Molecular modeling of antibody-antigen complexes between the Brucella abortus O-chain polysaccharide and a specific monoclonal antibody.

A molecular model of the binding site of an anti-carbohydrate antibody (YsT9.1) has been developed using computer-assisted modeling techniques and molecular dynamics calculations. Sequence homologies among YsT9.1 and the Fv regions of McPC603, J539 and human Bence--Jones protein REI, all of which have solved crystal structures, provided the basis for the modeling. The groove-type combining site model had a topography which was complementary to low energy conformers of the polysaccharide, a Brucella O-antigen, and the site could be almost completely filled by a pentasaccharide epitope in either of two docking modes. Putative interactions between this epitope and the antibody are consistent with the known structural requirements for binding and lead to the design of oligosaccharide inhibitors that probe the veracity of the modeled docked complex. Ultimately both the Fv model and the docked complex will be compared with independent crystal structures of YsT9.1 Fab with and without pentasaccharide inhibitor, currently at the stage of refinement.     

Molecular anatomy of the antibody binding site

The binding region of immunoglobulins, which includes the portion of the molecule having the most variability in its amino acid sequence, is shown to have a surprisingly constant structure that can be characterized in terms of a simple, well-defined model. The binding region is composed of the antigen combining site plus its immediate vicinity and arises by noncovalent association of the light and heavy chain variable domains (VL and VH, respectively). The antigen combining site itself consists of six polypeptide chain segments ("hypervariable loops") which comprise some 80 amino acid residues and are attached to a framework of VL and VH beta-sheet bilayers. Having analyzed refined x-ray crystallographic coordinates for three antigen-binding fragments (Fab KOL (Marquart, M., Deisenhofer, J., and Huber, R. (1980) J. Mol. Biol. 141, 369-391), MCPC 603 (Segal, D., Padlan, E. A., Cohen, G. H., Rudikoff, S., Potter, M., and Davies, D. R. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4298-4302), and NEW (Saul, F. A., Amzel, L. M., and Poljak, R. J. (1978) J. Biol. Chem. 253, 585-597] we use the results to introduce a general model for the VL-VH interface forming the binding region. The region consists of two closely packed beta-sheets, and its geometry corresponds to a 9-stranded, cylindrical barrel of average radius 0.84 nm with an average angle of -53 degrees between its two constituent beta-sheets. The barrel forms the bottom and sides of the antigen combining site. The model demonstrates that the structural variability of the binding region is considerably less than was thought previously. Amino acid residues which are part of the domain-domain interface and appear not to be accessible to solvent or antigen contribute to antibody specificity.     

Domain association in immunoglobulin molecules. The packing of variable domains

We have analyzed the structure of the interface between VL and VH domains in three immunoglobulin fragments: Fab KOL, Fab NEW and Fab MCPC 603. About 1800 A2 of protein surface is buried between the domains. Approximately three quarters of this interface is formed by the packing of the VL and VH beta-sheets in the conserved "framework" and one quarter from contacts between the hypervariable regions. The beta-sheets that form the interface have edge strands that are strongly twisted (coiled) by beta-bulges. As a result, the edge strands fold back over their own beta-sheet at two diagonally opposite corners. When the VL and VH domains pack together, residues from these edge strands form the central part of the interface and give what we call a three-layer packing; i.e. there is a third layer composed of side-chains inserted between the two backbone side-chain layers that are usually in contact. This three-layer packing is different from previously described beta-sheet packings. The 12 residues that form the central part of the three observed VL-VH packings are absolutely or very strongly conserved in all immunoglobulin sequences. This strongly suggests that the structure described here is a general model for the association of VL and VH domains and that the three-layer packing plays a central role in forming the antibody combining site.     

Antibody engineering: the use of Escherichia coli as an expression host.

