Camelid Single-domain Antibody Research Articles

Development of an Engineered Single-Domain Antibody for Targeting MET in Non-Small Cell Lung Cancer.

The Mesenchymal Epithelial Transition (MET) receptor tyrosine kinase is upregulated or mutated in 5% of non-small-cell lung cancer (NSCLC) patients and overexpressed in multiple other cancers. We sought to develop a novel single-domain camelid antibody with high affinity for MET that could be used to deliver conjugated payloads to MET expressing cancers. From a naïve camelid variable-heavy-heavy (VHH) domain phage display library, we identified a VHH clone termed 1E7 that displayed high affinity for human MET and was cross-reactive with MET across multiple species. When expressed as a bivalent human Fc fusion protein, 1E7-Fc was found to selectively bind to EBC-1 (MET amplified) and UW-Lung 21 (MET exon 14 mutated) cell lines by flow cytometry and immunofluorescence imaging. Next, we investigated the ability of [89Zr]Zr-1E7-Fc to detect MET expression in vivo by PET/CT imaging. [89Zr]Zr-1E7-Fc demonstrated rapid localization and high tumor uptake in both xenografts with a %ID/g of 6.4 and 5.8 for EBC-1 and UW-Lung 21 at 24 h, respectively. At the 24 h time point, clearance from secondary and nontarget tissues was also observed. Altogether, our data suggest that 1E7-Fc represents a platform technology that can be employed to potentially both image and treat MET-altered NSCLC.

Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22

Chimeric antigen receptor (CAR-) T cell therapies targeting B-cell restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B-cell malignancies, but relapse remains common. Camelid single domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28 to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, though only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies a lead CD22sdCAR-T was selected; achieving comparable complete responses to a benchmark short-linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular level specificity of the lead CD22-sdAb. This presents a complete report on pre-clinical development of a novel CD22sdCAR therapeutic.

Combination Screening of a Naïve Antibody Library Using E. coli Display and Single-Step Colony Assay

The use of single-domain camelid antibodies, termed VHHs or nanobodies, has found increasing application in diagnosis, pharmaceutical development, and research because of their superior properties, such as small size, elevated stability, high water solubility, and excellent affinity for the antigen. Antigen-specific VHHs are generated by screening VHH display libraries via bio-panning. However, the bio-panning step needs to be repeated multiple times, which is time-consuming and laborious. Here, we developed a simple and rapid screening method that combined Escherichia coli display and a single-step colony assay to successfully identify positive clones from a naïve VHH library. The library was constructed from peripheral blood mononuclear cells of alpaca, and VHHs were displayed on the surface of E. coli using the inverse autotransporter intimin. Libraries enriched by magnetic cell sorting were screened directly using a single-step colony assay. Colonies formed on the hydrophilic filter and antigen-coated membrane. The expression of VHHs was induced, and those bound to the antigen on the membrane were detected as positive clones. Screening and identification of positive clones required only two days, which saves considerable time and resources compared to existing protocols.

Physicochemical differences between camelid single-domain antibodies and mammalian antibodies.

In recent years, single-domain antibodies, also known as nanobodies, have emerged as an alternative to full immunoglobulin Gs (IgGs), due to their various advantages, including increased solubility, faster clearance, and cheaper production. Nanobodies are generally derived from the variable domain of the camelid heavy-chain-only immunoglobulin Gs (hcIgGs). Due to the high sequence homology between variable heavy chains of camelids (VHHs) and humans (VHs), hcIgGs are ideal candidates for nanobody development. However, further examination is needed to understand the structural differences between VHs and VHHs. This analysis is essential for nanobody engineering to mitigate potential immunogenicity, while preserving stability, functionality, and antigen specificity. We obtained the VH and VHH sequences of various camelid and non-camelid mammalian antibodies from public databases and used multiple sequence alignment based on the Chothia numbering scheme. Aligned sequences were subjected to diverse analyses encompassing paratope length, binding prediction, motif, disulfide bridge, salt bridge profiling, and physicochemical characteristic distribution. Logistic Regression coupled with the Boruta - Random Forest algorithm facilitated the comprehensive examination of physicochemical properties. Our findings revealed longer, less variable paratope sequences in VHHs, along with specific antigen binding residues with increased binding potential compared to VHs. Although the VHs showed more heterogeneous noncanonical disulfide bond patterns, the VHHs had a higher number of noncanonical disulfide bridges. Intriguingly, a typical salt bridge between the 94th and 101st positions in the VHs had a very low encounter rate in the VHHs. Surprisingly, we also identified notable differences in the physicochemical patterns of mostly conserved frameworks (FWs), especially the FW2 and FW3 regions, between VHs and VHHs. Our findings point to possible key sites in VHHs as candidate residues for nanobody engineering efforts.

