Display of somatostatin-related peptides in the complementarity determining regions of an antibody light chain

Peptide display in antibody complementarity determining regions (CDRs) offers several advantages over other peptide display systems including the potential to graft heterologous peptide sequences into multiple positions in the same backbone molecule. Despite the presence of six CDRs in an antibody variable domain, the majority of insertions reported have been made in heavy chain CDR3 (h-CDR3) which may be explained in part by the highly variable length and sequence diversity found in h-CDR3 in native antibodies. The ability to graft peptide sequences into CDRs is restricted by amino acids in these loops that make structural contacts to framework regions or are oriented towards the hydrophobic interior and are important for the proper folding of the antibody. To identify such positions in human kappa-light chain CDR1 (kappa-CDR1) and CDR2 (kappa-CDR2), we performed alignments of 1330 kappa-light chain variable region amino acid sequences and 19 variable region X-ray crystal structures. From analyses of these alignments, we predict insertion points where sequences can be grafted into kappa-CDR1 and kappa-CDR2 to prepare synthetic antibody molecules. We then tested these predictions by inserting somatostatin and somatostatin-related sequences into kappa-CDR1 and kappa-CDR2, and analyzing the expression and ability of the modified antibodies to bind to membranes containing somatostatin receptor 5. These results expand the repertoire of CDRs that can be used for the display of heterologous peptides in the CDRs of antibodies.     

Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions

A structure-based approach was used to design libraries of synthetic heavy chain complementarity determining regions (CDRs). The CDR libraries were displayed as either monovalent or bivalent single-chain variable fragments (scFvs) with a single heavy chain variable domain scaffold and a fixed light chain variable domain. Using the structure of a parent antibody as a guide, we restricted library diversity to CDR positions with significant exposure to solvent. We introduced diversity with tailored degenerate codons that ideally only encoded for amino acids commonly observed in natural antibody CDRs. With these design principles, we reasoned that we would produce libraries of diverse solvent-exposed surfaces displayed on stable scaffolds with minimal structural perturbations. The libraries were sorted against a panel of proteins and yielded multiple unique binding clones against all six antigens tested. The bivalent library yielded numerous unique sequences, while the monovalent library yielded fewer unique clones. Selected scFvs were converted to the Fab format, and the purified Fab proteins retained high affinity for antigen. The results support the view that synthetic heavy chain diversity alone may be sufficient for the generation of high-affinity antibodies from phage-displayed libraries; thus, it may be possible to dispense with the light chain altogether, as is the case in natural camelid immunoglobulins.     

AbRSA: A robust tool for antibody numbering

The remarkable progress in cancer immunotherapy in recent years has led to the heat of great development for therapeutic antibodies. Antibody numbering, which standardizes a residue index at each position of an antibody variable domain, is an important step in immunoinformatic analysis. It provides an equivalent index for the comparison of sequences or structures, which is particularly valuable for antibody modeling and engineering. However, due to the extremely high diversity of antibody sequences, antibody‐numbering tools cannot work in all cases. This article introduces a new antibody‐numbering tool named AbRSA, which integrates heuristic knowledge of region‐specific features into sequence mapping to enhance the robustness. The benchmarks demonstrate that, AbRSA exhibits robust performance in numbering sequences with diverse lengths and patterns compared with the state‐of‐the‐art tools. AbRSA offers a user‐friendly interface for antibody numbering, complementarity‐determining region delimitation, and 3D structure rendering. It is freely available at http://cao.labshare.cn/AbRSA .     

The structure of the anti-c-myc antibody 9E10 Fab fragment/epitope peptide complex reveals a novel binding mode dominated by the heavy chain hypervariable loops

The X-ray structure of the Fab fragment from the anti-c-myc antibody 9E10 was determined both as complex with its epitope peptide and for the free Fab. In the complex, two Fab molecules adopt an unusual head to head orientation with the epitope peptide arranged between them. In contrast, the free Fab forms a dimer with different orientation. In the Fab/peptide complex the peptide is bound to one of the two Fabs at the "back" of its extended CDR H3, in a cleft with CDR H1, thus forming a short, three-stranded antiparallel beta-sheet. The N- and C-terminal parts of the peptide are also in contact with the neighboring Fab fragment. Comparison between the CDR H3s of the two Fab molecules in complex with the peptide and those from the free Fab reveals high flexibility of this loop. This structural feature is in line with thermodynamic data from isothermic titration calorimetry.     

