Active site structure and antigen binding properties of idiotypically cross-reactive anti-fluorescein monoclonal antibodies

This report includes complete VH and V kappa nucleotide and deduced amino acid sequences of idiotypically cross-reactive monoclonal anti-fluorescein antibodies that differed greater than 10(5)-fold in affinity. High affinity monoclonal antibody 4-4-20 and intermediate affinity antibodies 10-25, 5-14, 9-40, 12-40, and 3-24 utilized greater than or equal to 90% homologous VHIIIC germ-line genes. Extensive D segment length and sequence variability were observed; however, compensatory germ-line JH4 (4-4-20 and 3-24) or JH3 (10-25, 5-14, 9-40, and 12-40) sequence lengths resulted in H chain CDR3 + FR4 to be a constant 18 amino acids. In addition, each antibody and low affinity 3-13 rearranged greater than or equal to 96% homologous V kappa II genes to J kappa 1, except for 10-25 (J kappa 5) and 3-13 (J kappa 4). Resolved crystal structure of complexed fluorescein and 4-4-20 Fab fragments revealed residues HisL27d, TyrL32, ArgL34, SerL91, TrpL96, and TrpH33 acted as hapten contact residues. Antibodies 5-14, 9-40, 12-40, and 3-24 primary structures possessed identical contact residues as 4-4-20 except for the substitution of HisL34 for ArgL34. Thus, ArgL34 was implicated in the increased affinity of monoclonal antibody 4-4-20. Finally, it was difficult to correlate extensive H chain CDR3 residue heterogeneity directly with fluorescein binding and idiotypy.     

Two induced anti-Z-DNA monoclonal antibodies use VH gene segments related to those of anti-DNA autoantibodies

The cDNA for H and L chain V regions of two anti-Z-DNA mAb, Z22 and Z44, were cloned and sequenced. These are the first experimentally induced anti-nucleic acid antibody sequences available for comparison with autoantibody sequences. Z22 and Z44 are IgG2b and IgG2a antibodies from C57BL/6 mice. They recognize different facets of the Z-DNA structure. They both use VH10 family genes and share 95% sequence base sequence identity in the VH and leader sequences; however, they differ in the 5'-untranslated region of the VH mRNA, indicating they arise from different germline genes. Both use JH4 segments. They differ from each other very extensively in the CDR3 of both H and L chains. The most closely related H chains in the current GenBank/EMBL data base are two mouse IgG anti-DNA autoantibodies, one from an MRL-lpr/lpr mouse (MRL-DNA4) and one from an NZB/NZW mouse (BV04-01). Z22 and Z44 share 95% sequence identity with these antibodies in the VH segment. In addition, Z22 is identical to MRL-DNA4 at 91% of the positions in the 5'-untranslated region of the H chain mRNA. The two antibodies share 95% base sequence identity in the V kappa segment. The most closely related L chains, with 97 to 98% sequence identity, are the V kappa 10b germline gene for Z22 and the V kappa 10a germ line gene, which is associated with A/J anti-arsonate antibodies and BALB/c anti-ABO blood group substance antibodies, for Z44. Z22 and Z44 share several structural features (similarities in VH, JH, and V kappa) but differ very markedly in the L chain CDR1 and both H and L chain CDR3 sequences; these regions may determine the differences in their specific interactions with Z-DNA.     

