti-human epidermal growth factor receptor 2 (Her2) antibodies with selective binding in the acidic tumor microenvironment. We used an affinity maturation platform in which dual-pH histidine-scanning mutagenesis was implemented for pH selectivity optimization. Testing of a small set of designs for binding to the recombinant Her2 ectodomain led to the identification of antigen-binding fragment (Fab) variants with the desired pH-dependent binding behavior. Binding selectivity toward acidic pH was improved by as much as 25-fold relative to the parental bH1-Fab. In vitro experiments on cells expressing intact Her2 confirmed that designed variants formatted as IgG1/k full-size antibodies have high affinity and inhibit the growth of tumor spheroids at a level comparable to that of the benchmark anti-Her2 antibody trastuzumab (Herceptin®) at acidic pH, whereas these effects were significantly reduced at physiological pH. In contrast, both Herceptin and the parental bH1 antibody exhibited strong cell binding and growth inhibition irrespective of pH. This work demonstrates the feasibility of computational optimization of antibodies for selective targeting of the acidic environment such as that found in many solid tumors.

Binding symmetry and surface flexibility mediate antibody self-association.

Solution stability is an important factor in the optimization of engineered biotherapeutic candidates such as monoclonal antibodies because of its possible effects on manufacturability, pharmacology, efficacy and safety. A detailed atomic understanding of the mechanisms governing self-association of natively folded protein monomers is required to devise predictive tools to guide screening and re-engineering along the drug development pipeline. We investigated pairs of affinity-matured full-size antibodies and observed drastically different propensities to aggregate from variants differing by a single amino-acid. Biophysical testing showed that antigen-binding fragments (Fabs) from the aggregating antibodies also reversibly associated with equilibrium dissociation constants in the low-micromolar range. Crystal structures (PDB accession codes 6MXR, 6MXS, 6MY4, 6MY5) and bottom-up hydrogen-exchange mass spectrometry revealed that Fab self-association occurs in a symmetric mode that involves the antigen complementarity-determining regions. Subtle local conformational changes incurred upon point mutation of monomeric variants foster formation of complementary polar interactions and hydrophobic contacts to generate a dimeric Fab interface. Testing of popular in silico tools generally indicated low reliabilities for predicting the aggregation propensities observed. A structure-aggregation data set is provided here in order to stimulate further improvements of in silico tools for prediction of native aggregation. Incorporation of intermolecular docking, conformational flexibility, and short-range packing interactions may all be necessary features of the ideal algorithm.

Binding and functional profiling of antibody mutants guides selection of optimal candidates as antibody drug conjugates.

An increasingly appreciated conundrum in the discovery of antibody drug conjugates (ADCs) is that an antibody that was selected primarily for strong binding to its cancer target may not serve as an optimal ADC. In this study, we performed mechanistic cell-based experiments to determine the correlation between antibody affinity, avidity, internalization and ADC efficacy. We used structure-guided design to assemble a panel of antibody mutants with predicted Her2 affinities ranging from higher to lower relative to the parent antibody, Herceptin. These antibodies were ranked for binding via SPR and via flow-cytometry on high-Her2 SKOV3 cells and low-Her2 MCF7 cells, the latter acting as a surrogate for low-Her2 normal cells. A subpanel of variants, representative of different Her2-binding affinities (2 strong, 2 moderate and 3 weak), were further screened via high-content imaging for internalization efficacies in high versus low-Her2 cells. Finally, these antibodies were evaluated in ADC cytotoxicity screening assays (using DM1 and MMAE secondary antibodies) and as antibody-drug conjugates (DM1 and PNU159682). Our results identified specific but weak Her2-binding variants as optimal candidates for developing DM1 and PNU ADCs since they exhibited high potencies (low to sub-nM) in high-Her2 SKOV3 cells and low toxicities in low-Her2 cells. The 2 strong-affinity variants were highly potent in SKOV3 cells but also showed significant toxicities in low-Her2 cells and therefore are predicted to be toxic in normal tissues. Our findings show that pharmacological profiling of an antibody library in multiple binding and functional assays allows for selection of optimal ADCs.

Enhanced anti-metastatic bioactivity of an IGF-TRAP re-engineered to improve physicochemical properties.

