Murine VH Research Articles

Camelization of a murine single-domain antibody against aflatoxin B1 and its antigen-binding analysis.

Aflatoxin B1 (AFB1), a highly toxic mycotoxin, always contaminated in a variety of agricultural products. Camelid variable domain of heavy chain antibody (VHH) is a noteworthy reagent in immunoassay, owing to its excellent characteristics. Immunization of camelid animals is a straightforward strategy to produce VHHs. In this study, to avoid the dependence on the large animals, the camelized, murine antibody (cVHs) against AFB1 was prepared in vitro based on the identities between murine VH and camelid VHH and then to develop an immunoassay for AFB1. A murine anti-AFB1 VH fragment (VH-2E6) was selected for camelization through replacement of conserved hydrophobic residues in framework region 2 (FR2) (cVH-FR2), point mutation at position 103 in the FR4 region (cVH-103), and CDR3-grafted with a high AFB1-affinity VHH (cVH-Nb26). The cVH-Nb26 had a yield of 5 mg/L as refolded protein expressed from Escherichia coli and 10 mg/L expressed from Pichia pastoris. Compared with anti-AFB1 single-chain fragment variable (scFv) 2E6, cVH-Nb26 performed more than 20-fold enhancement of AFB1-binding interactions. Although the AFB1-affinity of cVH-Nb26 cannot meet the application requirement in the present form, our study provides effective strategies for preparation of camelized antibody in vitro, which could be a promising immunoreagent for AFB1 detection.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12550-021-00433-z.

The Near Infra-Red (NIRF) molecular imaging of oxidised LDL in atherosclerosis with the native autoantibody LO1, and its molecularly expressed cysteine-tagged Fab construct (LO1-Fab-cys)

Purpose: Oxidation of low density lipoprotein in the arterial wall is a key molecular event on the pathogenesis of atherosclerosis. We aimed to develop a NIRF-based antibody-targeted molecular imaging approach for identifying oxidised LDL (oxLDL) in vivo. Methods and results: LO1 is a naturally occurring IgG3κ autoantibody isolated from an LDL receptor deficient hypercholesterolaemic mouse. It reacts with malondialdehyde-conjugated LDL (MDA-LDL), an epitope expressed in oxidised LDL and which is recognised as reflecting vulnerability in human atherosclerotic lesions. Immunohistochemical (IHC) staining of biotinylated LO1 showed colocalisation to advanced mouse atherosclerotic plaque and to necrotic core within culprit human carotid atherosclerotic plaque. We used the immunoglobulin SH groups to successfully label LO1 with a Near Infra Red Fluorescence (NIRF) agent for in vivo imaging studies. Using two optical imaging techniques (IVIS Spectrum combined with hybrid high resolution CT as well as Fluorescence Molecular Tomography (FMT)) we demonstrated that LO1 sensitively and specifically identifies atherosclerotic lesions in vivo in the aorta of the LDLr-/- atherosclerotic mouse model but not in control mice. LO1 also demonstrated superior specificity to lesional regions of interest (ROIs) than a caged MMP probe (MMP-sense). En face immunohistochemistry on aortae from mice injected with labelled LO1 and an anti CD-31 antibody showed localisation of the targeting antibody to the sub-endothelium of the aortic arch and root. LO1 was successfully sequenced and sequence verified as germline. It was then expressed in HEK cells as a chimeric Fab (∼50kD), with murine VH and VL and human CH1 and CL. The terminus of CH1 extends to the THTC of the hinge region to provide the free cysteine for NIRF labelling. In vitro characterisation of the construct revealed maintained function following affinity purification on an anti-human CH1 column and labelling with a NIRF agent. Initial in vivo work confirms localisation to atherosclerotic lesions within the LDLr-/- model. Conclusion: LO1 and its molecularly expressed human chimeric construct (LO1-Fab-cys) provide useful tools for imaging and therapeutic targeting of oxidised LDL in atherosclerosis. Further imaging studies will be enabled by the development new technologies such as the combined Optical Fluorescence Domain Imaging and Near Infrared Fluorescence (OFDI/NIRF) intravascular catheter.