Biophysical properties of human antibody variable domains

There are great demands on the stability, expression yield and resistance to aggregation of antibody fragments. To untangle intrinsic domain effects from domain interactions, we present first a systematic evaluation of the isolated human immunoglobulin variable heavy (V(H)) and light (V(L)) germline family consensus domains and then a systematic series of V(H)-V(L) combinations in the scFv format. The constructs were evaluated in terms of their expression behavior, oligomeric state in solution and denaturant-induced unfolding equilibria under non-reducing conditions. The seven V(H) and seven V(L) domains represent the consensus sequences of the major human germline subclasses, derived from the Human Combinatorial Antibody Library (HuCAL). The isolated V(H) and V(L) domains with the highest thermodynamic stability and yield of soluble protein were V(H)3 and V(kappa)3, respectively. Similar measurements on all domain combinations in scFv fragments allowed the scFv fragments to be classified according to thermodynamic stability and in vivo folding yield. The scFv fragments containing the variable domain combinations H3kappa3, H1bkappa3, H5kappa3 and H3kappa1 show superior properties concerning yield and stability. Domain interactions diminish the intrinsic differences of the domains. ScFv fragments containing V(lambda) domains show high levels of stability, even though V(lambda) domains are surprisingly unstable by themselves. This is due to a strong interaction with the V(H) domain and depends on the amino acid sequence of the CDR-L3. On the basis of these analyses and model structures, we suggest possibilities for further improvement of the biophysical properties of individual frameworks and give recommendations for library design.     

Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. IV. The less frequently expressed VL are heterogeneous

We previously demonstrated that the human anti-Haemophilus influenzae type b polysaccharide (Hib-PS) VL repertoire is dominated by a product of the V kappa II gene, A2, and that V kappa II-A2 anti-Hib-PS antibodies have little or no somatic mutation in VL. To further study this VL repertoire, we studied non-A2 anti-Hib-PS antibodies that were identified either serologically or by amino-terminal amino acid sequence analysis. Of 15 non-A2 anti-Hib-PS antibodies from 12 vaccinated adults, we found four V lambda, five V kappa I, one non-A2 V kappa II, four V kappa III, and one V kappa IV antibodies. As expected, all but two of these subjects also produced V kappa II-A2 antibodies. Interestingly, one of these subjects lacks the A2 gene in the germ line. However, both subjects who did not produce detectable V kappa II antibody did produce normal amounts of total anti-Hib-PS antibody after vaccination. Candidate V kappa genes for the non-A2 antibodies were identified by comparison of up to 60 VL amino acid residues, including CDR1 and CDR2, with all sequenced V kappa genes. V kappa I antibodies appear to be products of three newly sequenced V kappa I genes, O8, O18, and L11, that are reported here. The O8 and O18 genes encode identical amino acid sequences. The non-A2 V kappa II antibody is a likely product of the A1 or A17 genes, the V kappa III antibodies are likely products of the A27 gene, and the V kappa IV antibody is a product of the single V kappa IV gene, B3. Unlike V kappa II-A2 antibodies, the V kappa I, V kappa III, and V kappa IV antibodies differed by one to five CDR residues from the germ line product of the candidate genes, suggesting the presence of somatic mutations. Thus, anti-Hib-PS antibodies can be divided into two types, the most frequently observed A2 antibodies with little or no somatic mutation and non-A2 antibodies that likely contain somatic mutations.     

Sequence similarities and cross-idiotypic specificity of L chains among human monoclonal IgM kappa with anti-gamma-globulin activity

The complete amino acid sequence of five light chain variable (V) regions of human monoclonal IgM kappa rheumatoid factors (RF) was determined, and their cross-reactive idiotypes (CRI) were characterized with antibodies induced by immunization with synthetic peptides PSL2 and PSL3, corresponding to the second and third complementarity-determining regions (CDR) of the SIE light chain. Together with two additional RF studied previously, all seven RF belong to the V kappa IIIb sub-subgroup. The region encoded by the V kappa gene segment (positions 1 to 95) in all seven proteins was virtually identical in primary structure, whereas the sequence from positions 96 to 108 defined the usage of the J kappa 1 gene in three proteins and the J kappa 2 gene in four of them. Position 96 contributed by the recombination of the V kappa and J kappa gene segments showed the presence of four different amino acid residues. Both anti-PSL2 and anti-PSL3 bind efficiently to all separated L chains when analyzed by the Western blot technique, and the binding was inhibited specifically by the corresponding peptides. The results reveal that the majority of human IgM-RF light chains are derived from a single germ line V kappa gene or a family of closely related V kappa III germ line genes, and express two "primary structure-dependent" CRI, which are largely dependent on the amino acid sequence of the second and third light chain CDR.     

