VH Domain Research Articles

Disulfide linkage engineering for improving biophysical properties of human VH domains

To enhance their therapeutic potential, human antibody heavy chain variable domains (V(H)s) would benefit from increased thermostability. The highly conserved disulfide linkage that connects Cys23 and Cys104 residues in the core of V(H) domains is crucial to their stability and function. It has previously been shown that the introduction of a second disulfide linkage can increase the thermostability of camelid heavy-chain antibody variable domains (V(H)Hs). Using four model domains we demonstrate that this strategy is also applicable to human V(H) domains. The introduced disulfide linkage, formed between Cys54 and Cys78 residues, increased the thermostability of V(H)s by 14-18°C. In addition, using a novel hexa-histidine capture technology, circular dichroism, turbidity, size exclusion chromatography and multiangle light scattering measurements, we demonstrate reduced V(H) aggregation in domains with the Cys54-Cys78 disulfide linkage. However, we also found that the engineered disulfide linkage caused conformational changes, as indicated by reduced binding of the V(H)s to protein A. This indicates that it may be prudent to use the synthetic V(H) libraries harboring the engineered disulfide linkage before screening for affinity reagents. Such strategies may increase the number of thermostable binders.

The Non-Ig Parts of the VpreB and λ5 Proteins of the Surrogate Light Chain Play Opposite Roles in the Surface Representation of the Precursor B Cell Receptor

The VpreB and λ5 proteins, together with Igμ-H chains, form precursor BCRs (preBCRs). We established λ5(-/-)/VpreB1(-/-)/VpreB2(-/-) Abelson virus-transformed cell lines and reconstituted these cells with λ5 and VpreB in wild-type form or with a deleted non-Ig part. Whenever preBCRs had the non-Ig part of λ5 deleted, surface deposition was increased, whereas deletion of VpreB non-Ig part decreased it. The levels of phosphorylation of Syk, SLP65, or PLC-γ2, and of Ca(2+) mobilization from intracellular stores, stimulated by μH chain crosslinking Ab were dependent on the levels of surface-bound preBCRs. It appears that VpreB probes the fitness of newly generated VH domains of IgH chains for later pairing with IgL chains, and its non-Ig part fixes the preBCRs on the surface. By contrast, the non-Ig part of λ5 crosslinks preBCRs for downregulation and stimulation.

Understanding the molecular recognition between antibody fragments and protein A biomimetic ligand

Affinity chromatography with protein A from Staphylococcus aureus (SpA) is the most widespread and accepted methodology for antibody capture during the downstream process of antibody manufacturing. A triazine based ligand (ligand 22/8) was previously developed as an inexpensive and robust alternative to SpA chromatography (Li et al. [12] and Teng et al. [11]). Despite the experimental success, there is no structural information on the binding modes of ligand 22/8 to antibodies, namely to Immunoglobulin G (IgG) molecules and fragments. In this work, we addressed this issue by a molecular docking approach allied to molecular dynamics simulations. Theoretical results confirmed the preference of the synthetic ligand to bind IgG through the binding site found in the crystallographic structure of the natural complex between SpA and the Fc fragment of IgG. Our studies also suggested other unknown “hot-spots” for specific binding of the affinity ligand at the hinge between VH and CH1 domains of Fab fragment. The best docking poses were further analysed by molecular dynamics studies at three different protonation states (pH 3, 7 and 11). The main interactions between ligand 22/8 and the IgG fragments found at pH 7 were weaker at pH 3 and pH 11 and in these conditions the ligand start losing tight contact with the binding site, corroborating the experimental evidence for protein elution from the chromatographic adsorbents at these pH conditions.

A Second TCRδ Locus in Galliformes Uses Antibody-like V Domains: Insight into the Evolution of TCRδ and TCRμ Genes in Tetrapods

Analyses of the available avian genomes revealed the presence of a second TCRδ locus in the Galliformes. This second TCRδ locus is nonsyntenic to the conventional TCRα/δ and is unusual in that the V genes are more related to IgH V genes (VH) than to TCR V genes. The second TCRδ is not found in another avian lineage, the passerine zebra finch. Rather the finch's conventional TCRα/δ locus contains VH genes that are expressed with the conventional Cδ gene, similar to what has been found in amphibians. A comparison between Galliformes and Passeriformes genomic organization suggests an origin of the second TCRδ in the former lineage involving gene duplication. Expression of these atypical TCRδ transcripts with a VH domain paired with Cδ was found in lymphoid tissues of both avian lineages. The configuration of the second TCRδ in chicken and turkey is reminiscent of the TCRδ duplication that is present in nonplacental mammals and provides insight into the origin of the uniquely mammalian TCRμ locus.

