Evaluation of auditory discrimination in children with ADD and without ADD

BACKGROUND: Through antibody engineering, immunoglobulins can be tailored for their particular application. In this respect, small recognition units are desired for the targeting of antigens in obstructed locations like solid tumors. OBJECTIVES: To design efficient, minimum size recognition units, heavy chain variable regions (VH) had previously been modified for the use as antigen specific, single domain antibody fragments. To develop a rational approach to improve affinity, antigen binding is investigated here by analysing the effect of randomisations of CDR1 and 2 residues in VH domains specific for hapten and protein ligands. STUDY DESIGN: Randomised repertoires were displayed on phage and affinity selected to improve and analyse antigen binding. Affinities of newly selected VH domains were determined in their soluble format to assess the role of modified residues in binding. RESULTS: In four of five randomisation experiments, a new VH with an improved antigen affinity compared to the primary VH was selected. Dissociation constants decreased from 160 nM to 25 nM or 47 nM (CDR1 or CDR2 randomisation of an anti-Ox VH), from 300 nM to 31 nM (CDR2 randomisation of an anti-NIP VH) and from 3.1 microM to 1.6 microM (CDR2 randomisation of an anti-lysozyme VH). CONCLUSIONS: Thus the affinity of VH domains can be improved after site specific, secondary randomisations in CDR1 and CDR2, phage display and antigen selection. As differences in the CDR3 sequences had formed the only difference between the primary VH domains used in this study, the effect of CDR1 and CDR2 mutations of affinity is consistent with a participation of all three CDRs in antigen binding by single VH domains.     

Absolute conservation of residue 6 of immunoglobulin heavy chain variable regions of class IIA is required for correct folding

While studying the expression of single-chain antibodies (scFv) derived from several murine monoclonal antibodies, we found that residue 6 in Framework region 1 of the heavy chain variable domain plays a crucial role in antibody folding. Binding activity of three murine antibodies with a heavy chain variable region (VH) subgroup IIA was completely lost when at this position the wild-type residue glutamine (Q) was substituted by glutamate (E). Increased sensitivity towards trypsin digestion of soluble scFv suggested that the lack of binding activity was caused by incorrect folding of Q6E mutants. Grafting of the three additional class IA derived FR1 residues, based upon the comparison between both classes of VH sequences, on to the 'defect' subgroup IIA sequence, partially restored the antigen binding activity of the Q6E-containing scFv. Our results suggest that residue 6 of the heavy chain may be part of a folding nucleus, involving the first two beta-strands of Framework region 1. The evolutionary conservation of either glutamine or glutamate at position 6 in different antibody families may well indicate that within immunoglobulin VH domains, different family specific folding nuclei have evolved.     

Low rate of somatic hypermutations characterize progressive B-cell lymphomas

Immunoglobulin heavy (IgH) chain gene rearrangements were characterized in 40 samples from 15 patients with B-cell lymphomas at different time points during tumour progression. Using polymerase chain reaction (PCR) amplification and single strand conformation polymorphism (SSCP) analysis of variable heavy (VH) chain gene segments, we found that 6 cases displayed alterations in their IgH chain rearrangements at relapse. These alterations were mainly observed in follicular or transformed lymphomas, but no association to clinical features was found. Nucleotide sequence analysis revealed a low frequency of mutations in 3 cases, whereas 1 case displayed an extensive mutation rate in a compartment with transformed morphology at relapse. The mutations observed most probably resulted from somatic hypermutations. Further, the mutations were scattered randomly over the VH gene segment and no significant bias favouring amino acid substitutions was observed in 3 cases, suggesting that the tumour cells had not been subjected to antigen-driven selection. In 1 case, however, the mutation pattern indicated that the tumour cells had been affected by an antigen selection process. In the 2 remaining cases, the original V(H)DJ(H) rearrangement could no longer be detected by VH gene family specific PCR at relapse, but using primers specific for the framework region 2 or 3 altered rearrangements were demonstrated, implying that mutations had been introduced in framework region 1. However, the majority of the tumour cell clones analysed were relatively stable during tumour progression, which make them eligible for analysis of minimal residual disease using the VH gene regions as molecular markers.     

