Structural analysis,selection, and ontogeny of the shark new antigen receptor (IgNAR): identification of a new locus preferentially expressed in early development

The new antigen receptor (IgNAR) family has been detected in all elasmobranch species so far studied and has several intriguing structural and functional features. IgNAR protein, found in both transmembrane and secretory forms, is a dimer of heavy chains with no associated light chains, with each chain of the dimer having a single free and flexible V region. Four rearrangement events (among 1V, 3D, and 1J germline genes) generate an expressed NAR V gene, resulting in long and diverse CDR3 regions that contain cysteine residues. IgNAR mutation frequency is very high and "selected" mutations are found only in genes encoding the secreted form, suggesting that the primary repertoire is entirely CDR3-based. Here we further analyzed the two IgNAR types, "type 1" having one cysteine in CDR3 and "type 2" with an even number (two or four) of CDR3 cysteines, and discovered that placement of the disulfide bridges in the IgNAR V domain differentially influences the selection of mutations in CDR1 and CDR2. Ontogenetic analyses showed that IgNAR sequences from young animals were infrequently mutated, consistent with the paradigm that the shark immune system must become mature before high levels of mutation accompanied with selection can occur. Nevertheless, also in agreement with the idea that the IgNAR repertoire is entirely CDR3-based, but unlike studies in most other vertebrates, N-region diversity is present in expressed IgNAR clones at birth. During the investigation of this early IgNAR repertoire we serendipitously detected a third type of IgNAR gene that is expressed in all neonatal tissues; later in life its expression is perpetuated only in the epigonal organ, a tissue recently shown to be a (the?) primary lymphoid tissue in elasmobranchs. This "type 3" IgNAR gene still undergoes three rearrangement events (two D regions are "germline-joined"), yet CDR3 sequences were exactly of the same length and very similar sequence, suggesting that "type 3" CDR3s are selected early in ontogeny, perhaps by a self-ligand.     

Multi‐constraint computational design suggests that native sequences of germline antibody H3 loops are nearly optimal for conformational flexibility

The limited size of the germline antibody repertoire has to recognize a far larger number of potential antigens. The ability of a single antibody to bind multiple ligands due to conformational flexibility in the antigen‐binding site can significantly enlarge the repertoire. Among the six complementarity determining regions (CDRs) that generally comprise the binding site, the CDR H3 loop is particularly variable. Computational protein design studies showed that predicted low energy sequences compatible with a given backbone structure often have considerable similarity to the corresponding native sequences of naturally occurring proteins, indicating that native protein sequences are close to optimal for their structures. Here, we take a step forward to determine whether conformational flexibility, believed to play a key functional role in germline antibodies, is also central in shaping their native sequence. In particular, we use a multi‐constraint computational design strategy, along with the Rosetta scoring function, to propose that the native sequences of CDR H3 loops from germline antibodies are nearly optimal for conformational flexibility. Moreover, we find that antibody maturation may lead to sequences with a higher degree of optimization for a single conformation, while disfavoring sequences that are intrinsically flexible. In addition, this computational strategy allows us to predict mutations in the CDR H3 loop to stabilize the antigen‐bound conformation, a computational mimic of affinity maturation, that may increase antigen binding affinity by preorganizing the antigen binding loop. In vivo affinity maturation data are consistent with our predictions. The method described here can be useful to design antibodies with higher selectivity and affinity by reducing conformational diversity. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Recurrent somatic mutations in mouse antibodies to p-azophenylarsonate increase affinity for hapten

Two mouse mAb specific for the hapten p-azophenylarsonate and encoded by the same combination of germ-line V, D, and J genes differ 200-fold in affinity for hapten. We determined the amino acid sequences of the V regions of the high affinity antibody and compared them to the published sequences of the low affinity antibody which is not somatically mutated. Of 19 amino acid substitutions, two, Ile57 and Thr58 in the H chain, also occur, either alone or together, in other somatically mutated antibodies specific for p-azophenylarsonate; these antibodies have been independently isolated. Introduction of either one of these mutations alone into the low affinity antibody by oligonucleotide-directed mutagenesis increased the antibody affinity for hapten three- to fourfold, whereas introduction of both mutations together conferred an eightfold increase in affinity. These results support the hypothesis that somatic mutations are selected on the basis of the affinity for antigen that they confer, and suggest that even relatively small increases in affinity may be selected, probably in a sequential manner.     

