Heavy Chain Variable Domains Research Articles

Neutralizing VHH Antibodies Targeting the Spike Protein of PEDV

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that infect pigs’ intestinal epithelial cells, causing high morbidity and mortality. Due to the rapid mutation of PEDV, vaccine efficacy is uncertain, prompting exploration of alternative treatments. Nanobodies, also known as variable heavy chain domains of heavy chain-only antibodies (VHHs), offer significant potential in biomedical applications due to their small size and high specificity. In this study, yeast two-hybrid technology was employed to screen for eight specific VHH sequences targeting the PEDV S protein from a synthetically constructed nanobody yeast library. The VHH genes were then cloned into expression plasmids for recombinant protein production, and the resulting VHHs (termed PEDV S-VHHs) were purified. Indirect immunofluorescence assay (IFA) and Western blotting analysis confirmed that these VHHs specifically bind to both PEDV and its S protein. Neutralization assays demonstrated that seven PEDV S-VHHs exhibited potent neutralizing activity against PEDV. Additionally, a combination of these seven antibodies showed enhanced antiviral effects. Preliminary predictions were also made regarding the binding sites between these VHHs and PEDV. The PEDV S-VHHs described in this study hold potential as candidates for the prevention and treatment of PEDV infection.

Polybodies: Next-generation clinical antibodies

Conventional antibodies [full-length and fragments: F(ab′)2, fragment antigen-binding (Fab), single-chain variable fragment (scFv), variable heavy domain of heavy chain (VHH)] are monospecific first-generation antibodies that have dominated the biopharmaceuticals field. However, advanced protein engineering technology has led to the advent of the next-generation polybody, which is a significant improvement over the conventional antibody. Polybodies comprise polyspecific and polyvalent antibodies that enable a single antibody to target multiple specific antigens simultaneously. Polybodies are superior to first-generation antibodies (more efficacious, broad-spectrum, resistance resistant, customizable, etc.) and provide a cost-effective healthcare solution. This review thoroughly addresses developments in polybodies, highlighting their superiority over conventional antibodies and offering future perspectives to encourage the generation of innovative immunotherapies.

Development of nanobodies against the coat protein of maize chlorotic mottle virus.

Maize lethal necrosis (MLN) is a maize disease caused by the maize chlorotic mottle virus (MCMV), a potyvirus which causes yield losses of 30-100%. The present study aimed to isolate nanobodies against the MCMV coat protein (CP) for the diagnosis of MLN. MCMV CP expressed in Escherichia coli was used for llama immunization. VHH (i.e. variable heavy domain of heavy chain) gene fragments were prepared from blood drawn from the immunized llama and used to generate a library in E. coli TG1 cells. MCMV specific nanobodies were selected by three rounds of phage display and panning against MCMV CP. The selected nanobodies were finally expressed in E. coli WK6 cells and purified. Eleven MCMV-specific nanobodies were identified and shown to detect MCMV in infected maize plants. Thus, our results show that nanobodies isolated from llama immunized with MCMV CP can distinguish infected and healthy maize plants,potentially enabling development of affordable MCMV detection protocols.

Development of a novel bacterial production system for recombinant bioactive proteins completely free from endotoxin contamination.

Endotoxins, or lipopolysaccharides (LPS), are potent immunostimulatory molecules of critical concern in bacterial recombinant protein expression systems. The gram-negative bacterium Acinetobacter baumannii exhibits an interesting and unique phenotype characterized by the complete loss of LPS. In this study, we developed a novel system for producing recombinant proteins completely devoid of endotoxin contamination using LPS-deficient A. baumannii. We purified endotoxin-free functional green fluorescent protein, which reduced endotoxin contamination by approximately three orders of magnitude, and also purified the functional cytokine tumor necrosis factor (TNF)-α. Additionally, utilization of the Omp38 signal peptide of A. baumannii enabled the extracellular production of variable domain of heavy chain of heavy chain (VHH) antibodies. With these advantages, mNb6-tri-20aa, a multivalent VHH that specifically binds to the spike protein of severe acute respiratory syndrome coronavirus 2, was purified from the culture supernatant, and endotoxin contamination was reduced by a factor of approximately 2 × 105 compared with that in conventional expression systems. A virus neutralization assay demonstrated the functionality of the purified antibody in suppressing viral infections. Moreover, we applied our system to produce ozoralizumab, a multispecific VHH that binds to human TNF-α and albumin and are marketed as a rheumatoid arthritis drug. We successfully purified a functional antibody from endotoxin contamination. This system establishes a new, completely endotoxin-free platform for the expression of recombinant proteins, which distinguishes it from other bacterial expression systems, and holds promise for future applications.

