VL Sequences Research Articles

Characterisation of a fourth immunoglobulin light chain isotype in the common carp

Three isotypes of immunoglobulin (Ig) light (L) chain, designated L1A, L1B, and L3, have been characterised in the common carp ( Cyprinus carpio L.) to date. In this paper the molecular cloning of a fourth IgL isotype in carp, designated L2, is described. A reverse transcription–polymerase chain reaction (RT–PCR) method including 5′- and 3′-RACE was used to isolate carp L2 cDNA clones. The VL sequences could be divided into two distinct VL families, designated VL2-1 and VL2-2, most similar to rainbow trout (68% similarity) and zebrafish (78%) VL2 amino acid sequences, respectively. The CL amino acid sequences showed the highest similarity to zebrafish L2 (80%), and contained the characteristic cysteines necessary for intradomain or interchain disulphide bridges as did the VL sequences. Neither the VL nor CL sequences demonstrated such a high similarity to the other carp IgL isotypes, L1A, L1B, and L3. For JL segments, sequence variations appeared to be confined to a few positions. In the course of 5′- and 3′-RACE, cDNA clones containing recombination signal sequence (RSS), representatives of IgL sterile transcripts, were obtained. Southern blot analyses suggested that the locus of carp L2 has a cluster-like organisation. Phylogenetic analyses showed that both carp VL2 and CL2 amino acid sequences highly clustered with other teleost L2 sequences.

Human IgG Fc-binding phage antibodies constructed from synovial fluid CD38+ B cells of patients with rheumatoid arthritis show the imprints of an antigen-dependent process of somatic hypermutation and clonal selection.

The persistent presence of rheumatoid factors (RFs) in the circulation is a characteristic phenomenon in patients with rheumatoid arthritis (RA). Recent data indicate that RFs associated with seropositive RA are derived from terminally differentiated CD20-, CD38+ plasma cells (PCs) present in synovial fluids of the inflamed joints. These cells were shown to secrete RFs actively and are thought to originate from germinal centre (GC)-like structures present in the inflamed synovium. To obtain a representative image of the structural properties of IgM and IgG RFs associated with RA, phage antibody display libraries were constructed from CD38+ PCs isolated from the inflamed joints of RF-seropositive patients with RA. Subsequently, human IgG Fc-binding monoclonal phage antibodies were selected and analysed. The data suggest that RA-associated RFs are encoded by a diverse set of VL and a more restricted set of VH regions. VH gene family usage of PC-derived IgM- and IgG-RFs was found to be restricted to the VH1 and 3 gene families, with a preference for VH3, and many different VL genes were shown to contribute to RF specificity. Clonally related VH as well as VL sequences were identified, based on the presence of identical CDR3 regions and shared somatic mutations. In this B cell selection process base-pair substitutions as well as deletions of triplets in CDR regions, leaving the transcripts in frame, were involved. Together, these data provide further evidence for an Ag-driven immune response in the terminal differentiation into RF-producing PCs in patients with RA, including expansion of clonally related B cells, selection and isotype switching, all hallmarks of a GC reaction.

Specificity mapping of human anti-T cell receptor monoclonal natural antibodies: defining the properties of epitope recognition promiscuity.

The classical concept of antibody binding is defined as an exclusive and high-affinity interaction with one epitope. The emerging reality about antibody combing sites, however, is that some can bind unrelated determinants. The studies presented here define this quality as epitope recognition promiscuity by analyzing the capacity of monoclonal human autoantibodies to bind sets of overlapping peptides duplicating the complete structures of T cell receptor (TCR) alpha and beta chains and immunoglobulin lambda chain. We assessed the binding of these monoclonal antibodies (mAbs) to a set of homologous peptides corresponding to the CDR1 segments of human Vbeta gene products, a major epitope used in the selection of the antibodies. We present data on the binding characteristics of four human mAbs selected for the ability to bind TCR epitopes. These mAbs are IgM molecules with VH and VL sequences in germline configuration, but have diverse VH CDR3 regions. These studies aim to characterize the property of epitope promiscuity and show that the relationship between the binding site and its epitope is a complex interaction and unpredictable from antigen sequence alone. Our results support the conclusion that epitope recognition promiscuity is a genuine feature of antibody and TCR recognition.

