Antibody Variable Domains Research Articles

Rational Protein Engineering to Enhance MHC-Independent T-cell Receptors.

Chimeric antigen receptor (CAR)-based therapies have pioneered synthetic cellular immunity but remain limited in their long-term efficacy. Emerging data suggest that dysregulated CAR-driven T-cell activation causes T-cell dysfunction and therapeutic failure. To re-engage the precision of the endogenous T-cell response, we designed MHC-independent T-cell receptors (miTCR) by linking antibody variable domains to T-cell receptor constant chains. Using predictive modeling, we observed that this standard "cut and paste" approach to synthetic protein design resulted in myriad biochemical conflicts at the hybrid variable-constant domain interface. Through iterative modeling and sequence modifications, we developed structure-enhanced miTCRs which significantly improved receptor-driven T-cell function across multiple tumor models. We found that 41BB costimulation specifically prolonged miTCR T-cell persistence and enabled improved leukemic control in vivo compared with classic CAR T cells. Collectively, we have identified core features of hybrid receptor structure responsible for regulating function. Significance: Improving the durability of engineered T-cell immunotherapies is critical to enhancing efficacy. We used a structure-informed design to evolve improved miTCR function across several models. This work underscores the central role of synthetic receptor structure in T-cell function and provides a framework for improved receptor engineering.

Design, Construction, and Validation of a Yeast-Displayed Chemically Expanded Antibody Library.

In vitro display technologies, exemplified by phage and yeast display, have emerged as powerful platforms for antibody discovery and engineering. However, the identification of antibodies that disrupt target functions beyond binding remains a challenge. In particular, there are very few strategies that support identification and engineering of either protein-based irreversible binders or inhibitory enzyme binders. Expanding the range of chemistries in antibody libraries has the potential to lead to efficient discovery of function-disrupting antibodies. In this work, we describe a yeast display-based platform for the discovery of chemically diversified antibodies. We constructed a billion-member antibody library that supports the presentation of a range of chemistries within antibody variable domains via noncanonical amino acid (ncAA) incorporation and subsequent bioorthogonal click chemistry conjugations. Use of a polyspecific orthogonal translation system enables introduction of chemical groups with various properties, including photo-reactive, proximity-reactive, and click chemistry-enabled functional groups for library screening. We established conjugation conditions that facilitate modification of the full library, demonstrating the feasibility of sorting the full billion-member library in "protein-small molecule hybrid" format in future work. Here, we conducted initial library screens after introducing O-(2-bromoethyl)tyrosine (OBeY), a weakly electrophilic ncAA capable of undergoing proximity-induced crosslinking to a target. Enrichments against donkey IgG and protein tyrosine phosphatase 1B (PTP1B) each led to the identification of several OBeY-substituted clones that bind to the targets of interest. Flow cytometry analysis on the yeast surface confirmed higher retention of binding for OBeY-substituted clones compared to clones substituted with ncAAs lacking electrophilic side chains after denaturation. However, subsequent crosslinking experiments in solution with ncAA-substituted clones yielded inconclusive results, suggesting that weakly reactive OBeY side chain is not sufficient to drive robust crosslinking in the clones isolated here. Nonetheless, this work establishes a multi-modal, chemically expanded antibody library and demonstrates the feasibility of conducting discovery campaigns in chemically expanded format. This versatile platform offers new opportunities for identifying and characterizing antibodies with properties beyond what is accessible with the canonical amino acids, potentially enabling discovery of new classes of reagents, diagnostics, and even therapeutic leads.

Unveiling the multifaceted landscape of N-glycosylation in antibody variable domains: Insights and implications

N-glycosylation at the antibody variable domain has emerged as an important modification influencing antibody function. Despite its significance, information regarding its role and regulation remains limited. To address this gap, we comprehensively explored antibody structures housing N-glycosylation within the Protein Data Bank, yielding fresh insights into this intricate landscape. Our findings revealed that among 208 structures, N-glycosylation was more prevalent in human and mouse antibodies containing IGHV1–8 and IGHV2–2 germline genes, respectively. Moreover, our research highlights the potential for somatic hypermutation to introduce N-glycosylation sites by substituting polar residues (Ser or Thr) in germline variable genes with asparagine. Notably, our study underscores the prevalence of N-glycosylation in antiviral antibodies, especially anti-HIV. Besides antigen-antibody interaction, our findings suggest that N-glycosylation may impact antibody specificity, affinity, and avidity by influencing Fab dimer formation and complementary-determining region orientation. We also identified different glycan structures in HIV and SARS-CoV-2 antibody proteomic datasets, highlighting disparities from the N-glycan structures between PDB antibodies and biological repertoires further highlighting the complexity of N-glycosylation patterns. Our findings significantly enrich our understanding of the N-glycosylation's multifaceted characteristics within the antibody variable domain. Additionally, they underscore the pressing imperative for a more comprehensive characterization of its impact on antibody function.