YIA1: IMAGING BEYOND THE LUMEN: NEAR INFRA-RED IN VIVO MOLECULAR IDENTIFICATION OF OXIDISED LDL IN ATHEROSCLEROSIS USING MAB LO1, AND THE GENERATION AND DEVELOPMENT OF ITS MOLECULARLY EXPRESSED CYSTEINE-TAGGED CHIMERIC FAB CONSTRUCT (LO1-FAB-CYS)

<h3>Introduction</h3> The <i>in vivo</i> identification of vulnerable atherosclerotic plaque remains elusive despite current advances in intravascular morphological imaging technologies such as IVUS-VH and OCT. With the recent advent of intra-vascular catheter-based optical fluorescence domain imaging combined with near infra-red fluorescence (OFDI-NIRF), we aim to translationally develop a novel NIRF antibody-based technique for the identification of oxidised LDL in atherosclerosis. <h3>Methods and Results</h3> We isolated a previously unknown IgG3κ autoantibody against oxLDL (designated LO1) from an LDL receptor deficient hypercholesterolaemic mouse. Characterisation of LO1 revealed specificity for malondialdehyde-conjugated LDL (MDA-LDL), an epitope expressed in oxidised LDL and which is known to reflect vulnerability in human atherosclerotic lesions. Immunohistochemical (IHC) staining of biotinylated LO1 showed binding to advanced mouse atherosclerotic plaque and to necrotic core within culprit human carotid atherosclerotic plaque. We used the immunoglobulin SH groups to successfully label LO1 with a Near Infra-Red Fluorescence (NIRF) reporter VivoTag-S MAL 750 (designated LO1-750) for in vivo imaging studies. Using two optical imaging techniques (IVIS Spectrum combined with hybrid high resolution CT; Fluorescence Molecular Tomography (FMT)) we have demonstrated that intra-venously injected LO1-750 sensitively and specifically identifies atherosclerotic lesions <i>in vivo</i> in the aortae of LDLr^−/− mouse model (Figure&nbsp;1) but not in control mice. LO1-750 also demonstrated superior specificity for lesional regions of interest (ROIs) than a caged MMP probe. <i>In situ en face</i> IHC on extracted aortae with LO1-750 showed localisation to the aortic arch and root, with co localisation to the sub-endothelium as demonstrated by counter injection with an anti CD-31 antibody (Figure&nbsp;2). In order to facilitate future use in human subjects, LO1 was successfully sequenced and sequence verified as germline. It was then expressed in HEK cells as a chimeric Fab (∼50kD), with murine VH and VL and human CH1 and CL. The terminus of CH1 extends to the THTC of the hinge region to provide the free cysteine for NIRF labelling<i>. In vitro</i> characterisation of the construct revealed maintained function when purified over an anti-human CH1 column and labelled with Vivo Tag-S MAL 750. Injected LO1-cys-fab localised to the sub-endothelium of the diseased aortic arch of LDLr^−/− mice in a similar fashion to the native LO1 antibody. Initial FMT studies have confirmed localisation to atherosclerotic lesions within the LDLr^−/− model. <h3>Conclusion</h3> The in vivo optical imaging of oxidised LDL is possible with LO1 and the chimerically humanised LO1-Cys-Fab. Future work will aim to translate this to optical imaging of atherosclerosis in humans.

Humanization of an anti-CεmX monoclonal antibody 4B12 for potential clinical use in targeting mIgE+B cells (140.19)

Abstract The CεmX domain of 52 a.a. residues, located between the CH4 domain and membrane-anchor segment of human membrane-bound ? chain on mIgE+ B cells, is a unique antigenic site for the immunological targeting of those B cells. We have demonstrated that a chimeric form of anti-CεmX monoclonal antibody (mAb), 4B12, can induce apoptosis and ADCC against a human mIgE.Fc+ B cell transfectoma via effector cells from PBMC in vitro (presented by another abstract in this meeting). To enhance the clinical applicability of 4B12, we have humanized it by first grafting its six complementary-determining regions (CDRs) into human Ig VH subgroup IV and V? subgroup II frameworks (FWs). Those residues in the FWs of VH and VL of the parental murine 4B12 that are essential for Fv dimer interaction and CDR conformation were identified by analyzing the binding affinity of a number of designed Ab variants with site-directed mutations, using competitive ELISA and surface plasmon resonance analysis. A humanized 4B12 retaining the affinity of its parent was obtained by adopting all FWs of the selected human V? template without any change and all FWs of the selected VH with only one residue change. The Val at position 71 of the human VH was replaced by Arg at the corresponding position of the murine VH. Based on molecular modeling analysis, it is likely that such a change is probably crucial for maintaining the proper structural packing of Arg71 with Ile29 in the CDRH1 and Gly55 in the CDRH2. The KD of the chimeric and humanized 4B12 are 5.37×10-9 M-1 and 5.76×10-9 M-1, respectively. Affinity enhancement of humanized 4B12 by selective CDR mutations is in progress.

