Development of Shark Single Domain Antibodies Specific for Human α-Fetoprotein and the Multimerization Strategy in Serum Detection.

Sensitive detection of cancer biomarkers can contribute to the timely diagnosis and treatment of diseases. In this study, the whitespotted bamboo sharks were immunized with human α-fetoprotein (AFP), and a phage-displayed variable new antigen receptor (VNAR) single domain antibody library was constructed. Then four unique VNARs (VNAR1, VNAR11, VNAR21, and VNAR25) against AFP were isolated from the library by biopanning for the first time. All of the sequences belong to type II of VNAR, and the VNAR11 was much different from the rest of the three sequences. Then VNAR1 and VNAR11 were selected to fuse with the C4-binding protein α chain (C4bpα) sequence and efficiently expressed in the Escherichia coli system. Furthermore, a VNAR-C4bpα-mediated sandwich chemiluminescence immunoassay (VSCLIA) was developed for the detection of AFP in human serum samples. After optimization, the VSCLIA showed a limit of detection of 0.74 ng/mL with good selectivity and accuracy. Moreover, the results of clinical serum samples detected by the VSCLIA were confirmed by an automatic immunoanalyzer in the hospital, indicating its practical application in actual samples. In conclusion, the novel antibody element VNAR exhibits great potential for immunodiagnosis, and this study also provides a new direction and experimental basis for AFP detection.

Non-specific cytotoxic cell receptor protein-1 (NCCRP-1) is involved in anti-parasite innate CD8+ T cell-mediated cytotoxicity in ginbuna crucian carp.

CD8+ cytotoxic T cells (CTLs) are a main cellular component of adaptive immunity. Our previous research has shown that CD8+ cells demonstrate spontaneous cytotoxic activity against the parasite Ichthyophthirius multifiliis in ginbuna crucian carp, suggesting that CD8+ cells play an important role in innate immunity. Herein, we investigated the molecules and cellular signal pathways involved in the cytotoxic response of ginbuna crucian carp. We considered non-specific cytotoxic receptor protein-1 (NCCRP-1) as candidate molecule for parasite recognition. We detected NCCRP-1 protein in CD8+ cells and the thymus as well as in other cells and tissues. CD8+ cells expressed mRNA for NCCRP-1, Jak2, and T cell-related molecules. In addition, treatment with a peptide containing the presumed antigen recognition site of ginbuna NCCRP-1 significantly inhibited the cytotoxic activity of CD8+ cells against the parasites. The cytotoxic activity of CD8+ cells was significantly inhibited by treatment with the JAK1/2 inhibitor baricitinib. These results suggest that teleost CTLs recognize I. multifiliis through NCCRP-1 and are activated by JAK/STAT signaling.

Isolation and Characterization of Targeting-HBsAg VNAR Single Domain Antibodies from Whitespotted Bamboo Sharks (Chiloscyllium plagiosum).

Immunoglobulin new antigen receptor (IgNAR) is a naturally occurring antibody that consists of only two heavy chains with two independent variable domains. The variable binding domain of IgNAR, called variable new antigen receptor (VNAR), is attractive due to its solubility, thermal stability, and small size. Hepatitis B surface antigen (HBsAg) is a viral capsid protein found on the surface of the Hepatitis B virus (HBV). It appears in the blood of an individual infected with HBV and is widely used as a diagnostic marker for HBV infection. In this study, the whitespotted bamboo sharks (Chiloscyllium plagiosum) were immunized with the recombinant HBsAg protein. Peripheral blood leukocytes (PBLs) of immunized bamboo sharks were further isolated and used to construct a VNAR-targeted HBsAg phage display library. The 20 specific VNARs against HBsAg were then isolated by bio-panning and phage ELISA. The 50% of maximal effect (EC50) of three nanobodies, including HB14, HB17, and HB18, were 4.864 nM, 4.260 nM, and 8.979 nM, respectively. The Sandwich ELISA assay further showed that these three nanobodies interacted with different epitopes of HBsAg protein. When taken together, our results provide a new possibility for the application of VNAR in HBV diagnosis and also demonstrate the feasibility of using VNAR for medical testing.

Humanization of the Shark VNAR Single Domain Antibody Using CDR Grafting.