A humanised murine monoclonal antibody protects mice from Venezuelan equine encephalitis virus, Everglades virus and Mucambo virus when administered up to 48 h after airborne challenge

Currently there are no licensed antiviral treatments for the Alphaviruses Venezuelan equine encephalitis virus (VEEV), Everglades virus and Mucambo virus. We previously developed a humanised version of the mouse monoclonal antibody 1A3B-7 (Hu1A3B-7) which exhibited a wide range of reactivity in vitro and was able to protect mice from infection with VEEV. Continued work with the humanised antibody has now demonstrated that it has the potential to be a new human therapeutic. Hu1A3B-7 successfully protected mice from infection with multiple Alphaviruses. The effectiveness of the humanisation process was determined by assessing proliferation responses in human T-cells to peptides derived from the murine and humanised versions of the VH and VL domains. This analysis showed that the number of human T-cell epitopes within the humanised antibody had been substantially reduced, indicating that Hu1A3B-7 may have reduced immunogenicity in vivo.

Over 30% of patients with splenic marginal zone lymphoma express the same immunoglobulin heavy variable gene: ontogenetic implications

We performed an immunogenetic analysis of 345 IGHV-IGHD-IGHJ rearrangements from 337 cases with primary splenic small B-cell lymphomas of marginal-zone origin. Three immunoglobulin (IG) heavy variable (IGHV) genes accounted for 45.8% of the cases (IGHV1-2, 24.9%; IGHV4-34, 12.8%; IGHV3-23, 8.1%). Particularly for the IGHV1-2 gene, strong biases were evident regarding utilization of different alleles, with 79/86 rearrangements (92%) using allele (*)04. Among cases more stringently classified as splenic marginal-zone lymphoma (SMZL) thanks to the availability of splenic histopathological specimens, the frequency of IGHV1-2(*)04 peaked at 31%. The IGHV1-2(*)04 rearrangements carried significantly longer complementarity-determining region-3 (CDR3) than all other cases and showed biased IGHD gene usage, leading to CDR3s with common motifs. The great majority of analyzed rearrangements (299/345, 86.7%) carried IGHV genes with some impact of somatic hypermutation, from minimal to pronounced. Noticeably, 75/79 (95%) IGHV1-2(*)04 rearrangements were mutated; however, they mostly (56/75 cases; 74.6%) carried few mutations (97-99.9% germline identity) of conservative nature and restricted distribution. These distinctive features of the IG receptors indicate selection by (super)antigenic element(s) in the pathogenesis of SMZL. Furthermore, they raise the possibility that certain SMZL subtypes could derive from progenitor populations adapted to particular antigenic challenges through selection of VH domain specificities, in particular the IGHV1-2(*)04 allele.

Role of Structure-Based Changes due to Somatic Mutation in Highly Homologous DNA-Binding and DNA-Hydrolyzing Autoantibodies Exemplified by A23P Substitution in the VH Domain

Anti-DNA autoantibodies are responsible for tissue injury in lupus. A subset of DNA-specific antibodies capable of DNA cleavage can be even more harmful after entering the living cells by destroying nuclear DNA. Origins of anti-DNA autoantibodies are not fully understood, and the mechanism of induction of DNA-cleaving activity remains speculative. The autoantibody BV04-01 derived from lupus-prone mouse is the only DNA-hydrolyzing immunoglobulin with known 3D structure. Identification and analysis of antibodies homologous to BV04-01 may help to understand molecular bases and origins of DNA-cleaving activity of autoantibodies. BLAST search identified murine anti-DNA autoantibody MRL-4 with sequences of variable region genes highly homologous to those of autoantibody BV04-01. Despite significant homology to BV04-01, not only MRL-4 had no DNA-cleaving activity, but also reversion of its unusual P23 mutation to the germline alanine resulted in a dramatic loss of affinity to DNA. Contrary to this effect, transfer of the P23 mutation to the BV04-01 has resulted in a significant drop in DNA binding and almost complete loss of catalytic activity. In the present paper we analyzed the properties of two homologous autoantibodies and mutants thereof and discussed the implications of unusual somatic mutations for the development of autoantibodies with DNA-binding and DNA-hydrolyzing activity.