Characterization of immunoglobulin light chain isotypes in the common carp.

Two or three isotypes of immunoglobulin (Ig) light (L) chain have been demonstrated in several teleost species thus far. A reverse transcription-polymerase chain reaction (RT-PCR) strategy was used with the aim of isolating cDNA clones encoding IgL chain from the common carp ( Cyprinus carpio. L.) different from the previously isolated isotype, designated L1A. cDNA clones representing two novel isotypes, designated L1B and L3, were obtained in this study. For CL segments, L1B sequences were similar to type L1/G in various teleost species, as well as L1A, while L3 sequences were closely related with type L3/F in catfish and zebrafish. For VL segments, however, L1A sequences demonstrated higher similarity to L3 rather than L1B. These results were also supported by the phylogenetic study. The similarity found between L1A and L3 VL sequences was based on the very high degree of nucleotide identity in FR1, and/or FR2 to FR3, strongly suggesting the involvement of interlocus recombination between them. Southern blot analyses suggested that the locus of L1B has a cluster-like organization, but that those of L3 and L1A are currently unknown due to cross-hybridization between those VL probes. Northern blot analyses showed that CL mRNA of each isotype was expressed in lymphoid tissues examined, particularly strongly in pronephros, and was predominantly composed of approximately 1-kb and 0.6-kb transcripts, possibly corresponding to fully spliced (LVJC) and truncated (JC/J-C) forms, respectively.

Characterisation of immunoglobulin light chain cDNAs of the Atlantic salmon, Salmo salar L.; evidence for three IgL isotypes

By screening a cDNA library and analysis of DNA produced by a combined 3′RACE/5′-anchored PCR, we have isolated three isotypes of IgL in the Atlantic salmon. Two of the isotypes were homologous to rainbow trout IgL1 and L2 sequences, while the third represents a previously uncharacterised salmonid IgL. The novel type 3 CL region is homologous to spotted wolffish c1 and yellowtail sequences, while the VL region is more similar to channel catfish F class than to any other fish VL sequences. Southern analysis indicates that the gene segments of all three isotypes are organised in multiple clusters. In addition, the VL gene segments of type 3 are arranged in opposite orientation relative to the JL and CL segments, while gene segments in type 2 clusters are all in the same orientation. Although transcripts of type 1 and 3 were readily found in the spleen and head kidney, only minute amounts of type 2 transcripts were seen. The majority of type 3 messages were truncated, suggesting that spliced and full-length transcripts of this isotype probably are present at a low level compared to type 1 transcripts. The uniqueness of the type 3 VLJL sequences suggests that this isotype offers additional diversity to the antigen-binding site of Atlantic salmon immunoglobulins.

Sequence analysis of V(4-34)-encoded antibodies from single B cells of two patients with systemic lupus erythematosus (SLE).

SLE is an autoimmune disease characterized by the presence of autoantibodies against double-stranded (ds)DNA. A large proportion (approx. 40%) of patients with lupus also have increased levels of serum immunoglobulin encoded by the V(4-34) heavy chain gene, which often fluctuate with disease activity, and this gene is utilized by a subset of anti-dsDNA antibodies. In order to probe the nature of the V(4-34)-encoded immunoglobulin, B cells were isolated from the blood of two patients with active disease, using the 9G4 MoAb specific for the immunoglobulin gene product. Following cell picking, single-cell polymerase chain reaction (PCR) amplification of cDNA was used to investigate both V(H) and V(L) genes. Sequences were obtained from B cells synthesizing IgM (n = 10), IgG (n = 4) and IgA (n = 1). For V(H), all were derived from V(4-34) as expected, and the isotype-switched sequences and 2/6 IgM sequences were somatically mutated. In contrast, V(L) (12 kappa and 3 lambda) showed a low level of mutation, possibly indicating secondary rearrangements. The three most highly mutated V(H) sequences were associated with unmutated V(L) sequences. Analysis of the distribution of mutations revealed only minor clustering in complementarity-determining regions (CDRs) characteristic of antigen selection. The CDR3 lengths of V(H) ranged from five to 19 amino acids, and in 3/15 there was evidence of an excess of positively charged amino acids, compared with the normal expressed repertoire. Basic amino acids were also found at the V(L)-J(L) junctions in 4/15. These findings provide insight into the V(4-34)-V(L) gene combinations used by B cells in patients with SLE which might have clinical relevance.