Characterization of a novel format scFv×VHH single-chain biparatopic antibody against metal binding protein MtsA.

Biparatopic antibodies (bpAbs) are engineered antibodies that bind to multiple different epitopes within the same antigens. bpAbs comprise diverse formats, including fragment-based formats, and choosing the appropriate molecular format for a desired function against a target molecule is a challenging task. Moreover, optimizing the design of constructs requires selecting appropriate antibody modalities and adjusting linker length for individual bpAbs. Therefore, it is crucial to understand the characteristics of bpAbs at the molecular level. In this study, we first obtained single-chain variable fragments and camelid heavy-chain variable domains targeting distinct epitopes of the metal binding protein MtsA and then developed a novel format single-chain bpAb connecting these fragment antibodies with various linkers. The physicochemical properties, binding activities, complex formation states with antigen, and functions of the bpAb were analyzed using multiple approaches. Notably, we found that the assembly state of the complexes was controlled by a linker and that longer linkers tended to form more compact complexes. These observations provide detailed molecular information that should be considered in the design of bpAbs.

Identification of a Fully Human Antibody VH Domain Targeting Anaplastic Lymphoma Kinase (ALK) with Applications in ALK-Positive Solid Tumor Immunotherapy.

The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC50 of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer.

Engineering a Low-Immunogenic Mirror-Image VHH against Vascular Endothelial Growth Factor.

Immunogenicity is a major caveat of protein therapeutics. In particular, the long-term administration of protein therapeutic agents leads to the generation of antidrug antibodies (ADAs), which reduce drug efficacy while eliciting adverse events. One promising solution to this issue is the use of mirror-image proteins consisting of d-amino acids, which are resistant to proteolytic degradation in immune cells. We have recently reported the chemical synthesis of the enantiomeric form of the variable domain of the antibody heavy chain (d-VHH). However, identifying mirror-image antibodies capable of binding to natural ligands remains challenging. In this study, we developed a novel screening platform to identify a d-VHH specific for vascular endothelial growth factor A (VEGF-A). We performed mirror-image screening of two newly constructed synthetic VHH libraries displayed on T7 phage and identified VHH sequences that effectively bound to the mirror-image VEGF-A target (d-VEGF-A). We subsequently synthesized a d-VHH candidate that preferentially bound the native VEGF-A (l-VEGF-A) with submicromolar affinity. Furthermore, immunization studies in mice demonstrated that this d-VHH elicited no ADAs, unlike its corresponding l-VHH. Our findings highlight the utility of this novel d-VHH screening platform in the development of protein therapeutics exhibiting both reduced immunogenicity and improved efficacy.