Mutation pattern of paired immunoglobulin heavy and light variable domains in chronic lymphocytic leukemia B cells

B-cell chronic lymphocytic leukemia (CLL) patients display leukemic clones bearing either germline or somatically mutated immunoglobulin heavy variable (IGHV ) genes. Most information on CLL immunoglobulins (Igs), such as the definition of stereotyped B-cell receptors (BCRs), was derived from germline unmutated Igs. In particular, detailed studies on the distribution and nature of mutations in paired heavy- and light-chain domains of CLL clones bearing mutated Igs are lacking. To address the somatic hyper-mutation dynamics of CLL Igs, we analyzed the mutation pattern of paired IGHV-diversity-joining (IGHV-D-J ) and immunoglobulin kappa/lambda variable-joining (IGK/LV-J ) rearrangements of 193 leukemic clones that displayed ≥ 2% mutations in at least one of the two immunoglobulin variable (IGV ) genes (IGHV and/or IGK/LV ). The relationship between the mutation frequency in IGHV and IGK/LV complementarity determining regions (CDRs) and framework regions (FRs) was evaluated by correlation analysis. Replacement (R) mutation frequency within IGK/LV chain CDRs correlated significantly with mutation frequency of paired IGHV CDRs in λ but not κ isotype CLL clones. CDRs of IGKV-J rearrangements displayed a lower percentage of R mutations than IGHVs. The frequency/pattern of mutations in kappa CLL Igs differed also from that in κ-expressing normal B cells described in the literature. Instead, the mutation frequency within the FRs of IGHV and either IGKV or IGLV was correlated. Notably, the amount of diversity introduced by replaced amino acids was comparable between IGHVs and IGKVs. The data indicate a different mutation pattern between κ and λ isotype CLL clones and suggest an antigenic selection that, in κ samples, operates against CDR variation.     

Construction of an Artificially Randomized IgNAR Phage Display Library: Screening of Variable Regions that Bind to Hen Egg White Lysozyme

To develop a multi-antigen-specific immunoglobulin new antigen receptor (IgNAR) variable (V) region phage display library, CDR3 in the V region of IgNAR from banded houndshark (Triakis scyllium) was artificially randomized, and clones specific for hen egg white lysozyme (HEL) were obtained by the biopanning method. The nucleotide sequence of CDR3 in the V region was randomly rearranged by PCR. Randomized CDR3-containing segments of the V region were ligated into T7 phage vector to construct a phage display library and resulted in a phage titer of 3.7?×?10⁷ PFU/ml. Forty clones that contained randomized CDR3 inserts were sequenced and shown to have different nucleotide sequences. The HEL-specific clones were screened by biopanning using HEL-coated ELISA plates. After six rounds of screening, nine clones were identified as HEL-specific, eight of which showed a strong affinity to HEL in ELISA compared to a negative control (i.e., empty phage clone). The deduced amino acid sequences of CDR3 from the HEL-specific phage clones fell into four types (I?IV): type I contains a single cysteine residue and type II?IV contain two cysteine residues. These results indicated that the artificially randomized IgNAR library is useful for the rapid isolation of antigen-specific IgNAR V region without immunization of target antigen and showed that it is possible to isolate an antigen-specific IgNAR V region from this library.     

Dissecting contact potentials for proteins: relative contributions of individual amino acids

Mimotopes mimic the three-dimensional topology of an antigen epitope, and are frequently recognized by antibodies with affinities comparable to those obtained for the original antibody-antigen interaction. Peptides and anti-idiotypic antibodies are two classes of protein mimotopes that mimic the topology (but not necessarily the sequence) of the parental antigen. In this study, we combine these two classes by selecting mimotopes based on single domain IgNAR antibodies, which display exceptionally long CDR3 loop regions (analogous to a constrained peptide library) presented in the context of an immunoglobulin framework with adjacent and supporting CDR1 loops. By screening an in vitro phage-display library of IgNAR variable domains (V(NAR)s) against the target antigen monoclonal antibody MAb5G8, we obtained four potential mimotopes. MAb5G8 targets a linear tripeptide epitope (AYP) in the flexible signal sequence of the Plasmodium falciparum Apical Membrane Antigen-1 (AMA1), and this or similar motifs were detected in the CDR loops of all four V(NAR)s. The V(NAR)s, 1-A-2, -7, -11, and -14, were demonstrated to bind specifically to this paratope by competition studies with an artificial peptide and all showed enhanced affinities (3-46 nM) compared to the parental antigen (175 nM). Crystallographic studies of recombinant proteins 1-A-7 and 1-A-11 showed that the SYP motifs on these V(NAR)s presented at the tip of the exposed CDR3 loops, ideally positioned within bulge-like structures to make contact with the MAb5G8 antibody. These loops, in particular in 1-A-11, were further stabilized by inter- and intra- loop disulphide bridges, hydrogen bonds, electrostatic interactions, and aromatic residue packing. We rationalize the higher affinity of the V(NAR)s compared to the parental antigen by suggesting that adjacent CDR1 and framework residues contribute to binding affinity, through interactions with other CDR regions on the antibody, though of course definitive support of this hypothesis will rely on co-crystallographic studies. Alternatively, the selection of mimotopes from a large (<4 x 10(8)) constrained library may have allowed selection of variants with even more favorable epitope topologies than present in the original antigenic structure, illustrating the power of in vivo selection of mimotopes from phage-displayed molecular libraries.     