Human ScFv that block sodium ion channel activity of tetrodotoxin

Tetrodotoxin (TTX) is a heterocyclic guanidinium alkaloid (C11H17N3O8) with molecular mass of ∼320 Da. The TTX and toxic analogs block sodium ion activity of mammalian nerve cells resulting in failure to conduct nerve impulse which manifested clinically in host as variable degrees of organ paralysis. Human intoxication occurs after consuming food containing the toxins. Current treatment of the poisoning is supportive and symptomatic. There has been no specific drug or antidote for the TTX mediated malady. In this study, phage clones displaying human single chain antibody fragments (HuScFv) were selected from a human ScFv phage display library. HuScFv derived from phagemid transformed Escherichia coli clones (clones s16 and s35) bound to the TTX as tested by indirect ELISA and band shift assay. Homology modeling and molecular docking revealed that VL domain of the s16-HuScFv interacted with the hydroxyl groups of C6, C9, C10 and C11 of the TTX by means of Tyr 223, Ser226 and Tyr228, while the Asp53 and Asp55 of the VH domain of s35-HuScFv interacted with the positions 1 and 2 of the guanidinium group and the hydroxyl groups at C9 and C10 of the TTX. The s16- and s35-HuScFv neutralized the TTX bioactivity in nerve cell based- and mouse bio-assays. Moreover, the HuScFv could rescue the intoxicated mice from the TTX mediated lethality. Thus, the HuScFv derived from the transformed E. coli clones have high potential as a safe, effective and specific therapeutic remedy for TTX intoxication in humans and warrant further trials.

Structure of Fab fragment of malaria transmission blocking antibody 2A8 against P. vivax P25 protein

Understanding the structural basis of recognition between antigen and antibody requires the structural comparison of free and complexed components. Previously, we have reported the crystal structure of the complex between Fab fragment of murine monoclonal antibody 2A8 (Fab2A8) and Plasmodium vivax P25 protein (Pvs25) at 3.2Å resolution. We report here the crystallization and X-ray structure of native Fab2A8 at 4.0Å resolution. The 2A8 antibody generated against Pvs25 prevents the formation of P. vivax oocysts in the mosquito, when assayed in membrane feeding experiment.Comparison of native Fab2A8 structure with antigen bound Fab2A8 structure indicates the significant conformational changes in CDR-H1 and CDR-H3 regions of VH domain and CDR-L3 region of VL domain of Fab2A8. Upon complex formation, the relative orientation between VL and VH domains of Fab2A8 is conserved, while significant differences are observed in elbow angles of heavy and light chains. The combing site residues of complexed Fab2A8 exhibited the reduced temperature factor compared to native Fab2A8, suggesting a loss of conformational entropy upon antigen binding.

A case of convergence: why did a simple alternative to canonical antibodies arise in sharks and camels?