Recent advancement in targeted therapy and role of emerging technologies to treat cancer.

Cancer is a complex disease that causes abnormal cell growth and spread. DNA mutations, chemical or environmental exposure, viral infections, chronic inflammation, hormone abnormalities, etc., are underlying factors that can cause cancer. Drug resistance and toxicity complicate cancer treatment. Additionally, the variability of cancer makes it difficult to establish universal treatment guidelines. Next-generation sequencing has made genetic testing inexpensive. This uncovers genetic mutations that can be treated with specialty drugs. AI (artificial intelligence), machine learning, biopsy, next-generation sequencing, and digital pathology provide personalized cancer treatment. This allows for patient-specific biological targets and cancer treatment. Monoclonal antibodies, CAR-T, and cancer vaccines are promising cancer treatments. Recent trial data incorporating these therapies have shown superiority in clinical outcomes and drug tolerability over conventional chemotherapies. Combinations of these therapies with new technology can change cancer treatment and help many. This review discusses the development and challenges of targeted therapies like monoclonal antibodies (mAbs), bispecific antibodies (BsAbs), bispecific T cell engagers (BiTEs), dual variable domain (DVD) antibodies, CAR-T therapy, cancer vaccines, oncolytic viruses, lipid nanoparticle-based mRNA cancer vaccines, and their clinical outcomes in various cancers. We will also study how artificial intelligence and machine learning help find new cancer treatment targets.

Single domain Camelid antibody fragments for molecular imaging and therapy of cancer.

Despite innovations in cancer therapeutics, cancer remains associated with high mortality and is one of biggest health challenges worldwide. Therefore, developing precise cancer imaging and effective treatments is an unmet clinical need. A relatively novel type of therapeutics are heavy chain variable domain antibody fragments (VHHs) derived from llamas. Here, we explored the suitability of VHHs for cancer imaging and therapy through reviewing the existing literature. We searched the MEDLINE, EMBASE and Cochrane databases and identified 32 papers on molecular imaging and 41 papers on therapy that were suitable for comprehensive reviewing. We found that VHHs harbor a higher specificity and affinity compared to mAbs, which contributes to high-quality imaging and less side-effects on healthy cells. The employment of VHHs in cancer imaging showed remarkably shorter times between administration and imaging. Studies showed that 18F and 99mTc are two optimal radionuclides for imaging with VHHs and that site-specific labelling is the optimal conjugation modality for VHHs with radionuclide or fluorescent molecules. We found different solutions for reducing kidney retention and immunogenicity of VHHs. VHHs as anticancer therapeutics have been tested in photodynamic therapy, targeted radionuclide therapy, immunotherapy and molecular targeted therapy. These studies showed that VHHs target unique antigen epitopes, which are distinct from the ones recognized by mAbs. This advantage means that VHHs may be more effective for targeted anticancer therapy and can be combined with mAbs. We found that high cellular internalization and specificity of VHHs contributes to the effectiveness and safety of VHHs as anticancer therapeutics. Two clinical trials have confirmed that VHHs are effective and safe for cancer imaging and therapy. Together, VHHs seem to harbor several advantages compared to mAbs and show potential for application in personalized treatment for cancer patients. VHH-based imaging and therapy are promising options for improving outcomes of cancer patients.