The hypervariable loops of an antibody molecule are supported on the relatively conserved beta-sheeted frameworks of the heavy- and light-chain variable domains (designated VH and VL domains, respectively). Residues within and flanking these loops interact with antigen and confer the specificity and affinity of antigen binding on the immunoglobulin molecule. Thus, the isolation and expression of VH and VL domain genes are of particular interest both for analysis of the determinants of antibody specificity and for generation of fragments with binding affinities for use in therapy and diagnosis. The PCR can now be used to isolate diverse repertoires of antibody VH and VL domain genes from antibody-producing cells from different species, including humans and mice. The genes can be expressed as either secreted or surface-bound Fv or Fab fragments, using Escherichia coli expression systems, and the desired antigen-binding specificity screened for or, preferably, selected. The use of E. coli as an expression host allows the required antigen-binding specificity to be isolated in clonal form in a matter of days. The VH and VL domain genes can also be hypermutated and higher-affinity variants isolated by screening or selection. Thus, the use of this technology should allow the isolation of novel binding specificities or specificities that are difficult to generate by hybridoma technology. It will also facilitate the isolation of human-derived Fv/Fab fragments that may be less immunogenic in therapy. This approach therefore has almost unlimited potential in the generation of therapeutics with binding specificities to order. The fragments can be used either alone or linked to effector functions in the form of antibody-constant domains or toxins. The new technology could prove to be a method of choice for the rapid and convenient production of designer antibodies.     

A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds.     

Antiferritin VL homodimer binds human spleen ferritin with high specificity

The antiferritin variable light domain (VL) dimer binds human spleen ferritin ( approximately 85% L subunits) but with approximately 50-fold lower affinity, K(a)=4 x 10(7) x M(-1), than the parent F11 antibody (K(a)=2.1 x 10(9) x M(-1)). The VL dimer does not recognize either rL (100% L subunits) or rH (100% H subunits) human ferritin, whereas the parent antibody recognizes rL-ferritin. To help explain the differences in ferritin binding affinities and specificities, the crystal structure of the VL domain (2.8A resolution) was determined by molecular replacement and models of the antiferritin VL-VH dimer were made on the basis of antilysozyme antibody D1.3. The domain interface is smaller in the VL dimer but a larger number of interdomain hydrogen bonds may prevent rearrangement on antigen binding. The antigen binding surface of the VL dimer is flatter, lacking a negatively charged pocket found in the VL-VH models, contributed by the CDR3 loop of the VH domain. Loop CDR2 (VL dimer) is located away from the antigen binding site, while the corresponding loop of the VH domain would be located within the antigen binding site. Together these differences lead to 50-fold lower binding affinity in the VL dimer and to more restricted specificity than is seen for the parent antibody.     

The specificity properties that distinguish members of a set of homologous anti-digoxin antibodies are controlled by H chain mutations

Five murine A/J strain anti-digoxin mAb (35-20, 40-40, 40-120, 40-140, and 40-160) have highly homologous H and L chain V regions, only differing by somatic mutation, yet differ in affinity and specificity. The availability of the VH and VL genomic clones from one hybridoma, 40-140, has now allowed studies involving in vitro mutagenesis and chain recombination among these five hybridomas. To determine the relative contributions of the mutations found in either VH or VL to the overall binding properties of these antibodies, we recombined the 40-140VH with the VL of each hybridoma. The 40-140VH gene was transfected into hybridoma variants that produce only VL. The recombinant antibodies show that the mutations present in VH, rather than in VL, affect the fine specificity properties of these antibodies, whereas, the mutations among both VH and VL chains are important in determining antigen affinity. From mutations present in VH that affect fine specificity properties, the comparison of the antibody sequences, and from the previously measured binding properties, we predicted and tested selected VH mutations for their ability to alter specificity or affinity by doing site-directed in vitro mutagenesis. The results for the somatic mutations found in this group of antibodies show: 1) VH mutations control the fine specificity properties that distinguish different members of this group; 2) in particular, VH residues 54 and 55 in CDR2 control the distinguishing characteristics of specificities between these antibodies; and 3) by mutagenesis, we had the unusual result of being able to alter Ag specificity without affecting affinity. A computer model of the 40-140 antibody binding site was generated which indicates that VH residues 54 and 55 are highly accessible.     

