Contact residues and predicted structure of the reovirus type 3-receptor interaction.

Sequence similarity between the reovirus type 3 hemagglutinin (HA3) and a anti-idiotypic monoclonal antibody (87.92.6) has been shown to define the site of interaction with a neutralizing (idiotypic) monoclonal antibody (9B.G5) and the cellular receptor for the virus. A synthetic peptide (VL peptide) derived from the anti-idiotypic sequence inhibits viral binding to the receptor. In this study, variants of the VL peptide were utilized to probe specific amino acid residues involved in binding the neutralizing antibody and the receptor. These studies indicate that the--OH groups of several residues are involved in contacting the reovirus type 3 receptor, including Tyr49, Ser50, Ser52, and Thr53 in the anti-idiotypic sequence, corresponding to Tyr326, Ser327, Ser329, and Ser325 in HA3, respectively. In contrast, only Ser50 of the anti-idiotypic sequence, corresponding to Ser327 of HA3, significantly altered neutralizing antibody binding. Additional studies implicate sialic acid as a potential reovirus type 3 receptor on some cells. This includes inhibition of binding of reovirus type 3 and 87.92.6 to L cells by heavily sialylated glycoproteins. Sialic acid was therefore utilized as a candidate receptor to analyze potential interaction schemes with HA3/87.92.6. Sequence similarity to other immunoglobulin structures with similar sequences allowed modeling of the three-dimensional structure of these epitopes. These structures, in combination with peptide studies, allow the development of a model of the interaction of these epitopes with sialic acid, which serves as a reovirus type 3 receptor. These models reveal that similar amino acid residues and side-chain geometries may be utilized by the reovirus type 3 and influenza hemagglutinins in their interactions with cell-surface receptors.     

Cell receptors for the mammalian reovirus. IV. Reovirus-specific cytolytic T cell lines that have idiotypic receptors recognize anti-idiotypic B cell hybridomas

Cytotoxic T lymphocyte (Tc) cell lines specific for reovirus type 3 lysed an uninfected B cell hybridoma line, 87.92.6, that expresses and secretes an anti-idiotypic antibody that reacts with an anti-viral hemagglutinin monoclonal antibody, 9BG5. Monoclonal anti-idiotype 87.92.6 was shown by fluorescence analysis to specifically bind to reovirus Tc and to block reovirus-specific Tc from killing reovirus-infected target cells or the 87.92.6 hybridoma. An anti-LFA-1 monoclonal antibody, M17, interfered with Tc-mediated lysis of reovirus-infected targets and the 87.92.6 cells, indicating the similarity of cellular interactions mediated by LFA-1 structures when Tc bind to virally infected targets or 87.92.6 targets. Together with studies in which anti-H2 or monoclonal idiotypic antibodies were found to interfere with reovirus-specific Tc recognition of virally infected or 87.92.6 targets, these experiments indicate that some reovirus-specific Tc have conformations in their receptor that can be recognized by anti-idiotype.     

Design of bioactive peptides based on antibody hypervariable region structures. Development of conformationally constrained and dimeric peptides with enhanced affinity

The variable regions of antibody molecules bind antigens with high affinity and specificity. This binding is imparted largely by the hypervariable portions of the variable region. Hypervariable regions typically fold into reverse turn or loop structures. Peptides derived from antibody hypervariable region sequences can bind antigens with similar specificity, albeit with markedly lower affinity. In this study, cyclic and dimeric peptide analogs of an anti-idiotypic/antireceptor antibody hypervariable region were developed. This antibody (87.92.6) binds to reovirus type 3 receptors on cells as well as to a neutralizing anti-reovirus type 3 monoclonal antibody (9B.G5). The cyclic peptides were utilized to probe the optimal conformation for binding to both the receptor and 9B.G5. By dimerizing or constraining the conformation of these peptides, higher affinity binding was produced. By utilizing several different cyclic peptides, the optimal conformation for binding was established. The conformationally optimized cyclic peptide possessed greater than 40-fold higher affinity for the receptor and the idiotype than the linear analog. This study suggests that conformationally constrained and dimeric peptides derived from antibody hypervariable loop sequences can bind antigens (including receptors) with reasonable affinity. hypervariable loop sequences can bind antigens (including     

