Lampreys and hagfish belong to the Agnatha (jaw-less) branch of primitive vertebrates. Also called cyclostomes because of their circular oral opening, these eel-shaped fish bridge the gap between invertebrates and vertebrates. Their unique phylogenetic position makes these creatures excellent specimens for the study of the evolution of adaptive immunity [1]. Leukocytes in agnathan blood have been shown to be similar to mammalian lymphocytes and produce agglutinins [2] and opsonins [3]. It has also been known that soluble ‘antibody’ molecules are found in lamprey serum in response to antigenic stimulation [4].

The agglutinin and opsonin property of agnathan lymphocytes has been traced to the presence of variable lymphocyte receptors (VLRs) on the cell surface by Pancer et al [5]. The lymphocytes are specialised cells of the intestinal cavity of lamprey larvae (ammocoetes) and are concentrated in an invagination of the intestine called the typhlosole, one of the earliest hematopoietic organs [6]. In the adult, lymphocyte-like cells are mainly accumulated in the sub-epithelial layers of the digestive tract and in the branchial arches of the gills which are often referred to as the Agnathan ‘thymus” [7].

These small lymphocytes have a prominent nucleus with densely packed chromatin, reducing the cytoplasm to a mere 10% of the rim of the cell. Upon antigenic stimulation, these cells undergo clonal expansion and enlarge into lymphoblasts [8]. There are two types of lymphoblasts in the sea lamprey which resemble mammalian T and B lymphocytes [9, 10]. These can be distinguished based on the expression of the variable lymphocyte receptor (VLR) genes. VLRA and VLRC are expressed in the T-like lymphocytes [11]. The VLRB gene is expressed in the B-like lymphocytes which mount an adaptive humoral response with secreted, soluble multimeric VLRs to both carbohydrate and protein antigenic challenges [12].

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Figure 1. Diagrammatic representation of the domain organisation of VLRs (Lambodies).

Gene conversion generates the required diversity in response to both soluble and particulate antigens resulting in a recombination of LRR cassettes that can churn out up to 1014 different types of receptors [13]. The site of generation of VLRB+ lymphocytes is thought to be the typhlosole (spiral valve at the end of the intestine) although these lymphocytes can also be found in the kidney and the blood [12].

VLR antibodies are dimeric, with disulfide linkages holding two monomers. In circulation, they are found as oligomers of these dimeric units [12].

Transcriptome analysis of buffy coat leukocytes has revealed that VLRs are single polypeptide molecules that are organised into distinct domains [10] (see Fig 1). The distinguishing feature of mature VLRs is the leucine repeat region (LRR) which is similar to that found in the vertebrate von Willebrand factor receptor GpIb [14]. The N-terminal cap (LRRNT) precedes a variable number of LRRs (LRRVs). The first LRR after the N-terminal (LRR1) is followed by seven to eight LRRs that form the bend of the VLR molecule. The last LRR (LRRe) is linked to a connecting peptide (CP), a C-terminal cap (LRRCT). An invariant threonine-proline stalk connects the series of LRRs to a glycosyl-phosphatidyl inositol (GPI) anchor for membrane localisation of VLRA and VLRC. Multimerisation of secreted VLRs takes place via a cysteine rich hydrophobic region. If the anchor region is deleted, monomers of the VLRs are secreted [15].

The overall shape of the molecule is like a curved solenoid, with a concave surface made of rigid β-strands, involved in antigen binding. The leucine residues of the repeat sequences form a hydrophobic core, while conserved asparagine and phenylalanine residues generate a highly conserved backbone. The disulfide linkages found at the N and C termini are also conserved.

The Leucine Repeat Regions (LRRs) show conservation in length and sequence. The convex portion of the LRRs does not have pockets or grooves for antigen binding in their 310 helices or strands. However, short variable patches of residues are scattered on this surface of the molecule.

Sequence analysis has shown the presence of defined stretches of variable amino acid residues. In the concave surface, the clusters of such residues with a predominance of tyrosine form a significant hypervariable region [16].

When glycan antigens are injected into lamprey larvae, soluble VLRs can be detected in the blood collected from the animal after a period of 4 weeks. The lambody titre has been found to increase in a dose-dependent manner in response to booster injections of the antigen after 14 days [12].

cDNA expression libraries have established that VLRs are single polypeptide chains, with the cDNA corresponding to the entire length of the polypeptide [5, 13]. The expression of cDNA libraries can therefore be used to generate and screen for VLRs that bind to a specific antigen [14].