Structural requirements for a specificity switch and for maintenance of affinity using mutational analysis of a phage-displayed anti-arsonate antibody of Fab heavy chain first complementarity-determining region

We previously showed that a single mutation at heavy (H) position 35 of Abs specific for p-azophenylarsonate (Ars) resulted in acquisition of binding to the structurally related hapten p-azophenylsulfonate (Sulf). To explore the sequence and structural diversity of the H chain first complementarity-determining region (HCDR1) in modulating affinity and specificity, positions 30-36 in Ab 36-65 were randomly mutated and expressed as Fab in a bacteriophage display vector. Ab 36-65 is germline encoded, lacking somatic mutations. Following affinity selection on Sulf resins, 55 mutant Fab were isolated, revealing seven unique HCDR1 sequences containing different amino acids at position H:35. All Fab bound Sulf, but not Ars. Site-directed mutagenesis in a variety of HCDR1 sequence contexts indicates that H:35 is critical for hapten specificity, independent of the sequence of the remainder of HCDR1. At H:35, Asn is required for Ars specificity, consistent with the x-ray crystal structure of the somatically mutated anti-Ars Ab 36-71, while Sulf binding occurs with at least seven different H:35 residues. All Sulf-binding clones selected following phage display contained H:Gly33, observed previously for Ars-binding Abs that use the same germline V(H) sequence. Site-directed mutagenesis at H:33 indicates that Gly plays an essential structural role in HCDR1 for both Sulf- and Ars-specific Abs.     

Generation and application of type-specific anti-heparan sulfate antibodies using phage display technology. Further evidence for heparan sulfate heterogeneity in the kidney

Detailed analysis of various heparan sulfate (HS) species is seriously hampered by a lack of appropriate tools, such as antibodies. We adopted phage display technology to generate anti-HS antibodies. A "single pot" semisynthetic human antibody phage display library was subjected to four rounds of selection on HS from bovine kidney using panning methodology. Three different phage clones expressing anti-HS single chain variable fragment antibodies (HS4C3, HS4D10, and HS3G8) were isolated, with an amino acid sequence of the complementarity-determining region 3 of GRRLKD (VH3 gene, DP-38), SLRMNGCGAHQ (VH3 gene, DP-42), and YYHYKVN (VH1 gene, DP-8), respectively. The antibodies react with HS and heparin, but not with DNA or other glycosaminoglycans. Kd values for HS are about 0.1 microM. The three antibodies react differently toward various HS preparations and show different staining patterns on rat kidney sections, indicating recognition of different HS molecules. This also holds for two described mouse anti-HS IgMs (JM403 and 10E4; both generated by conventional hybridoma technique) and indicates the presence of at least 5 different HS species in the kidney. O- and N-sulfation are important for binding of HS to HS4C3 and HS3G8. The three single chain antibodies, but not JM403, block a basic fibroblast growth factor binding site of HS. It is concluded that phage display technology presents a powerful technique to generate antibodies specific for HS epitopes. This is the first time this technique has been successfully applied to obtain directly antibodies to (poly)saccharides.     

Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability

Folding stabilities of camelized human antibody VH domains were studied through the determination of their melting points in thermodenaturation experiments. The melting point of a VH domain originating from a synthetic library of human VHs, which had been optimized for the use as small recognition units through the mimicking of camelid antibody heavy chains occurring naturally without light chain, was 56.6 degrees C compared with 71.2 degrees C of the original human VH. Its stability was improved (melting point 61.6 degrees C) through three mutations to mimic camelid VHs even further: Va137 was replaced by phenylalanine and two cysteines were introduced at position 33 and 100b. The resulting VH folded properly and formed a second intradomain disulphide between the extra cysteines. The new mutations were then built constitutively into a phage-display VH library, from which antigen-specific VHs were selected. Two were analysed for stability with melting points of 72.6 and 75.3 degrees C. Thus secondary camelization enabled the isolation of VHs with improved folding stabilities exceeding even that of the original human VH. This indicates an effect on folding stability for some mutations specific in the light chain lacking camelid heavy chains.     

"Diabodies": small bivalent and bispecific antibody fragments

Bivalent and bispecific antibodies and their fragments have immense potential for practical application. Here we describe the design of small antibody fragments with two antigen-binding sites. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. As indicated by a computer graphic model of the dimers, the two pairs of domains can pack together with the antigen-binding sites pointing in opposite directions. The dimeric antibody fragments, or "diabodies," can be designed for bivalent or bispecific interactions. Starting from the monoclonal antibodies NQ11.7.22 (NQ11) and D1.3 directed against the hapten phenyloxazolone and hen egg lysozyme, respectively, we built bivalent fragments (VHNQ11-VLNQ11)2 and (VHD1.3-VLD1.3)2 and bispecific fragments VHNQ11-VLD1.3 and VHD1.3-VLNQ11. The fragments were expressed by secretion from bacteria and shown to bind specifically to the hapten and/or antigen. Those with 5- and 15-residue linkers had similar binding affinities to the parent antibodies, but a fragment with the VH domain joined directly to the VL domain was found to have slower dissociation kinetics and an improved affinity for hapten. Diabodies offer a ready means of constructing small bivalent and bispecific antibody fragments in bacteria.     