A specific nanobody-based affinity chromatography resin as a platform for small ubiquitin-related modifier fusion protein purification

As an excellent fusion tag for expressing heterologous proteins, yeast SUMO (small ubiquitin-related modifier) has unique advantages such as improving solubility, promoting stability, and reducing degradation, but it lacks a simple and rapid purification method. Camelid single-domain antibodies (VHHs or nanobodies) show great promise as an efficient tool in analytical application. In this study, VHHs against SUMO protein were isolated for the first time using biopanning of an immune camelid nanobody library. Among these nanobodies, VS2 demonstrated a high expression level (1.12 g L − 1), and a high affinity for SUMO (2.26 nM). Meanwhile, VHHs were coupled to agarose resins by cysteine at the C-terminal to form affinity chromatography resins. The VS2 resin showed excellent specificity and a dynamic binding capacity for SUMO, SUMO-DsbA (disulfide oxidoreductase) and SUMO-SAM (S-adenosylmethionine synthetase) were 2.41 mg/mL resin, 7.57 mg/mL resin and 16.23 mg/mL resin, respectively. Furthermore, the VS2 resin enabled one-step purification of SUMO-fusions [SUMO-Fc (human IgG1-Fc fragment), SUMO-IGF1 (human insulin-like growth factor 1), SUMO-FGF21 (human fibroblast growth factor 21), SUMO-G-CSF (human Granulocyte colony-stimulating factor), SUMO-PDGF (human platelet-derived growth factor) and SUMO-PAS200 (conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala-and Ser)], and maintained binding capacity and selectivity over 25 purification cycles, each including 15 min of cleaning-in-place with 0.1 M NaOH. This study demonstrated that the VS2 resin was a useful tool at the laboratory scale for one-step purification of various SUMO fusions from complex mixtures.

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.

Cryo-EM structure of antibacterial efflux transporter QacA from Staphylococcus aureus reveals a novel extracellular loop with allosteric role.

Efflux of antibacterial compounds is a major mechanism for developing antimicrobial resistance. In the Gram-positive pathogen Staphylococcus aureus, QacA, a 14 transmembrane helix containing major facilitator superfamily antiporter, mediates proton-coupled efflux of mono and divalent cationic antibacterial compounds. In this study, we report the cryo-EM structure of QacA, with a single mutation D411N that improves homogeneity and retains efflux activity against divalent cationic compounds like dequalinium and chlorhexidine. The structure of substrate-free QacA, complexed to two single-domain camelid antibodies, was elucidated to a resolution of 3.6 Å. The structure displays an outward-open conformation with an extracellular helical hairpin loop (EL7) between transmembrane helices 13 and 14, which is conserved in a subset of DHA2 transporters. Removal of the EL7 hairpin loop or disrupting the interface formed between EL7 and EL1 compromises efflux activity. Chimeric constructs of QacA with a helical hairpin and EL1 grafted from other DHA2 members, LfrA and SmvA, restore activity in the EL7 deleted QacA revealing the allosteric and vital role of EL7 hairpin in antibacterial efflux in QacA and related members.

Production and characterization of a camelid single domain anti-CD22 antibody conjugated to DM1.