The insulin-like growth factor (IGF) axis has been implicated in the progression of malignant disease and identified as a clinically important therapeutic target. Several IGF-1 receptor (IGF-1R) targeting drugs including humanized monoclonal antibodies have advanced to phase II/III clinical trials, but to date, have not progressed to clinical use, due, at least in part, to interference with insulin receptor signalling. We previously reported on the production of a soluble fusion protein consisting of the extracellular domain of human IGF-1R fused to the Fc portion of human IgG1 (first generation IGF-TRAP) that bound human IGF-1 and IGF-2 with a 3 log higher affinity than insulin. We showed that the IGF-TRAP had potent anti-cancer activity in several pre-clinical models of aggressive carcinomas. Here we report on the re-engineering of the IGF-TRAP with the aim of improving physicochemical properties and suitability for clinical applications. We show that cysteine-serine substitutions in the Fc hinge region of IGF-TRAP eliminated high-molecular-weight oligomerized species, while a further addition of a flexible linker, not only improved the pharmacokinetic profile, but also enhanced the therapeutic profile of the IGF-TRAP, as evaluated in an experimental colon carcinoma metastasis model. Dose-response profiles of the modified IGF-TRAPs correlated with their bio-availability profiles, as measured by the IGF kinase-receptor-activation (KIRA) assay, providing a novel, surrogate biomarker for drug efficacy. This study provides a compelling example of structure-based re-engineering of Fc-fusion-based biologics for better manufacturability that also significantly improved pharmacological parameters. It identifies the re-engineered IGF-TRAP as a potent anti-cancer therapeutic.

Optimization of a high-cell-density polyethylenimine transfection method for rapid protein production in CHO-EBNA1 cells.

For pre-clinical evaluation of biotherapeutic candidates, protein production by transient gene expression (TGE) in Chinese Hamster Ovary (CHO) cells offers important advantages, including the capability of rapidly and cost-effectively generating recombinant proteins that are highly similar to those produced in stable CHO clones. We have established a novel CHO clone (CHO-3E7) expressing a form of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) with improved TGE productivity relative to parental CHO cells. Taking advantage of a new transfection-compatible media formulation that permits prolonged, high-density culture, we optimized transfection parameters (cell density, plasmid vector and polyethylenimine concentrations) and post-transfection culture conditions to establish a new, high-performing process for rapid protein production. The growth media is chemically defined, and a single hydrolysate feed is added post-transfection, followed by periodic glucose supplementation. This method gave significantly higher yields than our standard low-cell density, F17-based CHO-3E7 TGE method, averaging several hundred mg/l for a panel of recombinant proteins and antibodies. Purified antibodies produced using the two methods had distinct glycosylation profiles but showed identical target binding kinetics by SPR. Key advantages of this new protein production platform include the cost-effectiveness of the transfection reagent, the commercial availability of the culture media and the ability to perform high-cell-density transfection without media change.

A Novel Affinity Tag, ABTAG, and Its Application to the Affinity Screening of Single-Domain Antibodies Selected by Phage Display.

ABTAG is a camelid single-domain antibody (sdAb) that binds to bovine serum albumin (BSA) with low picomolar affinity. In surface plasmon resonance (SPR) analyses using BSA surfaces, bound ABTAG can be completely dissociated from the BSA surfaces at low pH, over multiple cycles, without any reduction in the capacity of the BSA surfaces to bind ABTAG. A moderate throughput, SPR-based, antibody screening assay exploiting the unique features of ABTAG is described. Anti-carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) sdAbs were isolated from a phage-displayed sdAb library derived from the heavy chain antibody repertoire of a llama immunized with CEACAM6. Following one or two rounds of panning, enriched clones were expressed as ABTAG fusions in microtiter plate cultures. The sdAb-ABTAG fusions from culture supernatants were captured on BSA surfaces and CEACAM6 antigen was then bound to the captured molecules. The SPR screening method gives a read-out of relative expression levels of the fusion proteins and kinetic and affinity constants for CEACAM6 binding by the captured molecules. The library was also panned and screened by conventional methods and positive clones were subcloned and expressed for SPR analysis. Compared to conventional panning and screening, the SPR-based ABTAG method yielded a considerably higher diversity of binders, some with affinities that were three orders of magnitude higher affinity than those identified by conventional panning.