Protecting the piglet gut microbiota against ETEC-mediated post-weaning diarrhoea using specific binding proteins

Post-weaning diarrhoea (PWD) in piglets presents a widespread problem in industrial pig production and is often caused by enterotoxigenic E. coli (ETEC) strains. Current solutions, such as antibiotics and medicinal zinc oxide, are unsustainable and are increasingly being prohibited, resulting in a dire need for novel solutions. Thus, in this study, we propose and evaluate a protein-based feed additive, comprising two bivalent heavy chain variable domain (VHH) constructs (VHH-(GGGGS)3-VHH, BL1.2 and BL2.2) as an alternative solution to manage PWD. We demonstrate in vitro that these constructs bind to ETEC toxins and fimbriae, whilst they do no affect bacterial growth rate. Furthermore, in a pig study, we show that oral administration of these constructs after ETEC challenge reduced ETEC proliferation when compared to challenged control piglets (1-2 log10 units difference in gene copies and bacterial count/g faeces across day 2–7) and resulted in week 1 enrichment of three bacterial families (Prevotellaceae (estimate: 1.12 ± 0.25, q = 0.0054), Lactobacillaceae (estimate: 2.86 ± 0.52, q = 0.0012), and Ruminococcaceae (estimate: 0.66 ± 0.18, q = 0.049)) within the gut microbiota that appeared later in challenged control piglets, thus pointing to an earlier transition towards a more mature gut microbiota. These data suggest that such VHH constructs may find utility in industrial pig production as a feed additive for tackling ETEC and reducing the risk of PWD in piglet populations.

Abstract 2617: BCG022, a novel HER3 × MET bispecific antibody-drug conjugate (bsADC), demonstrates promising anti-tumor efficacy

Abstract HER3 and MET expression act as bypass resistance mechanisms, conferring resistance to multiple therapeutic agents, such as EGFR TKI treatment. HER3 and MET are also co-expressed at high prevalence in multiple tumor types, including gastric, colorectal, breast, and non-small-cell lung cancer (NSCLC). In addition, HER3 and MET are frequently overexpressed in liver metastases from patients with colorectal cancer, indicating that targeting both targets may provide clinical benefit. We previously reported generation of a fully human bispecific antibody (bsAb) targeting HER3 and MET using RenLite® mice, which contain the full human heavy chain variable domain with a common human kappa light chain to facilitate bispecific antibody assembly. This bsAb demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including NSCLC, gastric, colorectal, and hepatocellular cancer cell lines. The bsAb also showed enhanced internalization activity compared to parental monovalent antibodies in the NUGC-4 cell line expressing HER3 and MET. Here, we evaluated the efficacy of the bsAb when conjugated to two distinct payloads: vcMMAE and BLD1102, Biocytogen’s novel, proprietary linker/payload system composed of a DNA Topo I inhibitor payload (BCPT02) and a highly hydrophilic protease-cleavable linker. The in vivo efficacy of BCG022-vcMMAE and BCG022-BLD1102 was evaluated in cell-derived NSCLC and gastric cancer xenografts, and in patient-derived gastric and pancreatic xenograft models. Both demonstrated strong anti-tumor activity, with efficacy greater than or comparable to benchmark ADCs. Taken together, these promising preclinical results suggest that the BCG022 bsADC could be a novel treatment for HER3 and MET co-expressing tumors. Citation Format: Zhuolin Li, Chengzhang Shang, Zhenyan Han, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. BCG022, a novel HER3 × MET bispecific antibody-drug conjugate (bsADC), demonstrates promising anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2617.

Abstract 2619: A novel EGFR × HER3-targeting bispecific antibody drug-conjugate, BCG019, demonstrates robust anti-tumor efficacy in preclinical evaluation