Structural characterization of a cross-reactive idiotype shared by monoclonal antibodies specific for the human CD4 molecule.

A panel of mouse monoclonal anti-CD4 antibodies was characterized in terms of idiotypic expression by using specific anti-idiotypic antibody (anti-Id) reagents generated in rabbits immunized with anti-Leu3a, a monoclonal anti-CD4 which inhibits the human immunodeficiency virus (HIV) gp120 binding to CD4. Direct binding and competitive inhibition assays demonstrate that the majority of monoclonal anti-CD4 antibodies able to recognize CD4 epitopes overlapping the epitope recognized by anti-Leu3a expressed an antigen-combining site-related cross-reactive idiotype (IdX). Western blot analysis was used to demonstrate that this IdX is associated primarily with the light (L) chain of the monoclonal anti-CD4 antibodies. To further characterize the structural basis of the IdX, the nucleotide sequence of the variable region of the L kappa chain of anti-Leu3a was determined. Peptides corresponding to the first, second, and third complementarity determining regions (CDRs) of the L chain of anti-Leu3a were synthesized and used to immunize rabbits. All anti-peptide antisera recognized the immunizing peptide, the cognate anti-Leu3a molecule, and several other monoclonal anti-CD4 antibodies by direct binding assays. Western blot analysis utilizing the anti-CDR peptide reagents demonstrates that the reactivity to the monoclonal anti-CD4 antibodies was L chain-specific. The anti-Id generated by immunizing with the intact anti-Leu3a molecule failed to recognize the three L chain-derived CDR synthetic peptides, suggesting that the IdX requires the presence of the three-dimensional configuration of the L chain for its expression. The broad range of reactivity exhibited by the antipeptide antisera indicates that the majority of mouse monoclonal anti-CD4 antibodies characterized in this study utilize L chains encoded by a single germ line variable (V) region kappa (V kappa) chain gene or by V kappa genes that belong to the same gene family.     

Generation of a murine single chain Fv (scFv) antibody specific for cucumber mosaic virus (CMV) using a phage display library

With the long-term goal of generating CMV-resistant transgenic plants using antibody genes, a single-chain variable fragment (scFv) antibody that binds to the cucumber mosaic virus was isolated from a scFv phage display library by four rounds of affinity selection with CMV-Mf as an antigen. The scFv has the identical binding specificity to CMV as a monoclonal antibody that is generated by the hybridoma fusion technique, and recognized purified preparations of CMV isolates belonging to either subgroup I or II in immunoblotting. The nucleotide sequences of the recombinant antibody showed that a heavy chain variable region (V(H)) gene belonged to the VH3 subgroup and the kappa light chain variable region (V kappa) came from the Vkappa4 subgroup. Our results demonstrate that the scFv phage display library, an alternative approach to the traditional hybridoma fusion technique, has a potential applicability in the study of plant virus and plant pathology.     

Natural mouse and human antibodies bind to a peptide derived from a germline VH chain. Evidence for evolutionary conserved self-binding locus

Murine antibodies derived from the V1 S107/T15 germline structure combined with Vk 22 L chains express the property of self-binding. Previous studies have shown that the self-binding is mediated by the Fab fragment involving structures of the hapten binding site. The molecular locus of self-binding has also been identified by showing that a peptide derived from the CDR2/FR3 region of the V1 S107 H chain inhibits self-binding. We have addressed the question of whether self-binding antibodies interact with peptides that inhibit self-binding. We found that labeled TEPC15 (T15) binds to immobilized VH (50-73) peptide; the peptide binding is specific because different CDR peptides and other unrelated peptides do not inhibit this binding. Furthermore, the hapten phosphorylcholine is a potent inhibitor for the T15-peptide binding. We have demonstrated the presence of naturally occurring antibodies that bind to the T15H(50-73) peptide in the sera of different strains of mice and also in humans, indicating that the CDR2/FR3 sequence of T15 is a conserved Id determining region. We have isolated peptide-specific antibodies from pooled normal human Ig preparations. Human anti-peptide antibodies have self-binding properties similar to their murine counterparts. This interspecies conserved peptide binding of antibodies that are self-binding indicates the existence of an evolutionarily important and biologically active site.     