The variable domain of the new antigen receptor (VNAR ) of shark single domain antibodies is evolutionarily distant from the variable regions (VH ) of mammalian immunoglobulins, yet it still has complementarity-determining regions (CDRs) that are involved in antigen recognition, therefore making it possible to humanize by grafting these CDRs to the framework of human VH homologs. Here, we show the VNAR CDR based on an analysis of currently available VNAR -antigen structure complexes in the global Protein Data Bank archive of 3D structure data, and describe the detailed protocol to humanize VNAR by CDR grafting, using B6 (an anti-Pseudomonas exotoxin VNAR ), the most common type (Type II) of shark VNAR s, as an example. Ongoing efforts will further optimize the protocol for moving shark VNAR s to the clinic for treating cancer and other human diseases. Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol: Humanize shark VNAR sequence by CDR grafting Support Protocol 1: VNAR structure prediction and comparison Support Protocol 2: Measure binding kinetics of humanized VNAR using bio-layer interferometry (BLI).

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.

Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography.

Single-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.

Shark New Antigen Receptor (IgNAR): Structure, Characteristics and Potential Biomedical Applications.

Shark is a cartilaginous fish that produces new antigen receptor (IgNAR) antibodies. This antibody is identified with a similar human heavy chain but dissimilar sequences. The variable domain (VNAR) of IgNAR is stable and small in size, these features are desirable for drug discovery. Previous study results revealed the effectiveness of VNAR as a single molecule or a combination molecule to treat diseases both in vivo and in vitro with promising clinical applications. We showed the first evidence of IgNAR alternative splicing from spotted bamboo shark (Chiloscyllium plagiosum), broadening our understanding of the IgNARs characteristics. In this review, we summarize the discoveries on IgNAR with a focus on its advantages for therapeutic development based on its peculiar biochemistry and molecular structure. Proper applications of IgNAR will provide a novel avenue to understand its special presence in cartilaginous fishes as well as designing a number of drugs for undefeated diseases.

Biological characterisation, expression and functional analysis of non-specific cytotoxic cell receptor protein 1 in Nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cell receptor protein 1 (NCCRP-1) plays a role in recognition of target cell and activation of non-specific cytotoxic cell (NCC). In this study, the full length of Nile tilapia NCCRP-1 (On-NCCRP-1) was cloned. cDNA is composed of 1045 bp with a 90 bp of 5'-Untranslated Regions (UTR), 702 bp open reading frame (ORF) and 253 bp 3'-UTR, encoding 233 amino acids (GenBank accession no: MF162296). The On-NCCRP-1 genomic sequence is 4471 bp in length and contains six exons and five introns. On-NCCRP-1 possesses some inherent conservative domains, such as proline-rich motifs, antigen recognition site, and F-box-related domain. Subcellular localisation and Western blot analysis indicated that On-NCCRP-1 is located in the cell membrane. The transcript of On-NCCRP-1 was detected in all the examined tissues of healthy Nile tilapia by using qRT-PCR, with the highest expression levels in the liver. Following Streptococcus agalactiae challenged in vivo, the On-NCCRP-1 expression was up-regulated significantly in brain, intestines, head kidney and spleen. In the in vitro analysis, the On-NCCRP-1 expression in NCCs was up-regulated significantly from 8 h to 12 h after LPS challenge, and up-regulated significantly at 12 h after challenged with polyI:C. After NCCs were challenged with inactivated S. agalactiae, the On-NCCRP-1 expression was down-regulated significantly after 24 h. NF-кB pathway was strongly activated by the over-expression of On-NCCRP-1 in HEK-293T cells. These results indicate that On-NCCRP-1, as a membrane surface receptor of NCCs, may play an important role in immune response to pathogenic infection in Nile tilapia.

[Progress in shark single-domain antibody].

Monoclonal antibody (mAb) is an important biological macromolecule and widely used in immune detection, in vitro diagnostics, and drug discovery. However, the inherent properties of mAb restrict its further development, such as high molecular weight and complex structure. Therefore, there is an urgent need to develop alternatives for mAb. Various types of miniaturized antibodies have been developed, among which the variable domain of immunoglobulin new antigen receptor (VNAR) is very attractive. The shark single-domain antibody, also known as shark VNAR, is an antigen-binding domain obtained by genetic engineering technology based on the immunoglobulin new antigen receptor (IgNAR) that naturally exists in selachimorpha. It has a molecular weight of 12 kDa, which is the smallest antigen-binding domain found in the known vertebrates at present. Compared with mAb, the shark VNAR exhibits various superiorities, such as low molecular weight, high affinity, tolerance to the harsh environment, good water solubility, strong tissue penetration, and recognition of the hidden epitopes. It has attracted wide attention in the fields of immunochemical reagents and drug discovery. In this review, various aspects of shark VNAR are elaborated, including the structural and functional characteristics, generating and humanization techniques, affinity maturation strategies, application fields, advantages and disadvantages, and prospects.