Antibody drug conjugates (ADCs) with twelve FDA approved drugs, known as a novel category of anti-neoplastic treatment created to merge the monoclonal antibody specificity with cytotoxicity effect of chemotherapy. However, despite many undeniable advantages, ADCs face certain problems, including insufficient internalization after binding, complex structures and large size of full antibodies especially in targeting of solid tumors. Camelid single domain antibody fragments (Nanobody®) offer solutions to this challenge by providing nanoscale size, high solubility and excellent stability, recombinant expression in bacteria, in vivo enhanced tissue penetration, and conjugation advantages. Here, an anti-human CD22 Nanobody was expressed in E.coli cells and conjugated to Mertansine (DM1) as a cytotoxic payload. The anti-CD22 Nanobody was expressed and purified by Ni-NTA resin. DM1 conjugated anti-CD22 Nanobody was generated by conjugation of SMCC-DM1 to Nanobody lysine groups. The conjugates were characterized using SDS-PAGE and Capillary electrophoresis (CE-SDS), RP-HPLC, and MALDI-TOF mass spectrometry. Additionally, flow cytometry analysis and a competition ELISA were carried out for binding evaluation. Finally, cytotoxicity of conjugates on Raji and Jurkat cell lines was assessed. The drug-to-antibody ratio (DAR) of conjugates was calculated 2.04 using UV spectrometry. SDS-PAGE, CE-SDS, HPLC, and mass spectrometry confirmed conjugation of DM1 to the Nanobody. The obtained results showed the anti-CD22 Nanobody cytotoxicity was enhanced almost 80% by conjugation with DM1. The binding of conjugates was similar to the non-conjugated anti-CD22 Nanobody in flow cytometry experiments. Concludingly, this study successfully suggest that the DM1 conjugated anti-CD22 Nanobody can be used as a novel tumor specific drug delivery system.

Simple and Highly Efficient Detection of PSD95 Using a Nanobody and Its Recombinant Heavy-Chain Antibody Derivatives.

The post-synaptic density protein 95 (PSD95) is a crucial scaffolding protein participating in the organization and regulation of synapses. PSD95 interacts with numerous molecules, including neurotransmitter receptors and ion channels. The functional dysregulation of PSD95 as well as its abundance and localization has been implicated with several neurological disorders, making it an attractive target for developing strategies able to monitor PSD95 accurately for diagnostics and therapeutics. This study characterizes a novel camelid single-domain antibody (nanobody) that binds strongly and with high specificity to rat, mouse, and human PSD95. This nanobody allows for more precise detection and quantification of PSD95 in various biological samples. We expect that the flexibility and unique performance of this thoroughly characterized affinity tool will help to further understand the role of PSD95 in normal and diseased neuronal synapses.

Abstract 2339: Exploration of multiple immunotherapy modalities in ostesarcoma

Abstract Osteosarcoma (OS) is the most common primary bone malignancy affecting long bones in children and the third most frequent in adults in the United States. It is highly invasive and metastatic. The survival rates over the last 20 years are unchanged. Therefore, it is essential to develop novel, targeted therapeutic approaches to treat OS. In pursuit of that goal, novel models are needed. Due to the high similarity in cancer genetics, the spontaneous occurrence of tumors, and the presence of an intact immune system, dogs are deemed an excellent cancer immunotherapy model. OS is also the most common malignant bone tumor (80%) in dogs. Monotherapies like oncolytic viruses and immune checkpoint inhibitors have been shown to be insufficient for effective cancer treatment. Therefore, we propose a combination of immune checkpoint inhibition and oncolytic virotherapy to target osteosarcoma tumor cells. In this combined therapy, the oncolytic virus will serve to both stimulate an inflammatory response and transmit the checkpoint inhibitor, which in turn will block PD-1-directed down-regulation of T cells, allowing the adaptive immune system to respond better to tumors. In order to evaluate this approach in the canine model, we have developed an oncolytic virus (CAV2-AU-M2) based on canine adenovirus type 2 and armed with an immune checkpoint inhibitor, an anti-canine PD1 single domain camelid antibody (sdAb). Four canine osteosarcoma cell lines, D17, D22, MCKOS, and CF11, were cultured in two-dimensional (2D) and three-dimensional (3D) cultures and were infected with CAV2-AU-M2 to analyze the infective and lytic effects of the virus. CAV2-AU-M2 infected and lysed the cells in 2D. The virus also produced anti-PD1 sdAb upon infecting the cells. Similarly, results were evident in 3D cultures. The virus infected the spheroids in the outer layers on Day 4 and slowly diffused towards the core in 7 days. The spheroids infected started showing some cell death on day 4, which progressed significantly by Day 7. This approach to oncolytic virotherapy makes the tumor more visible to the immune system and abrogates tumor-induced immune suppression. Citation Format: Aliaa A. Ismail, Daniel J. Patton, Theresa A. Higgins, Maninder Sandey, Bruce F. Smith, Payal Agarwal. Exploration of multiple immunotherapy modalities in ostesarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2339.