Construction of a semisynthetic antibody library using trinucleotide oligos.

A semisynthetic antibody library composed of single chain Fv fragments (scFv) was constructed by replacing the heavy chain CDR3 region of a human scFv by a random sequence of eight amino acids using trinucleotide codons. After cloning into a phage display vector, an antibody library was generated with a complexity of 8 x 10(8) independent clones. The library was screened for binders to dinitrophenol, fluorescein isothiocyanate and 3-nitro-4-hydroxy-5-iodophenylacetic acid. scFv antibodies that specifically bound the antigen were obtained in each case.     

双峰驼重链抗体组库的基本特征研究

目的:利用二代高通量测序技术,了解双峰骆驼循环B细胞重链抗体(HCAbs)组库的组成和基本特征。方法:通过分离骆驼外周血单核细胞(PBMC),提取m RNA,利用多重PCR和Illumina Mi-seq高通量测序技术对三头双峰骆驼的重链抗体可变区进行深度测序,分析了重链抗体组库V、J基因组成、重排时末端基因删除数和V-J基因配对率,以及CDR3的长度、香农多样性指数(Shannon index)、氨基酸组成分布等基本特征。结果:鉴定出平均每头骆驼130000条有效数据和67561条独特CDR3序列,HCAbs含量较高的V基因为IGHV1S45、IGHV1S50和IGHV1S52,J基因为IGHJ4和IGHJ6,所对应的V-J基因配对含量大于40%;CDR3的长度主要分布在10-30个氨基酸之间,含量较高的氨基酸为丙氨酸、甘氨酸和半胱氨酸;CDR3区域70%以上的平均长度为20个氨基酸长度,其中V基因长度为3 bp,J基因长度分布在1-18 bp。结论:双峰骆驼B细胞重链抗体组库由巨大的、不均匀分布(以少数VJ基因克隆占大多数)的和具有高度多样性的多克隆抗体构成,较长CDR3和富含丙氨酸、甘氨酸和半胱氨酸是HCAbs的重要特征。     

High Inter-Individual Diversity of Point Mutations,Insertions, and Deletions in Human Influenza Virus Nucleoprotein-Specific Memory B Cells

The diversity of virus-specific antibodies and of B cells among different individuals is unknown. Using single-cell cloning of antibody genes, we generated recombinant human monoclonal antibodies from influenza nucleoprotein-specific memory B cells in four adult humans with and without preceding influenza vaccination. We examined the diversity of the antibody repertoires and found that NP-specific B cells used numerous immunoglobulin genes. The heavy chains (HCs) originated from 26 and the kappa light chains (LCs) from 19 different germ line genes. Matching HC and LC chains gave rise to 43 genetically distinct antibodies that bound influenza NP. The median lengths of the CDR3 of the HC, kappa and lambda LC were 14, 9 and 11 amino acids, respectively. We identified changes at 13.6% of the amino acid positions in the V gene of the antibody heavy chain, at 8.4 % in the kappa and at 10.6 % in the lambda V gene. We identified somatic insertions or deletions in 8.1% of the variable genes. We also found several small groups of clonal relatives that were highly diversified. Our findings demonstrate broadly diverse memory B cell repertoires for the influenza nucleoprotein. We found extensive variation within individuals with a high number of point mutations, insertions, and deletions, and extensive clonal diversification. Thus, structurally conserved proteins can elicit broadly diverse and highly mutated B-cell responses.     

Human lupus anti-DNA autoantibodies undergo essentially primary V kappa gene rearrangements.