Sequence structure character of IgNAR Sec in whitespotted bamboo shark (Chiloscyllium plagiosum).

Whitespotted bamboo shark (Chiloscyllium plagiosum) is a demersal cartilaginous fish with an adaptive immune system founded upon immunoglobulins. In this manuscript, we characterize the IgNAR of the whitespotted bamboo shark. A newly discovered alternative splicing form of IgNAR Sec (IgNARshort (ΔC2-C3) Sec) was identified, in which the C1 domain was spliced directly to the C4 domain, the process resulted in a molecule containing three constant domains. However, a single unpaired cysteine remains in the highly flexible hinge region, contributing in the formation of an interchain disulfide bond. Two types of C1 domain were found, and the one lacking a short α-helix showed lower proportion. This finding suggests that short α-helices might be important to the stability of IgNAR. High-throughput sequencing revealed that the percentage of VNAR types significantly vary between the diverse species of sharks. The variable region of IgNAR (the VNAR) with small size and stabilization is a potential candidate for immunotherapeutic agents. The structure and stability analysis in this manuscript may be useful in future biomedical applications.

The Innate Biologies of Adaptive Antigen Receptors.

Nonclonal innate immune responses mediated by germ line-encoded receptors, such as Toll-like receptors or natural killer receptors, are commonly contrasted with diverse, clonotypic adaptive responses of lymphocyte antigen receptors generated by somatic recombination. However, the Variable (V) regions of antigen receptors include germ line-encoded motifs unaltered by somatic recombination, and theoretically available to mediate nonclonal, innate responses, that are independent of or largely override clonotypic responses. Recent evidence demonstrates that such responses exist, underpinning the associations of particular γδ T cell receptors (TCRs) with specific anatomical sites. Thus, TCRγδ can make innate and adaptive responses with distinct functional outcomes. Given that αß T cells and B cells can also make nonclonal responses, we consider that innate responses of antigen receptor V-regions may be more widespread, for example, inducing states of preparedness from which adaptive clones are better selected. We likewise consider that potent, nonclonal T cell responses to microbial superantigens may reflect subversion of physiologic innate responses of TCRα/ß chains.

Identification, functional characterization and the potential role of variable lymphocyte receptor EsVLRA from Eriocheir sinensis in response to secondary challenge after Vibrio parahaemolyticus vaccine.

Variable lymphocyte receptors (VLRs) play an important role via their antigen-special reorganization in jawless vertebrates (agnathans) adaptive immune response. In the present study, the open reading frame (ORF) of Eriocheir sinensis VLRA (designated as EsVLRA) was identified. EsVLRA comprised a 799-amino-acid polypeptide with one LRR_NT domain, thirteen LRR domains and one LRR_CT domain, which showed a high domain consistency of the VLR genes in lamprey (Petromyzon marinus). The transcript of EsVLRA was detected in all examined tissues with the highest level detected in hepatopancreas. Notably, the expression of EsVLRA in hepatopancreas, gonads, gill and intestine of male crabs was significantly higher than that in females. The recombinant EsVLRA exhibited strong bacteria-binding activity rather than antibacterial activity, suggesting its crucial role in immune recognition. Furthermore, 6 h earlier response and a significantly higher peak of EsVLRA mRNA expression was observed after challenge with live Vibrio parahaemolyticus (240.6-fold, P < 0.01, crabs receive secondary challenge after V. parahaemolyticus vaccine to the carbs only receive twice PBS injection, N = 6), compared with those only received first injection with formalin-inactivated V. parahaemolyticus (39.7-fold, P < 0.01, challenge 6 h to vaccination 12 h). The findings of this study together demonstrated that EsVLRA plays an important role in the immune system of E. sinensis, serving as a pattern recognition receptor and involving in the immune priming.

Enhancers as regulators of antigen receptor loci three-dimensional chromatin structure.

Enhancers are defined as regulatory elements that control transcription in a cell-type and developmental stage-specific manner. They achieve this by physically interacting with their cognate gene promoters. Significantly, these interactions can occur through long genomic distances since enhancers may not be near their cognate promoters. The optimal coordination of enhancer-regulated transcription is essential for the function and identity of the cell. Although great efforts to fully understand the principles of this type of regulation are ongoing, other potential functions of the long-range chromatin interactions (LRCIs) involving enhancers are largely unexplored. We recently uncovered a new role for enhancer elements in determining the three-dimensional (3D) structure of the immunoglobulin kappa (Igκ) light chain receptor locus suggesting a structural function for these DNA elements. This enhancer-mediated locus configuration shapes the resulting Igκ repertoire. We also propose a role for enhancers as critical components of sub-topologically associating domain (subTAD) formation and nuclear spatial localization.