The success of our adaptive humoral immune response, mediated by B cells, relies on its capacity to produce multiple antigen-recognition specificities against any possible invader. This antigen-recognition specificity is attributed to antibodies, which display remarkable flexibility and diversity. That diversity is generated by cutting and pasting of immunoglobulin (Ig) gene segments during B-cell development to generate functional variable (V) genes, which are selected from two separate regions of the genome to yield so-called “heavy” (H) and “light” (L) chains (Figure 1; see Section 1 in Text S1). Ig H and L chain V genes have been expanded in various vertebrate taxa to yield multiple families and subfamilies [1]. Given the obvious advantage of the diversity afforded by such multichain antibodies, it was extremely surprising to discover that some species—notably cartilaginous fish (holocephalins, e.g., chimeras, and elasmobranchs, e.g., sharks, skates, and rays) and camelids (e.g., camels and llamas)—developed functional H-chain-only antibodies (HCAbs) (Figures 1 and ​and2)2) [2],[3]. Loss of the combinatorial H-L diversification of heterotetrameric (H2L2) antibodies should result in a handicapped, less diversified antigen-binding repertoire in HCAbs; yet, contrary to expectations, high-affinity, antigen-reactive HCAbs can be elicited to all studied antigens [4],[5]. Even more peculiar, the HCAbs function alongside conventional H2L2 antibodies. What explains this remarkable evolutionary convergence of functional HCAbs in sharks and camels? What forces drove such distantly related species to produce antibodies with this simple structure? And, if they are so effective why is their prevalence not more widespread in evolution? Figure 1 Schematic of classical H2L2 and H2 homodimeric HCAbs. Figure 2 Simplified vertebrate phylogenetic tree emphasizing those taxa possessing single-domain Igs or TCRs. Occurrence of Functional Heavy Chain-Only Antibodies in Camelids and Shark The antigen-binding fragment of camelid HCAbs consists of a single domain known as a variable domain of the heavy chain of HCAbs (VHH) (Figure 1) [2]. It was demonstrated that HCAbs from dromedaries (one-humped camels) infected with trypanosomes are capable of associating tightly and specifically with these parasite antigens [2]. Subsequently, HCAbs were elicited to many different foreign antigens. HCAbs are present in all living species of the mammalian family Camelidae, but not in other ungulates (hoofed mammals). Approximately equal levels of HCAbs and H2L2 antibodies are present in camel blood [6]. The origin of camelid Ig genes encoding HCAbs is unambiguous. Phylogenetic analyses have demonstrated that the HCAb-dedicated γ genes were derived from γ genes coding for conventional antibodies. The genes emerged and diverged ∼25 million years ago [6],[7], after Tylopoda split from other mammals (60–80 million years ago) and before the camel and llama speciation (∼11 million years ago) (Figure 2) [6]. Also, the VHH germline genes descended from the classical variable region of a heavy-chain (VH) genes, and the diversity (D) and JH genes are even shared in the formation of the VH or VHH domains. Thus, the emergence of HCAbs was a relatively recent event in these species. In the nurse shark (Ginglymostoma cirratum), a novel secreted antigen receptor was reported and named the new antigen receptor (NAR) [3]. It too is composed of two covalently associated H-chains with no associated L-chains, but having six domains (Figure 1). Since the molecule shares several functional features with Ig isotypes, it was renamed IgNAR [8], and its N-terminal V domain is known as V-NAR. Later studies showed IgNAR to be induced by immunization with protein antigens with similar kinetics as in a typical IgM response [9]. Although the origin of shark IgNAR is poorly understood, IgNAR is found in all elasmobranchs and thus emerged at least 220 million years ago (Figure 2). However, the single-domain V is also found as a T-cell receptor (TCR) in all living cartilaginous fish (so-called NAR-TCR) [10],[11], showing that this immune system feature appeared at least 350 million years ago (Figure 2). In addition, V-NAR gene clusters actually are present in the TCR-δ loci of all cartilaginous fish and thus it remains uncertain whether they originated as a TCR, an Ig, or perhaps a common antigen receptor ancestor [10].

Limiting CDR-H3 Diversity Abrogates the Antibody Response to the Bacterial Polysaccharide α 1→3 Dextran

Anti-polysaccharide Ab responses in mice are often oligoclonal, and the mechanisms involved in Ag-specific clone production and selection remain poorly understood. We evaluated the relative contribution of D(H) germline content versus N nucleotide addition in a classic oligoclonal, T-independent Ab response (α 1→3 dextran [DEX]) by challenging adult TdT-sufficient (TdT(+/+)) and TdT-deficient (TdT(-/-)) gene-targeted mice, limited to the use of a single D(H) gene segment (D-limited mice), with Enterobacter cloacae. D-limited mice achieved anti-DEX-specific levels of Abs that were broadly comparable to those of wild-type (WT) BALB/c mice. Sequence analysis of the third CDR of the H chain intervals obtained by PCR amplification of V(H) domain DNA from DEX-specific plasmablasts revealed the near universal presence of an aspartic acid residue (D99) at the V-D junction, irrespective of the composition of the D(H) locus. Although WT mice were able to use germline D(H) (DQ52, DSP, or DST) gene segment sequence, TdT activity, or both to produce D99, all three D-limited mouse strains relied exclusively on N addition. Additionally, in the absence of TdT, D-limited mice failed to produce a DEX response. Coupled with previous studies demonstrating a reduced response to DEX in TdT(-/-) mice with a WT D(H) locus, we concluded that in the case of the anti-DEX repertoire, which uses a short third CDR of the H chain, the anti-DEX response relies more intensely on sequences created by postnatal N nucleotide addition than on the germline sequence of the D(H).