Nucleotide and translated amino acid sequences of cDNA coding for the variable regions of the light and heavy chains of mouse hybridoma antibodies to blood group A and B substances

This is the first report of nucleotide and translated amino acid sequences of the variable region light (VL) and heavy (VH) chains of mouse monoclonal hybridoma anti-blood group A and B substances, the combining sites of which have been mapped. Monoclonal hybridoma anti-A and anti-B produced in BALB/c mice by immunization with A or B blood group substances, with A1 erythrocytes, and water-soluble blood group A substance or with synthetic B determinants coupled to bovine serum albumin or to O erythrocytes have been characterized immunochemically. To relate the immunochemical properties of the monoclonals to their primary structures, we have cloned and sequenced cDNAs of variable regions of light and heavy chains of two anti-A and two anti-B. The anti-A hybridomas have very similar combining site specificities and have almost identical VH sequences belonging to the J558 germ-line family, but their VL are from different germ-line VK gene families. The two anti-B hybridomas have different combining site specificities and use the same VL which differs completely from the anti-A VL; their VH are derived from different VH germ-line genes belonging to the J606 family. The results suggest that the heavy chains play a major role in determining the specificities of the antibody combining sites, with only minor contribution of VL. Additional sequence data on monoclonal antibodies of defined specificity for blood group substances are needed for further insights into the genetic and structural basis for their specificities.     

Prediction of VH–VL domain orientation for antibody variable domain modeling

The antigen‐binding site of antibodies forms at the interface of their two variable domains, VH and VL, making VH–VL domain orientation a factor that codetermines antibody specificity and affinity. Preserving VH–VL domain orientation in the process of antibody engineering is important in order to retain the original antibody properties, and predicting the correct VH–VL orientation has also been recognized as an important factor in antibody homology modeling. In this article, we present a fast sequence‐based predictor that predicts VH–VL domain orientation with Q² values ranging from 0.54 to 0.73 on the evaluation set. We describe VH–VL orientation in terms of the six absolute ABangle parameters that have recently been proposed as a means to separate the different degrees of freedom of VH–VL domain orientation. In order to assess the impact of adjusting VH–VL orientation according to our predictions, we use the set of antibody structures of the recently published Antibody Modeling Assessment (AMA) II study. In comparison to the original AMAII homology models, we find an improvement in the accuracy of VH–VL orientation modeling, which also translates into an improvement in the average root‐mean‐square deviation with regard to the crystal structures. Proteins 2015; 83:681–695. © 2015 Wiley Periodicals, Inc.     

Gene cloning, bacterial expression, in vitro refolding, and characterization of a single-chain Fv antibody against PreS1(21-47) fragment of HBsAg

The murine monoclonal antibody 125E11 is an IgG which recognizes PreS1(21-47) fragment of large hepatitis B surface antigen. It has been successfully used for clinical detection of HBV virion in serum of hepatitis B patients. In present study, the genes of variable region in heavy chain (VH) and light chain (VL) of 125E11 have been cloned. Sequence analysis of cloned VH gene and VL gene showed that they had general characterization of immunoglobin variable region genes. According to Kabat classification, VH gene and VL gene belong to VH10 family, subgroup IIID and Vkappa family subgroup I, respectively. An expression vector of 125E11 single-chain Fv antibody fusion protein, in which VH and VL peptide were connected by a flexible linker (Gly(4)Ser)(3), was constructed. The scFv fusion protein was highly expressed in Escherichia coli mainly in inclusion body form. Using urea and pH gradient gel filtration method, the refolding of scFv was efficiently achieved. The refolding efficiency reached about 11% and 2.7 mg refolded scFv was obtained from 1L of culture. The binding activity and specificity of 125E11 scFv against PreS1(21-47)-containing antigen were also analyzed.     

