Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. II. IgG antibodies contain VH genes from a single VH family and VL genes from at least four VL families

To define the V gene family repertoire of human IgG anti-Haemophilus influenzae type b polysaccharide antibodies, we purified six IgG1 and nine IgG2 anti-Hib-PS antibodies to monoclonality from immune serum of six individuals and performed N-terminal amino acid sequence analysis. Of the 15 clonal antibodies we examined, all H chain V regions were of the VHIII family. In contrast, the L chains of these antibodies were clearly from at least four different VL families; VKI, VKII, VKIII, and V lambda. Interestingly. VL family expression correlated with the cross-reactivity of these antibodies to the capsular carbohydrate of Escherichia coli K100. VKII antibodies did not cross-react, whereas antibodies expressing V lambda, VKI, or VKIII generally cross-reacted. We conclude that L chain V regions are very important contributors to the limited heterogeneity in this antibody repertoire.     

VH and VL gene usage by murine IgG antibodies that bind autologous insulin

To assess the recognition structures of antibodies that bind a self-Ag, we used mRNA analysis to identify the V region genes of IgG antibodies that bind autologous insulin. Four anti-insulin mAb from primary immunization of BALB/c mice use different combinations of H and L chain V region genes. Two VH genes are from the V-gam 3-2 and V-gam 3-8 families that are infrequently expressed in adult BALB/c mice, and two VH genes are members of the J558 family. Each anti-insulin antibody uses a different Vk gene family. Two antibodies express common Vk genes (Ox1 and Vk21C), whereas two other Vk genes are unusual in BALB/c mice. One Vk gene may represent a BALB/c equivalent of the VkOx2 subfamily and another is identical to a Vk used by anti-idiotypic antibodies from C57Bl/6 mice. When compared with known germ-line counterparts, all of the Vk sequences are close to germ-line configuration. In contrast, the germ-line counterparts for the anti-insulin VH genes are not known, however, they differ only in five to seven predicted amino acids from VH of other expressed antibodies. One antibody (mAb 123) differs in one amino acid in complementarity-determining regions 1 and 2 from the VH of the murine tumor BCL1, and another (mAb 126) employs an unmutated DFL16.1 germ-line D segment. These data suggest that antibodies binding autologous insulin use V gene components that are not extensively mutated, even when derived by immunization with heterologous insulin.     

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.     

Specific interaction between VH and VL regions of human monoclonal immunoglobulins

Preferential reassociation of immunoglobulin H and L chains was investigated using a method of competitive hybridization. A model was built in which two monoclonal light chains, one autologous, L_A, and one heterologous, L_B, were competitively reassociated with a given monoclonal heavy chain, H_A. A total of 12 human myeloma proteins were used with known isotypic, allotypic and variability subgroups: 44 distinct combinations were studied by competitive hybridizations. It was found that a preferential reassociation occurred between the complementary H and L chains that were associated in the native molecule in 80% of the cases. It was clearly established that the subgroups had no influence on the preferential reassociations that seem, therefore, to rely exclusively on individual (“idiotypic”) structural differences. It was shown that, although H and L chains had been fully reduced and denatured, the same degree of preferential reassociation was observed after the chains had been reoxidized and refolded. These experiments suggest, therefore, that the observed preferential reassociations are the consequence of an antigen-independent selection process that must have taken place during the differentiation of the antibody-forming cells.     

VL:VH domain rotations in engineered antibodies: Crystal structures of the Fab fragments from two murine antitumor antibodies and their engineered human constructs

The crystal structures of two pairs of Fab fragments have been determined. The pairs comprise both a murine and an engineered human form, each derived from the antitumor antibodies A5B7 and CTM01. Although antigen specificity is maintained within the pairs, antigen affinity varies. A comparison of the hypervariable loops for each pair of antibodies shows their structure has been well maintained in grafting, supporting the canonical loop model. Detailed structural analysis of the binding sites and domain arrangements for these antibodies suggests the differences in antigen affinity observed are likely to be due to inherent flexibility of the hypervariable loops and movements at the V_L:V_H domain interface. The four structures provide the first opportunity to study in detail the effects of protein engineering on specific antibodies. Proteins 29:161–171, 1997. © 1997 Wiley-Liss, Inc.     