Construction of a circularly connected VHH bispecific antibody (cyclobody) for the desirable positioning of antigen-binding sites.

A bispecific antibody (bsAb) is an emerging class of next-generation biological therapeutics. BsAbs are engineered antibodies possessing dual antigen-binding paratopes in one molecule. The circular backbone topology has never been demonstrated, although an enormous number of bispecific constructs have been proposed. The circular topology is potentially beneficial for fixing the orientation of two paratopes and protection from exopeptidase digestion. We construct herein a circularly connected bispecific VHH, termed cyclobody, using the split-intein circular ligation of peptides and proteins. The constructed cyclobodies are protected from proteolysis with a retained bispecificity. The anti-EGFR × anti-GFP cyclobody can specifically stain EGFR-positive cells with GFP. The anti-EGFR × anti-CD16 cyclobody shows cytotoxic activity against EGFR-positive cancer cells with comparative activity of a tandem VHH construct. Successful demonstration of a new topology for the bispecific antibody will expand the construction strategy for developing antibody-based drugs and reagents.

In Vitro Maturation of a Humanized Shark VNAR Domain to Improve Its Biophysical Properties.

VNAR domains are the binding regions of new antigen receptor proteins (IgNAR) which are unique to sharks, skates, and rays (Elasmobranchii). Individual VNAR domains can bind antigens independently and are the smallest reported adaptive immune recognition entities in the vertebrate kingdom. Sharing limited sequence homology with human immunoglobulin domains, their development and use as biotherapeutic agents require that they be humanized to minimize their potential immunogenicity. Efforts to humanize a human serum albumin (HSA)-specific VNAR, E06, resulted in protein molecules that initially had undesirable biophysical properties or reduced affinity for cognate antigen. Two lead humanized anti-HSA clones, v1.10 and v2.4, were subjected to a process of random mutagenesis using error-prone PCR. The mutated sequences for each humanized VNAR variant were screened for improvements in affinity for HSA and biophysical properties, achieved without a predicted increase in overall immunogenicity.

Coordinated regulation of scaffold opening and enzymatic activity during CARD11 signaling.

The activation of key signaling pathways downstream of antigen receptor engagement is critically required for normal lymphocyte activation during the adaptive immune response. CARD11 is a multidomain signaling scaffold protein required for antigen receptor signaling to NF-κB, c-Jun N-terminal kinase, and mTOR. Germline mutations in the CARD11 gene result in at least four types of primary immunodeficiency, and somatic CARD11 gain-of-function mutations drive constitutive NF-κB activity in diffuse large B cell lymphoma and other lymphoid cancers. In response to antigen receptor triggering, CARD11 transitions from a closed, inactive state to an open, active scaffold that recruits multiple signaling partners into a complex to relay downstream signaling. However, how this signal-induced CARD11 conversion occurs remains poorly understood. Here we investigate the role of Inducible Element 1 (IE1), a short regulatory element in the CARD11 Inhibitory Domain, in the CARD11 signaling cycle. We find that IE1 controls the signal-dependent Opening Step that makes CARD11 accessible to the binding of cofactors, including Bcl10, MALT1, and the HOIP catalytic subunit of the linear ubiquitin chain assembly complex. Surprisingly, we find that IE1 is also required at an independent step for the maximal activation of HOIP and MALT1 enzymatic activity after cofactor recruitment to CARD11. This role of IE1 reveals that there is an Enzymatic Activation Step in the CARD11 signaling cycle that is distinct from the Cofactor Association Step. Our results indicate that CARD11 has evolved to actively coordinate scaffold opening and the induction of enzymatic activity among recruited cofactors during antigen receptor signaling.

Membrane Organization and Physical Regulation of Lymphocyte Antigen Receptors: A Biophysicist's Perspective.