Cloning, sequencing and analyzing of the heavy chain V region genes of human polyreactive antibodies

The heavy chain variable region genes of 5 human polyreactive mAbs generated in our laboratory have been cloned and sequenced using polymerase chain reaction(PCR) technique.We found that 2 and 3 mAbs utilized genes of the VHIV and VHⅢ families,respectively.The former 2 VH segments were in germline configuration.A common VH segment,with the best similarity of 90.1% to the published VHⅢ germline genes,was utilized by 2 different rearranged genes encoding the V regions of other 3 mAbs.This strongly suggests that the common VH segment is a unmutated copy of an unidentified germline VHⅢ gene.All these polyreactive mAbs displayed a large NDN region(VH-D-JH junction).The entire H chain V regions of these polyreactive mAbs are unusually basic.The analysis of the charge properties of these mAbs as well as those of other poly-and mono-reactive mAbs from literatures prompts us to propose that the charged amino acids with a particular distribution along the H chain V region,especially the binding sites(CDRs),may be an important structural feature involved in antibody polyreactivity.     

Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle

Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.     

Thermodynamic consequences of mutations in vernier zone residues of a humanized anti-human epidermal growth factor receptor murine antibody, 528

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.     

Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle

Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.     

Variable domain antibodies specific for viral hemorrhagic septicemia virus (VHSV) selected from a randomized IgNAR phage display library

Phage display libraries are used to screen for nucleotide sequences that encode immunoglobulin variable (V) regions that are specific for a target antigen. We previously constructed an immunoglobulin new antigen receptor (IgNAR) phage display library. Here we used this library to obtain an IgNAR V region that is specific for viral hemorrhagic septicemia virus (VHSV). A phage clone (clone 653) was found to be specific for VHSV by the biopanning method. The V region of clone 653 was used to construct a 6 × His tagged recombinant IgNAR-653 V protein (rIgNAR-653) using the Escherichia coli pET system. The rIgNAR-653 protein bound specifically to VHSV, confirming its activity.     

A functional antibody mutant with an insertion in the framework region 3 loop of the VH domain: implications for antibody engineering.

We have studied the effects of a four residue insertion into the FR3 loop of the heavy chain variable region from the anti-NP antibody B1-8. The insertion mutant is obtained as secreted antibody without major defects in biosynthesis, indicating that antibody variable domains can accommodate length variation not only in complementarity determining regions (CDRs), but also in framework region (FR) loops. The B1-8 antigen binding site is not affected by the change in a neighbouring loop. FR3 insertions represent a new method of antibody engineering with a potential to obtain strong antigen binding by designing additional antigen contacting residues.     

Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype

The new antigen receptor (IgNAR) antibodies from sharks are disulphide bonded dimers of two protein chains, each containing one variable and five constant domains. Three types of IgNAR variable domains have been discovered, with Type 3 appearing early in shark development and being overtaken by the antigen-driven affinity-matured Type 1 and 2 response. Here, we have determined the first structure of a naturally occurring Type 2 IgNAR variable domain, and identified the disulphide bond that links and stabilizes the CDR1 and CDR3 loops. This disulphide bridge locks the CDR3 loop in an "upright" conformation in contrast to other shark antibody structures, where a more lateral configuration is observed. Further, we sought to model the Type 3 isotype based on the crystallographic structure reported here. This modeling indicates (1) that internal Type 3-specific residues combine to pack into a compact immunoglobulin core that supports the CDR loop regions, and (2) that despite apparent low-sequence variability, there is sufficient plasticity in the CDR3 loop to form a conformationally diverse antigen-binding surface.     

NMR evidence for mechanical coupling of phosphate B(I)-B(II) transitions with deoxyribose conformational exchange in DNA

2a2 is the most commonly rearranged gene in the human V(lambda )locus. It has been postulated that certain immunoglobulin genes (including 2a2) are rearranged preferentially because their germline sequences encode structures capable of binding to a range of antigens. Somatic mutation could then increase the specificity and affinity of binding to a particular antigen.We studied the properties of five IgG molecules in which the same heavy chain was paired with different light chains derived from 2a2. The pattern of somatic mutations in 2a2 was shown to be crucial in conferring the ability to bind DNA, but two different patterns of mutation each conferred this ability.Computer-generated models of the three-dimensional structures of these antibodies illustrate the ability of 2a2 to form a DNA binding site in different ways. Somatic mutations at the periphery of the DNA binding site were particularly important. In two different light chains, mutations to arginine at different sites in the complementarity determining regions (CDRs) enhanced binding to DNA. In a third light chain, however, mutation to arginine at a different site blocked binding to DNA.     