Thermodynamics of Fab-ssDNA interactions: contribution of heavy chain complementarity determining region 3.

The recombinant anti-ssDNA Fab, DNA-1, and 16 heavy chain complementarity determining region 3 (HCDR3) mutant variants were selected for thermodynamic characterization of ssDNA binding. The affinity of Fab to (dT)(15) under different temperatures and cation concentrations was measured by equilibrium fluorescence quenching titration. Changes in the standard Gibbs free binding energy (DeltaG degrees ), enthalpy (DeltaH degrees ), entropy (DeltaS degrees ), and the number of ionic pairs (Z) formed upon interaction were determined. All Fab possessed an enthalpic nature of interaction with ssDNA, that was opposite to the previously reported entropically driven binding to dsDNA [Tanha, J., and Lee, J. S. (1997) Nucleic Acids Res. 25, 1442-1449]. The contribution of separate residues of HCDR3 to ssDNA interaction was investigated. Analysis of the changes in DeltaH degrees and TDeltaS degrees, induced by substitutions in HCDR3, revealed a complete entropy/enthalpy compensation. Mutations R98A and D108A at the ends of the HCDR3 loop produced increases in TDeltaS degrees ( )()by 10.4 and 15.9 kcal/mol, respectively. Substitution of proline for arginine at the top of HCDR3 resulted in a new electrostatic contact with (dT)(15). The observed linear correlation of Z and DeltaG degrees ( )()of nonelectrostatic interactions (DeltaG degrees (nonel)) at the anti-ssDNA combining site was used for the estimation of the specific DeltaG degrees (nonel) [-20 to -25 cal/(mol.A(2))], the average contact area (450-550 A(2)), the maximal Z (6-7), and the limit in affinity under standard cation concentrations [(0.5-1) x 10(8) M(-)(1)] for this family of Fab. Results suggested that rational engineering of HCDR3 could be utilized to control the affinity and likely the specificity of Ab-DNA interactions.     

Isomerization in the CDR2 of a monoclonal antibody: Binding analysis and factors that influence the isomerization rate

Isomerization of a monoclonal antibody is one of the common routes of protein degradation. An isomerization in the complementarity‐determining region (CDR) was found previously and is investigated in depth in this work. Affinity analysis proves that the antibody with one isomerized heavy chain has lower binding. Binding constants were determined, and exhibited a slower on‐rate in conjunction with a faster off‐rate for this isomerization. To determine the role of the buffer on the rate of isomerization, this antibody was incubated in various matrices and the amount of isomerized antibody was determined by hydrophobic interaction chromatography (HIC). The rate was found to be dependent on the pH as well as the net negative charge of the buffer components that can act as proton acceptors. An Arrhenius plot was performed to predict the levels of isomerization and a comparison of real samples proved the model was correct. This work affirms that isomerization in the CDR of a therapeutic antibody is important to monitor and the formulation buffer plays a significant role in the rate of the isomerization. Biotechnol. Bioeng. 2010; 105: 515–523. © 2009 Wiley Periodicals, Inc.     

Contribution of a single heavy chain residue to specificity of an anti-digoxin monoclonal antibody.

Two distinct spontaneous variants of the murine anti-digoxin hybridoma 26-10 were isolated by fluorescence-activated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26-10. Replacement of Asn with Gln reduced affinity for digoxin 10-fold relative to the wild-type antibody, but maintained wild-type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90-fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16-acetylated haptens and haptens with a saturated lactone. The X-ray crystal structure of the 26-10 Fab in complex with digoxin (Jeffrey PD et al., 1993, Proc Natl Acad Sci USA 90:10310-10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds with 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild-type Asn. We therefore performed molecular modeling experiments which suggested that substitution of Gln or Asp can maintain these hydrogen bonds whereas the other substituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in binding of the wild-type and Gln-mutant antibodies, however, suggest that these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of this antibody. The Ala mutant eliminates the wild-type hydrogen bonding, and molecular modeling suggests that the reduced side-chain volume also provides space that can accommodate a congener with a 16-acetyl group or saturated lactone, accounting for the altered fine specificity of this antibody.     