Protein engineering of antibody fragments for pharmaceutical production

Antibody fragments without the Fc region are attracting attention in the pharmaceutical industry due to their high ability to penetrate solid tissues, cost-effective expression using microbial expression systems, and distinctive modes of action compared to those of full-size antibodies. Based on these characteristics, several antibody fragment agents have been approved. However, developing platform engineering methodologies to accelerate their development is important. In this review, we summarize and discuss protein engineering strategies for preparing therapeutic antibody fragments composed of antibody variable domains. Three (introduction of high-solubility tag systems, complementarity-determining region grafting, and domain arrangements) and two (introduction of purification tag systems and mutagenesis studies for protein L- or protein A-binding) protein engineering strategies have been reported for the cultivation and purification processes, respectively. Fusion tags might negatively impact molecular folding, function, immunogenicity, and final yield. If the production behavior of antibody fragments is not improved through complementarity-determining region grafting, domain arrangements, or human sequence-based mutagenesis, using additional fusion tag systems should be considered, with careful attention to the points described above. This summarized knowledge regarding protein engineering strategies for effectively producing antibody fragments will further accelerate therapeutic antibody fragment development.

KA-Search, a method for rapid and exhaustive sequence identity search of known antibodies

Antibodies with similar amino acid sequences, especially across their complementarity-determining regions, often share properties. Finding that an antibody of interest has a similar sequence to naturally expressed antibodies in healthy or diseased repertoires is a powerful approach for the prediction of antibody properties, such as immunogenicity or antigen specificity. However, as the number of available antibody sequences is now in the billions and continuing to grow, repertoire mining for similar sequences has become increasingly computationally expensive. Existing approaches are limited by either being low-throughput, non-exhaustive, not antibody specific, or only searching against entire chain sequences. Therefore, there is a need for a specialized tool, optimized for a rapid and exhaustive search of any antibody region against all known antibodies, to better utilize the full breadth of available repertoire sequences. We introduce Known Antibody Search (KA-Search), a tool that allows for the rapid search of billions of antibody variable domains by amino acid sequence identity across either the variable domain, the complementarity-determining regions, or a user defined antibody region. We show KA-Search in operation on the sim 2.4 billion antibody sequences available in the OAS database. KA-Search can be used to find the most similar sequences from OAS within 30 minutes and a representative subset of 10 million sequences in less than 9 seconds. We give examples of how KA-Search can be used to obtain new insights about an antibody of interest. KA-Search is freely available at https://github.com/oxpig/kasearch.

Effect of posttranslational modifications and subclass on IgG activity: from immunity to immunotherapy.

Humoral immune responses are characterized by complex mixtures of polyclonal antibody species varying in their isotype, target epitope specificity and affinity. Posttranslational modifications occurring during antibody production in both the antibody variable and constant domain create further complexity and can modulate antigen specificity and antibody Fc-dependent effector functions, respectively. Finally, modifications of the antibody backbone after secretion may further impact antibody activity. An in-depth understanding of how these posttranslational modifications impact antibody function, especially in the context of individual antibody isotypes and subclasses, is only starting to emerge. Indeed, only a minute proportion of this natural variability in the humoral immune response is currently reflected in therapeutic antibody preparations. In this Review, we summarize recent insights into how IgG subclass and posttranslational modifications impact IgG activity and discuss how these insights may be used to optimize therapeutic antibody development.

Identification of nurse shark VNAR single-domain antibodies targeting the spike S2 subunit of SARS-CoV-2.

SARS-CoV-2 is the etiological agent of the COVID-19 pandemic. Antibody-based therapeutics targeting the spike protein, specifically the S1 subunit or the receptor binding domain (RBD) of SARS-CoV-2, have gained attention due to their clinical efficacy in treating patients diagnosed with COVID-19. An alternative to conventional antibody therapeutics is the use of shark new antigen variable receptor domain (VNAR ) antibodies. VNAR s are small (<15 kDa) and can reach deep into the pockets or grooves of the target antigen. Here, we have isolated 53 VNAR s that bind to the S2 subunit by phage panning from a naïve nurse shark VNAR phage display library constructed in our laboratory. Among those binders, S2A9 showed the best neutralization activity against the original pseudotyped SARS-CoV-2 virus. Several binders, including S2A9, showed cross-reactivity against S2 subunits from other β coronaviruses. Furthermore, S2A9 showed neutralization activity against all variants of concern (VOCs) from alpha to omicron (including BA1, BA2, BA4, and BA5) in both pseudovirus and live virus neutralization assays. Our findings suggest that S2A9 could be a promising lead molecule for the development of broadly neutralizing antibodies against SARS-CoV-2 and emerging variants. The nurse shark VNAR phage library offers a novel platform that can be used to rapidly isolate single-domain antibodies against emerging viral pathogens.