A single VH gene is utilized predominantly in anti-BrMRBC hybridomas derived from purified Ly-1 B cells. Definition of the VH11 family

By establishing hybridomas from two distinct surface IgM+ splenic B cell populations, Ly-1 B cells and "conventional" (Ly-1-) B cells, we found that the Ly-1 B population includes a 30 to 70 times higher frequency (1 to 2%) of cells with specificity for bromelain treated autologous red blood cells (anti-BrMRBC) when compared with conventional B cells (0.03%). We cloned and sequenced the V genes encoding anti-BrMRBC antibody from two hybridomas made with Ly-1 B cells sorted from the spleen of SM/J mice. The VH sequence (for both) is identical with the previously reported sequence associated with this specificity and belongs to a new VH gene family. This gene family, defined here as VH11, has only two members and is the predominant VH rearranged in a collection of Ly-1 B derived anti-BrMRBC hybridomas, always in association with a single VL gene (a member of the V kappa 9 family). Furthermore, analysis of hybridomas made with Ly-1 B cells sorted from the peritoneum reveals a yet higher increased frequency of VH11-encoded anti-BrMRBC specificity (30%). This variation in frequency of anti-BrMRBC in the Ly-1 population depending on location, together with the repeated association of VH11 with a particular V kappa gene suggest that antigen driven selection is (at least in part) responsible for the biased V gene expression seen in this population. Furthermore, a mechanism that might contribute to biased expression, preferential rearrangement due to close proximity to J (as seen in pre-B lines), is excluded by localization of VH11 5' to several of the more J-proximal families (Q52, 7183).     

Ten percent of normal B cells and plasma cells share A VH determinant(s) (J606-GAC) with a distinct subset of murine VHIII plasmacytomas

Our findings indicate that a subset of VHIII antibodies, which we refer to as J606-GAC, contains a determinant(s) that is present on 5 to 15% of normal splenic B cells and plasma cells as detected by immunofluorescence. This subpopulation is detected by purified antibody, 0-1, which was prepared against a murine anti-group A carbohydrate (anti-GAC) hybridoma antibody. The J606-GAC subset includes the beta 2, 1 fructosan myelomas J606, EPC109, W3082, ABPC4, and UPC61, as well as 13 anti-GAC hybridomas. The 0-1 antiserum failed to react with hybridoma and myeloma immunoglobulins from murine VH groups I and II or other VHIII antibodies. By Western blot analysis, it was observed to react with isolated heavy, but not light, chains of J606-GAC-bearing antibodies. 0-1 failed to react with myelomas XRPC44 and J539, which have the same J region as J606 but a very different VH region. These observations indicate that 0-1 is detecting a VH region determinant. The J606-GAC marker recognized by 0-1 was expressed as early as 4 days after birth and was expressed at similar frequencies in germfree and conventional mice. Immunoprecipitation of both surface and biosynthetically labeled proteins from spleen cells or J606-GAC-positive hybridoma cell lines, respectively, confirmed that 0-1 was recognizing an immunoglobulin determinant.     

Rescue of HIV-1 broad neutralizing antibody-expressing B cells in 2F5 VH x VL knockin mice reveals multiple tolerance controls