Characterization of the human Ig heavy chain antigen binding complementarity determining region 3 using a newly developed software algorithm, JOINSOLVER

We analyzed 77 nonproductive and 574 productive human V(H)DJ(H) rearrangements with a newly developed program, JOINSOLVER. In the productive repertoire, the H chain complementarity determining region 3 (CDR3(H)) was significantly shorter (46.7 +/- 0.5 nucleotides) than in the nonproductive repertoire (53.8 +/- 1.9 nucleotides) because of the tendency to select rearrangements with less TdT activity and shorter D segments. Using criteria established by Monte Carlo simulations, D segments could be identified in 71.4% of nonproductive and 64.4% of productive rearrangements, with a mean of 17.6 +/- 0.7 and 14.6 +/- 0.2 retained germline nucleotides, respectively. Eight of 27 D segments were used more frequently than expected in the nonproductive repertoire, whereas 3 D segments were positively selected and 3 were negatively selected, indicating that both molecular mechanisms and selection biased the D segment usage. There was no bias for D segment reading frame (RF) use in the nonproductive repertoire, whereas negative selection of the RFs encoding stop codons and positive selection of RF2 that frequently encodes hydrophilic amino acids were noted in the productive repertoire. Except for serine, there was no consistent selection or expression of hydrophilic amino acids. A bias toward the pairing of 5' D segments with 3' J(H) segments was observed in the nonproductive but not the productive repertoire, whereas V(H) usage was random. Rearrangements using inverted D segments, DIR family segments, chromosome 15 D segments and multiple D segments were found infrequently. Analysis of the human CDR3(H) with JOINSOLVER has provided comprehensive information on the influences that shape this important Ag binding region of V(H) chains.     

A computational approach for studying antibody–antigen interactions without prior structural information: the anti‐testosterone binding antibody as a case study

Given the increasing exploitation of antibodies in different contexts such as molecular diagnostics and therapeutics, it would be beneficial to unravel the atomistic level properties of antibody‐antigen complexes with the help of computational modeling. Thus, here we have studied the feasibility of computational tools to gather atomic scale information regarding the antibody‐antigen complexes solely starting from an amino acid sequence. First, we constructed a homology model for the anti‐testosterone binding antibody based on the knowledge based classification of complementary determining regions (CDRs) and implicit solvent molecular dynamics simulations. To further examine whether the generated homology model is suitable for studying antibody‐antigen interactions, docking calculations were carried out followed by binding free‐energy simulations. Our results indicate that with the antibody modeling approach presented here it is possible to construct accurate homology models for antibodies which correctly describes the antibody‐antigen interactions, and produces absolute binding free‐energies that are comparable with experimental values. In addition, our simulations suggest that the conformations of complementary determining regions (CDRs) may considerably change from the X‐ray configuration upon solvation. In conclusion, here we have introduced an antibody modeling workflow that can be used in studying the interactions between antibody and antigen solely based on an amino acid sequence, which in turn provides novel opportunities to tune the properties of antibodies in different applications. Proteins 2017; 85:322–331. © 2016 Wiley Periodicals, Inc.     

Length-independent structural similarities enrich the antibody CDR canonical class model

Complementarity-determining regions (CDRs) are antibody loops that make up the antigen binding site. Here, we show that all CDR types have structurally similar loops of different lengths. Based on these findings, we created length-independent canonical classes for the non-H3 CDRs. Our length variable structural clusters show strong sequence patterns suggesting either that they evolved from the same original structure or result from some form of convergence. We find that our length-independent method not only clusters a larger number of CDRs, but also predicts canonical class from sequence better than the standard length-dependent approach.

To demonstrate the usefulness of our findings, we predicted cluster membership of CDR-L3 sequences from 3 next-generation sequencing datasets of the antibody repertoire (over 1,000,000 sequences). Using the length-independent clusters, we can structurally classify an additional 135,000 sequences, which represents a ～20% improvement over the standard approach. This suggests that our length-independent canonical classes might be a highly prevalent feature of antibody space, and could substantially improve our ability to accurately predict the structure of novel CDRs identified by next-generation sequencing.     

人源抗EPO基因工程抗体重链可变区的克隆和鉴定

利用噬菌体抗体显示技术筛选 EPO的人源抗体 ,得到了抗 EPO的人源抗体的重链基因。此抗体基因在噬菌体表面呈现的抗体分子具有良好的抗体活性和特异性。为制备完整的、具有更高亲和力的抗体打下了基础。     

Generation of humanized anti-DNA hydrolyzing catalytic antibodies by complementarity determining region grafting

Humanization of nonhuman antibodies (Abs) has been carried out mainly for Abs which bind to antigen without catalytic activity. Here we report humanization of mouse-originated 3D8 (m3D8) mAbs (scFv, VH, and VL) with DNA hydrolyzing catalytic activity by grafting the complementarity determining regions (CDRs) into the corresponding regions of a fixed human framework scaffold, generating humanized 3D8 (h3D8) Abs in the respective format of scFv, VH, and VL. h3D8 Abs retained comparable DNA binding and hydrolyzing activities to those of the corresponding m3D8 Abs. Our results suggest that CDRs of anti-DNA hydrolyzing Abs might possess the intrinsic properties of DNA binding and hydrolyzing activities.     