Role of MHC II affinity and molecular mimicry in defining anti-HER-2/neu MAb-3 linear peptide epitope

With the aid of computational biology, we have studied the possibility of predicting the peptides able to evoke humoral immune response by using as experimental model the human HER-2/neu breast cancer-associated antigen. We already demonstrated that HER-2/neu peptides, that are the target of humoral human and mouse immune responses, correspond to those sequences having a low degree of sequence similarity to host's proteome. Here we report that the linear peptide determinant of the anti-HER-2/neu MAb-3 is characterized by a low degree of sequence similarity to mouse proteome in combination with high binding potential to specific MHC II molecule.     

An improved model of association for VH-VL immunoglobulin domains: asymmetries between VH and VL in the packing of some interface residues

The antibody-binding site is formed as a result of the association between VH and VL domains. Several studies have shown that this association plays an important role in the mechanism of antigen-antibody interaction (Stanfield et al. Structure 1: 83-93, 1993). Considering this, we propose that variations in the VH-VL association are part of the diversification strategy of the antibody repertoires. Previously, a model of association for VH-VL domains based on geometrical characteristics of the packing at the interface was developed by Chothia et al. (J. Mol. Biol. 186: 61-663, 1985). This model includes a common association form for antibodies and a three-layer structure for the interface. In the present work, a complementary model is introduced to account for the general geometrical restrictions of the VH-VL interface, and particular arrangements related to the chemical properties or the side-chain orientations of participating residues. Groups of residues assume common side-chain orientations, which are apparently related to particular functions of different interface zones. Analyses of amino acid usage and network are in agreement with the side-chain orientation patterns. Based on these observations, a three-zone model has evolved to illuminate geometrical and functional restrictions acting over the VH-VL interface. Additionally, this study has revealed the asymmetrical relationships between VH and VL residues important for the association of the two domains.     

Anti-idiotypic antibody identifies the cellular receptor of reovirus type 3

The binding and subsequent infectivity of reovirus to target cells are mediated by interaction with specific cell surface viral receptors. To gain a more detailed understanding of the biochemistry of the reovirus receptor and the cellular consequences of viral attachment, we have studied the binding of type 3 reovirus (Dearing strain) in a quantitative manner utilizing an antiidiotypic antibody probe. A syngeneic monoclonal antiidiotypic antibody (87.92.6) was prepared by immunization with hybridoma cells which secrete an antireovirus hemagglutinin-specific antibody. This antiidiotypic antibody was previously shown to specifically recognize the cell surface receptor for reovirus type 3. In this report, we demonstrate that antiidiotype mimicked reovirus tropism in binding to murine thymomas; antiidiotype inhibited the binding of reovirus to specific targets, but not the binding of anti-H-2; and cross linking of receptor-bound antiidiotype by antiimmunoglobulin induced patching, but not capping of reovirus receptors. Utilizing radiolabeled antiidiotype, we next quantitate the number of reovirus receptors on R1.1 and YAC thymoma cells and, finally, report on the preliminary identification of the reovirus receptor as a 67,000-Da membrane glycoprotein.     

Minimum requirements for immunogenic and antigenic activities of homologs of a synthetic peptide of influenza virus hemagglutinin.