The affinity and specificity of binding of VLRs to sugars is similar to that of monoclonal antibodies [17]. The term “lambody” (lamprey antibody) was therefore introduced by Hong et al [18] to describe these high affinity binding soluble variable lymphocyte receptor molecules.

Lambodies differ from mammalian antibodies in their size and structure. The differences between regular antibodies and lambodies are listed in Table 1.

The generation of lambodies involves the following steps.

American brook lamprey (Lampetra appendix) and Northern brook lamprey (Ichthyomyzon fossor) have been sourced from Lamprey Services, Ludington, MI [5] or Hammond Bay Biological station. Pacific hagfish Eptatretus stoutii (30–60 cm long) have been purchased from Marinus (Long Beach, CA) [19].

The ammocoete larvae are sedated and the antigen (in PBS) is injected intraperitoneally. Weekly injections are given for up to four weeks. Particulate antigens elicit the best response. Soluble antigens should be attached to a carrier bead for a good response [14].

Buffy cost leukocytes are obtained from blood drained from the larva by making a cut in the tail [5]. Alternatively, the larvae are dissected from the ventral side and the whole intestine as well as the typhlosole (spiral valve) is removed. The tissue is macerated in a solution of diluted PBS (1 part water+ 2 parts PBS) between two glass slides [8].

Lymphocyte-like cells are isolated using their typical light scatter characteristics due to their smaller size and the rim of cytoplasm surrounding a large nucleus [20] or through gradient centrifugation with Percoll [21].

Total RNA is extracted from approximately 1x 106 cells, reverse transcribed and amplified using PCR. cDNAs larger than 500bp are pooled and cloned into a suitable expression vector such as the phagemid TriplEx2 [10] or plasmid pKINGeo/ccdB [21]. The recombinant clones are packaged into phage particles and transfected into a suitable E.coli host and plated. The plaques obtained are picked, PCR amplified and sequenced.

Insert sequences are subjected to BLASTX searches against the nonredundant protein database at the National Center for Biotechnology Information (NCBI). Immunologically relevant sequences are matched in public databases and grouped into clusters of identical or overlapping sequences [8].

cDNA fragments have been cloned into various expression vectors and display libraries have been screened for their specificity and affinity of binding to different antigens. The display platforms used till date are a baculovirus system [22], yeast surface display [18, 23], human cell line HEK-293T [24] and M13 phage display [25]. The cells can be treated with a glycosidase in order to improve the enrichment of antigen-binding clones using FACS and biotinylated antigens [18].

The lambodies secreted by the expression library can be tested for specificity, affinity and avidity using ELISAs [23].

Recombinant lambodies from cDNA expression libraries have been primarily used to study glycan antigen binding. Larvae injected with the human O-type erythrocytes generated lambodies that recognised a-L-Fucp-(1→2)-b-D-Galp-OR, (R is glycoprotein or glycolipid) in Chinese hamster ovary cells transfected with fucosyltransferase [22]. Other antigens that are bound by lambodies include blood group antigens on erythrocytes [26], hen egg lysozyme [9, 27] and the major coat protein from the spores of the bacterium Bacillus anthracis [28].The binding of these molecules to sulphated and sialylated glycans has also been demonstrated by using Tn4+ B cells and human milk as the injected antigen [23].

Secreted monoclonal lambodies are tetramers or pentamers of dimers, held together by disulfide bonds. This property of oligomerisation as well as improvement in specificity, avidity or affinity can be altered by directed mutagenesis [28]. This kind of molecular engineering can therefore, generate custom-made lambodies.

Given the complex nature of glycans, there are very few methods available for their detection and analysis. These methods also lack specificity since most anti-glycan antibodies are low affinity IgM antibodies that may also be cross-reactive with poorly defined epitopes [29]. In fact, the currently available antibodies recognize less than 4% of mammalian glycan determinants [18].

In addition to glycan epitopes, lambodies have also been shown to bind to protein antigens [9, 27, 28]. More recently, lambodies were found to bind to another molecule of the mammalian immune system, TLR5 [30].

Secreted VLRs are oligomeric, comprising four or five disulfide-linked dimeric subunits, somewhat akin to vertebrate IgM antibodies. They retain antigen-binding activity without aggregation or oligomerisation after elution from a BclA affinity column at pH > 11 with a yield of at least 2-10mg/L. They are extremely stable after storage for one year in the refrigerator, one month at room temperature or 36 h at 56 °C without any change in specificity or affinity [28].

