Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.     

Guided selection of a pan carcinoma specific antibody reveals similar binding characteristics yet structural divergence between the original murine antibody and its human equivalent

Antibody engineering provides an excellent tool for the generation of human immunotherapeutics for the targeted treatment of solid tumours. We have engineered and selected a completely human antibody to epithelial glycoprotein-2 (EGP-2), a transmembrane glycoprotein present on virtually all human simple epithelia and abundantly expressed on a variety of human carcinomas. We chose to use the procedure of "guided selection" to rebuild a high-affinity murine antibody into a human antibody, using two consecutive rounds of variable domain shuffling and phage library selection. As a starting antibody, the murine antibody MOC-31 was used. After the first round of guided selection, where the V(H) of MOC-31 was combined in Fab format with a human V(L)C(L) library, a small panel of human light chains was identified, originating from a segment of the VkappaIII family, whereas the MOC-31 V(L) is more homologous to the VkappaII family. Nevertheless, one of the chimaeric Fabs, C3, displayed an off-rate similar to MOC-31 scFv. Combining the V(L) of C3 with a human V(H) library, while retaining the V(H) CDR3 of MOC-31, clones were selected using human V(H) genes originating from the rarely used V(H)7 family. The best clone, 9E, shows over 13 amino acid mutations from the germline sequence, has an off-rate comparable to the original antibody and specifically binds to the "MOC-31"-epitope on EGP-2 in specificity and competition ELISA, FACS analysis and immunohistochemistry. In both V(L) and V(H) of antibody 9E, three germline mutations were found creating the MOC-31 homologue residue. Structural modelling of both murine and human antibodies reveals that one of the germline mutations, 53Y in V(H) CDR2, is likely to be involved in antigen binding. We conclude that, although they may bind the same epitope and have similar binding affinity to the antigen as the original murine antibody, human antibodies derived by guided selection unlike CDR-grafted antibodies, may retain only some of the original key elements of the binding site chemistry. The selected human anti-EGP-2 antibody will be a suitable reagent for tumour targeting.     

The ability of cytotoxic T cells to recognize HLA-A2.1 or HLA-B7 antigens expressed on murine cells correlates with their epitope specificity

Two groups of human and murine cytotoxic T lymphocyte (CTL) clones specific for human leukocyte antigen (HLA)-A2 or -B7 can be distinguished based on their ability to kill murine transfectants expressing these molecules. The clones which do not recognize murine transfectants exhibited greatly reduced conjugate formation with these cells, indicating that the inability to lyse these cells occurs in recognition and binding. No systematic differences in inhibitory titer between the two types of CTL clones were seen with anti-CD8 (Lyt-2), anti-LFA-1, or monoclonal antibodies against HLA class I molecules. However, blocking with anti-HLA class I monoclonal antibodies suggested that different CTL clones recognized spatially separate epitopes on HLA-A2 and -B7. In addition, a correlation between the inability to recognize murine transfectants and fine specificity was seen. Eight of nine clones which did not lyse murine transfectants also failed to recognize human cells expressing HLA-A2.2 or -A2.3. In contrast only 5 of 12 clones which lysed transfectants failed to recognize the variant molecules. Analogous data were obtained with human CTL clones raised against HLA-A2.1. These findings suggest that CTL clones that do not lyse murine cells expressing appropriate antigens recognize epitopes that have been altered or lost as a consequence of expression on the murine cell surface. It is suggested that the loss of HLA-associated epitopes on the murine cell surface may be due to differences between mouse and human cells in the processing or presentation of class I-associated peptides.     

Pathogenic autoantibodies in systemic lupus erythematosus are derived from both self-reactive and non-self-reactive B cells