Receptors at the membrane of immune cells are the central players of innate and adaptative immunity, providing effective defence mechanisms against pathogens or cancer cells. Their function is intimately linked to their position at and within the membrane which provides accessibility, mobility as well as membrane proximal cytoskeleton anchoring, all of these elements playing important roles in the final function and links to cellular actions. Understanding how immune cells integrate the specific signals received at their membrane to take a decision remains an immense challenge and a very active field of fundamental and applied research. Recent progress in imaging and micromanipulation techniques have led to an unprecedented refinement in the description of molecular structures and supramolecular assemblies at the immune cell membrane, and provided a glimpse into their dynamics and regulation by force. Several key elements have been scrutinized such as the roles of relative sizes of molecules, lateral organisation, motion in the membrane of the receptors, but also physical cues such as forces, mediated by cellular substrates of different rigidities or applied by the cell itself, in conjunction with its partner cell. We review here these recent discoveries associated with a description of the biophysical methods used. While a conclusive picture integrating all of these components is still lacking, mainly due to the implication of diverse and different mechanisms and spatio-temporal scales involved, the amount of quantitative data available opens the way for physical modelling and numerical simulations and new avenues for experimental research.

Oriented attachment of VNAR proteins, via site-selective modification, on PLGA-PEG nanoparticles enhances nanoconjugate performance.

Herein we report the construction of a nanoparticle-based drug delivery system which targets a key regulator in tumour angiogenesis. We exploit a Variable New Antigen Receptor (VNAR) domain, conjugated using site-specific chemistry, to direct poly lactic acid-co-glycolic acid-polyethylene glycol (PLGA-PEG) nanoparticles to delta like canonical Notch ligand 4 (DLL4). The importance of site-specific chemistry is demonstrated.

Transcriptomic analysis of immunoglobulin novel antigen receptor (IgNAR) heavy chain constant domains of brownbanded bamboo shark (Chiloscyllium punctatum).

Cartilaginous fish are the evolutionarily oldest group of animals which possess antibodies, T cell receptors and major histocompatibility complex (MHC). The immunoglobulin novel antigen receptor (IgNAR) found in cartilaginous fish is a heavy chain homodimer which lacks light chain. The presence of non-canonical cysteine molecules and lack of CDR2 region make it more significant. To synthesize active binding domains based on variable region of IgNAR (VNAR), knowledge on the constant region dynamics play a significant role. The IgNAR exhibit species variations in its primary sequence features; hence, this study was conducted to determine the IgNAR heavy chain constant domain of the brownbanded bamboo shark (Chiloscyllium punctatum). Peripheral blood leukocytes (PBL) isolated from adult bamboo sharks were used to synthesize a cDNA library. A total of four billion residues of two million sequences (average length 218.41 bp) were obtained. Assembled sequences were aligned with published cartilaginous fish IgNAR constant region sequences. Transcriptome analysis revealed two distinct types of IgNAR in the brownbanded bamboo shark. Also, constant-1 domain sequences displayed 13 unique sequences which may reflect the least number of IgNAR gene clusters. The phylogenetic analysis revealed the closest relationship with the nurse shark (Ginglymostoma cirratum) followed by the wobbegong shark (Orectolobus maculatus) which belong to the same order Orectolobiformes. Analysis of the constant domains of the brownbanded bamboo shark IgNAR revealed an evolutionarily conserved nature and this knowledge can be used to design primers for VNAR cloning. Furthermore, knowledge on the structural features in IgNAR constant domains that increase the stability could be useful in the process of stabilizing human immunoglobulins.

Next-generation flexible formats of VNAR domains expand the drug platform's utility and developability.

Therapeutic mAbs have delivered several blockbuster drugs in oncology and autoimmune inflammatory disease. Revenue for mAbs continues to rise, even in the face of competition from a growing portfolio of biosimilars. Despite this success, there are still limitations associated with the use of mAbs as therapeutic molecules. With a molecular mass of 150 kDa, a two-chain structure and complex glycosylation these challenges include a high cost of goods, limited delivery options, and poor solid tumour penetration. There remains an urgency to create alternatives to antibody scaffolds in a bid to circumvent these limitations, while maintaining or improving the therapeutic success of conventional mAb formats. Smaller, less complex binders, with increased domain valency, multi-specific/paratopic targeting, tuneable serum half-life and low inherent immunogenicity are a few of the characteristics being explored by the next generation of biologic molecules. One novel 'antibody-like' binder that has naturally evolved over 450 million years is the variable new antigen receptor (VNAR) identified as a key component of the adaptive immune system of sharks. At only 11 kDa, these single-domain structures are the smallest IgG-like proteins in the animal kingdom and provide an excellent platform for molecular engineering and biologics drug discovery. VNAR attributes include high affinity for target, ease of expression, stability, solubility, multi-specificity, and increased potential for solid tissue penetration. This review article documents the recent drug developmental milestones achieved for therapeutic VNARs and highlights the first reported evidence of the efficacy of these domains in clinically relevant models of disease.