Clinical utility of immunoglobulin heavy chain gene rearrangement identification for tumour cell detection in multiple myeloma

To clarify the cellular origin of de novo CD5+ diffuse large B-cell lymphoma (CD5+ DLBL), particularly in comparison with other CD5+ B-cell neoplasms such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), we analyzed the nucleotide sequence of the Ig heavy chain variable region (IgVH) genes of de novo CD5+ DLBL cases. All 4 cases examined had extensive somatic mutations in contrast with CLL or MCL. The VH gene sequences of de novo CD5+ DLBL displayed 86.9% to 95.2% homology with the corresponding germlines, whereas those of simultaneously analyzed CLL and MCL displayed 97.6% to 100% homology. The VH family used was VH3 in 1 case, VH4 in 2 cases, and VH5 in 1 case. In 2 of 4 examined cases, the distribution of replacement and silent mutations over the complementarity determining region and framework region in the VH genes was compatible with the pattern resulting from the antigen selection. Clinically, CD5+ DLBL frequently involved a variety of extranodal sites (12/13) and lymph node (11/13). Immunophenotypically, CD5+ DLBL scarcely expressed CD21 and CD23 (3/13 and 2/13, respectively). These findings indicate that de novo CD5+ DLBL cells are derived from a B-1 subset distinct from those of CLL or MCL.     

A comparison of the crystallographic structures of two catalytic antibodies with esterase activity

The crystallographic structure of the Fab fragment of the catalytic antibody, 29G11, complexed with an (S)-norleucine phenyl phosphonate transition state analog was determined at 2.2 A resolution. The antibody catalyzes the hydrolysis of norleucine phenyl ester with (S)-enantioselectivity. The shape and charge complementarity of the binding pocket for the hapten account for the preferential binding of the (S)-enantiomer of the substrate. The structure is compared to that of the more catalytically efficient antibody, 17E8, induced by the same hapten transition state analog. 29G11 has different residues from 17E8 at eight positions in the heavy chain, including four substitutions in the hapten-binding pocket: A33V, S95G, S99R and Y100AN, and four substitutions at positions remote from the catalytic site, I28T, R40K, V65G and F91L. The two antibodies show large differences in the orientations of their variable and constant domains, reflected by a 32 degrees difference in their elbow angles. The VL and VH domains in the two antibodies differ by a rotation of 8.8 degrees. The hapten binds in similar orientations and locations in 29G11 and 17E8, which appear to have catalytic groups in common, though the changes in the association of the variable domains affect the precise positioning of residues in the hapten-binding pocket.     

Anti-CD40 plus interleukin-4-activated human naive B cell lines express unmutated immunoglobulin genes with intraclonal heavy chain isotype variability

Combination of anti-CD40 antibody and interleukin-4 (IL-4) induces B cell clonal expansion reminiscent of the T-dependent proliferation following antigenic challenge in vivo. We have analyzed the usage of CH genes and the presence or absence of somatic mutations within the progeny of a single human naive B cell activated with anti-CD40 + IL-4. To address this issue, single-cell cultures of naive (sIgD+) tonsillar B lymphocytes expressing the VH1-restricted G8 idiotype were set up. After culture and RNA extraction, VH1+ Ig mRNA were reverse-transcribed, amplified by polymerase chain reaction and sequenced. A single sIgD+ B cell could generate clones expressing mu, gamma 1, gamma 3, or epsilon, illustrating that the progeny of a single cell can express different isotypes in response to the same stimulus in vitro. The rate of somatic mutations affecting the immunoglobulin variable heavy chain gene was indistinguishable from the background of errors introduced by Taq polymerase.     