Previous studies have shown that both murine and human anti-double-stranded DNA (anti-dsDNA) antibodies can develop from non-DNA-reactive B cells and suggest a crucial role for somatic mutation in dsDNA binding. However, since only a limited number of human anti-dsDNA antibodies have been analyzed previously, we could not exclude other mechanisms for the generation of anti-dsDNA antibodies in patients with systemic lupus erythematosus (SLE). Therefore, we isolated IgM anti-dsDNA antibodies from peripheral blood B cells of a patient with SLE. Three somatically mutated IgM anti-DNA antibodies with pathogenic potential (glomerular binding) were reverted to their germline configuration. Although all three IgM anti-dsDNA antibodies came from the same lupus patient, they displayed different profiles. Reversion to the germline sequence of autoantibodies A9 and B5 resulted in decreased dsDNA binding. In contrast, the germline form of G3-recognized dsDNA as well as the mutated counterpart. These results suggest that mutated IgM anti-dsDNA antibodies may develop from both DNA- and non-DNA-reactive B cells. The implications are that B cell activation occurs in response to self and non-self antigens, while selection after activation may be mediated by self antigen in SLE. Moreover, ineffective tolerance checkpoints may exist before and after antigen activation in SLE.     

Discovery of human antibodies against the C5aR target using phage display technology

Phage display technologies have been increasingly utilized for the generation of therapeutic, imaging and purification reagents for a number of biological targets. Using a variety of different approaches, we have developed antibodies with high specificity and affinity for various targets ranging from small peptides to large proteins, soluble or membrane-associated as well as to activated forms of enzymes. We have applied this approach to G-protein coupled receptors (GPCRs), often considered difficult targets for antibody therapeutics and targeting. Here we demonstrate the use of this technology for the identification of human antibodies targeting C5aR, the chemoattractant GPCR receptor for anaphylatoxin C5a. The N-terminal region (residues 1-31) of C5aR, one of the ligand binding sites, was synthesized, biotinylated and used as the target for selection. Three rounds of selection with our proprietary human Fab phage display library were performed. Screening of 768 isolates by phage ELISA identified 374 positive clones. Based on sequence alignment analysis, the positive clones were divided into 22 groups. Representative Fab clones from each group were reformatted into IgGs and tested for binding to C5aR-expressing cells, the differentiated U-937 cells. Flow cytometric analysis demonstrated that nine out of 16 reformatted IgGs bound to cells. Competition with a C5aR monoclonal antibody S5/1 which recognizes the same N-terminal region showed that S5/1 blocked the binding of positive cell binders to the peptide used for selections, indicating that the identified cell binding IgGs were specific to C5aR. These antibody binders represent viable candidates as therapeutic or imaging agents, illustrating that phage display technology provides a rapid means for developing antibodies to a difficult class of targets such as GPCRs.     

Humanization of rabbit monoclonal antibodies via grafting combined Kabat/IMGT/Paratome complementarity-determining regions: Rationale and examples

Rabbit monoclonal antibodies (RabMAbs) can recognize diverse epitopes, including those poorly immunogenic in mice and humans. However, there have been only a few reports on RabMAb humanization, an important antibody engineering step usually done before clinical applications are investigated. To pursue a general method for humanization of RabMAbs, we analyzed the complex structures of 5 RabMAbs with their antigens currently available in the Protein Data Bank, and identified antigen-contacting residues on the rabbit Fv within the 6 Angstrom distance to its antigen. We also analyzed the supporting residues for antigen-contacting residues on the same heavy or light chain. We identified “HV4” and “LV4” in rabbit Fvs, non-complementarity-determining region (CDR) loops that are structurally close to the antigen and located in framework 3 of the heavy chain and light chain, respectively. Based on our structural and sequence analysis, we designed a humanization strategy by grafting the combined Kabat/IMGT/Paratome CDRs, which cover most antigen-contacting residues, into a human germline framework sequence. Using this strategy, we humanized 4 RabMAbs that recognize poorly immunogenic epitopes in the cancer target mesothelin. Three of the 4 humanized rabbit Fvs have similar or improved functional binding affinity for mesothelin-expressing cells. Interestingly, 4 immunotoxins composed of the humanized scFvs fused to a clinically used fragment of Pseudomonas exotoxin (PE38) showed stronger cytotoxicity against tumor cells than the immunotoxins derived from their original rabbit scFvs. Our data suggest that grafting the combined Kabat/IMGT/Paratome CDRs to a stable human germline framework can be a general approach to humanize RabMAbs.     