Abstract LB211: Discovery of RenNano®-derived human heavy-chain-only antibodies that cross the blood-brain barrier

Abstract Therapeutic antibodies have been successfully used to treat several diseases, such as cancers and autoimmune diseases. However, the utility of conventional antibodies for neurological conditions is limited by the blood-brain barrier (BBB). Several strategies to address this issue have been reported, including receptor-mediated transcytosis (RMT) of antibodies using transferrin receptors. We hypothesize that this strategy could be further improved by the use of single-domain antibodies (sdAbs), such as the variable domain of heavy-chain-only antibodies (HCAbs), or variable new antigen receptors (VNARs), which are significantly smaller, and therefore could be used to more efficiently transport drugs of interest across the BBB. To this end, we developed anti-transferrin receptor 1 (TFR1) HCAbs utilizing our fully human heavy-chain-only antibody mice (RenNano®). We immunized RenNano® mice with recombinant TFR1 proteins, isolated the B cells from spleen and lymph nodes, and performed single B cell antibody screening using the Beacon® Optofluidic system. Most of the antibodies tested were cross-reactive to human and monkey TFR1. Furthermore, even though the antigen specificity relies on the VHH domain instead of a conventional antibody variable domain, the affinity of these HCAbs can reach 10−8-10−9 (KD). Of the 7 HCAbs tested, 6 were internalized into the human brain microvascular endothelial cell line, hCMEC/D3. To assess brain penetration of these antibodies in vivo, mice expressing human TFR1 (hTFR1 mice) received a tail vein injection with either isotype control, positive control pabinafusp alfa (a BBB penetrating anti-TFR1 monoclonal antibody enzyme conjugate) analog or RenNano®-derived HCAbs. After 0.5, 6, 24, and 72 h of exposure, mice brains were dissected for the quantification of HCAbs and for immunohistochemical analyses. The levels of anti-TFR1 HCAbs in the parenchyma was significantly higher than isotype controls and pabinafusp alfa analog. In brain sections, HCAbs were clearly observed in the parenchyma, and were colocalized with TFR1-expressing cells. These results demonstrate that HCAbs developed from RenNano® mice are able to penetrate the BBB. Taken together, these data highlight the tremendous potential for HCAbs and its variable domain sdAbs for transporting cargo across the BBB. Due to their smaller size and simpler structure, sdAbs could ultimately provide therapeutic benefit for neurodegenerative diseases, and offer promising potential for tumor penetration. Citation Format: Yiqing Hu, Lijun Zhang, Wenying Wang, Huizhen Zhao, Jiawei Yao, Chunhui Lv, Yunsheng Yao, Li Hui, Qingcong Lin, Taolin Liu, Yuelei Shen. Discovery of RenNano®-derived human heavy-chain-only antibodies that cross the blood-brain barrier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB211.

Structural Characterization of Nanobodies during Germline Maturation.

Camelid heavy-chain antibody variable domains (VHH), nanobodies, are the smallest-known functional antibody fragments with high therapeutic potential. In this study, we investigate a VHH binding to hen egg-white lysozyme (HEL). We structurally and dynamically characterized the conformational diversity of four VHH variants to elucidate the antigen-binding process. For two of these antibodies, not only are the dissociation constants known, but also the experimentally determined crystal structures of the VHH in complex with HEL are available. We performed well-tempered metadynamics simulations in combination with molecular dynamics simulations to capture a broad conformational space and to reconstruct the thermodynamics and kinetics of conformational transitions in the antigen-binding site, the paratope. By kinetically characterizing the loop movements of the paratope, we found that, with an increase in affinity, the state populations shift towards the binding competent conformation. The contacts contributing to antigen binding, and those who contribute to the overall stability, show a clear trend towards less variable but more intense contacts. Additionally, these investigated nanobodies clearly follow the conformational selection paradigm, as the binding competent conformation pre-exists within the structural ensembles without the presence of the antigen.

Structure-guided design of a potent Clostridiodes difficile toxin A inhibitor.