T-cells engineered with a novel VHH-based chimeric antigen receptor against CD19 exhibit comparable tumoricidal efficacy to their FMC63-based counterparts.

Chimeric antigen receptor (CAR)-T cell therapy has established itself as a potent therapeutic option for certain patients with relapsed/refractory (R/R) hematologic malignancies. To date, four CD19-redirected CAR-T cell products have been granted the United States Food and Drug Administration (FDA) approval for medical use. However, all of these products are equipped with a single-chain fragment variable (scFv) as their targeting domains. Camelid single-domain antibodies (VHH or nanobody) can also be used as alternatives to scFvs. In this study, we developed VHH-based CD19-redirected CAR-Ts, and compared them with their FMC63 scFv-based counterpart. Human primary T cells were transduced to express a second-generation 4-1BB-CD3ζ-based CAR construct whose targeting domain was based on a CD19-specific VHH. The expansion rate, cytotoxicity, and secretion of proinflammatory cytokines (IFN-γ, IL-2, and TNF-α) of the developed CAR-Ts were assessed and compared with their FMC63 scFv-based counterpart as they were co-cultured with CD19-positive (Raji and Ramos) and CD19-negative (K562) cell lines. VHH-CAR-Ts showed an expansion rate comparable to that of the scFv-CAR-Ts. In terms of cytotoxicity, VHH-CAR-Ts mediated cytolytic reactions against CD19-positive cell lines, comparable to those of their scFv-based counterparts. Moreover, both VHH-CAR-Ts and scFv-CAR-Ts secreted remarkably higher and similar levels of IFN-γ, IL-2, and TNF-α upon co-cultivation with Ramos and Raji cell lines compared with while cultured alone or co-cultured with K562 cells. Our results demonstrated that our VHH-CAR-Ts could mediate CD19-dependent tumoricidal reactions as potently as their scFv-based counterparts. Moreover, VHHs could be applied as the targeting domains of CAR constructs to overcome the issues associated with the use of scFvs in CAR-T therapies.

Isolation and Characterization of Single-Domain Antibodies from Immune Phage Display Libraries.

Naturally occurring heavy chain antibodies (HCAbs) in Camelidae species were a surprise discovery in 1993 by Hamers et al. Since that time, antibody fragments derived from HCAbs have garnered considerable attention by researchers and biotechnology companies. Due to their biophysico-chemical advantages over conventional antibody fragments, camelid single-domain antibodies (sdAbs, VHHs, nanobodies) are being increasingly utilized as viable immunotherapeutic modalities. Currently there are multiple VHH-based therapeutic agents in different phases of clinical trials in various formats such as bi- and multivalent, bi- and multi-specific, CAR-T, and antibody-drug conjugates. The first approved VHH, a bivalent humanized VHH (caplacizumab), was approved for treating rare blood clotting disorders in 2018 by the EMA and the FDA in 2019. This was followed by the approval of an anti-BCMA VHH-based CAR-T cell product in 2022 (ciltacabtagene autoleucel; CARVYKTI™) and more recently a trivalent antitumor necrosis factor alpha-based VHH drug (ozoralizumab; Nanozora®) in Japan for the treatment of rheumatoid arthritis. In this chapter we provide protocols describing the latest developments in isolating antigen-specific VHHs including llama immunization, construction of phage-displayed libraries, phage panning and screening of the soluble VHHs by ELISA, affinity measurements by surface plasmon resonance, functional cell binding by flow cytometry, and additional validation by immunoprecipitation. We present and discuss comprehensive, step-by-step methods for isolating and characterization of antigen-specific VHHs. This includes protocols for expression, biotinylation, purification, and characterization of the isolated VHHs. To demonstrate the feasibility of the entire strategy, we present examples of VHHs previously isolated and characterized in our laboratory.