Stepwise in vitro affinity maturation of Vitaxin, an alphav beta3-specific humanized mAb

A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based mutagenesis was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the alphav beta3 integrin complex, was used as a model system. Specifically, focused mutagenesis was used in a stepwise fashion to rapidly improve the affinity of the antigen binding fragment by greater than 90-fold. In the complete absence of structural information about the Vitaxin-alphav beta3 interaction, phage-expressed antibody libraries for all six Ig heavy and light chain complementarity-determining regions were expressed and screened by a quantitative assay to identify variants with improved binding to alphav beta3. The Vitaxin variants in these libraries each contained a single mutation, and all 20 amino acids were introduced at each complementarity-determining region residue, resulting in the expression of 2,336 unique clones. Multiple clones displaying 2- to 13-fold improved affinity were identified. Subsequent expression and screening of a library of 256 combinatorial variants of the optimal mutations identified from the primary libraries resulted in the identification of multiple clones displaying greater than 50-fold enhanced affinity. These variants inhibited ligand binding to receptor more potently as demonstrated by inhibition of cell adhesion and ligand competition assays. Because of the limited mutagenesis and combinatorial approach, Vitaxin variants with enhanced affinity were identified rapidly and required the synthesis of only 2,592 unique variants. The use of such small focused libraries obviates the need for phage affinity selection approaches typically used, permitting the use of functional assays and the engineering of proteins expressed in mammalian cell culture.     

Generation of a panel of related human scFv antibodies with high affinities for human CEA

BACKGROUND: A human single chain Fv (scFv) specific for human carcinoembryonic antigen (CEA) has been isolated from a 2.0 x 10(9) phage display library from unimmunised human donors. The dissociation constant of the scFv has been measured by surface plasmon resonance (SPR) and found to be 7.7 x 10(-9) M, with an off-rate component of 6.2 x 10(-3) s-1. In order to investigate directly whether increased affinity leads to improved targeting of CEA-positive tumours, this scFv has been affinity matured by both targeted mutagenesis of the CDRs of heavy and light chains, and by light chain shuffling. STUDY DESIGN: A partial randomisation scheme, biased towards amino acids commonly found as somatic mutations of germline antibody sequences, was used for directed diversification of VH and VL CDR3s. Diversification of the entire VL region was also introduced by light chain shuffling of the parental anti-CEA scFv. Selection of the mutagenised repertoires was carried out to enrich for antibodies with a reduced koff. RESULTS: Sequencing the selected clones identified a number of amino acid changes in the VH CDR3, one of which gave a four-fold reduction in koff. Stringent selection of the light chain shuffled library resulted in several clones with a two- to three-fold reduction in koff. It has been possible to combine the selected changes from both mutagenesis approaches by using the mutagenised heavy chain and a light chain derived by shuffling to give a human scFv with a dissociation constant for human CEA of 6.0 x 10(-10) M. CONCLUSION: A panel of human anti-CEA scFvs has been generated with differing dissociation constants for antigen, which will allow the correlation between tumour targeting efficiency in relation to binding affinity to be assessed directly. The scFv panel will be valuable in the optimisation of human antibodies for immunotherapy.     

Specific V gene usage in anti-phosphorylcholine IgE antibodies

Three hybridomas from phosphorylcholine(PC)-KLH immunized BALB/c mice producing IgE antibodies against the PC hapten were investigated for their fine specificity to the hapten and usage of V gene segments in H- and L-chains. All three IgE antibodies recognize the entire azophenyl-PC hapten. They are T15 Id negative and do not bind to the natural PC determinant expressed by the Streptococcus carbohydrate R36A. T15 Id positive IgE antibodies could neither be elicited by immunization in detectable amounts nor generated by the cell fusion technique. By using the Southern blot technique and nucleotide sequence analysis of PCR amplified VHDJH and VLJL rearrangements, we have demonstrated that the three IgE anti-PC hybridomas use the VH1-DSP2-JH2, the VHOX1-DSP2-JH3 or the VH36-60-D-JH2 gene segment combinations for the H chain together with the V kappa 1C-J kappa 1, V kappa 1C-J kappa 2 or V lambda 1-J lambda 1 genes for the L chains. Except for the VH36-60, the same gene segments were found in different combinations in anti-PC antibodies of other Ig classes than IgE. However, high rates of somatic mutations are expressed in both VH1 of the H chain and in V kappa 1C of the L chain. The VH36-60 is expressed in antibodies with the major Id of the azophenyl-arsonate (Ars) response and VHOX1 generally contributes to the phenyl-oxazolone specificity. This suggests that these V genes are involved in the recognition of the azophenyl moiety of the coupled PC hapten. Thus PC-KLH specific IgE antibodies utilize mutated VH1 and/or VH/VL gene segment combinations which are involved in binding of the azophenyl spacer. These IgE are therefore specific for azophenyl-phosphorylcholine, unlike antibodies normally expressed against the Streptococcus PC determinant in mice. The genetic diversity and the high mutation rates indicate that the specific B cells develop later in the immune response. Thus, they represent newly generated specificities of so-called group II anti-PC antibodies and are not isotype-switch descendants from already existing T15 Id positive IgM antibodies.     