The relative ease of generation and screening of lambodies, their stability and broad spectrum of antigen recognition makes them an attractive alternative to monoclonal antibodies. Some advantages of lambodies over monoclonal antibodies are listed in Table 2.

In addition to the above, VLRs / lambodies make an attractive option for custom made diagnostics because they can be manipulated in vitro to improve affinity and avidity of binding. These improved recombinant lambodies have been given the name “lampribodies” [25].

It was anticipated that one major disadvantage with lambodies/lampribodies would be a severe adverse immune response in humans elicited by Agnathan proteins [31]. However, a vaccine stabilised by LRRs from lampreys has been demonstrated to elicit a high titre of antibodies against the target antigen(s) and low titres against the LRR stabiliser derived from lamprey VLRs [32].

Lambodies are primarily sugar (glycan) binding proteins. In this way, they resemble lectins and can therefore be used in the same way [33].

Detection of the expression of aberrant glycan chains as a diagnostic in the progression of tumour growth is an emerging field in the treatment of cancer [34, 35]. The first reported application of lambodies in the detection of a glycan epitope in cancer done for the Thomsen-Friedenreich pancarcinoma carbohydrate antigen [36]. based on the correlation between erythrocyte agglutination as a symptom of this condition [37]. Lambodies have also been demonstrated as suitable for the diagnosis of chronic lymphocytic leukemia (CLL) [24].

VLR / lambody binding to avian influenza virus hemagglutinin [38], CD38 plasma cells [39] and inactivated simian immunodeficiency virus (SIV) [23] have also been reported.

Parts of VLRs, such as the leucine repeat region have been used to block peptides. Such truncated lambodies (called ‘repebodies’) against IL-6 have been shown to inhibit the IL6/STAT3 pathway in non-small cell lung cancer [40]. Another repebody against human VEGF has similarly been used to suppress choroidal neovascularisation, making it an attractive therapeutic in the context of age-related macular degeneration [41]. Drug delivery using tagged lambodies has been studied in the brain extracellular matrix [42].

A novel vaccine against several four different cancer-related markers has been generated using the LRR backbone of lambodies. This single vaccine elicits a high antibody titre against all four target antigens [32].

For research purposes, these versatile molecules have been successfully used to visualise in vivo protein-protein interaction and protein stabilisation. A detailed list of applications is given by Waters and Shusta (2018) [31] and Hassan et al (2019) [25].

Recently, McKitrick et al have developed modified lamprey VLRBs which have the Fc region of IgG molecules could be used to identify a wide spectrum of glycan epitopes including the stem cell SSEA markers, cancer antigens like CA-19 and the GD2 ganglioside, and 3-O-sulfated galactose [23, 43, 44]. The availability of glycan microarrays turn these chimeric molecules into smart anti-glycan reagents (SAGRs) which could revolutionise the detection of glycan epitopes in research and diagnostics. TR McKitrick et al developed a method for generating glycan-specific chimeric VLR-IgG-Fc proteins [45].

Lampreys thrive as both marine and freshwater species but have been declared endangered in most European countries. However, they are an invasive species in the Laurentian lakes on the Canadian – American border where farming and aquaculture of these fishes is allowed in and around the Great Lakes. This is also beneficial to the indigenous population of fish since the lamprey is an ectoparasite which has earlier led to a significant decrease in the population of fish [46]. Hence, the use of lampreys to generate lambodies does not encounter ecological or ethical problems in this part of the world. At least one company – Apolllo LLC- has also advertised the commercial manufacture of lambodies on its website.

The hagfish is a marine creature, mostly benthic and found on the soft silt of the sea floor even at greater depths. Unlike lamprey, they enter the body of their prey (which could be alive or dead and decaying) and consume the soft tissue [47]. They are also an endangered species and maintained in sea water aquaria. With respect to lambody generation, one drawback with hagfish is the copious amount of slime produced by these creatures when provoked [48]. This may make dissection and lymphocyte sorting messy and difficult.

When monoclonal antibodies were first reported in 1975, they were hailed as the most accurate molecules to target specific epitopes. Almost three decades later, the elucidation of the leucine repeat region (LRR) containing variable lymphocyte receptors (VLRs) of the Agnathan adaptive immune system opened a whole new dimension in this field. With increasing number of reports where lambodies / lampribodies / repebodies and now VLRB-Ig chimeras as smart anti-glycan reagents (SAGRs) have been used to study protein-glycan interaction, these molecules are poised to dominate the future of diagnostics, academic research and possibly therapeutics.