Synthetic peptides of increasing length and corresponding in sequence to the C-terminal end of the HA1 molecule of influenza virus were constructed and examined for their immunogenic and antigenic properties. Peptides containing at least the four C-terminal amino acids, when coupled to keyhole limpet hemocyanin, were capable of eliciting antibody in BALB/c mice that bound to the 24-residue parent peptide H3 HA1 (305 to 328). In the absence of a carrier, the C-terminal decapeptide was the shortest peptide capable of eliciting antibody. The specificity of this antibody was indistinguishable from that of a monoclonal antibody to the parent peptide which recognizes an epitope encompassed by the C-terminal seven residues. All peptides containing at least the C-terminal four residues were able to inhibit completely the binding of this monoclonal antibody to the parent peptide. Taken together, these results indicate that (i) the tetrapeptide is capable of eliciting specific antibody when coupled to a carrier, (ii) this tetrapeptide possesses all of the antigenic information necessary to occupy the paratope of a monoclonal antibody elicited by the longer parent peptide, and (iii) the decapeptide contains all of the information necessary to elicit a specific immune response and therefore carries an epitope recognized by T cells as well as one recognized by B cells.     

The structure of an antigenic determinant in a protein

The immunogenic and antigenic determinants of a synthetic peptide and the corresponding antigenic determinants in the parent protein have been elucidated. Four determinants have been defined by reactivity of a large panel of antipeptide monoclonal antibodies with short, overlapping peptides (7-28 amino acids), the immunizing peptide (36 amino acids), and the intact parent protein (the influenza virus hemagglutinin, HA). The majority of the antipeptide antibodies that also react strongly with the intact protein recognize one specific nine amino acid sequence. This immunodominant peptide determinant is located in the subunit interface in the HA trimeric structure. The relative inaccessibility of this site implies that antibody binding to the protein is to a more unfolded HA conformation. This antigenic determinant differs from those previously described for the hemagglutinin and clearly demonstrates the ability of synthetic peptides to generate antibodies that interact with regions of the protein not immunogenic or generally accessible when the protein is the immunogen.     

Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains

The antigen-binding site of antibodies from vertebrates is formed by combining the variable domains of a heavy chain (VH) and a light chain (VL). However, antibodies from camels and llamas are an important exception to this in that their sera contain, in addition, a unique kind of antibody that is formed by heavy chains only. The antigen-binding site of these antibodies consists of one single domain, referred to as VHH. This article reviews the mutations and structural adaptations that have taken place to reshape a VH of a VH-VL pair into a single-domain VHH with retention of a sufficient variability. The VHH has a potent antigen-binding capacity and provides the advantage of interacting with novel epitopes that are inaccessible to conventional VH-VL pairs.     

DNA binding by the VH domain of anti-Z-DNA antibody and its modulation by association of the VL domain

mAb Z22 is a highly selective IgG anti-Z-DNA Ab from an immunized C57BL/6 mouse. Previous studies showed that heavy chain CDR3 amino acids are critical for Z-DNA binding by the single chain variable fragment (scFv) comprising both V region heavy chain (VH) and V region light chain (VL) of mAb Z22 and that the VH domain alone binds Z-DNA with an affinity similar to that of whole variable fragment (Fv). To determine whether Z-DNA binding by VH alone and by Fv involves identical complementarity determining region residues, we tested effects of single or multiple amino acid substitutions in recombinant VH, scFv, and associated VH-VL heterodimers. Each recombinant product was a fusion protein with a B domain of Staphylococcal protein A (SPA). Z22VH-SPA alone was not highly selective; it bound strongly to other polynucleotides, particularly polypyrimidines, and ssDNA as well as to Z-DNA. In contrast, scFv-SPA or associated VH-VL dimers bound only to Z-DNA. VL-SPA domains bound weakly to Z-DNA; SPA alone did not bind. Introduction of multiple substitutions revealed that the third complementarity determining region of the heavy chain (CDR3H) was critical for both VH and scFv binding to Z-DNA. However, single substitutions that eliminated or markedly reduced Z-DNA binding by scFv instead caused a modest increase or no reduction in binding by VH alone. Association of VH-SPA with Z22VL-SPA restored both the effects of single substitutions and Z-DNA selectivity seen with Fv and intact Ab. Polypyrimidine and ssDNA binding by the isolated VH domain of immunization-induced anti-Z-DNA Ab resembles the activity of natural autoantibodies and suggests that VH-dependent binding to a ligand mimicked by polypyrimidines may play a role in B cell selection before immunization with Z-DNA.     