Nanobodies targeting ABCC3 for immunotargeted applications in glioblastoma

The cancer “omics” reveal many clinically relevant alterations that are transforming the molecular characterization of glioblastomas. However, many of these findings are not yet translated into clinical practice due, in part, to the lack of non-invasive biomarkers and the limitations imposed by the blood–brain barrier. Nanobodies, camelid single-domain antibody fragments, emerge as a promising tool for immunotargeted applications for diagnosing and treating glioblastomas. Performing agnostic bioinformatic analysis from glioblastoma patient datasets, we identified ATP Binding Cassette subfamily C member 3 (ABCC3) as a suitable target for immunotargeted applications. The expression of ABCC3 is associated with poor survival and impaired response to temozolomide. Importantly, high expression of ABCC3 is restricted to glioblastoma, with negligible levels in healthy brain tissue, and further correlates with tumor grade and stemness markers. We identified three immunogenic epitopes of ABCC3 which were used to isolate nanobodies from a glioblastoma-specific phage-display nanobody library. Two nanobodies targeting ABCC3 (NbA42 and NbA213) were further characterized and demonstrated in vivo selective recognition of ABCC3 in glioblastoma xenograft mouse models upon systemic administration. We designate NbA42 and NbA213 as new candidates to implement immunotargeted applications guiding a more personalized and precise diagnosis, monitoring, and treatment of glioblastoma patients.

Strategies to enhance immune cell activation & cancer cell killing

Abigail Pinchbeck, Assistant Editor, BioInsights, speaks to Karine Breckpot, Director, Laboratory for Molecular and Cellular Therapy (LMCT), Vrije Universiteit BrusselKarine Breckpot obtained a MSc in Biomedical Sciences in 1998 and a PhD in Medical Sciences in 2004, all at the Vrije Universiteit Brussel (VUB, Belgium). Following a postdoctoral stay at the Division of Infection and Immunity at the University College London (United Kingdom), she became a group leader and Assistant Professor in 2010 at the Laboratory for Molecular and Cellular Therapy (LMCT), VUB. She became a Tenured Professor in 2014 and Full Professor in 2019 at which point she became director of the research group LMCT. Her research is focused on cancer immunology and immunotherapy, designing novel immunotherapies focused on T cell activation using technologies such as mRNA, lentiviral vectors and antibodies, including camelid single domain antibodies. She was laureate of the Fund for Biotechnology award in 2005, received the Dr Karel-Lodewijk Verleysen award of the Belgian Royal Academy of Medicine in 2011, the Melanoma Research Alliance young investigator award in 2017 and the BCLAS award in honor of Jean-René Maisin in 2019. She is (co)promotor of 15 defended and 16 ongoing doctoral theses and author on more than 135 Q1 publications.Technologies such as mRNA, lentiviral vectors, and nanobodies are vital tools in the immuno-oncology arsenal. In this article, Karine Breckpot, the director of the Laboratory for Molecular and Cellular Therapy (LMCT), explains how she applies the latest technology to her work in engineering dendritic cells for immune activation and T cells for cancer cell rejection – and her predictions for 2023 and beyond.