Preferential use of the VH4 Ig gene family by diffuse large-cell lymphoma

Ig heavy chain variable region (VH) genes expressed by human diffuse large-cell lymphoma (DLC) and follicular lymphoma (FL) were identified and analyzed with respect to germline gene families. In 67 cases of FL, VH region genes were expressed in a pattern similar to that of normal B cells, with a predominance of the large VH3 gene family being used. In contrast, of the 17 cases of DLC, there was an extremely biased use of VH genes. Of these DLC tumors, 88% expressed genes from the small VH4 gene family; and even among these tumors, there was a limited use of genes, with 11 cases producing Igs derived from the VH4.21 germline gene. Although most of the VH genes expressed by DLC tumor cells contained mutations with respect to their germline counterparts, almost all of these mutations occurred before the clonal expansion of the tumor. This contrasts with our previous findings of ongoing mutations in FL and represents a fundamental difference between these two malignancies. This preferential gene use implies an important role for the VH4 gene family, and specifically for VH4.21, in the genesis of DLC.     

Directing phage selections towards specific epitopes

It is possible to direct selections from antibody repertoires displayed on filamentous phage towards unique epitopes on protein antigens by competing with related molecules. A phage display repertoire of human single chain Fvs (scFvs) was panned three times against foetal haemoglobin (HbF). The selection was dominated by one clone with a Kd of 10 nM but yielded at least 17 others, all of which bound HbF but crossreacted with adult haemoglobin (HbA). To direct selection towards HbF-specific epitopes, the repertoire was preincubated with HbA in solution before each panning. Crossreactive scFvs can form complexes with the soluble HbA and thereby be prevented from binding the immobilized HbF. Four clones with preferential binding to HbF emerged under these conditions. One of these (Hb-1), with a Kd of 6 microM, had exquisite specificity for HbF and could distinguish cells expressing HbF from those expressing HbA by immunocytochemistry and flow cytometry. This antibody has an affinity that is 600-fold lower than the dominant crossreactive clone, and so only emerged under conditions of 'competitive deselection'. Thus, competitive deselection is a viable means for directing selections towards useful epitopes. It permits a more effective 'search' of phage display repertoires and allows the emergence of lower affinity clones with useful specificities. These clones may be useful in themselves or may serve as leads for in vitro affinity maturation.     

Coupling protein design and in vitro selection strategies: improving specificity and affinity of a designed beta-protein IL-6 antagonist

The minibody is a designed small beta-protein conceived to enable the construction of large libraries of minimal discontinuous epitopes displayed on the surface of filamentous phage. The 61 residue molecule consists of three strands from each of the two beta-sheets of the variable domain of immunoglobulins packed face to face, along with the exposed H1 and H2 hypervariable regions. We have previously shown that from a minibody repertoire of more than 50 million molecules displayed on phage, we were able to select a minibody with micromolar affinity for human interleukin-6 that behaves as a selective cytokine antagonist. The minibody exposes a surface composed of two constrained loops, which provides the possibility of improving IL-6 binding and specificity by swapping the hypervariable regions, followed by further selection. We established experimental conditions for "stringent" selection such as monovalent phage display, competitive selection and epitope masking. Here, we show that by virtue of the optimization/selection process, we have isolated a minibody with improved antagonistic potency and greater specificity. Furthermore, using hIL-6 mutants carrying amino acid substitutions in distinct surface sites it was possible to carefully define the cytokine region that binds the minibody.     