Abstract EGFR and HER3 are receptor tyrosine kinases that are highly expressed in multiple epithelial tumors. EGFR point mutations and small insertions within the kinase domain are common in lung cancer. HER3 is overexpressed in patients who are resistant to EGFR-TKI therapy, and is one of the common resistance mechanisms of EGFR and HER2-targeted therapy. We hypothesize that simultaneous targeting of EGFR and HER3 with a bispecific ADC will have the potential to overcome the resistance caused by HER3 after EGFR-targeted therapy and obtain better efficacy than the combination of EGFR monotherapy and HER3 monotherapy. We first generated fully human bispecific antibodies (bsAbs) targeting EGFR and HER3 using RenLite® mice, which contain the full human heavy chain variable domain with a common human kappa light chain to facilitate bispecific antibody assembly. The anti-EGFR × HER3 bsAb demonstrated reactivity to human and cynomolgus monkey antigens and binding to multiple cancer cell lines, including NSCLC, gastric, pancreatic, colorectal, and breast cancer cell lines. The anti-EGFR × HER3 bsAb also showed high internalization activity in cell lines expressing EGFR and HER3. The anti-EGFR × HER3 bsAb was then conjugated with vc-MMAE (valine-citrulline-monomethyl auristatin E) for proof-of-concept studies, as well as BLD1102, Biocytogen’s novel, proprietary linker/payload system composed of a DNA Topo I inhibitor payload (BCPT02) and highly hydrophilic protease-cleavable linker, to generate EGFR- and HER3-targeting bispecific ADC candidates BCG019. In vivo efficacy of the bsADC candidates were evaluated in cell-derived NSCLC and gastric cancer xenografts and in patient-derived gastric and colorectal xenograft models. Both bsADCs showed superior anti-tumor activity when compared to benchmark ADCs. Collectively, these results suggest that our novel anti-EGFR × HER3 bsADC has therapeutic potential for EGFR and HER3 co-expressing tumors. Citation Format: Zhuolin Li, Chengzhang Shang, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. A novel EGFR × HER3-targeting bispecific antibody drug-conjugate, BCG019, demonstrates robust anti-tumor efficacy in preclinical evaluation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2619.

Molecular and structural basis of anti-DNA antibody specificity for pyrrolated proteins

Anti-DNA antibodies (Abs), serological hallmarks of systemic lupus erythematosus (SLE) and markers for diagnosis and disease activity, show a specificity for non-nucleic acid molecules, such as N-pyrrolated proteins (pyrP) containing Nε-pyrrole-L-lysine (pyrK) residues. However, the detailed mechanism for the binding of anti-DNA Abs to pyrP remains unknown. In the present study, to gain structural insights into the dual-specificity of anti-DNA Abs, we used phage display to obtain DNA-binding, single-chain variable fragments (scFvs) from SLE-prone mice and found that they also cross-reacted with pyrP. It was revealed that a variable heavy chain (VH) domain is sufficient for the recognition of DNA/pyrP. Identification of an antigenic sequence containing pyrK in pyrP suggested that the presence of both pyrK and multiple acidic amino acid residues plays important roles in the electrostatic interactions with the Abs. X-ray crystallography and computer-predicted simulations of the pyrK-containing peptide-scFv complexes identified key residues of Abs involved in the interaction with the antigens. These data provide a mechanistic insight into the molecular basis of the dual-specificity of the anti-DNA Abs and provide a basis for therapeutic intervention against SLE.

Extensive antibody search with whole spectrum black-box optimization

In designing functional biological sequences with machine learning, the activity predictor tends to be inaccurate due to shortage of data. Top ranked sequences are thus unlikely to contain effective ones. This paper proposes to take prediction stability into account to provide domain experts with a reasonable list of sequences to choose from. In our approach, multiple prediction models are trained by subsampling the training set and the multi-objective optimization problem, where one objective is the average activity and the other is the standard deviation, is solved. The Pareto front represents a list of sequences with the whole spectrum of activity and stability. Using this method, we designed VHH (Variable domain of Heavy chain of Heavy chain) antibodies based on the dataset obtained from deep mutational screening. To solve multi-objective optimization, we employed our sequence design software MOQA that uses quantum annealing. By applying several selection criteria to 19,778 designed sequences, five sequences were selected for wet-lab validation. One sequence, 16 mutations away from the closest training sequence, was successfully expressed and found to possess desired binding specificity. Our whole spectrum approach provides a balanced way of dealing with the prediction uncertainty, and can possibly be applied to extensive search of functional sequences.