Fluorescence Polarization Immunoassay of Human Lactoferrin in Milk Using Small Single-Domain Antibodies

Due to its unique structure and properties, human breast milk lactoferrin (hLF) has many nutritional and health-promoting functions in infants, including protection against inflammation and bacterial infections. The lack of LF in breastmilk or formula can result in the weakening of the infant’s immune system. Noncompetitive polarization fluorescence immunoassay (FPIA) is a promising method for hLF quantification in milk and dairy products, which does not require the separation of the bound and free protein and allows to avoid time-consuming sample preparation. The use of fluorescently labeled single-domain camelid antibodies (nanobodies) for protein recognition in FPIA makes it possible to quantify relatively large antigens, in particular, hLF. In this work, we used previously obtained fluorescein isothiocyanate (FITC)-conjugated anti-hLF5 and anti-hLF16 nanobodies, which selectively recognized two different human lactoferrin epitopes, but did not bind to goat lactoferrin. The kinetics of hLF interaction with the FITC-labeled nanobodies was studied. The dissociation constant (KD) for the anti-LF5 and anti-LF16 nanobodies was 3.2 ± 0.3 and 4.9 ± 0.4 nM, respectively, indicating the high-affinity binding of these nanobodies to hLF. We developed the FPIA protocol and determined the concentration of FITC-labeled anti-hLF5 and anti-hLF16 nanobodies that provided the optimal fluorescence signal and stable fluorescence polarization value. We also studied the dependence of fluorescence polarization on the hLF concentration in the noncompetitive FPIA with FITC-anti-hLF5 nanobody. The detection limit for hLF was 2.1 ± 0.2 µg/ml and the linear range for determining the hLF concentration was 3-10 µg/ml. FPIA is commonly used to assay low-molecular-weight substances; however, the use of fluorescently labeled nanobodies allows quantification of high-molecular-weight proteins. Here, we demonstrated that FPIA with fluorescently labeled nanobodies can be used for hLF quantification in milk.

Neutralizing epitopes on Clostridioides difficile toxin A revealed by the structures of two camelid VHH antibodies.

Toxin A (TcdA) and toxin B (TcdB) are two key virulence factors secreted by Clostridioides difficile, which is listed as an urgent threat by the CDC. These two large homologous exotoxins are mainly responsible for diseases associated with C. difficile infection (CDI) with symptoms ranging from diarrhea to life threatening pseudomembranous colitis. Single-domain camelid antibodies (VHHs) AH3 and AA6 are two potent antitoxins against TcdA, which when combined with two TcdB-targeting VHHs showed effective protection against both primary and recurrent CDI in animal models. Here, we report the co-crystal structures of AH3 and AA6 when they form complexes with the glucosyltransferase domain (GTD) and a fragment of the delivery and receptor-binding domain (DRBD) of TcdA, respectively. Based on these structures, we find that AH3 binding enhances the overall stability of the GTD and interferes with its unfolding at acidic pH, and AA6 may inhibit the pH-dependent conformational changes in the DRBD that is necessary for pore formation of TcdA. These studies reveal two functionally critical epitopes on TcdA and shed new insights into neutralizing mechanisms and potential development of epitope-focused vaccines against TcdA.

B7-H3 Trike Enhances Killing of Myeloid Derived Suppressor Cells in Multiple Myeloma