A Highly Conserved Interspecies VH in the Human Genome

Idiotype conservation between human and mouse antibodies has been observed in association with various infectious and autoimmune diseases. We have isolated a human anti-idiotypic antibody to a mouse monoclonal anti-IgE antibody (BSW17) suggesting a conserved interspecies idiotype associated with an anti-IgE response. To find the homologue of BSW17 in the human genome we applied the guided selection strategy. Combining VH of BSW17 with a human VL repertoire resulted in three light chains. The three VL chains were then combined with a human VH repertoire resulting in three clones specific for human IgE. Surprisingly, one clone, Hu41, had the same epitope specificity and functional in vitro activity as BSW17 and VH complementarity-determining regions identical with that of BSW17. Real-time PCR analysis confirmed the presence of the Hu41 VH sequence in the human genome. These data document the first example of the isolation of a human antibody where high sequence similarity to the original murine VH sequence is associated with common antigen and epitope specificity.

Mutation of a single conserved residue in VH complementarity-determining region 2 results in a severe Ig secretion defect.

During an immune response, somatic mutations are introduced into the VH and VL regions of Ig chains. The consequences of somatic mutation in highly conserved residues are poorly understood. Ile51 is present in 91% of murine VH complementarity-determining region 2 sequences, and we demonstrate that single Ile51-->Arg or Lys substitutions in the PCG1-1 Ab are sufficient to severely reduce Ig secretion (1-3% of wild-type (WT) levels). Mutant H chains, expressed in the presence of excess L chain, associate with Ig binding protein (BiP) and GRP94 and fail to form HL and H2L assembly intermediates efficiently. The mutations do not irreversibly alter the VH domain as the small amount of mutant H chain, which assembles with L chain as H2L2, is secreted. The secreted mutant Ab binds phosphocholine-protein with avidity identical with that of WT Ab, suggesting that the combining site adopts a WT conformation. A computer-generated model of the PCG1-1 variable region fragment of Ig (Fv) indicates that Ile51 is buried between complementarity-determining region 2 and framework 3 and does not directly contact the L chain. Thus, the Ile51-->Arg or Ile51-->Lys mutations impair association with the PCG1-1 L chain via indirect interactions. These interactions are in part dependent on the nature of the L chain as the PCG1-1 VH single Ile51-->Arg or Ile51-->Lys mutants were partially rescued when expressed with the J558L lambda1 L chain. These results represent the first demonstration that single somatic mutations in V(H) residues can impair Ig secretion and suggest one reason for the conservation of Ile51 in so many Ig VH.

Immunoglobulin VH gene sequence analysis of spontaneous murine immunoglobulin-secreting B-cell tumours with clinical features of human disease.

The 5T series of multiple myelomas (MM) and Waldenstrsöm's macroglobulinaemia-like lymphomas (WM), which developed spontaneously in ageing mice of the C57BL/KaLwRij strain, shows clinical and biological features that closely resemble their corresponding human diseases. In order to compare the patterns of somatic mutation in VH genes of mouse tumours with those of human counterparts, we have determined and analysed sequences of immunoglobulin VH genes in five cases of murine MM, two of WM and one of biclonal benign monoclonal gammopathy (BMG). Four of five MM and 2/2 WM cases used VH genes of the large J558 family; one MM used a gene of the VGAM3.8 family, and both clones of the BMG used genes of the 36-60 family. N-region insertions were observed in all cases, but D-segment genes were only identified in 6/9 cases, which were all from the D-SP family and translated in reading frame 3. Compared with human MM, in which the VH genes have been found to be consistently hypermutated (mean% +/- SD = 8.8 +/- 3.2), the degree of somatic mutation in the murine tumours was significantly lower (mean% +/- SD = 2.9 +/- 2.3). There was no significant evidence of clustering of replacement mutations in complementarity determining regions (CDR), a feature considered to be characteristic of antigen-selected sequences. However, one clone of the biclonal BMG case showed intraclonal variation, a feature described in some cases of human BMG. These results indicate that murine VH genes in mature tumours differ from human counterparts in the level and distribution of somatic mutations, but support the concept that BMG may be distinct from MM.