We have recently characterized the heavy chain variable region (VH) genes expressed by a panel of human anti-DNA antibodies derived from four patients with systemic lupus erythematosus and expressing an idiotypic marker representative of a subset of pathogenic autoantibodies. Here, we have cloned and sequenced the kappa chain variable region genes (V kappa) of the clones whose VH genes had been previously analysed. All the V kappa genes utilized map to the 280 kb portion of the 3' end of the locus, suggesting that they represent essentially the products of primary rearrangements. This proximal clustering of the V kappa genes used contrasts with the broad distribution of immunization-induced human antibody V kappa genes over 1400 kb of the locus. In addition, lupus autoantibodies show no tendency to express the downstream junctional (J kappa) exons--another indication of infrequent secondary variable gene assembly. Since successive rearrangements may extinguish high-affinity recognition of self antigens, we propose that this bias in V kappa and J kappa expression reflects a low rate of secondary light chain rearrangements among lupus autoantibodies. We also postulate that the corrective mechanism capable of editing potentially aggressive, self-reactive antibodies in these patients may be deficient--a deficit that could be genetically determined and/or somatically acquired.     

Inhibition of Cryptosporidium parvum infection of a mammalian cell culture by recombinant scFv antibodies

Two phage display antibody libraries (Tomlinson I and J) were screened against the whole oocysts of Cryptosporidium parvum to select for scFv (single chain variable fragment) antibodies. Three scFv antibodies were selected that bound to C. parvum oocysts as determined by monoclonal phage ELISA. DNA sequencing revealed that clone A11 lacked the majority of its V (H) chain. Clone B10 had a stop codon in the first framework region of the V (H) chain. We changed this stop codon to Gly by site-directed mutagenesis, and designated the variant mutB10. Clone B9 had a complete scFv gene with no internal stop codons. These antibody genes were individually subcloned into the pET-20b expression vector for soluble scFv antibody production. C. parvum infectivity was determined by infection of HCT-8 tissue culture monolayers and quantified by the foci detection method. By incubating C. parvum oocysts with individual scFv antibodies for 1 h at 37 degrees C prior to infecting the HCT-8 cells with the oocyst-scFv mixture, the infectivity of C. parvum was reduced in a dose-dependant manner. At the highest soluble scFv concentration tested (4 nmol), the mean number of infectious foci was reduced by 82%, 73% and 94% for the A11, B9 and mutB10 scFv, respectively. This inhibition of oocyst infectivity was abolished when the scFvs were exposed to boiling water. The results showed that the 3 selected scFvs bound to C. parvum oocysts, and their ability to neutralize infectivity may have potential therapeutic potential against cryptosporidiosis.     

Molecular basis of an isogeneic anti-idiotypic response.

The nucleotide sequences of the variable region genes expressed in the heavy and light chains of six isogeneic anti-idiotope antibodies recognizing idiotopes on two closely related antibodies with specificity for the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) were determined. In two independently derived anti-idiotope cell lines the same or strongly homologous V kappa, VH and D region genes had originally been rearranged. The two lines express long and partly homologous N sequences (presumed to be not of germ line origin) at the border of D, resulting in CDR3s of unusual length. An unusually long CDR3, partly encoded by N sequences, is also present in the heavy chain of a third anti-idiotope antibody. The VH regions of the three remaining anti-idiotope antibodies originate from a single VH gene which belongs to the same VH group as the VH genes expressed in the other anti-idiotopes. Two of these antibodies, expressing similar V, D and J elements, had been isolated from the same mouse and appear to have diverged from the same B cell precursor by at least two rounds of somatic mutation. Somatic point mutations have occurred in most, if not all anti-idiotope V region sequences. In two instances somatic mutations in J increase the structural homology between anti-idiotopes. The anti-idiotypic response in this system is thus genetically restricted and may depend upon the selection of non-germ line sequences, suggesting an explanation for the low frequency at which anti-idiotope antibodies are expressed in this system.     

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

The 3D8 single chain variable fragment (3D8 scFv) is an anti-nucleic acid antibody that can hydrolyze nucleic acids and enter the cytosol of cells without reaching the nucleus. The Tat peptide, derived from the basic region of the HIV-1 Tat protein, translocates to cell nuclei and has TAR RNA binding activity. In this study, we generated a Tat-grafted antibody (_H3Tat-3D8) by replacing complementarity-determining region 3 (CDR3) within the VH domain of the 3D8 scFv with a Tat_48–60 peptide (GRKKRRQRRRPPQ). _H3Tat-3D8 retained the DNA-binding and DNA-hydrolyzing activity of the scFv, and translocated to the nuclei of HeLa cells and preferentially recognized TAR RNA. Thus, the properties associated with the Tat peptide were transferred to the antibody via Tat-grafting without loss of the intrinsic DNA-binding and hydrolyzing activities of the 3D8 scFv antibody.     