Crystal structures of camelid heavy-chain antibody variable domains (VHHs) bound to fragments of the combined repetitive oligopeptides domain of Clostridiodes difficile toxin A (TcdA) reveal that the C-terminus of VHH A20 was located 30 Å away from the N-terminus of VHH A26. Based on this observation, we generated a biparatopic fusion protein with A20 at the N-terminus, followed by a (GS)6 linker and A26 at the C-terminus. This A20-A26 fusion protein shows an improvement in binding affinity and a dramatic increase in TcdA neutralization potency (>330-fold [IC 50]; ≥2,700-fold [IC 99]) when compared to the unfused A20 and A26 VHHs. A20-A26 also shows much higher binding affinity and neutralization potency when compared to a series of control antibody constructs that include fusions of two A20 VHHs, fusions of two A26 VHHs, a biparatopic fusion with A26 at the N-terminus and A20 at the C-terminus (A26-A20), and actoxumab. In particular, A20-A26 displays a 310-fold (IC 50) to 29,000-fold (IC 99) higher neutralization potency than A26-A20. Size-exclusion chromatography-multiangle light scattering (SEC-MALS) analyses further reveal that A20-A26 binds to TcdA with 1:1 stoichiometry and simultaneous engagement of both A20 and A26 epitopes as expected based on the biparatopic design inspired by the crystal structures of TcdA bound to A20 and A26. In contrast, the control constructs show varied and heterogeneous binding modes. These results highlight the importance of molecular geometric constraints in generating highly potent antibody-based reagents capable of exploiting the simultaneous binding of more than one paratope to an antigen.

Construction of Rabbit Immune Antibody Libraries.

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from non-murine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

P859: ANTI-CD38 NANOBODY JK36 ALLOWS RELIABLE MRD DETECTION IN DARATUMUMAB TREATED MULTIPLE MYELOMA PATIENTS

Background: The introduction of immunotherapies such as daratumumab, a CD38 monoclonal antibody has improved survival in multiple myeloma (MM), however a few considerations on minimal residual disease (MRD) assessment by flow cytometry have been raised and deserve our attention. In flow cytometry CD38 is frequently used as a gating marker of plasma cells (PCs), but daratumumab can hampering myeloma cell detection. A strategy to overcome this interference is to staining PCs with anti-CD38 nanobody JK36. Nanobodies are single variable domain antibody fragments (VHH) that often expose a long complementarity-determining region 3 (CDR3), feature that allows anti-CD38 nanobody JK36 recognizing a cryptic epitope not masked by anti-CD38 therapies. Aims: To compare the median fluorescence intensity (MFI) of CD38 multiepitope (ME) antibody and anti-CD38 nanobody in bone marrow samples from MM patients not treated with daratumumab. Secondly, to compare flow cytometric MRD data obtained using CD38-ME and anti-CD38 nanobody from daratumumab treated MM patients. Methods: Samples were obtained from 10 MM patients not treated with daratumumab to compare the MFI of CD38-ME vs CD38-nanobody at baseline and after incubation with daratumumab at a concentration of 10 nM for 60 min at room temperature. Marrow samples were stained using panel CD38/CD56/CD19/CD138/CD45 with variable CD38, CD38-ME vs anti-CD38 nanobody. Data were analyzed using Kaluza 2.1.1 software (Beckman Coulter). Moreover, 17 bone marrow samples were processed using bulk lysis and subsequently stained using the MM-MRD EuroFlow 8-color antibody combination panel and tubes 1 and 2 with variable CD38, CD38-ME vs anti-CD38 nanobody. Samples were acquired on CytoFlex flow cytometer (Beckman Coulter) and data were analyzed using Infinicyt 2.0 software (Cytognos). Statistical analysis was performed using SPSS version 24, verifying the assumption of normality of the variable, pairwise comparisons were carried out using the T-tests for related samples. Results: The MFI values of CD38 ME on samples from daratumumab not treated patients was higher than samples after incubation with daratumumab (p=0.003). For CD38 nanobody, no significant differences were observed for the MFI values of samples, independent of daratumumab incubation (p=0,139) (figure 1A). The percentage of loss of MFI were higher in the CD38 ME group (-36,32%) than in the CD38 nanobody group (-0,35%). Regarding 17 patients treated with anti-CD38 therapies, 14 were treated with daratumumab and 3 with isatuximab. Sixteen samples were MRD positive with a sensitivity of 10-5 or 10-6 by both approaches. As shown in figure 1B, CD38-ME and CD38 nanobody both showed high and comparable MFI on PCs from patients treated with anti-CD38 therapies. Image:Summary/Conclusion: The anti-CD38 nanobody JK36 construct has lower coefficient of variation than CD38 ME antibody. It is a viable alternative to CD38 ME antibody to gate PCs by flow cytometry allowing reliable MRD detection to identify PCs in the presence of anti CD38 biologics. Nanobody technology open new avenues in the detection capability of MRD flow cytometry studies in the era of immunotherapy.