Guided selection of antibody fragments specific for human interferon γ receptor 1 from a human VH- and VL-gene repertoire

Objective: The guided selection strategy for isolation of human antibody (Ab) fragments specific for human interferon γ receptor 1 (IFNGR-1) from a cloned Ab VH and VL repertoire has been investigated. In order to identify recombinant Abs binding to soluble antigen, a novel method termed affinity sedimentation was introduced here. Results and conclusions: The VH region of murine monoclonal Ab (IRγ-1) against human IFNGR-1 was combined with human VL repertoire and used for selection of human VL regions. One of these human VL regions (κ2) possesses high homology to the murine template VL region, also in CDR3 (77%). A chimeric Fab consisting of κ2 and the murine IRγ-1 VH region was highly IFNGR-1 specific and exerted the same epitope specificity and a comparable binding affinity as the parental murine Fab. In a further step, the selected human VL region κ2 was combined with a human VH repertoire and led by guided selection to the generation of a completely human Fab (1b5) specific for human IFNGR-1. The overall VH region homology of 1b5 compared to the parental antibody IRγ-1 was 81%, with a rather low homology in CDR3. Binding competition studies revealed that the epitope recognized by 1b5 differs from the parental Ab IRγ-1.

Use of a single VH family and long CDR3s in the variable region of cattle Ig heavy chains.

We have analyzed transcripts encoding the variable regions of Ig heavy chains from adult and fetal bovine splenocytes and bovine x mouse heterohybridomas. The 13 adult, seven fetal and two heterohybridomas transcripts as well as the six genes that were sequenced had > 83% identity to each other in the VH-encoded regions (FRs 1-3 and CDRs 1 and 2). By this criterion, all the bovine sequences were assigned to one family, which corresponds to the bovine homolog of the murine Q52 family. Southern blot analysis of genomic DNA demonstrated that homologs of other murine VH families such as 7183, S107 and 36-60 were present in the genome, but transcripts from these families were not detected in rapid amplification of cDNA ends (RACE)-PCR amplified products or in individual clones. The sequences of the adult transcripts using the mu isotype showed extensive somatic mutation indicating that the process of somatic hypermutation begins earlier in development of the bovine B cell. The length of CDR3 from V(D)J rearrangements averaged 21 amino acids, which is larger than other mammalian CDR3s. Analysis of CDR3s from 23 fetal transcripts revealed a preference for a reading frame in the putative D genes which is rich in glycine and tyrosine, and is also extensively mutated in adults. The bovine immune system appears to utilize Ig VH genes of a single family, but generates antibody diversity by extensive somatic mutation and long CDR3s which are subsequently hypermutated.

Cloning and Expression of cDNAs Encoding the Variable Domains of the Antibreast Carcinoma Antibody Mc5

Mc5, a murine monoclonal antibody that binds to human breast epithelial mucin (BEM), has been shown to be a promising reagent in the diagnosis of breast cancer. We have cloned cDNAs encoding both variable regions of Mc5 (VL and VH) as well as the CL and CH1 constant regions. Mc5 is an IgG1, kappa antibody. We have constructed an IgG1, kappa human/mouse chimeric antibody (by inserting the murine VH and VL-encoding cDNAs into plasmids encoding human constant domains), and expressed it in SP2/0-Ag14 mouse myeloma cells. The affinity of chimeric Mc5 (chMc5) for BEM is 4.4 x 10(8) M-1. Mc5 binds BEM with an affinity constant of 2.8 x 10(8) M-1. Purified chMc5 and purified Mc5 gave similar competition curves when tested against either 125I-labeled Mc5 or 125I-labeled chMc5 for binding to BEM in a competition radioimmunodetection format. Additionally, chMc5 used in breast carcinoma tissue staining stained as well as the original Mc5.

Rapid and reliable cloning of antibody variable regions and generation of recombinant single chain antibody fragments.