Single domain antibodies with VH hallmarks are positively selected during panning of llama ( Lama glama) naïve libraries

Independent variable domains with VH hallmarks have been repeatedly identified in immune and pre-immune VHH libraries. In some cases, stable independent VH domains have been also isolated in mouse and human recombinant antibody repertoires. However, we have come to realize that VHs were selected with a higher efficiency than VHHs during biopanning of a pre-immune (VHH) library. The biochemical and biophysical comparison did not indicate a presence of any feature that would favor the VH binders during the selection process. In contrast, selected VHHs seemed to be more stable than the VHs, ruling out the existence of a thermodynamically – favored VH sub-class. Therefore, we reasoned that a certain degree of thermodynamic instability may be beneficial for both displaying and expression of VH(H)s when the Sec-pathway is used for their secretion to avoid the cytoplasmic trapping of fast-folding polypeptides. Indeed, VHHs, but not VHs, were accumulated at higher concentrations when expressed fused to the dsbA leader peptide, a sequence that drives the linked polypeptides to the co-translational SRP secretion machinery. These data suggest that the thermodynamically favored VHHs can be lost during biopanning, as previously observed for DARPins and in contrast to the recombinant antibodies in scFv format.

Probing the stability-limiting regions of an antibody single-chain variable fragment: a molecular dynamics simulation study

Antibody single-chain variable fragments (scFvs) offer particular advantages over the full-size antibodies, including easy expression, efficient local concentration and fast body clearance. However, scFvs typically show low thermal stability that limits their biomedical and biotechnological applications. In this study, we examined the thermal stability of the human and murine vascular endothelial growth factor antibody scFv fragment by molecular dynamics simulations. A consistent observation was the dissociation of the light-chain (VL) and heavy-chain (VH) domains and loss of the native structures of both domains in the simulations at the elevated temperatures. The stability-limiting structural elements in the protein were revealed from the detailed analyses on the native contacts. We found that dissociation of the VL-VH domains was the first event leading to the unfolding of the native structure of the protein and the disruption of the VL-VH interface was largely due to the break of the interfacial hydrophobic and aromatic interactions while the hydrogen-bonding interaction between Gln38 in VL and Gln39 in VH remained. Within the β-barrel structure of the VL and VH domains, β-strands β6, β2 and β11 appeared to be the least stable. In addition, we found that the VH domain was more thermally resistant than the VL domain. Based on these findings, we discussed potential strategies to improve the stability of this therapeutically important scFv fragment.

Synthetic single-framework antibody library integrated with rapid affinity maturation by VL shuffling

Affinity maturation is often applied to improve the properties of antibodies isolated from universal antibody libraries in vitro. A synthetic human scFv antibody library was constructed in single immunoglobulin framework to enable rapid affinity maturation by updated Kunkel's mutagenesis. The initial diversity was generated predominantly in the V(H) domain combined with only 36 V(L) domain variants yielding 3 × 10(10) unique members in the phage-displayed library. After three rounds of panning the enriched V(H) genes from the primary library selections against lysozyme were incorporated into a ready-made circular single-stranded affinity maturation library containing 7 × 10(8) V(L) gene variants. Several unique antibodies with 0.8-10 nM (K(d), dissociation constant) affinities against lysozyme were found after panning from the affinity maturation library, contrasted by only one anti-lysozyme scFv clone with K(d) <20 nM among the clones panned from the primary universal library. The presented single-framework strategy provides a way to convey significant amount of functional V(H) domain diversity to affinity maturation without bimolecular ligation leading to a diverse set of antibodies with binding affinities in the low nanomolar range.