Human Germline Antibody Gene Segments Encode Polyspecific Antibodies

Structural flexibility in germline gene-encoded antibodies allows promiscuous binding to diverse antigens. The binding affinity and specificity for a particular epitope typically increase as antibody genes acquire somatic mutations in antigen-stimulated B cells. In this work, we investigated whether germline gene-encoded antibodies are optimal for polyspecificity by determining the basis for recognition of diverse antigens by antibodies encoded by three V_H gene segments. Panels of somatically mutated antibodies encoded by a common V_H gene, but each binding to a different antigen, were computationally redesigned to predict antibodies that could engage multiple antigens at once. The Rosetta multi-state design process predicted antibody sequences for the entire heavy chain variable region, including framework, CDR1, and CDR2 mutations. The predicted sequences matched the germline gene sequences to a remarkable degree, revealing by computational design the residues that are predicted to enable polyspecificity, i.e., binding of many unrelated antigens with a common sequence. The process thereby reverses antibody maturation in silico. In contrast, when designing antibodies to bind a single antigen, a sequence similar to that of the mature antibody sequence was returned, mimicking natural antibody maturation in silico. We demonstrated that the Rosetta computational design algorithm captures important aspects of antibody/antigen recognition. While the hypervariable region CDR3 often mediates much of the specificity of mature antibodies, we identified key positions in the V_H gene encoding CDR1, CDR2, and the immunoglobulin framework that are critical contributors for polyspecificity in germline antibodies. Computational design of antibodies capable of binding multiple antigens may allow the rational design of antibodies that retain polyspecificity for diverse epitope binding.     

Monoclonal antibodies to bovine UDP-galactosyltransferase. Characterization, cross-reactivity, and utilization as structural probes

A series of mouse monoclonal antibodies has been developed against a soluble form of bovine UDP-galactose:N-acetylglucosamine galactosyltransferase purified to apparent chemical homogeneity by a combination of affinity and immunoadsorption chromatography. The purified enzyme consists of two molecular mass variants of 42 and 48 kDa. Individual monoclonal antibodies were selected for by their ability to recognize immobilized affinity-purified galactosyltransferase and were not reactive against bovine alpha-lactalbumin and bovine immunoglobulins. Based on competitive binding assays and Western blot analysis with either galactosyltransferase or lactose synthetase (covalently cross-linked alpha-lactalbumin galactosyltransferase), these monoclonal antibodies can be subdivided into four groups. Group A (3 clones) recognize an epitope at or near the alpha-lactalbumin binding site. In addition, this group is cross-reactive with soluble galactosyltransferase from human milk and pleural effusion. Group B (6 clones) and D (1 clone) appear to recognize two different epitopes on the 6-kDa fragment which is released when the 48-kDa galactosyltransferase polypeptide is converted to the 42-kDa form, apparently by proteolysis. Groups A and C (1 clone) recognize epitopes found on both the 48- and 42-kDa polypeptide. Interestingly, immunofluorescence studies indicate that only two monoclonal antibody groups (C and D) are able to decorate membrane-bound galactosyltransferase (Golgi-associated) in formalin-fixed, methanol-, or detergent-permeabilized cells. Thus, these groups of monoclonal antibodies appear to identify four separate structural/functional domains on soluble galactosyltransferase, two of which are not readily accessible for binding in situ.     

Characterization of a high-affinity human antibody with a disulfide bridge in the third complementarity-determining region of the heavy chain

Disulfide bridges are common in the antigen-binding site from sharks (new antigen receptor) and camels (single variable heavy-chain domain, VHH), in which they confer both structural diversity and domain stability. In human antibodies, cysteine residues in the third complementarity-determining region of the heavy chain (CDR-H3) are rare but naturally encoded in the IGHD germline genes. Here, by panning a phage display library designed based on human germline genes and synthetic CDR-H3 regions against a human cytokine, we identified an antibody (M3) containing two cysteine residues in the CDR-H3. It binds the cytokine with high affinity (0.4?nM), recognizes a unique epitope on the antigen, and has a distinct neutralization profile as compared with all other antibodies selected from the library. The two cysteine residues form a disulfide bridge as determined by mass spectrometric peptide mapping. Replacing the cysteines with alanines did not change the solubility and stability of the monoclonal antibody, but binding to the antigen was significantly impaired. Three-dimensional modeling and dynamic simulations were employed to explore how the disulfide bridge influences the conformation of CDR-H3 and binding to the antigen. On the basis of these results, we envision that designing human combinatorial antibody libraries to contain intra-CDR or inter-CDR disulfide bridges could lead to identification of human antibodies with unique binding profiles.     