Single chain antibody variable region fragments (sFv), by virtue of their size and method of construction are potentially useful as therapeutic reagents and as tools for exploring cell surface receptor function. sFv offer several advantages over the intact immunoglobulin molecule. For instance, they are expressed from a single transcript and can be molecularly linked to other proteins to generate bispecific sFv molecules or single-chain immunotoxins. The relatively small size of sFv is an advantage in allowing for easier penetrance into tissue spaces, and their clearance rate is exceedingly rapid. sFv are useful for gene therapy since they can be directed to a specific cellular localization and can be fused to retroviral env genes to control viral host range. To prepare sFv to murine and human leukocyte CD antigens, we devised a method for rapid cloning and expression that can yield functional protein within 2-3 weeks of RNA isolation from hybridoma cells. The variable regions were cloned by poly-G tailing the first strand cDNA followed by anchor PCR with a forward poly-C anchor primer and a reverse primer specific for constant region sequence. Both primers contain flanking restriction sites for insertion into PUC19. Sets of PCR primers for isolation of murine, hamster and rat VL and VH genes were generated. Following determination of consensus sequences for a specific VL and VH pair, the VL and VH genes were linked by DNA encoding an intervening peptide linker [usually (Gly4Ser)3] and the VL-link-VH gene cassettes were transferred into the pCDM8 mammalian expression vector. The constructs were transfected into COS cells and sFvs were recovered from spent culture supernatant. We have used this method to generate functional sFv to human CD2, CD3, CD4, CD8, CD28, CD40, CD45 and to murine CD3 and gp39, from hybridomas producing murine, rat, or hamster antibodies. Initially, the sFvs were expressed as fusion proteins with the hinge-CH2-CH3 domains of human IgG1 to facilitate rapid characterization and purification using goat anti-human IgG reagents or protein A. We also found that active sFv could be expressed with a small peptide > or = tag > or = or in a tail-less form. Expression of CD3 (G19-4) sFv tail-less or Ig tailed forms demonstrated increased cellular signalling activity and suggested that sFv have potential for activating receptors.

Effect of VH and VL consensus sequence-specific primers on the binding and neutralizing potential of a single-chain FV directed towards HuIFN-γ

We have previously reported on the cloning and bacterial expression of a biologically active scFv antibody fragment (scFv-D9D10) derived from the mouse anti-human interferon-gamma (HuIFN-γ) antibody, D9D10. Since the variable (V) regions were isolated by means of VH and VL consensus sequence-specific PCR primers and cloned in an expression vector relying on primer-incorporated restriction sites, some amino acids (aa) at the N-and C-terminal ends of the cloned V domains were expected to differ from the corresponding ones in the natural D9D10 antibody. Therefore, the naturally occuring sequences of both V domains were isolated by means of traditional cDNA synthesis procedures. In comparison with scFv-D9D10, the “natural” V sequences differed in three aa in VH and three in VL. The V domains of scFv-D9D10 were adapted to their natural sequence by means of PCR-directed mutagenesis to yield scFv-D9D10N. Comparison of the binding and neutralizing potentials of both antibody fragments did not reveal differences in either of both activities. In addition, their affinities of HuIFN-γ were found to be equal. These results show that murine VH and VL consensus-specific primers can yield antibody fragments having functional properties equivalent to those of the natural scFv. Information on the impact of the use of V-specific primers on kinetics of interaction between the recombinant antibody and the corresponding antigen is important for the development of most engineered antibodies or their fragments.

A heavy-chain grafted antibody that recognizes the tumor-associated TAG72 antigen.

Murine anti-TAG72 antibodies react with more than 85% of human colorectal, gastric and ovarian carcinomas. However, the therapeutic utility of murine antibodies in human is severely restricted by their immunogenicity. The construction of humanized antibodies can potentially circumvent this problem. The latter antibodies can be generated by grafting CDRs from a murine antibody onto human immunoglobulin FRs followed by combining the resultant product with human immunoglobulin constant regions. In this study, we constructed a heavy-chain humanized anti-TAG72 antibody that we designated hmM4. This was achieved by the transplantation of CDRs from the murine VH of the ccM4 antibody into FRs of the human myeloma protein NEWM. The humanized antibody hmM4 retained its binding reactivity for the TAG72 antigen as measured by ELISA and Western blotting analysis respectively. However, it showed considerably less immunoreactivity for the TAG72 antigen than the original chimeric antibody ccM4. These results indicate that the murine anti-TAG72 specificity can be grafted to human immunoglobulin, and that the choice of the human immunoglobulin framework for the grafted antibody may be critical in maintenance of the immunoreactivity.

Optimal humanization of 1B4, an anti-CD18 murine monoclonal antibody, is achieved by correct choice of human V-region framework sequences.