The HIV-1 broadly neutralizing Ab (bnAb) 2F5 has been shown to be poly-/self-reactive in vitro, and we previously demonstrated that targeted expression of its VDJ rearrangement alone was sufficient to trigger a profound B cell developmental blockade in 2F5 V(H) knockin (KI) mice, consistent with central deletion of 2F5 H chain-expressing B cells. In this study, we generate a strain expressing the entire 2F5 bnAb specificity, 2F5 V(H) × V(L) KI mice, and find an even higher degree of tolerance control than observed in the 2F5 V(H) KI strain. Although B cell development was severely impaired in 2F5 V(H) × V(L) KI animals, we demonstrate rescue of their B cells when cultured in IL-7/BAFF. Intriguingly, even under these conditions, most rescued B cell hybridomas produced mAbs that lacked HIV-1 Envelope (Env) reactivity due to editing of the 2F5 L chain, and the majority of rescued B cells retained an anergic phenotype. Thus, when clonal deletion is circumvented, κ editing and anergy are additional safeguards preventing 2F5 V(H)/V(L) expression by immature/transitional B cells. Importantly, 7% of rescued B cells retained 2F5 V(H)/V(L) expression and secreted Env-specific mAbs with HIV-1-neutralizing activity. This partial rescue was further corroborated in vivo, as reflected by the anergic phenotype of most rescued B cells in 2F5 V(H) × V(L) KI × Eμ-Bcl-2 transgenic mice and significant (yet modest) enrichment of Env-specific B cells and serum Igs. The rescued 2F5 mAb-producing B cell clones in this study are the first examples, to our knowledge, of in vivo-derived bone marrow precursors specifying HIV-1 bnAbs and provide a starting point for design of strategies aimed at rescuing such B cells.     

Recognition of DNA by VH and Fv domains of an IgG anti-poly(dC) antibody with a singly mutated VH domain

Secondary antigen stimulation usually produces IgG antibodies with hypermutated V segments. Studying a strong secondary response to the polynucleotide antigen poly(dC), however, we found a highly selective IgG antibody (mAb dC7) with only one mutation (a conservative Leu to Ileu substitution) throughout the whole VH domain. To investigate the roles of VH and VL domains in selective binding by this mAb, we prepared its VH, VL and single-chain Fv (scFv) fragments. A bacterial expression system produced soluble monomeric V region proteins. CD spectra confirmed that they had the beta-secondary structure expected for Ig domains. Both the scFv and VH fragments bound to single-stranded non-protonated poly(dC) and to ssDNA but not to protonated, more structured poly(dC) or dsDNA. The VL domain alone did not bind to nucleic acids, but VL association modified the VH binding, giving the scFv a 10-fold higher affinity than the VH for poly(dC) and greatly increasing the cytosine-dependent selectivity. Non-ionic interactions were prominent in the Fv reaction with a (dC)( n) sequence. Ionic interactions were revealed in Fv cross-reactions with ssDNA, and were more prominent in binding of either poly(dC) or ssDNA by VH alone, consistent with the lesser base selectivity of the VH. Thus, the Fv and VH alone bind to a single antigen, poly(dC), but mechanistic differences result from additional subsites in the Fv. Generation of a selective IgG with very few CDR mutations in either VH or VL, which was accompanied by IgM antibodies with unmutated V regions, also suggests that nucleic acid binding activity is a property of the B cell repertoire even before immunization.     

Probing VH gene-family utilization in human peripheral B cells by in situ hybridization

Recent studies on the genomic organization of the human Ig VH locus have revealed the presence of an important proportion of VH pseudogenes and a high degree of interspersion among VH gene-family members. The mechanisms of selection of VH genes expressed by differentiating B cells remain to be elucidated. We have made use of RNA-RNA in situ hybridization to probe the repertoire of VH gene-families assembled in peripheral B cells of normal adults. Cells were in vitro activated with mitogens and then hybridized to [35S]-labeled anti-sense RNA probes specific for C mu and C gamma genes, and for the six known human VH gene-families. We found that the numbers of cells expressing C mu and C gamma mRNA were similar to the total numbers of cells expressing members of the six VH gene-families. Therefore, the six known VH gene-families represent essentially the human VH locus. We also found that expression of VH gene-families does not closely correlate with their relative genomic complexity. This apparently biased expression may suggest that in some VH gene-families the ratio of pseudogenes/functional genes is particularly high, and/or that regulatory mechanisms play a major role in shaping the available VH gene repertoire in differentiating B cells.     

Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals

Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.     

Amino acid sequence of the VH region of a human myeloma immunoglobulin (IgG New).