Design, construction, and characterization of a large synthetic human antibody phage display library

Advances in proteomic research allow the identification of several hundred protein components in complex biological specimens. Structural information is typically lost during proteomic investigations. For this reason, the rapid isolation of monoclonal antibodies specific to proteins of interest would allow the study of structurally intact biological specimens, thus providing complementary proteomic information. Here, we describe the design, construction, characterization, and use of a large synthetic human antibody phage display library (ETH-2-Gold) containing three billion individual antibody clones. A large repertoire of antibodies with similar biochemical properties was produced by appending short variable complementarity-determining region 3 (CDR3) onto three antibody germline segments (DP47, DPK22, and DPL16), which are frequently found in human antibodies. The ETH-2-Gold library exhibits efficient display of antibody fragments on filamentous phage, as assessed by immunoblot. Furthermore, the library is highly functional, since >90% of clones express soluble antibodies in bacteria and since good quality monoclonal antibodies have been isolated against 16 different antigens. The usefulness of the library as a tool for generating monoclonal antibodies for biomedical applications was tested using the C-domain of tenascin-C (a marker of angiogenesis) as antigen and showing that specific antibodies to this target were able to stain vascular structures in tumor sections.     

Generation and selection of immunized Fab phage display library against human B cell lymphoma

The approval of using monoclonal antibodies as a targeted therapy in the management of patients with B cell lymphoma has led to new treatment options for this group of patients. Production ofmonoclonal antibodies by the traditional hybridoma technology is costly, and the resulting murine antibodies often have the disadvantage of triggering human anti-mouse antibody （HAMA） response. Therefore recombinant Fab antibodies generated by the phage display technology can be a suitable alternative in managing B cell lymphoma. In this study, we extracted total RNA from spleen cells of BALB/c mice immunized with human B lymphoma cells, and used RT-PCR to amplify cDNAs coding for the κ light chains and Fd fragments of heavy chains. After appropriate restriction digests, these cDNA fragments were successively inserted into the phagemid vector pComb3H-SS to construct an immunized Fab phage display library. The diversity of the constructed library was approximately 1.94× 10^7. Following five rounds of biopanning, soluble Fab antibodies were produced from positive clones identified by ELISA. From eight positive clones, FabC06, FabC21, FabC43 and FabC59 were selected for sequence analysis. At the level of amino acid sequences, the variable heavy domains （VH） and variable light domains （VL） were found to share 88-92% and 89-94% homology with sequences coded by the corresponding murine germline genes respectively. Furthermore, reactivity with membrane proteins of the B cell lymphoma was demonstrated by immunohistochemistry and western blotting. These immunized Fab antibodies may provide a valuable tool for further study of B cell lymphoma and could also contribute to the improvement of disease therapy.     

Hybridomas specific for carbohydrates; synthetic human blood group antigens for the production, selection, and characterization of monoclonal typing reagents

A general method for the production of carbohydrate-specific hybridoma antibodies is illustrated by generation of monoclonal antibody to the antigenic determinant of human blood group B. This trisaccharide determinant was chemically synthesized and covalently coupled to bovine serum albumin and human blood group O red cells. Soluble protein antigen and the 'artificial' B red cells were used to immunize BALB/c mice before fusion of spleen cells with the Sp2/0 plasmacytoma cell line. ELISA screening of putative hybrids for B-specific binding activity was facilitated by the availability of a second synthetic conjugate, B-horse hemoglobin. IgM-producing clones were identified by class-specific ELISA reagents and by hemagglutination assay. In this way, clones suitable for blood typing were rapidly identified. The precise antigenic specificity and Ig class of such monoclonal antibodies were defined by inhibition of precipitation and by gel filtration. Hybridoma antibodies were obtained from two separate fusion experiments. One of these, clone 3E-4, was of the IgM class and possessed a binding site that was completely satisfied (100% inhibition) by the trisaccharide determinant of the B blood group. This antibody is shown to be suitable for use in blood typing.     