Development of a c-MET x CD137 bispecific antibody for targeted immune agonism in cancer immunotherapy

BackgroundTargeting the costimulatory receptor CD137 has shown promise as a therapeutic approach for cancer immunotherapy, resulting in anti-tumor efficacy demonstrated in clinical trials. However, the initial CD137 agonistic antibodies, urelumab and utomilumab, faced challenges in clinical trials due to the liver toxicity or lack of efficacy, respectively. Concurrently, c-MET has been identified as a highly expressed tumor-associated antigen (TAA) in various solid and soft tumors. MethodsIn this study, we aimed to develop a bispecific antibody (BsAb) that targets both c-MET and CD137, optimizing the BsAb format and CD137 binder for efficient delivery of the CD137 agonist to the tumor microenvironment (TME). We employed a monovalent c-MET motif and a trimeric CD137 Variable Heavy domain of Heavy chain (VHH) for the BsAb design. ResultsOur results demonstrate that the c-MET x CD137 BsAb provides co-stimulation to T cells through cross-linking by c-MET-expressing tumor cells. Functional immune assays confirmed the enhanced efficacy and potency of the c-MET x CD137 BsAb, as indicated by activation of CD137 signaling, target cell killing, and cytokine release in various tumor cell lines. Furthermore, the combination of c-MET x CD137 BsAb with Pembrolizumab showed a dose-dependent enhancement of target-induced T cell cytokine release. ConclusionOverall, the c-MET x CD137 BsAb exhibits a promising developability profile as a tumor-targeted immune agonist by minimizing off-target effects while effectively delivering immune agonism. It has the potential to overcome resistance to anti-PD-(L)1 therapies.

Application of N-Terminal Site-Specific Biotin and Digoxigenin Conjugates to Clinical Anti-drug Antibody Assay Development.

Biotin- and digoxigenin (DIG)-conjugated therapeutic drugs are critical reagents used for the development of anti-drug antibody (ADA) assays for the assessment of immunogenicity. The current practice of generating biotin and DIG conjugates is to label a therapeutic antibody with biotin or DIG via primary amine groups on lysine or N-terminal residues. This approach modifies lysine residues nonselectively, which can impact the ability of an ADA assay to detect those ADAs that recognize epitopes located at or near the modified lysine residue(s). The impact of the lysine modification is considered greater for therapeutic antibodies that have a limited number of lysine residues, such as the variable heavy domain of heavy chain (VHH) antibodies. In this paper, for the first time, we report the application of site-specifically conjugated biotin- and DIG-VHH reagents to clinical ADA assay development using a model molecule, VHHA. The site-specific conjugation of biotin or DIG to VHHA was achieved by using an optimized reductive alkylation approach, which enabled the majority of VHHA molecules labeled with biotin or DIG at the desirable N-terminus, thereby minimizing modification of the protein after labeling and reducing the possibility of missing detection of ADAs. Head-to-head comparison of biophysical characterization data revealed that the site-specific biotin and DIG conjugates demonstrated overall superior quality to biotin- and DIG-VHHA prepared using the conventional amine coupling method, and the performance of the ADA assay developed using site-specific biotin and DIG conjugates met all acceptance criteria. The approach described here can be applied to the production of other therapeutic-protein- or antibody-based critical reagents that are used to support ligand binding assays.

Human Single Domain Antibody-Based CAR-T Cells Targeting BAFF-R Demonstrate Promising Preclinical Activity in B Cell Malignancies