The amino acid sequence of the heavy-chain variable region of the human immunoglobulin. New has been determined. Since the amino terminus of the heavy chain was blocked, the sequence of residues 1-69 was established by digesting the appropriate CNBr fragment separately with trypsin, chymotrypsin, and thermolysin and sequencing the resulting peptides. The region from residues 70 to 120 was present in another CNBr fragment which was submitted directly to automatic Edman degradation. The result of this experiment extended the sequence to residue 100. The primary structure of the remaining portion of the VH region was determined by automatic Edman degradation of a lysine-blocked tryptic peptide derived from this region which included residues 98-214. The sequence of the VH region of New corresponds most closely to VH sequences of proteins in the VH II subgroup. This primary structure makes it possible to construct a model from the high-resolution electron-density map of protein New.     

Improved Protein-A separation of VH3 Fab from Fc after Papain Digestion of Antibodies

Antibody-binding fragments (Fab) are generated from whole antibodies by treatment with papain and can be separated from the Fc component using Protein-A affinity chromatography. Commercial kits are available, which facilitate the production and purification of Fab fragments; however, the manufacturer fails to report that this method is inefficient for antibodies with V_H3 domains as a result of the intrinsic variable region affinity for Protein-A. A commercially available, modified Protein-A resin (MabSelect SuRe) has been engineered for greater stability. Here, we report that an additional consequence of the modified resin is the ability to purify V_H3 family Fab fragments, which cannot be separated effectively from other components of the papain digest by traditional Protein-A resin. This improvement of a commonly used procedure is of significance, as increasingly, therapeutic antibodies are being derived from human origin, where V_H3 is the most abundantly used variable region family.     

Chimeric B72.3 mouse/human (IgG1) antibody directs the lysis of tumor cells by lymphokine-activated killer cells

Summary Chimeric mouse/human B72.3 (cB72.3) antibodies having a human IgG1 (1) or IgG4 (4) constant region were compared to the native murine IgG1 B72.3 (nB72.3) monoclonal antibody (mAb) for their ability to participate with human effector cells in antibody-dependent cellular cytotoxicity (ADCC). Because the TAG-72 antigen recognized by B72.3 is poorly expressed on tissuecultured tumor cell lines, the xenografted OVCAR-3 human ovarian carcinoma ascites was used as a cytotoxicity target. The lytic activity of the cB72.3(1) mAb with peripheral blood lymphocytes was 1.5- to 50-fold greater than that of the nB72.3 mAb and usually the cB72.3(4) mAb. However, lymphocytes from some donors had similar ADCC activity with either the cB72.3(1) or cB72.3(4) mAb. The cB72.3(1) and the murine anti-colon carcinoma CO17-1A mAb had comparable activity in mediating ADCC against the OVCAR-3 tumor. Exposure of lymphoid cells to interleukin-2 (IL-2) (100–500 U/ml) for 24 h to generate lymphokine-activated killer (LAK) cells augmented ADCC mediated by the cB72.3(1) mAb 2- to 22-fold. By contrast, LAK cells from most donors expressed weak non-specific cytotoxicity against OVCAR-3 ascites tumor cells. The cB72.3(1), and to a lesser extent, the cB72.3(4) chimera also participated with monocytes in mediating ADCC, but the antibody-dependent lytic potency of monocytic effectors was much weaker than that of IL-2-activated lymphoid cells. These studies show that the cB72.3(1) mAb has appreciable ADCC-mediating properties, suggesting a potential role for its incorporation into treatment strategies utilizing adoptive killer cell and/or lymphokine therapy. Offprint requests to: J. Schlom, to whom reprint requests should be sent at 9000 Rockville Pike, Building 10, Room 8B07, Bethesda, MD 20892, USA     

High-affinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold

Phage-displayed synthetic antibody libraries were built on a single human framework by introducing synthetic diversity at solvent-exposed positions within the heavy chain complementarity-determining regions (CDRs). The design strategy of mimicking natural diversity using tailored codons had been validated previously with scFv libraries, which produced antibodies that bound to antigen, murine vascular endothelial growth factor (mVEGF), with affinities in the 100nM range. To improve library performance, we constructed monovalent and bivalent antigen-binding fragment (Fab) libraries, and explored different CDR-H3 diversities by varying the amino acid composition and CDR length. A Fab with sub-nanomolar affinity for mVEGF was obtained from a library with CDR-H3 diversity designed to contain all 20 naturally occurring amino acids. We then expanded the library by increasing the variability of CDR-H3 length and using tailored codons that mimicked the amino acid composition of natural CDR-H3 sequences. The library was tested against a panel of 13 protein antigens and high-affinity Fabs were obtained for most antigens. Furthermore, the heavy chain of an anti-mVEGF clone was recombined with a library of light chain CDRs, and the affinity was improved from low nanomolar to low picomolar. The results demonstrated that high-affinity human antibodies can be generated from libraries with completely synthetic CDRs displayed on a single scaffold.     