Generation and characterization of protective antibodies to Marburg virus

Marburg virus (MARV) and Ebola virus (EBOV) have been a source of epidemics and outbreaks for several decades. We present here the generation and characterization of the first protective antibodies specific for wild-type MARV. Non-human primates (NHP), cynomolgus macaques, were immunized with viral-replicon particles expressing the glycoproteins (GP) of MARV (Ci67 isolate). An antibody fragment (single-chain variable fragment, scFv) phage display library was built after four immunogen injections, and screened against the GP_1-649 of MARV. Sequencing of 192 selected clones identified 18 clones with distinct V_H and V_L sequences. Four of these recombinant antibodies (R4A1, R4B11, R4G2, and R3F6) were produced in the scFv-Fc format for in vivo studies. Mice that were challenged with wild-type Marburg virus (Ci67 isolate) receiving 100 µg of scFv-Fc on days ?1, 1 and 3 demonstrated protective efficacies ranging from 75–100%. The amino-acid sequences of the scFv-Fcs are similar to those of their human germline counterparts, sharing an identity ranging between 68 and 100% to human germline immunoglobulin. These results demonstrate for the first time that recombinant antibodies offer protection against wild-type MARV, and suggest they may be promising candidates for further therapeutic development especially due to their human homology.     

Construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes

Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig) variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR) step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3). The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx). Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N) protein of SARS-associated coronavirus (SCoV) were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.     

Fine specificity analysis of human influenza-specific cloned cell lines

Influenza-specific human-T-cell clones, proliferating in the presence of virus-infected cells with restriction by class II molecules and displaying class II-restricted CTL activity or specific helper activity in antibody synthesis, have been analyzed for antigenic specificities. All of them were obtained by in vitro stimulation against influenza A/Texas virus. In all cases the virus specificity appeared identical in cytolytic and proliferative responses. Three of the clones were broadly cross-reactive, recognizing all or almost all type A influenza strains. The three remaining clones were subtype specific when tested with human strains and recognized the surface glycoproteins of influenza virus. One of these lines reacted with an epitope of the neuraminidase N2 while the other two recognized the hemagglutinin H3. By using a large panel of mammalian and avian influenza strains, it can be demonstrated that hemagglutinin-specific human T cells can recognize a cross-reacting determinant shared by H3 and H4 subtypes of hemagglutinin which has never been detected with antibodies.     

Activation of cloned human natural killer cells via Fc gamma RIII

The Fc gamma RIII (CD16) Ag on human NK cells involved in antibody-dependent cellular cytotoxicity has been demonstrated to be an important activation structure. The present studies were carried out to further characterize the functional role of the CD16 Ag and the mechanisms whereby cytotoxicity is activated by using human NK clones. In phenotypic studies Fc gamma RIII was found to be expressed heterogeneously on various human cloned NK cells. Expression on CD3- and CD3+ clones varied with the donor and mAb used for detection. Functional data demonstrated that cytotoxicity against NK-resistant target cells can be induced in CD3-CD16+ NK clones and CD3+CD16+ clones with NK activity when various CD16 mAb were used. CD16 antibodies but not reactive isotype control antibodies induced cytotoxicity. In contrast to complete CD16 antibodies F(ab')2 fragments were not able to activate the cytotoxic mechanism. Both an antibody against FcR on the target cell (Fc gamma RII) and a CD11a antibody blocked induction of cytotoxicity. These results suggest that three steps are critical for activation of CD16+ cells via Fc gamma RIII: 1) specific binding of CD16 antibodies to Fc gamma RIII on effector cells irrespective of the epitope recognized; 2) cross-linking of effector cell CD16 Ag through binding of the Fc site of CD16 antibodies via corresponding FcR on the target cell membrane; and 3) interaction of CD11a/18 molecules with the target cell membrane.     