Multiple myeloma (MM) is sensitive to Natural Killer (NK) cell-mediated killing but develops methods to attenuate NK cell function. One method to enhance NK cell specificity against myeloma is antibody dependent cellular cytotoxicity through the CD16 receptor. We developed a tri-specific killer engager (TriKE®) with a camelid single-domain antibody (sdAb) fragment against CD16 (on NK cells) as well as a linker region containing IL-15, to enhance NK cell survival. Delivery of IL-15 by TriKE supports NK cell proliferation while avoiding off-target effects on T cells. The second sdAb targets B7-H3 (CD276), a tumor antigen that is expressed on myeloma and is associated with decreased progression free survival. B7-H3 is differentially expressed on myeloma tumor lines MM1S, RPMI-8226, U266, and H929 but all four MM lines are significantly killed by peripheral blood NK cells in the presence of 3nM B7-H3 TriKE compared to NK cells alone. Bone marrow aspirates from 5 MM patients (3 newly diagnosed and 2 relapsed) were analyzed by flow cytometry and showed 58-96% expression of B7-H3 on CD138+ plasma cells. The bone marrow biopsy aspirates also showed monocytic myeloid derived suppressor cells (Mo-MDSC, CD14+, CD11b+) that expressed B7-H3 (38-87%) as well as the damage associated molecular pattern (DAMP) S100A8/9. In comparison, CD14+ cells from the peripheral blood of healthy donors expressed very low levels of B7-H3 or S100A8/9 (<5%). Mo-MDSC are important to the pathogenesis of MM because they support MM growth and contribute to suppression of NK in the tumor microenvironment. B7-H3 is highly expressed on mature osteoclasts and MDSC can develop into osteoclasts in mouse models of MM and breast cancer. We saw that Mo-MDSC expressing B7-H3 also co-expressed RANK, a receptor necessary for signaling osteoclastic development. We developed Mo-MDSC from CD33+ myeloid cells from healthy donors using IL-6 and GM-CSF or co-culture with MM cell lines at 1:1000 ratio. Both types of MDSC suppressed NK proliferation. Peripheral blood derived MDSC expressed >80% B7-H3 and RANK. We separated B7-H3 positive and negative populations by flow cytometry and attempted to develop osteoclasts with M-CSF and RANK-L over 14 days. Tartrate resistant acid phosphatase (TRAP) staining was performed but both groups were negative and exhibited no phenotypic changes by light microscopy. We then isolated CD14+ cells from the bone marrow of an MM patient who was in complete remission (CR) after autologous stem cell transplant but had a distinct population of B7-H3+, RANK+, S100A8/9+ Mo-MDSC in her surveillance bone marrow aspirate. After fourteen days of culture with M-CSF and RANK-L, TRAP staining was positive, and cells had grown larger, consistent with osteoclastic differentiation (Figure 1A). We tested the ability of B7-H3 TriKE to eliminate MDSC by NK cells, therefore diminishing there immune suppression. Cytotoxicity assays were conducted over 48 hours with NK cells with or without B7-H3 TriKE. Killing was significantly enhanced by B7-H3 TriKE (Figure 1B). Since MDSC are known to suppress NK cell function, we assessed metabolic fitness in NK cells with MDSC at 1:1 ratio for 48 hours with or without 3nM B7-H3 TriKE and found that B7-H3 TriKE improved both the oxygen consumption and extracellular acidification rates. These data show that B7-H3 TriKE enhances NK cell killing of MM through direct targeting and eliminates suppressive Mo-MDSC. B7-H3+ Mo-MDSC can differentiate into osteoclasts and persist even in patients who are in CR, possibly contributing to relapse. B7-H3 TriKE may be particularly useful for patients with bone lesions or as consolidative therapy. Commercial manufacturing of B7-H3 TriKE (GTB-5550) has begun, and a phase I trial is expected in late 2023. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular basis for thermal stability and affinity in a VHH: Contribution of the framework region and its influence in the conformation of the CDR3.

The camelid single domain antibody, referred to VHH or Nanobody, is considered a versatile tool for various biotechnological and clinical applications because of its favorable biophysical properties. To take advantage of these characteristics and for its application in biotechnology and therapy, research on VHH engineering is currently vigorously conducted. To humanize a camelid VHH, we performed complementarity determining region (CDR) grafting using a humanized VHH currently in clinical trials, and investigated the effects of these changes on the biophysical properties of the resulting VHH. The chimeric VHH exhibited a significant decrease in affinity and thermal stability and a large conformational change in the CDR3. To elucidate the molecular basis for these changes, we performed mutational analyses on the framework regions revealing the contribution of individual residues within the framework region. It is demonstrated that the mutations resulted in the loss of affinity and lower thermal stability, revealing the significance of bulky residues in the vicinity of the CDR3, and the importance of intramolecular interactions between the CDR3 and the framework‐2 region. Subsequently, we performed back‐mutational analyses on the chimeric VHH. Back‐mutations resulted in an increase of the thermal stability and affinity. These data suggested that back‐mutations restored the intramolecular interactions, and proper positioning and/or dynamics of the CDR3, resulting in the gain of thermal stability and affinity. These observations revealed the molecular contribution of the framework region on VHHs and further designability of the framework region of VHHs without modifying the CDRs.