Aligning,analyzing, and visualizing sequences for antibody engineering: Automated recognition of immunoglobulin variable region features

The analysis and comparison of large numbers of immunoglobulin (Ig) sequences that arise during an antibody selection campaign can be time‐consuming and tedious. Typically, the identification and annotation of framework as well as complementarity‐determining regions (CDRs) is based on multiple sequence alignments using standardized numbering schemes, which allow identification of equivalent residues among different family members but often necessitate expert knowledge and manual intervention. Moreover, due to the enormous length variability of some CDRs the benefit of conventional Ig numbering schemes is limited and the calculation of correct sequence alignments can become challenging. Whereas, in principle, a well established set of rules permits the assignment of CDRs from the amino acid sequence alone, no currently available sequence alignment editor provides an algorithm to annotate new Ig sequences accordingly. Here we present a unique pattern matching method implemented into our recently developed ANTIC ALIgN editor that automatically identifies all hypervariable and framework regions in experimentally elucidated antibody sequences using so‐called “regular expressions.” By combination of this widely supported software syntax with the unique capabilities of real‐time aligning, editing and analyzing extended sets of amino acid and/or nucleotide sequences simultaneously on a local workstation, ANTIC ALIgN provides a powerful utility for antibody engineering. Proteins 2016; 85:65–71. © 2016 Wiley Periodicals, Inc.     

Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires

Studies of antibodies of known three-dimensional structure have revealed that insertion and deletion of amino acids at the hypervariable loops change the canonical structures, thus generating differences in the antigen-binding site topography. Such differences determine the size of the antigen with which the antibody interacts. Here, 59 unique antibodies determined at a resolution of 3.0 A or below, including 19 in complex with proteins, 18 with peptides and 22 with haptens, were analyzed to identify and characterize differences in the residues that are directly involved in the interaction with antigen, so-called specificity-determining residues (SDRs). It was found that antibodies use a similar number of SDRs to recognize proteins and peptides but contact haptens with five SDRs less. By using a score of SDR usage, differences in the location of the SDRs, depending on the type of antigen recognized, were then identified with precision. An analysis of the surface generated by the SDRs usage indicates that the differences found correlate well with the size of the antigen. Anti-protein antibodies have the largest SDR surface, with SDRs of high usage located in the edge of the surface. The SDR surface of anti-hapten antibodies is the smallest, with hot spots of contacts in the interior of the binding surface and buried in the V(L):V(H) interface. The SDR surface of anti-peptide antibodies has a size in between anti-protein and anti-hapten antibodies, with the SDRs of high usage located in the interior of the antigen-binding site but do not buried as in anti-hapten antibodies. These findings led to a fine-tuning of the model correlating differences in the antigen-binding site topography with its preference to recognize antigens of different size. Therefore, it is discussed how this knowledge should help to design antibody repertoires biased toward the recognition of antigens of predefined size.     

Analyzing the "degree of humanness" of antibody sequences

Genetically engineered mouse antibodies are now commonly in clinical use. However, their development is limited because the human immune system tends to regard them as foreign and this triggers an immune response. The solution is to make engineered antibodies appear more human. Here, we propose a method to assess the "degree of humanness" of antibody sequences providing a tool that may contribute to predictions of antigenicity. We analyzed sequences of antibodies belonging to various chains/classes in human and mouse. Our analysis of metrics based on percentage sequence identity between antibody sequences shows distinct differences between human and mouse sequences. Based on mean sequence identity and standard deviation, we calculated Z-scores for data sets of antibody sequences extracted from the Kabat database. We applied the analysis to a set of humanized and chimeric antibodies and to human germline sequences. We conclude that this approach may aid in the selection of more suitable mouse variable domains for antibody engineering to render them more human but in general, we find that typicality of a sequence compared with the expressed human repertoire is not well correlated with antigenicity. We have provided a Web server allowing humanness to be assigned for a sequence.     