Humanization of an anti-human IL-6 mouse monoclonal antibody glycosylated in its heavy chain variable region

Interleukin-6 (IL-6) inhibitors are good potential therapeutic agents in human patients, and anti-IL-6 antibodies are among the best candidates. Here, we have successfully humanized mouse monoclonal antibody SK2, which specifically binds to IL-6 and strongly inhibits IL-6 functions. Since this antibody possesses N-linked carbohydrates on Asn-30 of VH region, which seems to be very close to an antigen-binding site, influence of these carbohydrates on antigen-binding was investigated. A biosensor study showed that the mouse SK2 Fab and its deglycosylated fragments had almost equal Kd (Kon/Koff), 26.8 nM (1.05 x 10(6)/2.81 x 10(-2)) and 24.7 nM (1.28 x 10(6)/3.15 x 10(-2)), respectively. Furthermore, a mutant chimeric SK2 antibody, in which the N-glycosylation site was removed from the VH region, showed a Kd of 11 nM, almost similar to that of the original chimeric SK2 antibody, determined by Scatchard analysis with 125I-IL-6. These data indicate the carbohydrates of mouse SK2 VH region do not significantly influence antigen-binding activity. In the next step, two versions of each humanized SK2 VL and VH regions were carefully designed based on the amino acid sequences of human REI and DAW, respectively. Only one alteration, Tyr to Phe, was made at position 71 in the two light chains, according to the canonical residue for LI. A N-glycosylation site was introduced on the two heavy chains, by changing Ser to Asn at position 30. All four combinations of humanized light and heavy chains could bind to IL-6 as well as the chimeric SK2 antibody. The light chain first version, however, could not efficiently inhibit IL-6 binding to its receptor, indicating the importance of the LI loop conformation for the inhibitory activity of SK2 antibody. In contrast, both versions of the heavy chains were comparable, in yielding good humanized SK2 antibodies, suggesting that the glycosylation of the SK2 VH region has no influence in recreating a functional antigen-binding site in this humanization.     

Molecular basis for the interaction between human IgM and staphylococcal protein A

To examine the relationship between VH gene usage and reactivity of immunoglobulins, we cloned B cells from peripheral blood from adults and from human neonatal cord blood by EBV transformation. Nearly one-third of the B cell clones from both sources produced IgM reactive with staphylococcal protein A (SPA). None of such IgM reacted with other antigens, except for the crude extract of Staphylococcus aureus. All of 22 B cell clones producing IgM reactive with SPA expressed VH3 genes, while none of the control 15 clones secreting IgM nonreactive with SPA expressed VH3. The IgM proteins reactive with SPA could be clearly divided into two subjects based on the differential binding avidity to solid-phase SPA. Both kappa and lambda light chains were used in each subset of SPA-reactive IgM. Sequence analysis of the PCR products from seven VH3-IgM clones revealed that the VH3 genes were used in nearly germline configuration. The D and J gene usage was diverse. Comparison of amino acid sequences between antibodies with high and low avidity to SPA suggests that the differential avidity is related to amino acid sequence differences in the complementarity determining region 2 and framework region 3. The high frequencies of B cells committed to the production of SPA-reactive IgM in normal blood and the restricted use of VH3 heavy chain genes in nearly germline configuration in these cells support the notion that SPA behaves like a superantigen toward human B cells.     

Heavy chain variable region, light chain variable region, and heavy chain CDR3 influences on the mono- and polyreactivity and on the affinity of human monoclonal rheumatoid factors

Monoreactive high affinity pathologic autoantibodies were supposed previously to derive through somatic mutation from polyreactive low affinity autoantibodies that are encoded by a small set of unmutated V region genes in fetal and neonatal B cells. However, recent data exploring the physiologically expressed Ab repertoire and the importance of the stochastically generated heavy chain CDR3 (H-CDR3) in autoreactivity suggest that this scheme is incomplete. Here we analyzed via gene-swapping experiments and site-directed mutagenesis the relative contributions of the mutations in the light chain variable region (VL) and the heavy chain variable region (VH) domains and of the H-CDR3 in the autoreactivity of two IgM rheumatoid factors (RF), one a polyreactive low affinity Ab, the other a monoreactive high affinity Ab. These two RFs derived from the same V kappa III (humkv325) and VH1 (51p1) genes, but differed from each other by a few mutations and by the structure of the H-CDR3. The analysis of the reactivity patterns of different combinations of wild-type and in vitro engineered hybrid gene products clearly demonstrates the main influence of the H-CDR3 in the autoAb activity profiles. The results directly demonstrate the previously proposed hypothesis, namely, that the H-CDR3 plays a critical role in distinguishing poly- from monospecific RF. However, the data also indicate that self polyreactivity is a very fragile property and is dependent upon the primary structure of the VH segment.