An alpaca single-domain antibody (VHH) phage display library constructed by CDR shuffling provided high-affinity VHHs against desired protein antigens.

Antigen-combining sites of the camelid heavy-chain antibody variable domain (VHH) are constructed by three complementarity-determining regions (CDR1, CDR2 and CDR3). We prepared cDNA using mRNA extracted from peripheral lymphocytes of alpacas that had been non-immunized or immunized with human serum albumin (HSA). The VHH gene fragments encoding the amino-terminal half-containing CDR1 as well as CDR2 and the carboxy-terminal half-containing CDR3 were amplified independently by PCR, and then full-length VHH gene fragments were generated by overlap extension PCR and cloned into the phagemid vector. This protocol, referred to as CDR shuffling, allowed us to construct an alpaca VHH phage display library possessing repertoires different from those naturally occurring in animals. We asked, first, whether this library was able to provide the functional VHH fragments against HSA, an immunized antigen, and obtained 29 anti-HSA VHH clones, 41% possessed KD values of lower than 10-8 M, 5 of which had KD values of 10-10 M. We also obtained VHH clones against non-immunized protein antigens such as cardiac troponin T and I, Ebola virus glycoprotein 1 and human immunoglobulin G by biopanning. We compared the amino acid sequences and affinities and found that 43% of VHHs had KD values of less than 10-8 M, although those having KD values of 10-10 M were unavailable. These results suggested that the CDR-shuffled VHH phage display library could potentially provide VHHs against non-immunized protein antigens with similar levels of affinities to those against immunized antigens.

IgG Anti-Citrullinated Protein Antibody Variable Domain Glycosylation Increases Before the Onset of Rheumatoid Arthritis and Stabilizes Thereafter: A Cross-Sectional Study Encompassing ~1,500 Samples.

ObjectiveThe autoimmune response in rheumatoid arthritis (RA) is marked by the presence of anti–citrullinated protein antibodies (ACPAs). A notable feature of IgG ACPA is the abundant expression of N‐linked glycans in the variable domain. However, the presence of ACPA variable domain glycosylation (VDG) across disease stages, and its response to therapy, are poorly described. To understand its dynamics, we investigated the abundance of IgG ACPA VDG in 1,498 samples from individuals in different clinical stages.MethodsUsing liquid chromatography, we analyzed IgG ACPA VDG profiles in 7 different cohorts from Japan, Canada, The Netherlands, and Sweden. We assessed 106 healthy individuals, 228 individuals with presymptomatic RA, 277 individuals with arthralgia, 307 patients with new‐onset/early RA, and 117 RA patients after prespecified treatment regimens. Additionally, we measured VDG in 234 samples from patients with RA who did or did not achieve long‐term drug‐free remission (DFR) during up to 16 years follow‐up.ResultsIgG ACPA VDG significantly increased (P < 0.0001) toward disease onset and was associated with ACPA levels and epitope spreading prior to diagnosis. A slight increase in VDG was observed in patients with established RA, with a moderate influence of treatment (P = 0.007). In patients in whom DFR was later achieved, IgG ACPA VDG was already reduced at the time of RA onset.ConclusionThe abundance of IgG ACPA VDG increases toward RA onset and correlates with maturation of the ACPA response. While IgG ACPA VDG levels are fairly stable in established disease, a lower degree of VDG at RA onset correlates with DFR. Although the underlying biologic mechanisms remain elusive, our data support the concept that VDG relates to an expansion of the ACPA response in the pre‐disease phase and contributes to disease development.

A Strategy for Accessing Nanobody-Based Electrochemical Sensors for Analyte Detection in Complex Media.