A Combinatorial Peptide Library Around Variation of the Human Immunode ficiency Virus (HIV-1) V3 Domain Leads to Distinct T Helper Cell Responses

The hypervariable domain of the HIV gp120, the V3 loop domain, represents a target for neutralizing antibodies and for HIV vaccine strategies. In this study, we have investigated in murine species the potential cross-reactivity of immune responses elicited by immunization either with individual V3 peptides, derived from distinct HIV sequences (BRU, RF, SF2, MN and ELI sequences), or with a V3 combinatorial peptide library. We observed that individual V3 peptides are immunogenic but elicit a specific B- and T-cell immune response that is mainly restricted to the sequence of the immunizing peptide. In particular, T-cell responses that depend on T-cell receptor recognition of peptides bound to the molecules encoded by the major histocompatibility complex were significantly influenced by small differences in the peptide amino acid sequence. The combinatorial V3 peptide library, previously described as B- and T-cell immunogens, induced a more broadly reactive immune response, specially when T-cell cytokine secretion was used as a readout for restimulation of T-cells with individual V3 peptides. These data suggest that amino acid variations in the sequence of an antigenic peptide could lead to the induction of different transducing signals in the primed T-cell population and to the activation of T-cells with distinct cytokine secretion properties. These observations may have implications in the understanding of antigenic variability and in the design of vaccine strategies.     

Analysis of variable region genes encoding a human anti-DNA antibody of normal origin. Implications for the molecular basis of human autoimmune responses

In order to investigate the genetic basis for natural anti-DNA immune responses, we isolated and sequenced the variable gene elements (VH and VL) encoding an anti-DNA antibody expressed by a human hybridoma of normal origin (Kim4.6) and compared these sequences with those reported for four other human anti-DNA antibodies. The Kim4.6 antibody leader and VH segments were identical in nucleotide sequence with the VH1.9III germ-line VH3 gene, and the Kim4.6VL segment showed 98% nucleotide sequence identity with a V lambda I subgroup gene expressed in a Burkitt's lymphoma. Comparative analysis of Kim4.6 and other human hybridoma anti-DNA antibodies indicated that anti-DNA immune responses are diverse in terms of VH and VL gene utilization but may exhibit a bias toward rearrangement of VH genes that are over-represented in the fetal pre-B cell repertoire. Moreover, Kim4.6 and three of four other sequenced human anti-DNA antibodies appear to use a germ-line diversity gene, DXP'1, which may represent a counterpart of the DFL16.1 segment utilized in murine responses to the hapten nitrophenyl. Taken together, our findings indicate that anti-DNA immune responses can be encoded by nonmutated VH genes and that the elements and molecular mechanisms which engender this response are essentially the same among natural and lupus-associated anti-DNA antibodies. Our data also suggest that natural autoimmune responses originate early in B cell ontogeny as is consistent with the hypothesis that autoreactivity plays a major role in shaping the normal immune repertoire.     

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。     

Effects on interaction kinetics of mutations at the VH-VL interface of Fabs depend on the structural context

The influence of framework residues belonging to VH and VL modules of antibody molecules on antigen binding remains poorly understood. To investigate the functional role of such residues, we have performed semi-conservative amino acid replacements at the VH-VL interface. This work was carried out with (i) variants of the same antibody and (ii) with antibodies of different specificities (Fab fragments 145P and 1F1h), in order to check if functional effects are additive and/or similar for the two antibodies. Interaction kinetics of Fab mutants with peptide and protein antigens were measured using a BIACORE instrument. The substitutions introduced at the VH-VL interface had no significant effects on k(a) but showed small, significant effects on k(d). Mutations in the VH module affected k(d) not only for the two different antibodies but also for variants of the same antibody. These effects varied both in direction and in magnitude. In the VL module, the double mutation F(L37)L-Q(L38)L, alone or in combination with other mutations, consistently decreased k(d) about two-fold in Fab 145P. Other mutations in the VL module had no effect on k(d) in 145P, but always decreased k(d) in 1F1h. Moreover, in both systems, small-magnitude non-additive effects on k(d) were observed, but affinity variations seemed to be limited by a threshold. When comparing functional effects in antibodies of different specificity, no general rules could be established. In addition, no clear relationship could be pointed out between the nature of the amino acid change and the observed functional effect. Our results show that binding kinetics are affected by alteration of framework residues remote from the binding site, although these effects are unpredictable for most of the studied changes.     

Tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity

Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We previously identified several fully human neutralizing anti-VEGF receptor 2 (or kinase inserting domain-containing receptor (KDR)) antibodies from a large antibody phage display library. These antibodies bind specifically to KDR, block VEGF/KDR interaction, and inhibit VEGF-induced proliferation of human endothelial cells and migration of KDR+ leukemia cells. Three of these antibodies, interestingly, share an identical heavy chain variable (VH) sequence. In this report, we constructed a new library comprising the single VH paired with the variable light chain (VL) repertoire obtained from the original na?ve human library. Initial in vitro selection revealed that the single VH could pair with a number of different VL while retaining its specificity for KDR. However, a consensus VH/VL pair, clone 1121, was identified after three or four rounds of selection by tailoring the stringency of the panning conditions. Clone 1121 showed a >30-fold higher binding affinity to KDR (Kd, 100 pm) because of improvement on both association and dissociation constants and blocked VEGF/KDR interaction with an IC50 of approximately 1 nm, compared with that of 3-4 nm for the parent Fab fragments. Further, clone 1121 was more potent in inhibiting VEGF-stimulated KDR phosphorylation in endothelial cells. A binding epitope mapping study on clone 1121 and one of the parent clones, 2C6, demonstrated that both antibodies interacted with the third immunoglobulin domain within the extracellular region of KDR. Several peptide phage display libraries were utilized to further examine the fine binding specificities of the two antibodies. All of the 2C6-binding peptides are cysteine-constrained, whereas clone 1121 binds to both cysteine-constrained and linear peptides. It is noteworthy that most of the 2C6-binding peptides also cross-react with clone 1121, but none of the clone 1121-specific peptides binds to 2C6, indicating that clone 1121 retained part of the original binding epitope(s) of 2C6 while gaining new binding specificity. Taken together, our observation suggests that clone 1121 may have great clinical potential in anti-angiogenesis therapy. It further underscores the efforts to identify antibodies of high affinity for enhanced biological activities.     

β淀粉样肽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β淀粉样肽单克隆抗体轻链与重链可变区基因。方法:从分泌抗β淀粉样肽单克隆抗体的杂交瘤细胞株A8中提取总RNA,根据恒定区序列设计基因特异性引物,通过5′RACE法扩增抗体的轻链和重链可变区基因,测定并分析可变区基因序列,并克隆入pMD18-T载体。结果:重链可变区基因序列全长450bp,编码150个氨基酸残基;轻链可变区基因序列全长429bp,编码143个氨基酸残基。在GeneBank中对氨基酸序列进行比对分析,二者均符合小鼠IgG可变区基因的特征。根据Kabat法则对A8抗体轻链和重链可变区氨基酸序列基因进行分析并确定了3个抗原互补决定区(CDR)、4个框架区(FR)和信号肽。结论:通过5′RACE法得到了抗β淀粉样肽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,以及对该抗体进行人源化改造奠定了基础。     

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.     