Establishment and characterization of monoclonal antibody against androgen receptor

Hybrid cell lines were prepared by the fusion of BALB/c myeloma NS-1 cells with the lymphocytes of BALB/c mice that were immunized with partially purified androgen receptor (AR) from human prostates. Nine clones of the hybrid progeny were determined for the production of antibodies against AR by immunoprecipitation assay. One of the clones, referred to as "5F4", was chosen for analysis of the detailed specificity. The clone "5F4" secreted IgM class antibodies against AR. Competition study demonstrated that "5F4" antibody inhibited androgen binding of AR, suggesting that the antibody identifies androgen binding site of AR. Immunoblotting analysis showed that the antibody identified the ARs as two proteins, 95 kD and 41 kD proteins, on a sodium dodecyl sulfate polyacrylamide gel. It is suspected that a 95 kD protein should be a monomeric AR and a 41 kD protein is a proteolytic fragment of AR. Immunohistochemical analysis demonstrated that androgen-dependent tissues--human prostatic hypertrophy tissues, an AR abundant prostatic cancer tissue and fibroblast cells from human genital skin--were stained intensely with "5F4" monoclonal antibody, while androgen-independent tissues--fibroblast cells from lymph nodes, an AR deficient prostatic cancer tissue and human prostatic cancer cell line, PC-3--showed no staining. These results also support the specificity of the antibody for AR.     

Anti-idiotype-mediated epitope spreading and diminished phagocytosis by a human monoclonal antibody recognizing late-stage apoptotic cells

Apoptotic cells are considered an important auto-antigenic source in diseases such as systemic lupus erythematosus (SLE). A human monoclonal antibody demonstrating exquisite specificity towards late-stage apoptotic cells was generated from an SLE patient. Polyreactive recognition of ribonucleoproteins Ro52 and Ro60 was observed. The antibody significantly diminished the phagocytosis of apoptotic cells and a concomitant decrease in transforming growth factor-beta (TGF-beta) secretion was observed. Light and heavy chain sequencing revealed the antibody to be in essentially germline configuration. Elicited anti-idiotypic antibodies bound distinct self-antigens and showed augmented reactivity towards apoptotic cells as well. Thus, near-germline encoded antibodies recognizing antigens externalized during the process of apoptosis can mediate a variety of potentially pathogenic effects; decreases in the phagocytic uptake of dying cells would constitute a disease-perpetuating event and stimulation of the idiotypic network could lead to intermolecular epitope spreading, increasing the range of molecular targets..     

Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus

Immunoglobulin genes are generated somatically through specialized mechanisms resulting in a vast repertoire of antigen-binding sites. Despite the stochastic nature of these processes, the V-genes that encode most of the antigen-combining site are under positive evolutionary selection, raising the possibility that V-genes have been selected to encode key structural features of binding sites of protective antibodies against certain pathogens. Human, neutralizing antibodies to human cytomegalovirus that bind the AD-2S1 epitope on its gB envelope protein repeatedly use a pair of well-conserved, germline V-genes IGHV3-30 and IGKV3-11. Here, we present crystallographic, kinetic and thermodynamic analyses of the binding site of such an antibody and that of its primary immunoglobulin ancestor. These show that these germline V-genes encode key side chain contacts with the viral antigen and thereby dictate key structural features of the hypermutated, high-affinity neutralizing antibody. V-genes may thus encode an innate, protective immunological memory that targets vulnerable, invariant sites on multiple pathogens.     

Aberrant maturational characteristics of the immune responses of NZB mice to autologous and heterologous erythrocyte antigens

The maturational characteristics of the humoral immune responses of C3H and NZB mice to autologous and heterologous erythrocyte antigens were investigated. Clonal selection of antibody-secreting B lymphocytes was examined at the plaque-forming cell level of analysis of changes in mean antibody affinity and the heterogeneity of binding affinities. The primary immune response of C3H mice to SRBC exhibited a progressive temporal increase in mean relative antibody affinity and a concomitant restriction in the heterogeneity of binding affinities consistent with clonal selection and restriction of B lymphocytes to high affinity antibody-secreting cells. By contrast, the anti-SRBC immune response of NZB mice displayed aberrant maturational characteristics with a progressive decrease in mean relative antibody affinity but also clonal restriction with selection of clones of cells secreting low affinity antibodies. The spontaneous autoimmune responses of NZB mice to autologous erythrocyte surface autoantigens X and HB were different from the response to heterologous erythrocytes in that there was neither a progressive change in mean relative binding affinity nor evidence of progressive clonal restriction. Although the precise mechanisms responsible for the aberrant selection and derepression of B lymphocyte clones in NZB mice have not been identified, the very nature of the aberration suggests the existence of one or more defects which may be intrinsic to the B lymphocytes of NZB mice.