Nanobodies are single variable domain antibodies isolated from camelids and are rapidly distinguishing themselves as ideal recognition elements in biosensors due to their comparative stability, ease of production and isolation, and high binding affinities. However, transducing analyte binding by nanobodies in real time is challenging, as most nanobodies do not directly produce an optical or electrical signal upon target recognition. Here, we report a general strategy to fabricate sensitive and selective electrochemical sensors incorporating nanobodies for detecting target analytes in heterogeneous media, such as cell lysate. Graphite felt can be covalently functionalized with recombinant HaloTag-modified nanobodies. Subsequent encapsulation with a thin layer of a hydrogel using a vapor deposition process affords encapsulated electrodes that directly display a decrease in current upon antigen binding, without added redox mediators. Differential pulse voltammetry affords clear and consistent decreases in electrode current across multiple electrode samples for specific antigen concentrations. The change in observed current vs increasing antigen concentration follows Langmuir binding characteristics, as expected. Importantly, selective and repeatable target binding in unpurified cell lysate is only demonstrated by the encapsulated electrode, with an antigen detection limit of ca. 30 pmol, whereas bare electrodes lacking encapsulation produce numerous false positive signals in control experiments.

Overview, Generation, and Significance of Variable New Antigen Receptors (VNARs) as a Platform for Drug and Diagnostic Development.

The approval of the first VHH-based drug caplacizumab (anti-von Willebrand factor) has validated a two-decade long commitment in time and research effort to realize the clinical potential of single-domain antibodies. The variable domain (VNAR) of the immunoglobulin new antigen receptor (IgNAR) found in sharks provides an alternative small binding domain to conventional monoclonal antibodies and their fragments and heavy-chain antibody-derived VHHs. Evolutionarily distinct from mammalian antibody variable domains, VNARs have enhanced thermostability and unusual convex paratopes. This predisposition to bind cryptic and recessed epitopes has facilitated both the targeting of new antigens and new (neutralizing) epitopes on existing antigens. Together these unique properties position the VNAR platform as an alternative non-antibody binding domain for therapeutic drug, diagnostic and reagent development. In this introductory chapter, we highlight recent VNAR advancements that further underline the exciting potential of this discovery platform.

Application of phage display technology for the production of antibodies against Streptococcus suis serotype 2.

Streptococcus suis (S. suis) serotype 2 infection is a problem in the swine industry and responsible for most cases of human infection worldwide. Since current multiplex PCR cannot differentiate between serotypes 2 and 1/2, then serotype-specific antibodies (Abs) are required for serotype identification to confirm infection by serotype 2. This study aimed to generate Abs specific to S. suis serotype 2 by phage display from a human heavy chain variable domain (VH) antibody library. For biopanning, whole cells of S. suis serotype 2 were used as the target antigen. With increasing selection stringency, we could select the VH Abs that specifically bound to a S. suis serotype 2 surface antigen, which was identified as the capsular polysaccharide (CPS). From ELISA analysis, the specific phage clone 47B3 VH with the highest binding activity to S. suis serotype 2 was selected and shown to have no cross-reactivity with S. suis serotypes 1/2, 1, and 14 that shared a common epitope with serotype 2 and occasionally cause infections in human. Moreover, no cross-reactivity with other bacteria that can be found in septic blood specimens was also observed. Then, 47B3 VH was successfully expressed as soluble 47B3 VH in E. coli TG1. The soluble 47B3 VH crude extract was further tested for its binding ability in a dose-dependent ELISA assay. The results indicated that the activity of phage clone 47B3 was still retained even when the Ab occurred in the soluble form. A quellung reaction demonstrated that the soluble 47B3 VH Ab could show bioactivity by differentiation between S. suis serotypes 2 and 1/2. Thus, it will be beneficial to use this VH Ab in the diagnosis of disease or discrimination of S. suis serotypes Furthermore, the results described here could motivate the use of phage display VH platform to produce serotyping antibodies.