Accelerated Neuronal Cell Recovery from Botulinum Neurotoxin Intoxication by Targeted Ubiquitination

Botulinum neurotoxin (BoNT), a Category A biodefense agent, delivers a protease to motor neuron cytosol that cleaves one or more soluble NSF attachment protein receptors (SNARE) proteins involved in neurotransmission to cause a flaccid paralysis. No antidotes exist to reverse symptoms of BoNT intoxication so severely affected patients require artificial respiration with prolonged intensive care. Time to recovery depends on toxin serotype because the intraneuronal persistence of the seven known BoNT serotypes varies widely from days to many months. Our therapeutic antidote strategy is to develop ‘targeted F-box’ (TFB) agents that target the different intraneuronal BoNT proteases for accelerated degradation by the ubiquitin proteasome system (UPS), thus promoting rapid recovery from all serotypes. These agents consist of a camelid heavy chain-only VH (VHH) domain specific for a BoNT protease fused to an F-box domain recognized by an intraneuronal E3-ligase. A fusion protein containing the 14 kDa anti-BoNT/A protease VHH, ALcB8, joined to a 15 kDa F-box domain region of TrCP (D5) was sufficient to cause increased ubiquitination and accelerate turnover of the targeted BoNT/A protease within neurons. Neuronal cells expressing this TFB, called D5-B8, were also substantially resistant to BoNT/A intoxication and recovered from intoxication at least 2.5 fold quicker than control neurons. Fusion of D5 to a VHH specific for BoNT/B protease (BLcB10) led to accelerated turnover of the targeted protease within neurons, thus demonstrating the modular nature of these therapeutic agents and suggesting that development of similar therapeutic agents specific to all botulinum serotypes should be readily achievable.

Effective targeting of tumor vasculature with an antibody against tumor endothelial marker 7-R (TEM7R).

e13040 Background: Tumor associated blood vessels offer numerous tumor specific targets for therapy. St. Croix and colleagues reported the existence of novel genes expressed in human and murine tumor endothelium1,2. We report on the isolation, production and characterization of a single chain (scFv) and its corresponding IgG1 monoclonal antibody specific for murine TEM7R (mTEM7R) and present preliminary data that this antibody cross reacts with human TEM7R (hTEM7R). Methods: The cDNA encoding the extracellular domain (ECD) of mTEM7R was amplified by RT-PCR, cloned into the pSecTag2/Hygro A vector (Invitrogen) and stably transfected into HEK293 cells. A naïve human scFv phage display library comprised of 1010 independent clones3 was used to isolate scFv molecules reactive with mTEM7R. Using a solid phase panning strategy,4 we identified 5 families of anti-TEM7R scFv based on fingerprint analysis. Of the 5 families of scFv, 2 specifically bound to the mTEM7R ECD as confirmed by surface plasmon resonance (SPR) and by ELISA. One clone exhibited moderate affinity for purified ECD (10-8M) as measured by SPR and was chosen for further studies. This scFv was converted into a human IgG1 format by subcloning its Vh and Vl domains into the pMAZ-VH and pMAZ-Vl vectors (gift of Dr. Benhar, Tel-Aviv University), respectively. The resulting plasmids were co-transfected into HEK-T cells and the resulting IgG (BB1) was purified by Protein A affinity chromatography. Results: As measured by biodistribution and immuno-PET imaging studies, BB1 antibody demonstrated significant tumor uptake and prolonged tumor retention in SCID mice harboring s.c. tumor xenografts derived from five tumor cell lines of varying origin. Consistent with mTEM7R’s predicted vascular distribution, IHC and IF performed on fresh-frozen A431-derived tumor xenografts showed co-localization of BB1 and anti-CD31 antibodies to the tumor vasculature. In addition, BB1 demonstrated in vivo therapeutic efficacy against F9 (murine teratocarcinoma) and A431 tumor xenografts. Conclusions: The combined tumor targeting and therapeutic efficacy of BB1 antibody suggests TEM7R is a promising tumor vascular target and should be further investigated.

A Tat-grafted anti-nucleic acid antibody acquires nuclear-localization property and a preference for TAR RNA

The 3D8 single chain variable fragment (3D8 scFv) is an anti-nucleic acid antibody that can hydrolyze nucleic acids and enter the cytosol of cells without reaching the nucleus. The Tat peptide, derived from the basic region of the HIV-1 Tat protein, translocates to cell nuclei and has TAR RNA binding activity. In this study, we generated a Tat-grafted antibody ( H3Tat-3D8) by replacing complementarity-determining region 3 (CDR3) within the VH domain of the 3D8 scFv with a Tat 48–60 peptide (GRKKRRQRRRPPQ). H3Tat-3D8 retained the DNA-binding and DNA-hydrolyzing activity of the scFv, and translocated to the nuclei of HeLa cells and preferentially recognized TAR RNA. Thus, the properties associated with the Tat peptide were transferred to the antibody via Tat-grafting without loss of the intrinsic DNA-binding and hydrolyzing activities of the 3D8 scFv antibody.