Background: B-cell activating factor-receptor (BAFF-R) is a potential B-cell specific target, highly expressed in B cell malignancies and regulates B cell proliferation and survival. Single-chain antibody-based CAR-T cells targeting BAFF-R (PMB101) has demonstrated antitumor effects against human B cell malignancies and can overcome CD19 antigen loss (Qin et al. Sci Transl Med. 2019;11(511)). However, CARs built on antigen-specific single chain antibody variable fragment (scFv) may have some immunological risks. Recently, single variable domain of heavy chain antibodies are becoming an alternative to scFv to construct CARs. To improve the function of BAFF-R CAR-T cells, we designed CARs with only a fully human heavy-chain variable domain. Methods: A fully human antibody phage display library (IMARS, IASO Biotherapeutics) was used to select anti-BAFF-R clones using optimal protein/cell alternate panning. The enzyme-linked immunosorbent assay (ELISA) and flow cytometry were applied to evaluate the specificity of clones. The screened clones were grafted into a second-generation CAR with CD8α hinge, 4-1BB costimulatory and CD3ζ intracellular signaling domains to generate CAR-T cells. PMB101 and CAR-T cells constructed with VAY736 were used as positive controls. Non-transduced T cells from the same donor were used as a negative control. Results: Four candidate clones including clone 1, 5, 77 and 80 were finally screened by in vitro functional evaluation, among which clone 5, 77 and 80 were variable domain heavy chain antibodies. The expression levels of the degranulation marker CD107a on CAR-T cells constructed with the four candidate clones were significantly upregulated after coculture with BAFF-R positive tumor cells, while BAFF-R negative tumor cells could not effectively activate CAR-T cells (data not shown). The four clones exhibited similar cytotoxicity in CAR-T cells compared with the PMB101 and VAY736 CAR-T cells using the luciferase-based cytotoxicity assay (data not shown). Xenografts were established in NPG mice following intravenous injection of 1 x 10 6 jeko-1 cells expressing the firefly luciferase gene on day - 5, and a single dose of 4 x 10 6 CAR T cells were infused IV on day 0. Compared to the other groups, no progressive tumor growth was discovered in mice treated with clone 5 CAR-T cells until day 49. Clone 5 CAR-T cells showed excellent in vivo antitumor activity to PMB101 and conferred long-term survival (data not shown). Conclusion: We developed a novel BAFF-R CAR-T cell product structured on single-domain antibody. This product demonstrated promising preclinical activity, and it may provide an alternative choice for patients with B cell malignancies.

B-318 Development of Non-competitive Immunoassay System for Cyclosporine a Using Alpaca Naïve Library-derived VHH

Abstract Background Cyclosporine A is a potent immunosuppressive drug. It has a narrow therapeutic index, and therefore the measurement of cyclosporine’s blood concentration is essential to obtain optimal therapy. Cyclosporine A, a small cyclic polypeptide, is generally measured by competitive immunoassay, which is theoretically inferior to noncompetitive sandwich immunoassay in terms of sensitivity and specificity. In this study, we report a novel sandwich immunoassay for cyclosporine A using an alpaca naïve library-derived variable domain of heavy chain of heavy chain antibody (VHH) developed against an immunocomplex of cyclosporine A and anti-cyclosporine A mouse monoclonal antibody. Methods We generated a VHH against an anti-cyclosporine A mouse monoclonal antibody complexed with cyclosporine A by alpaca naïve VHH phage display library. A bivalent tandem VHH was generated using the established VHH, and a new sandwich immunoassay for measuring cyclosporine A was constructed using the tandem VHH conjugated with alkaline phosphatase. A prototype fully automated sandwich immunoassay was developed on a fully automated chemiluminescence analyzer using magnetic particles immobilized with the mouse monoclonal antibody and the tandem VHH conjugated with alkaline phosphatase. The prototype assay was further developed by implementing an automated whole blood pretreatment process with strong denaturing agents. The performances of the prototype immunoassays were investigated. Results The established VHH showed a highly specific reaction to the complex of cyclosporine A and the anti-cyclosporine A mouse monoclonal antibody, and a sandwich immunoassay for cyclosporine A was successfully developed using the VHH and the mouse monoclonal antibody. The tandem bivalent VHH exhibited higher reactivity to the complex than the monomeric VHH. The fully automated reagent prototype achieved a measurement range of 30 to 1500 ng/mL. Measurement of cyclosporine A in whole blood samples by the prototype reagent showed a correlation coefficient of 0.96 with mass spectrometry, and a correlation coefficient of 0.95 with a commercial IVD reagent. Conclusion We successfully developed a sandwich reagent prototype for cyclosporine A using VHH established by alpaca naïve VHH phage display library. This is the first report of a fully automated reagent for cyclosporine A measurement including an automated whole blood pretreatment process.