Anti-estradiol-17beta single-chain Fv fragments: Generation, characterization, gene randomization, and optimized phage display

A single-chain Fv fragment (scFv) against estradiol-17beta (E(2)) was generated to begin the construction of a library of various mutated anti-steroid antibodies with an improved affinity and/or specificity. A hybridoma clone secreting a specific anti-E(2) antibody (Ab#E4-4) was established by the cell fusion using splenocytes from a mouse immunized with an immunogenic E(2)-carrier conjugate. DNA fragments encoding the variable heavy and light domains (V(H) and V(L)) of the Ab#E4-4 were cloned and combined to give the scFv gene fragment encoding the sequence 5'-V(H)-(GGGGS)(3)-V(L)-3'. Compared to the Ab#E4-4 Fab fragment, soluble scFv (scFv#E4-4) protein showed a similar affinity to E(2) (K(a)=8.6x10(7)M(-1)) and a similar cross-reaction profile. To further study the fundamentals for creating a comprehensive library of mutated scFvs, the scFvV(H) and V(L) genes were amplified using error-prone PCR conditions and the frequency and pattern of incorporated mutations were investigated. For this, regular Taq polymerase was used in the presence of unequal concentrations of dNTPs. At 1.0mM MnCl(2), the error frequency reached to 8.5% and 11% for the V(H) and V(L) respectively, although a significant transition/transversion bias was observed. ScFv#E4-4 and the mutated polyclonal scFvs were then displayed on filamentous phage under various packaging conditions. Cultivation of the transformed bacteria was more suitable at 25 degrees C than at higher temperatures for the packaging of scFv-bearing phagemid particles. Based on these experimental conditions, an scFv-displaying phage library, each scFv member in which has mutated complementarity-determining region (CDR) H2, H3, L1, and L3, was constructed. A soluble scFv clone (scFv#m1-e7) with a mutated amino acid (I-->V) in CDR L1, isolated from this library, showed threefold higher affinity (K(a)=2.6 x 10(8)M(-1)) than that of scFv#4-4.     

ALTHEA Gold Libraries™: antibody libraries for therapeutic antibody discovery

We describe here the design, construction and validation of ALTHEA Gold Libraries?. These single-chain variable fragment (scFv), semisynthetic libraries are built on synthetic human well-known IGHV and IGKV germline genes combined with natural human complementarity-determining region (CDR)-H3/J_H (H3J) fragments. One IGHV gene provided a universal V_H scaffold and was paired with two IGKV scaffolds to furnish different topographies for binding distinct epitopes. The scaffolds were diversified at positions identified as in contact with antigens in the known antigen-antibody complex structures. The diversification regime consisted of high-usage amino acids found at those positions in human antibody sequences. Functionality, stability and diversity of the libraries were improved throughout a three-step construction process. In a first step, fully synthetic primary libraries were generated by combining the diversified scaffolds with a set of synthetic neutral H3J germline gene fragments. The second step consisted of selecting the primary libraries for enhanced thermostability based on the natural capacity of Protein A to bind the universal V_H scaffold. In the third and final step, the resultant stable synthetic antibody fragments were combined with natural H3J fragments obtained from peripheral blood mononuclear cells of a large pool of 200 donors. Validation of ALTHEA Gold Libraries? with seven targets yielded specific antibodies in all the cases. Further characterization of the isolated antibodies indicated K_D values as human IgG1 molecules in the single-digit and sub-nM range. The thermal stability (Tm) of all the antigen-binding fragments was 75°C–80°C, demonstrating that ALTHEA Gold Libraries? are a valuable source of specific, high affinity and highly stable antibodies.