Over 30% of Patients with Splenic Marginal Zone Lymphoma Express Distinctive Antigen Receptors Utilizing a Single Immunoglogulin Variable Gene: Implications for the Origin and Selection of the Neoplastic Cells

AbstractAbstract 634We systematically explored the immunoglobulin (IG) gene repertoire in 337 cases with splenic marginal-zone lymphoma (SMZL), by far the largest series yet. To resolve classification uncertainties, we included in the analysis only cases with a diagnosis of SMZL based on spleen histopathological findings or cases fulfilling the 2008 SBLG criteria (Matutes et al. Leukemia 2008). We here report that the IG heavy variable (IGHV) gene repertoire in SMZL is remarkably biased, with only three genes accounting for 45.8% of cases (IGHV1-2, 24.9%; IGHV4-34, 12.8%; IGHV3-23: 8.1%, respectively), significantly extending previous similar observations. Particularly for the IGHV1-2 gene, strong biases became evident at the level of utilization of different alleles, since 79/86 rearrangements (92%) utilized allele *04 vs. only 7/86 rearrangements (8%) that utilized allele *02. This is noteworthy, taking into consideration that these two alleles differ in a single nucleotide, leading to a single amino acid change in framework region (FR)-3. The repertoire biases became more pronounced when the analysis was focused on 171 rearrangements from 163 cases classified as SMZL based on splenic histopathology, according to the 2008 WHO criteria. Within this subgroup, 56/171 cases (32.7%) utilized IGHV1-2*04. Noticeably, only 1/17 cases with a diagnosis of splenic diffuse red pulp lymphoma utilized IGHV1-2*04 (p<0.02 for comparison to SMZL). The IGHV1-2*04 rearrangements carried significantly longer heavy complementarity-determining region-3 (VH CDR3) than all other cases (median, 22 vs. 17 amino acids, respectively; p<0.001). In addition, 52/79 IGHV1-2*04 cases (65.8%) employed one of the IGHD3-3, IGHD3-9 or IGHD3-10 genes. In 28/32 IGHV1-2*04/IGHD3 rearrangements, the IGHD gene was utilized in the same reading frame, leading to VH CDR3s with common “IGHD-derived” amino acid (AA) motifs. Using bioinformatics tools previously applied to CLL, biased associations of IGHV, IGHD and IGHJ genes with stereotyped VH CDR3s were identified in 25/345 sequences (7.2%). Noticeably, only 10/28 IGHV1-2*04/IGHD3-3 rearrangements with “IGHD-derived” VH CDR3 amino acid motifs could be assigned to “stereotyped” clusters. Despite exhibiting restricted usage of the IGHV1-2*04 and IGHD3-3 genes leading to great overall VH domain similarity, the remaining cases did not fulfill the established criteria for VH CDR3 “stereotypy”, as defined in other lymphoid malignancies, namely CLL. Based on somatic hypermutation (SHM) analysis, the sequences were divided into three groups: (i) truly unmutated (100% germline identity, GI): 46/345 sequences (13.3%); (ii) minimally/borderline mutated (97-99.9% GI): 130/345 sequences (37.7%); and (iii) significantly mutated (<97% identity): 169/345 sequences (49%). At the individual gene level, the distribution of rearrangements of IGHV genes according to SHM status varied significantly. In particular, 56/79 IGHV1-2*04 rearrangements (71%) were predominantly “borderline mutated”, whereas the majority (>67%) of rearrangements utilizing the IGHV3-23, IGHV3-30 and IGHV3-7 genes were “significantly mutated”; finally, IGHV4-34 gene rearrangements were evenly distributed to the three mutational subgroups. Shared (“stereotyped”) AA changes were identified for IGHV1-2*04 rearrangements, with certain FR2 and FR3 codons emerging as “hotspots” for recurrent, conservative AA changes. In conclusion, we demonstrate that more than 30% of cases with a histopathological diagnosis of SMZL on the spleen express IGHV1-2*04 receptors with unusually long VH CDR3s, biased usage of the IGHD3-3 gene, leading to shared “IGHD-derived” VH CDR3 motifs, and very precise molecular features of SHM. The biased expression of a distinctive germline-encoded VH specificity might be considered as evidence for heavy chain dominance in the clonogenic IG receptors in SMZL. These findings allude to selection by specific (super)antigenic element(s) in the pathogenesis of at least a major subset of SMZL. In addition, they raise the intriguing possibility that certain subtypes of SMZL could derive from progenitor cell populations adapted to particular antigenic challenges through cellular selection of VH domain specificities. Disclosures:No relevant conflicts of interest to declare.