Mutational analysis of avidity and fine specificity of anti-levan antibodies

Using the polyfructose, bacterial levan, as a model polysaccharide, we analyzed how V regions affect binding in anti-polysaccharide mAbs. Previously, panels of mAb were constructed from bacterial levan-immunized BALB/c and CBA/Ca mice. The BALB/c mAb were mostly germline VHJ606:Vkappa11, and a subset contained presumed somatic mutations in the complementarity-determining regions (CDRs) that correlated with increases in avidity for the beta(2-->1) inulin linkage of levan. The CBA/Ca mAb were more heterogeneous in V gene usage, but a subset of inulin-nonreactive mAb were VHJ606:Vlambda and had VH sequence differences in the CDRs from the VHJ606 regions of the BALB/c mAb. In this report, VHJ606 Abs containing various combinations of specifically mutated H and L chains were produced by engineered transfectants and tested for inulin avidity and levan binding. Two presumed somatic mutations seen in CDRs of the BALB/c hybridomas were shown to directly cause marked increases in avidity for inulin (VH N53H, 9-fold; VL N53I, 20-fold; together, 46-fold) but not for beta(2-->6) levan. Exchange of either positions 50 or 53 in VH or the H3 loop between the BALB/c and CBA/Ca mAb resulted in either fine specificity shift or total loss of bacterial levan binding. Three-dimensional models of the V regions suggested that residues that affect binding to inulin alone are near the edge of the CDR surface, while residues involved with binding both forms of levan and affecting fine specificity are in the VH:VL junctional area.     

Deep mutational scanning of an antibody against epidermal growth factor receptor using mammalian cell display and massively parallel pyrosequencing

We developed a method for deep mutational scanning of antibody complementarity-determining regions (CDRs) that can determine in parallel the effect of every possible single amino acid CDR substitution on antigen binding. The method uses libraries of full length IgGs containing more than 1000 CDR point mutations displayed on mammalian cells, sorted by flow cytometry into subpopulations based on antigen affinity and analyzed by massively parallel pyrosequencing. Higher, lower and neutral affinity mutations are identified by their enrichment or depletion in the FACS subpopulations. We applied this method to a humanized version of the anti-epidermal growth factor receptor antibody cetuximab, generated a near comprehensive data set for 1060 point mutations that recapitulates previously determined structural and mutational data for these CDRs and identified 67 point mutations that increase affinity. The large-scale, comprehensive sequence-function data sets generated by this method should have broad utility for engineering properties such as antibody affinity and specificity and may advance theoretical understanding of antibody-antigen recognition.     

Human Germline Antibody Gene Segments Encode Polyspecific Antibodies

Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three V_H gene segments. Panels of somatically mutated antibodies encoded by a common V_H gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the V_H gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.     

Isolation and characterization of an IgNAR variable domain specific for the human mitochondrial translocase receptor Tom70.

The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (VNAR), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15-18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (VNAR 12F-11) after four rounds of in vitro selection. VNAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3 mg x L(-1). Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected beta-sheet conformation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd = 2.2 +/- 0.31 x 10(-9) m-1) indicating that these VNAR domains can be efficiently displayed as bacteriophage libraries, and selected against target antigens with an affinity and stability equivalent to that obtained for other single domain antibodies. As an initial step in producing 'intrabody' variants of 12F-11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High affinity binding was only retained in the wild-type protein, which combined with our inability to affinity mature 12F-11, suggests that this particular VNAR is critically dependent upon precise CDR loop conformations for its binding affinity.     

A novel TNFalpha antagonizing peptide-Fc fusion protein designed based on CDRs of TNFalpha neutralizing monoclonal antibody

The variable regions of antibody molecules bind antigens with high affinity and specificity. The binding sites are imparted largely to the hypervariable portions (i.e., CDRs) of the variable region. Peptides derived from CDRs can bind antigen with similar specificity acting as mimic of antibody and become drug-designing core, although with markedly lower affinity. In order to increase the affinity and bioactivity, in this study, a novel peptide (PT) designed on CDRs of a TNFalpha neutralizing monoclonal antibody Z12 was linked with Fc fragment of human IgG1. The interaction mode of PT-linker-Fc (PLF) with TNFalpha was analyzed with computer-guided molecular modeling method. After expression in Escherichia coli and purification, recombinant PT-linker-Fc could bind directly with the TNFalpha coated on the ELISA plates. Furthermore, PLF could competitively inhibit the binding of Z12 to TNFalpha and also inhibit the TNFalpha-induced cytotoxicity on L929 cells. The TNFalpha antagonizing activity of PLF was significantly higher than that of the free peptide. This study highlights the potential of human Fc to enhance the potency of peptides designed on the CDRs of antibodies and could be useful in developing new TNFalpha antagonists.