IgG subclass expression by human B lymphocytes and plasma cells: B lymphocytes precommitted to IgG subclass can be preferentially induced by polyclonal mitogens with T cell help

The human IgG subclasses expressed by circulating B lymphocytes, tissue plasma cells, and plasma cells generated from B cell precursors in response to the polyclonal mitogens LPS and PWM were examined by immunofluorescence using subclass-specific monoclonal antibodies. The subclass distribution observed for circulating B lymphocytes was IgG2 (48%) greater than IgG1 (40%) greater than IgG3 (8%) greater than IgG4 (1%), while the distribution among IgG plasma cells in bone marrow, blood, spleen, and tonsils was IgG1 (64%) greater than IgG2 (26%) greater than IgG3 (8%) greater than IgG4 (1%). Multiple IgG isotypes were not observed on B cells or in plasma cells. Although IgG plasma cell responses to both LPS and PWM were T cell dependent, the distributions of IgG subclasses elicited were strikingly different. In control and LPS-stimulated cultures of blood mononuclear cells, the induced plasma cells expressed the IgG subclass distribution: IgG2 greater than 80%, IgG1 less than 20%, IgG3 less than 1%, IgG4 less than 1%. In PWM-stimulated cultures, the subclass distribution, IgG1 approximately 65%, IgG2 approximately 25%, IgG3 approximately 7%, IgG4 approximately 1%, was in perfect concordance with the in vivo subclass distribution of IgG plasma cells. Selective inhibition of suppressor T cell activity by x-irradiation and mitomycin C treatment did not alter the IgG subclass distribution pattern induced by LPS and PWM. Monoclonal antibodies were used to deplete selectively the B cell precursors bearing IgG1, IgG2, or IgG3 before PWM stimulation of blood mononuclear cells. In each instance, a reduction was observed only in the subpopulation of plasma cells producing the homologous IgG subclass. The results indicate that T cells can preferentially influence the terminal differentiation of B cells that are precommitted to different IgG subclasses.     

Impact of HIV-1 infection on VH3 gene repertoire of naive human B cells

B cells of the largest Ig variable heavy chain gene (VH) family, VH3, are reportedly decreased in patients with late stage HIV-1 disease. This deficit may contribute to their impaired responses to infections and vaccines. We confirmed that the VH3 family was underrepresented in serum IgM proteins, with a 45% decrease in patients with advanced HIV-1 disease. However, the proportion of VH3 within VH(1-6) IgM mRNA from peripheral B cells did not differ from that of control subjects (mean +/- SD, 57.1 +/- 9.7 vs 61.1 +/- 8. 7%). Similarly, within VH(1-6) IgD mRNA, which even more closely represents the unstimulated naive repertoire, the relative expression of VH3 mRNA was comparable in the two groups. Moreover, the frequency of individual genes within the VH3 family for IgD, particularly genes which encode putative HIV-1 gp120 binding sites, also was normal in HIV-1-infected patients. However, VH3 family expression for IgG mRNA was significantly decreased (17%) and VH4 IgG was increased (33%) relative to other VH families in advanced HIV-1-infected patients. Thus, the changes in VH family expression were more readily apparent in previously activated IgG "memory" B cell populations and, likely, in cells actively producing IgM rather than in resting naive cells. The presence of a relatively normal naive VH3 IgM and IgD mRNA repertoire in resting cells supports the prospect that with proper stimulation, particularly in conjunction with effective antiviral therapy, vigorous humoral immune responses to infections and vaccines may be elicited in this high-risk population.