T Cells Expressing Anti-B-Cell Maturation Antigen (BCMA) Chimeric Antigen Receptors with Antigen Recognition Domains Made up of Only Single Human Heavy Chain Variable Domains Specifically Recognize Bcma and Eradicate Tumors in Mice

Multiple myeloma (MM) is an almost-always incurable malignancy of plasma cells. Chimeric antigen receptors (CARs) are artificial fusion proteins that incorporate antigen-recognition domains and T-cell signaling domains. CAR T cells have been shown to have substantial activity against leukemia and lymphoma, which has generated great interest in developing CAR T-cell therapies for MM. CAR T cells targeting B-cell maturation antigen (BCMA) have been shown to have substantial activity against MM (Ali et al. Blood. 2016. 128(13). 1688-1700). Improvements in CAR T-cell therapies for MM are needed. One possible limitation of CAR T-cell therapies is generation of recipient anti-CAR immune responses. Recipient anti-CAR immune responses have been reported in patients on clinical trials of anti-CD19 CARs (Turtle et al. Journal of Clinical Investigation. 2016. 126 (6). 2123-3138). Recipient anti-CAR immune responses could potentially develop against murine antibody variable region domains that are included in the single-chain variable fragments (scFvs) of most CARs. In addition, recipient anti-CAR immune responses could develop against the artificial linker domain in the scFv or against junctions between different CAR components.We designed CARs with antigen-recognition domains of single fully-human heavy-chain-only variable domains (FHVH). The FHVH CARs do not contain a light chain variable region domain or a linker. The fully-human variable region, lack of a linker, and the minimized number of junctions should all lead to reduced immunogenicity for the FHVH antigen-recognition domain compared with a standard scFv.We constructed CARs with 4 different FHVH domains designated FHVH32, FHVH33, FHVH74, and FHVH93. We designed CARs with each of these FHVH domains along with a CD8-alpha hinge and transmembrane domain, either a CD28 or a 4-1BB costimulatory domain, and a CD3-zeta T-cell activation domain. DNA encoding these CARs was ligated into a gamma-retroviral vector, and primary human T cells were transduced with gamma-retroviruses encoding each of the CARs. We tested the T cells for CAR expression and a variety of T-cell functions. We assessed T-cell surface CAR expression by staining with phycoerythrin-labeled BCMA protein. T cells from 3 MM patients were transduced with genes encoding each of the CD28-containing CARs. The following mean percentages of T cells expressed each CAR after transduction: FHVH32-CD828Z (47.3%), FHVH33-CD828Z (49.6%), FHVH74-CD828Z (51.3%), and FHVH93-CD828Z (46.6%). The mean T-cell surface CAR expression of the 11D5-3-CD828Z CAR containing a standard murine scFv with a heavy chain variable domain and a light chain variable domain connected by a linker was 60.4%. Importantly, we found that each of the FHVH CARs specifically recognized BCMA in functional assays. Specific interferon gamma production by the CARs is shown in the Table. We also demonstrated BCMA-specific tumor-necrosis factor-alpha release, cytotoxicity, and proliferation by T cells expressing each of the 4 FHVH CARs containing CD28. We have previously reported clinical anti-myeloma responses with infusions of T cells expressing the 11D5-3-CD828Z CAR (Ali et al. Blood. 2016. 128(13). 1688-1700). T cells expressing FHVH CARs with CD28 domains, especially FHVH33-CD828Z and FHVH74-CD828Z, had BCMA-specific functions including cytokine release that were equal to or superior to those of T cells expressing 11D5-3-CD828Z (Table). We have demonstrated that human T cells expressing either FHVH33-CD828Z or FHVH74-CD828Z can eliminate established tumors of BCMA-expressing human RPMI8226 cells from immune-deficient mice.We also assessed FHVH CARs with 4-1BB costimulatory domains. Of the 4-1BB CARs, FHVH33-CD8BBZ CAR had the highest percentage of T cells with surface CAR expression (mean 52.1%, n=3). T cells expressing FHVH33-CD8BBZ exhibited BCMA-specific cytokine-release, degranulation, cytotoxicity, and proliferation.In summary, we have designed, constructed, and tested a novel type of CAR with an antigen-recognition domain made up of only single fully-human heavy-chain variable region domains. These CARs specifically recognized BCMA in vitro, and they eradicated tumors from mice. The simple, fully-human antigen-recognition domains in these CARs offer a potential advantage of decreased immunogenicity compared to other CAR designs. [Display omitted] DisclosuresTrinklein:TeneoBio: Employment. Buelow:TeneoBio: Employment. Kochenderfer:Bluebird bio: Research Funding; N/A: Patents & Royalties: I have multiple patents in the CAR field.; Kite Pharma: Research Funding.