The murine anti-CD18 mAb 1B4 has been humanized using CDR grafting. Three VH (Gal, Jon, and New) and two VL (Rei and Len) human frameworks, whose selection was based exclusively on their sequence identity with m1B4, were used to construct five human gamma 4/kappa recombinant antibodies: Gal/Rei, Gal/Len, Jon/Rei, and New/Rei, and a "hemichimeric" antibody pairing the VH of m1B4 with grafted Rei. Each of these h1B4 constructs completely inhibited the binding of m1B4 to activated human leukocytes with avidities (IC50) ranging from 1.5 to 8.0 nM, compared to 0.5 nM for m1B4. Replacement of three VH residues in the best VH framework, Gal, with the corresponding m1B4 "packing" (nonsolvent exposed) residues gave an h1B4 (mutant Gal/Rei) with the same avidity as m1B4. Avidity correlated with overall percent identity between the human and murine VH frameworks and, in particular, with conservation of "packing" residues. Rei and Len VL frameworks proved to be interchangeable. Further characterization showed that the Gal/Rei prototype was equipotent to m1B4 in blocking adhesion of polymorphonuclear leukocytes and monocytes to human vascular endothelium in vitro, and polymorphonuclear leukocyte extravasation into C5a-injected rabbit or monkey skin sites. Dual-label immunofluorescence microscopy of bone marrow cells with Gal/Rei h1B4 and m1B4 demonstrated that the fine specificity of the combining sites had not been altered by humanization. Reduced immunogenicity was demonstrated in rhesus monkeys that tolerated weekly treatment with h1B4 for 6 wk, whereas m1B4 induced profound anaphylaxis at 3 wk. Anti-1B4 titers in h1B4-treated rhesus were 50 to 66% lower and developed 1 wk later than in m1B4-treated monkeys. Crucially, the anti-h1B4 antibodies were anti-idiotypic while the anti-m1B4 antibodies were directed against constant and framework regions. We conclude that sequence identity searches are sufficient to identify suitable human frameworks for CDR-grafting of m1B4, yielding functionally equivalent humanized antibodies that are tolerated better in primates.

A peptide derived from an autoantibody can stimulate T cells in the (NZB x NZW)F1 mouse model of systemic lupus erythematosus.

To assess the ability of peptides derived from anti-DNA to stimulate syngeneic T lymphocytes and influence lupus in (NZB x NZW)F1 (NZB/W) mice. We synthesized (by Geysen pin method) overlapping 12-mer peptides recapitulating the amino acid sequence of the VH region of a nephritogenic monoclonal IgG2a anti-DNA antibody (A6.1) from an NZB/W mouse. Splenic T cells were cultured with the peptides; multiple experiments assayed 12-mer and 16-mer peptides which contained a triple-base motif (KFKGK). We immunized 20-week-old NZB/W mice with the 12-mer and evaluated them for evidence of nephritis and for quantities of antibodies in plasma and glomeruli. Three clusters of peptides caused proliferation of spleen cells from young, nonimmunized mice. Both the 12-mer FYNQKFKGKATL and the 16-mer GDTFYNQKFKGKATLT peptides stimulated purified T cells. The KXKXK motif occurs in 15% of murine Ig VH (NBRF protein database), compared with 100% (6 of 6) of NZB/W anti-DNA monoclonal antibody. Immunization with the 12-mer peptide increased plasma levels of IgG, anti-DNA, and immune complexes, and levels of anti-DNA in glomeruli; nephritis was accelerated. NZB/W anti-DNA contain peptide sequences in their VH regions that stimulate self-T cells. At least one motif is frequent in NZB/W anti-DNA. If some activated T cells provide help, this mechanism may contribute to sustained up-regulation of autoantibodies in murine lupus.

Variable region differences affect antibody binding to immobilized but not soluble antigen

We have examined the antigen binding characteristics of two chimeric IgG1 antibodies that differ only in the heavy chain variable region. Antibodies 10B and B11 were expressed from two different anti-(Tyr,Glu)-Ala-Lys murine VH genes joined to human IgG1 constant region genes in a murine anti-(Tyr,Glu)-Ala-Lys heavy chain loss variant hybridoma. The binding characteristics of the antibodies to (Tyr,Glu)-Ala-Lys and to a peptide conjugate, CYYYEEEEY:BSA, were measured in solution and solid phase assays. The antibodies exhibited similar affinities and binding characteristics when assayed in solution assays. However, when we measured binding of antibodies to immobilized antigens, we found that antibody affinity depended on the epitope density in the immobilized immune complexes. The binding of antibody 10B and of B11 to immobilized (Tyr,Glu)-Ala-Lys and to CYYYEEEEY:BSA were similar at high antigen density, but antibody B11 bound less well at lower antigen density. Fab fragments of 10B bound to immobilized (Tyr,Glu)-Ala-Lys and CYYYEEEEY:BSA, but Fab fragments of B11 did not bind to (Tyr,Glu)-Ala-Lys and bound less well to CYYYEEEEY:BSA than 10B Fabs.