2.9 A resolution structure of an anti-dinitrophenyl-spin-label monoclonal antibody Fab fragment with bound hapten

The crystal structure of the Fab fragment of the murine monoclonal anti-dinitrophenyl-spin-label antibody AN02 complexed with its hapten has been solved at 2.9 A resolution using a novel molecular replacement method. Prior to translation searches, a large number of the most likely rotation function solutions were subjected to a rigid body refinement against the linear correlation coefficient between intensities of observed and calculated structure factors. First, the overall orientation of the search model and then the orientations and positions of the four Fab domains (VH, VL, CH1 and CL) were refined. This procedure clearly identified the correct orientation of the search model. The refined search model was then subjected to translation searches which unambiguously determined the enantiomer and position in the unit cell of the crystal. The successful search model was refined 2.5 A crystal structure of the Fab fragment of HyHel-5 from which non-matching residues in the variable domains had been removed. HyHel-5 is a murine monoclonal antibody whose heavy and light chains are of the same subclass (gamma 1, kappa, respectively) as AN02. After molecular replacement the structure of the AN02 Fab has been refined using simulated annealing in combination with model building and conjugate gradient refinement to a current crystallographic R-factor of 19.5% for 12,129 unique reflections between 8.0 and 2.9 A. The root-mean-square (r.m.s.) deviation from ideal bond lengths is 0.014 A, and the r.m.s. deviation from ideal bond angles is 3.1 degrees. The electron density reveals the hapten sitting in a pocket formed by the loops of the complementarity determining region. The dinitrophenyl ring of the hapten is sandwiched between the indole rings of Trp96 of the heavy-chain and Trp91 of the light-chain. The positioning of the hapten and general features of the combining site are in good agreement with the results of earlier nuclear magnetic resonance experiments.     

抗人大肠癌重组单链抗体的研制

应用重组噬菌体抗体系统制备了重组单链抗体。首先从抗人结肠癌ND-1单抗杂交瘤细胞中提取mRNA,利用反转录多聚酶链反应(RT-PCR)扩增出单抗重链可变区(V_H)及轻链可变区(V_L)片段,再通过连接DNA合成单链抗体可变区片段(ScFv),然后经双酶切后克隆到pCANTAB5E载体中,在E.coliTGI细胞中表达出噬菌体融合蛋白,用抗原阳性噬菌体感染E.coliHB2151细胞,产生单链抗体,该单链抗体既保持了原单抗的特异性,应用上又优于原单抗。     

肝癌细胞特异性结合单链抗体三维结构的同源模建

用MSI公司开发的计算机辅助分子设计系统模建肝癌细胞表面抗原特异性单链抗体三维结构。先分别模建VH(variable region of the heave chain)和VL(variable region of the light chain)两个结构域,然后搭建出scFv(single chain variable fragment)片段的整体三维结构,并对模建的结构进行分子力学和动力学优化;对结构的合理性验证显示模建结构是合理的。文章可为预测该特异性单链抗体的生物活性以及研制高亲和力、高特异性的双价抗体提供结构信息。     

Modelling of the combining sites of three anti-lysozyme monoclonal antibodies and of the complex between one of the antibodies and its epitope.

Models of the antigen combining sites of three monoclonal antibodies, which recognise different but overlapping epitopes within the 'loop' region of hen egg lysozyme (HEL), have been generated from the cDNA sequences of their Fv regions (the VL and VH domains) and the known crystal structures of immunoglobulin fragments. The alpha-carbon backbone of the structurally conserved framework region has been derived from the IgG myeloma protein NEW, and models for the hypervariable loop regions have been selected on the basis of length and maximum sequence homology. The model structures have been refined by energy minimisation. Both the size and chemical nature of the predicted combining site models correlate broadly with the epitope boundaries previously determined by affinity studies. A model of the complex formed between one antibody and the corresponding lysozyme epitope is described, and contact residues are identified for subsequent testing by oligonucleotide-directed site-specific mutagenesis.     

抗人前列腺干细胞抗原单克隆抗体可变区基因的5＇RACE扩增及序列分析

目的：克隆并分析抗人前列腺干细胞抗原单克隆抗体轻链和重链的可变区基因。方法：从分泌抗人前列腺干细胞抗原单克隆抗体的杂交瘤细胞株中提取总RNA,根据小鼠IgG恒定区序列设计特异性引物,通过5’RACE法扩增其轻链和重链的可变区基因,克隆入pMD18-T载体,测序并分析其可变区序列。结果：3株抗人前列腺干细胞抗原单克隆抗体的重链可变区基因序列全长均为423bp,编码141个氨基酸残基;轻链可变区基因序列全长均为393bp,编码131个氨基酸残基;在GenBank中对氨基酸序列进行比对分析,均符合小鼠IgG可变区基因的特征;根据Kabat法则对3株抗体轻链和重链可变区氨基酸序列进行分析,确定了3个抗原互补决定区、4个框架区和前导肽。结论：通过5＇RACE法得到了3株抗人前列腺干细胞抗原单克隆抗体轻链与重链可变区基因,为进一步研究抗体三维结构、人源化改造奠定了基础。     