Ranking the Susceptibility of Disulfide Bonds in Human IgG1 Antibodies by Reduction, Differential Alkylation, and LC−MS Analysis

One of the basic structural features of human IgG1 is the arrangement of the disulfide bond structure, 4 inter chain disulfide bonds in the hinge region and 12 intra chain disulfide bonds associated with twelve individual domains. Disulfide bond structure is critical for the structure, stability, and biological functions of IgG molecules. It has been known that inter chain disulfide bonds are more susceptible to reduction than intra chain disulfide bonds. However, a complete ranking of the susceptibility of disulfide bonds in IgG1 molecules is lacking. A method including reduction, differential alkylation, and liquid chromatography-mass spectrometry (LC-MS) analysis was developed and employed to investigate the complete ranking order of the susceptibility of disulfide bonds in two recombinant monoclonal antibodies. The results confirmed that inter chain disulfide bonds were more susceptible than intra chain disulfide bonds. In addition, it was observed that the disulfide bonds between the light chain and heavy chain were more susceptible than disulfide bonds between the two heavy chains. The upper disulfide bond of the two inter heavy chain disulfide bonds was more susceptible than the lower one. Furthermore, disulfide bonds in the CH2 domain were the most susceptible to reduction. Disulfide bonds in VL, CL, VH, and CH1 domains had similar and moderate susceptibility, while disulfide bonds in the CH3 domain were the least susceptible to reduction. Interestingly, a difference between IgG1kappa and IgG1lambda was also observed. The difference in the susceptibility of inter light heavy chain disulfide bonds and inter heavy chain disulfide bonds was smaller in IgG1kappa than in IgG1lambda. The intra chain disulfide bonds in the Fab region of IgG1kappa were also less susceptible than disulfide bonds in the Fab region of IgG1lambda.

Isolation of a Novel Nanobody Against HER-2/neuUsing Phage Displays Technology

In 1993, Hamers-Casterman and colleagues discovered a novel type of IgG antibodies.1 Camelid serum contains functional heavy-chain homodimeric antibodies without light chains. The variable domain of these single chain antibodies are named as VHHs (nanobodies). The binding ability of VHH to antigens was found to be similar or even better than classical IgG.2 Single-domain antibody was also identified in special cartilaginous fish by Greenberg and colleagues.3 It was also found that they are suitable for expression as multivalent formats like bispecific, enzyme, or toxin-VHH fusions to increase avidity without using current linkers like those that existed between VL and VH domains, which often result in aggregation and decrease affinity because of mispairing of VL and VH domains.4 Lacking of the mispairing problems allows designing more flexible linkers and successfully achieving functional trivalent-bispecific VHHs.5,6 Sequencing and crystallographic analysis of camel VHH has showed homology to the human VHH. The above facts prepare some advantages for biotechnological applications of VHH, such as facile genetic manipulation, increased functional size of libraries, high physicochemical stability, ease of production of multivalent forms, high stability, recognition of hidden antigenic sites, well expression, rapid tissue penetration, and fast blood clearance.7 Soluble VHHs have been produced in Escherichia coli (E. coli), filamentous fungi, Saccharomyces cerevisiae, and Pichia pastoris. 8–11 VHH single-domains can be used whenever high stability is required, such as use in shampoo for preventing dandruff.12 They were used in immunoaffinity purification as capturing reagents and in biosensor technology.13,14 VHH is also reported to be a good candidate for oral immunotherapy because of its resistance against high pH, and its ability to bind to the target in the presence of high concentrations of many other agents.15 This type of functional VHH antibody …