Characterization of a murine immunoglobulin VH gene segment in subgroup III: A new member of the 7183 gene family

A new gene for the variable region of the immunoglobulin heavy chain (VH gene) has been isolated from BALB/c adult liver DNA using a cDNA plasmid probe containing a mouse VH sequence. The complete nucleotide sequence of this germline gene (VH10-19), shows that it belongs to the 7183 gene family. The VH gene appears to contain an intervening 104-base-long sequence and displays the same recombination signal sequences that those observed in the germline 8 IX. The presence of an internal heptamer at the 3' end of the VH10-19 coding region let an alternative recombination event that could increase the representation of this gene in the immature repertoire.

Organization of the murine immunoglobulin VH complex: Placement of two new VH families (VH10 and VH11) and analysis of VH family clustering and interdigitation

During B cell development, there is an ordered expression of heavy chain variable region (VH) genes during ontogeny such that JH proximal VH genes are rearranged and expressed before the more JH distal VH genes. Thus, the relative chromosomal position of VH genes is biologically significant. We have previously employed deletion mapping to order the nine described murine VH gene families as follows: 3609-J558-(J606/VGAM3-8/S107)-3660-(X24/Q52/7183). (Families within parentheses were not mapped relative to each other.) In this report we continue this analysis by mapping two recently described heavy chain variable region gene families (VH10 and VH11). VH10 is located at the JH proximal end of the major cluster of J558 VH gene segments. VH11 (a very small family) is intermingled with the 3660 family. Although in general VH genes are thought to be clustered, we and others have reported some interspersion between families. To further address this issue, we have analyzed 80 recombinant phage clones containing J558 VH gene segments for the presence of other VH family genes. Our data indicate that the J558 and 3609 VH families are extensively intermingled as has recently been described for the most JH proximal Q52 and 7183 families.

Autoantibodies and the Fetal Antibody Repertoire

Developing fetal B cells preferentially rearrange a restricted subset of the encoded antibody gene segments. There are striking structural similarities between elements expressed early in man and in mouse, most evident on comparison of murine VH elements from the VH7183 family to human VH elements of the VH3 family. The similarity is pronounced in two framework regions which together encode a possible binding site that is distinct from the classical antigen-combining site. By comparing all known human and murine VH gene sequences, we have demonstrated that these regions have been conserved in a family-specific manner throughout the mammalian radiation. The "non-conserved" spacer of the recombinase recognition signal is also highly conserved in a family-specific manner, suggesting a mechanism by which the expression of family-dependent features may be regulated. The evidence that such features contribute to the high incidence of self- and poly-specificity in the fetal antibody repertoire is reviewed.

Complete nucleotide sequence of an immunoglobulin heavy-chain gene and analysis of immunoglobulin gene organization in a primitive teleost species.

The immunoglobulin heavy-chain variable region (VH) locus in a phylogenetically primitive teleost (Elops saurus) has been characterized by a strategy that relied initially on cross-hybridization between genomic VH segments and a murine VH probe. Using a homologous (Elops) VH probe and DNA sequencing, this gene family has been shown to be complex and to contain overt pseudogenes. A homologous probe also has been used to isolate a full copy length cDNA containing constant (CH) as well as joining (JH) and VH regions. Genomic analyses using CH-, JH-, and VH-specific probes have demonstrated the presence of only a single hybridizing CH and several JH elements. JH-CH linkage is less than or equal to 3.6 kilobases (kb) and VH-CH linkage is less than or equal to 100 kb, as estimated by field-inversion gel electrophoresis. An additional VH family sharing less than 50% nucleotide identity with the prototype Elops VH sequence is described. Taken together, these results suggest that the immunoglobulin VH locus in a comparatively primitive teleost resembles the VH locus in mammals, but not that found in the more phylogenetically distant elasmobranchs. The evolutionary radiations of cartilaginous and bony fishes are associated with a dramatic change in the organization and, presumably, regulation of immunoglobulin genes. The origins of the modern VH gene locus can be traced to the primitive teleost fishes.