Programmable Attenuation of Antigenic Sensitivity for a Nanobody-Based EGFR Chimeric Antigen Receptor Through Hinge Domain Truncation.

Epidermal growth factor family receptor (EGFR) is commonly overexpressed in many solid tumors and an attractive target for chimeric antigen receptor (CAR)-T therapy, but as EGFR is also expressed at lower levels in healthy tissues a therapeutic strategy must balance antigenic responsiveness against the risk of on-target off-tumor toxicity. Herein, we identify several camelid single-domain antibodies (also known as nanobodies) that are effective EGFR targeting moieties for CARs (EGFR-sdCARs) with very strong reactivity to EGFR-high and EGFR-low target cells. As a strategy to attenuate their potent antigenic sensitivity, we performed progressive truncation of the human CD8 hinge commonly used as a spacer domain in many CAR constructs. Single amino acid hinge-domain truncation progressively decreased both EGFR-sdCAR-Jurkat cell binding to EGFR-expressing targets and expression of the CD69 activation marker. Attenuated signaling in hinge-truncated EGFR-sdCAR constructs increased selectivity for antigen-dense EGFR-overexpressing cells over an EGFR-low tumor cell line or healthy donor derived EGFR-positive fibroblasts. We also provide evidence that epitope location is critical for determining hinge-domain requirement for CARs, as hinge truncation similarly decreased antigenic sensitivity of a membrane-proximal epitope targeting HER2-CAR but not a membrane-distal EGFRvIII-specific CAR. Hinge-modified EGFR-sdCAR cells showed clear functional attenuation in Jurkat-CAR-T cells and primary human CAR-T cells from multiple donors in vitro and in vivo. Overall, these results indicate that hinge length tuning provides a programmable strategy for throttling antigenic sensitivity in CARs targeting membrane-proximal epitopes, and could be employed for CAR-optimization and improved tumor selectivity.

Anti-Metalloprotease P-I Single-Domain Antibodies: Tools for Next-Generation Snakebite Antivenoms.

In order to address the global antivenom crisis, novel antivenoms need to present high therapeutic efficacy, broad neutralization ability against systemic and local damage, sufficient safety, and cost-effectiveness. Due to biological characteristics of camelid single-domain antibodies (VHH) such as high affinity, their ability to penetrate dense tissues, and facility for genetic manipulation, their application in antivenoms has expanded considerably. VHHs that are active against the metalloprotease BjussuMP-II from the snake Bothrops jararacussu were selected. After isolation of BjussuMP-II, a camelid was immunized with the purified toxin in order to construct the recombinant phage library. Following a round of biopanning, 52% of the selected clones were able to recognize BjussuMP-II in an ELISA assay. After sequencing, seven sequence profiles were identified. One selected clone (VHH61) showed cross-reactivity to B. brazili venom, but did not recognize the Crotalus and Lachesis genera, indicating specificity for the Bothrops genus. Through in vitro tests, the capacity to neutralize the toxicity triggered by BjussuMP-II was observed. Circular dichroism spectroscopy indicated a robust secondary structure for VHH61, and the calculated melting temperature (TM) for the clone was 56.4°C. In silico analysis, through molecular docking of anti-BjussuMP-II VHHs with metalloprotease, revealed their potential interaction with amino acids present in regions critical for the toxin's conformation and stability. The findings suggest that anti-BjussuMP-II VHHs may be beneficial in the development of next-generation antivenoms.