A V kappa-J kappa junctional change in an antidigoxin recombinant antibody destroys digoxin-binding activity

A set of high affinity antidigoxin antibodies were previously identified with high homologous V kappa 1A L chain sequences but were associated with two entirely different VH regions and two dramatically different specificities for digoxin analogs. Antibodies 40-20, 40-60, 40-90, and 40-100 displayed similar binding specificities but differed from that of antibody 26-10. In a previous study using somatic cell fusion for Ig chain recombination we demonstrated that a recombinant antibody consisting of the H chain of antibody 26-10 and the L chain of antibody 40-20 retained digoxin binding and the 26-10 Id, but displayed a binding specificity pattern dominated by the 26-10 H chain donor. In the present study we produced three additional chain recombinant antibodies that contain the 26-10 H chain recombined with each of the L chains of antibodies 40-60, 40-90, and 40-100. All four recombinants expressed the 26-10 Id indistinguishably from the 26-10 antibody. Two of the recombinants (using the 40-60 and 40-90 L chains) bind digoxin; however, the recombinant using the 40-100 L chain failed to bind digoxin. Complete sequence analyses of the 40-20, 40-60, 40-90, and 40-100 VH and VL regions were performed. Antibodies 40-90 and 40-100 have identical VH region sequences but differed only in their L chains at position 96 (proline/leucine). This single difference at the VK-JK junction abolished digoxin binding in the context of one H chain (26-10), but does not cause a significant change in binding in association with the "normal" parental chains 40-90 and 40-100. Thus, structurally closely related VL regions can recombine with different VH regions to form digoxin binding sites of different specificity; in one binding site the identity of a L chain junctional residue is critical whereas in the second binding site that residue is unimportant. Molecular modeling studies revealed major differences between calculated binding site structures for 26-10 when leucine is substituted for proline at position 96 in the 26-10 VL region.     

Bacterial single-chain antibody fragments, specific for carcinoembryonic antigen.

We have produced single-chain antibody (scFv) fragments in bacteria specific for carcinoembryonic antigen (CEA). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions (VH and VL) of the mouse monoclonal antibody (MAb) CB-CEA.1. A 14-amino acid linker was used in the synthesis of the scFv gene. The VH and VL regions were amplified from cDNA by PCR using 5' end FR1 and 3' end constant region primers, and then sequenced. VH was then amplified by PCR using an exact 5' end FR1 primer, and a phosphorylated (PP) 3' end primer for J2 that also encoded the first 7 amino acids of the linker. VL was amplified with a PP 5' end primer for FR1, also encoding the remaining 7 amino acids of the linker, and a 3' end primer for J5, plus a stop codon and a BglII restriction site. The fragments were ligated and reamplified with the PP VH 5' and VL 3' end primers. The VH-linker-VL structure was blunt-cloned into expression vectors bearing the tryptophan promoter and pelB or ompA signal peptide sequences. Culture supernatant, bacteria pellet and periplasm preparations were assayed in Western blot and a protein of about 27 kDa was identified with rabbit antibodies specific for the Fab of CB-CEA.1. Bacterial supernatant and periplasm preparations also inhibited the recognition of CEA by HRP-labeled CB-CEA.1 in enzyme-linked immunosorbent assay (ELISA). Periplasm preparations were purified by affinity chromatography with specific anti-idiotypic MAbs. The Western blot of the eluates identified a protein of approximately 27 kDa that blocked the recognition of CEA by HRP-labeled CB-CEA.1 in ELISA. The VH-linker-VL structure was cloned into a vector bearing the lacZ promoter and the pelB signal peptide. The recombinant bacterial clones also expressed about 27 kDa scFv, specific for CEA.