High-volume hybridoma sequencing on the NeuroMabSeq platform enables efficient generation of recombinant monoclonal antibodies and scFvs for neuroscience research

The Neuroscience Monoclonal Antibody Sequencing Initiative (NeuroMabSeq) is a concerted effort to determine and make publicly available hybridoma-derived sequences of monoclonal antibodies (mAbs) valuable to neuroscience research. Over 30 years of research and development efforts including those at the UC Davis/NIH NeuroMab Facility have resulted in the generation of a large collection of mouse mAbs validated for neuroscience research. To enhance dissemination and increase the utility of this valuable resource, we applied a high-throughput DNA sequencing approach to determine immunoglobulin heavy and light chain variable domain sequences from source hybridoma cells. The resultant set of sequences was made publicly available as a searchable DNA sequence database (neuromabseq.ucdavis.edu) for sharing, analysis and use in downstream applications. We enhanced the utility, transparency, and reproducibility of the existing mAb collection by using these sequences to develop recombinant mAbs. This enabled their subsequent engineering into alternate forms with distinct utility, including alternate modes of detection in multiplexed labeling, and as miniaturized single chain variable fragments or scFvs. The NeuroMabSeq website and database and the corresponding recombinant antibody collection together serve as a public DNA sequence repository of mouse mAb heavy and light chain variable domain sequences and as an open resource for enhancing dissemination and utility of this valuable collection of validated mAbs.

Preclinical assessment of a novel human antibody VH domain targeting mesothelin as an antibody-drug conjugate

Mesothelin (MSLN) has been a validated tumor-associated antigen target for several solid tumors for over a decade, making it an attractive option for therapeutic interventions. Novel antibodies with high affinity and better therapeutic properties are needed. In the current study, we have isolated and characterized a novel heavy chain variable (VH) domain 3C9 from a large-size human immunoglobulin VH domain library. 3C9 exhibited high affinity (KD [dissociation constant] <3nM) and binding specificity in a membrane proteome array (MPA). In a mouse xenograft model, 3C9 fused to human IgG1 Fc was detected at tumor sites as early as 8h post-infusion and remained at the site for over 10days. Furthermore, 3C9 fused to a human Fc domain drug conjugate effectively inhibited MSLN-positive tumor growth in a mouse xenograft model. The X-ray crystal structure of full-length MSLN in complex with 3C9 reveals interaction of the 3C9 domains with two distinctive residue patches on the MSLN surface. This newly discovered VH antibody domain has a high potential as a therapeutic candidate for MSLN-expressing cancers.

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.

Shark VNAR phage display libraries: An alternative source for therapeutic and diagnostic recombinant antibody fragments.

The development of recombinant antibody fragments as promising alternatives to full-length immunoglobulins offers vast opportunities for biomedicine. Antibody fragments have important advantages compared with conventional monoclonal antibodies that make them attractive for the biotech industry: superior stability and solubility, reduced immunogenicity, higher specificity and affinity, capacity to target the hidden epitope and cross the blood-brain barrier, the ability to refold after heat denaturation and inexpensive and easy large-scale production. Different antibody formats such as antigen-binding fragments (Fab), single-chain fragment variable (scFv) consisting of the antigen-binding domains of Ig heavy (VH) and light (VL) chain regions connected by a flexible peptide linker, single-domain antibody fragments (sdAbs) like camelid heavy-chain variable domains (VHHs) and shark variable new antigen receptor (VNARs), and bispecific antibodies (bsAbs) are currently being evaluated as diagnostics or therapeutics in preclinical studies and clinical trials. In the present review, we summarize and discuss studies on VNARs, the smallest recombinant antibody fragment, obtained after the screening of different types of phage display antibody libraries. Results published until March 2023 are discussed.