Many variable region genes are utilized in the antibody response of BALB/c mice to the influenza virus A/PR/8/34 hemagglutinin

We have examined how many different H chain variable (VH) and kappa-chain variable (Vk) germ-line genes are used in the antibody response to the influenza virus A/PR/8/34 hemagglutinin (PR8 HA), and have assessed how the expression of individual VH and/or Vk genes contributes to the generation of specificity for the HA. A panel of 51 hybridoma antibodies that recognize two antigenic regions on the HA were compared for the sequence of their Ig H and L chain V regions. The hybridomas were obtained from 28 individual BALB/c mice that had been immunized with PR8 under a variety of primary and secondary response immunization protocols. The degree and pattern of sequence similarity suggests that 29 different VH genes drawn from seven different VH gene families, and 25 different Vk genes drawn from 12 different Vk gene families were used in this panel. Based on current estimates of the total numbers of VH and Vk genes in the mouse, this suggests that between 2.5 and 10% of the entire VH and Vk germ-line repertoires were used by these hybridomas. Despite this extensive diversity, some V genes were repetitively identified among these hybridomas, and were most often expressed in the context of specific VH/Vk combinations. Because antibodies that used identical VH/Vk combinations also usually displayed similar reactivity patterns with a panel of mutant viruses, this indicates that VH/Vk pairing can be important in establishing the specificity of antibodies for the HA.     

The critical role of arginine residues in the binding of human monoclonal antibodies to cardiolipin

Previously we reported that the variable heavy chain region (VH) of a human beta2 glycoprotein I-dependent monoclonal antiphospholipid antibody (IS4) was dominant in conferring the ability to bind cardiolipin (CL). In contrast, the identity of the paired variable light chain region (VL) determined the strength of CL binding. In the present study, we examine the importance of specific arginine residues in IS4VH and paired VL in CL binding. The distribution of arginine residues in complementarity determining regions (CDRs) of VH and VL sequences was altered by site-directed mutagenesis or by CDR exchange. Ten different 2a2 germline gene-derived VL sequences were expressed with IS4VH and the VH of an anti-dsDNA antibody, B3. Six variants of IS4VH, containing different patterns of arginine residues in CDR3, were paired with B3VL and IS4VL. The ability of the 32 expressed heavy chain/light chain combinations to bind CL was determined by ELISA. Of four arginine residues in IS4VH CDR3 substituted to serines, two residues at positions 100 and 100 g had a major influence on the strength of CL binding while the two residues at positions 96 and 97 had no effect. In CDR exchange studies, VL containing B3VL CDR1 were associated with elevated CL binding, which was reduced significantly by substitution of a CDR1 arginine residue at position 27a with serine. In contrast, arginine residues in VL CDR2 or VL CDR3 did not enhance CL binding, and in one case may have contributed to inhibition of this binding. Subsets of arginine residues at specific locations in the CDRs of heavy chains and light chains of pathogenic antiphospholipid antibodies are important in determining their ability to bind CL.     

A strategy to identify linker-based modules for the allosteric regulation of antibody-antigen binding affinities of different scFvs

Antibody single-chain variable fragments (scFvs) are used in a variety of applications, such as for research, diagnosis and therapy. Essential for these applications is the extraordinary specificity, selectivity and affinity of antibody paratopes, which can also be used for efficient protein purification. However, this use is hampered by the high affinity for the protein to be purified because harsh elution conditions, which may impair folding, integrity or viability of the eluted biomaterials, are typically required. In this study, we developed a strategy to obtain structural elements that provide allosteric modulation of the affinities of different antibody scFvs for their antigen. To identify suitable allosteric modules, a complete set of cyclic permutations of calmodulin variants was generated and tested for modulation of the affinity when substituting the linker between VH and VL. Modulation of affinity induced by addition of different calmodulin-binding peptides at physiologic conditions was demonstrated for 5 of 6 tested scFvs of different specificities and antigens ranging from cell surface proteins to haptens. In addition, a variety of different modulator peptides were tested. Different structural solutions were found in respect of the optimal calmodulin permutation, the optimal peptide and the allosteric effect for scFvs binding to different antigen structures. Significantly, effective linker modules were identified for scFvs with both VH-VL and VL-VH architecture. The results suggest that this approach may offer a rapid, paratope-independent strategy to provide allosteric regulation of affinity for many other antibody scFvs.