Engineering of conserved residues near antibody heavy chain complementary determining region 3 (HCDR3) improves both affinity and stability

Affinity and stability are crucial parameters in antibody development and engineering approaches. Although improvement in both metrics is desirable, trade-offs are almost unavoidable. Heavy chain complementarity determining region 3 (HCDR3) is the best-known region for antibody affinity but its impact on stability is often neglected. Here, we present a mutagenesis study of conserved residues near HCDR3 to elicit the role of this region in the affinity-stability trade-off. These key residues are positioned around the conserved salt bridge between V_H-K94 and V_H-D101 which is crucial for HCDR3 integrity. We show that the additional salt bridge at the stem of HCDR3 (V_H-K94:V_H-D101:V_H-D102) has an extensive impact on this loop's conformation, therefore simultaneous improvement in both affinity and stability. We find that the disruption of π-π stacking near HCDR3 (V_H-Y100E:V_L-Y49) at the V_H-V_L interface cause an irrecoverable loss in stability even if it improves the affinity. Molecular simulations of putative rescue mutants exhibit complex and often non-additive effects. We confirm that our experimental measurements agree with the molecular dynamic simulations providing detailed insights for the spatial orientation of HCDR3. V_H-V102 right next to HCDR3 salt bridge might be an ideal candidate to overcome affinity-stability trade-off.     

Recombinant expression and purification of human placental growth factor 1 and specific camel heavy chain polyclonal antibody preparation

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family. Unlike VEGF, PlGF is dispensable for normal cell development as well as playing various roles in pathological angiogenesis which occurs in tissue ischemia, inflammation, and malignancy. The PlGF-1 has been considered as a potential candidate for the diagnosis and targeting of pathological angiogenesis. Camelidae serum contains an important fraction of functional antibodies, called heavy-chain antibodies (HcAbs) that are naturally devoid of light chains. Camelid HcAbs recognize their cognate antigens by a single variable-domain, referred to as VHH or Nanobody.Here, we describe the expression and purification of recombinant human PlGF-1 (rhPlGF-1). This protein was subsequently used for the preparation of camel heavy chain polyclonal antibody against rhPlGF-1.The recombinant expression plasmid pET-26b-hPlGF-1 was introduced into Escherichia coli BL21 cells to express the rhPlGF-1 protein. Purified rhPlGF-1 was used to immunize camel, the specific reactivity of HcAb was determined with ELISA and western blot. Western blot analysis indicated that the antiserum specifically reacted to the recombinant protein. The rhPlGF-1 protein and its antibody may be used for the development of detection assays needed for clinical research.     

X-ray structures of fragments from binding and nonbinding versions of a humanized anti-CD18 antibody: Structural indications of the key role of VH residues 59 to 65

X-ray crystal structures of fragments from two different humanized antiCD18 antibodies are reported. The Fv fragment of the nonbinding version has been refined in space group C2 with a=64.2 Å, b=61.3 Å, c=51.8 Å, and β=99° to an R-value of 18.0% at 1.9 Å, and the Fab fragment of the tight-binding version has been refined in space group P3 with a=101. Å and c=45.5 Å to an R-value of 17.8% at 3.0 Å resolution. The very large difference in their binding affinity (>1000-fold) is attributed to large and local structural differences in the C-terminal part of CDR-H2, and from this we conclude there is direct contact between this region and antigen when they combine. X-ray structures of antibody–antigen complexes available in the literature have yet to show this part of CDR-H2 in contact with antigen, despite its hypervariable sequence. Implications of this result for antibody humanization are discussed. © 1994 John Wiley & Sons, Inc.     

A Two-in-One antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions

A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.     

A Two-in-One antibody engineered from a humanized interleukin 4 antibody through mutation in heavy chain complementarity-determining regions

A mono-specific antibody may recruit a second antigen binding specificity, thus converting to a dual-specific Two-in-One antibody through mutation at the light chain complementarity-determining regions (CDRs). It is, however, unknown whether mutation at the heavy chain CDRs may evolve such dual specificity. Herein, we examined the CDRs of a humanized interleukin 4 (IL4) antibody using alanine scanning and structural modeling, designed libraries of mutants in regions that tolerate mutation, and isolated dual specific antibodies harboring mutation at the heavy chain CDRs only. We then affinity improved an IL4/IL5 dual specific antibody to variants with dissociation constants in the low nanomolar range for both antigens. The results demonstrate the full capacity of antibodies to evolve dual binding specificity.