Influences of amino acid sequences in FR1 region on binding activity of the scFv and Fab of an antibody to human gastric cancer cells

Murine anti-gastric cancer mAb 3H11 has promising clinical applications. In this work, engineering of 3H11 scFv and Fab was conducted to increase its usefulness. 3H11 scFv and Fab were constructed by PCR amplification of the V-domains with degenerate primers for FR1. The bacterially expressed 3H11 Ab fragments showed no antigen binding activity. Then the N-terminal sequences of V regions were mutated to the 3H11 original sequence. The expressed scFv and Fab in bacterial culture supernatant showed binding activity to gastric cancer cells. Comparing the expression of unmutated and mutated 3H11 Fab, we found that the sequence changes of the V region N terminus introduced by PCR may seriously affect antigen binding but not the expression of antibody. Correction of either VL or VH N-terminal sequences can partially restore the antigen binding activity (61% for VL and 73% for VH).

Co-secretion of two distinct kappa light chains by the mu-9 hybridoma.

Mu-9 is a monoclonal antibody (MAb) specific for the CSAp antigen (Ag) expressed by colorectal cancers. By using variable (V)-region-specific primers, the respective VH and VL sequences of Mu-9 were polymerase chain reaction (PCR)-amplified. However, chimeric Ab (cMu-9-1) constructed from these PCR-amplified V sequences failed to bind the CSAp Ag. Although the light chain of murine Mu-9 was not glycosylated, that of cMu-9-1 was found to be O-glycosylated, as confirmed by reducing SDS-PAGE analyses, glycoprotein blotting and O-linked specific deglycosylation studies. Removal of O-linked oligosaccharides either by enzymatic digestion or by blocking O-glycosylation with a specific inhibitor did not restore the immunoreactivity of cMu-9-1, indicating that light chain O-glycosylation was not the cause for lack of immunoreactivity. We reported earlier that screening of a Mu-9 cDNA library uncovered the presence of an additional light chain sequence that was later proven to be the authentic light chain of Mu-9. Analyses of the cDNA sequence encoding the nonimmunoreactive light chain, however, revealed no defects that would preclude the sequence from being translated and secreted by the murine hybridoma. By adapting the Mu-9 hybridoma culture to serum-free conditions, we confirmed the secretion of low levels of O-glycosylated light chain. The biological significance of the O-glycosylation as well as the cosecretion of both light chains with respect to allelic exclusion are discussed.

Inhibition of human immunodeficiency virus type 1 replication in vitro in acutely and persistently infected human CD4+ mononuclear cells expressing murine and humanized anti-human immunodeficiency virus type 1 Tat single-chain variable fragment intrabodies.

We have previously reported that a murine anti-Tat sFv intrabody, termed sFvtat1Ck, directed against the proline-rich N-terminal activation domain of HIV-1, is a potent inhibitor of HIV-1 replication [Mhashilkar, A. M., et al. (1995). EMBO J. 14, 1542-1551]. In this study, the protective effect of sFvtat1Ck expression on HIV-1 replication in both acutely infected and persistently infected CD4+ cells was examined. Stably transfected CD4+ SupT1 cells were resistant to HIV-1 infection at high MOI with both the laboratory isolate HxB2 and six syncytium-inducing (SI) primary isolates. Persistently infected U1 cells, which can be induced to increase HIV-1 mRNA synthesis on addition of PMA or TNF-alpha, showed decreased production of HIV-1 in the presence of sFvtat1Ck. In transduced CD4+-selected, CD8+-depleted, and total PMBCs, the sFvtat1Ck-expressing cells showed marked inhibition of HIV-1 replication. The anti-Tat sFv was subsequently humanized by substituting compatible human framework regions that were chosen from a large database of human V(H) and V(L) sequences on the basis of high overall framework matching, similar CDR length, and minimal mismatching of canonical and V(H)/V(L) contact residues. One humanized anti-Tat sFv intrabody, termed sFvhutat2, demonstrated a level of anti-HIV-1 activity that was comparable to the parental murine sFv when transduced PBMCs expressing the murine or humanized sFv intrabodies were challenged with HxB2 and two SI primary isolates. Because Tat is likely to have both direct and indirect effects in the pathogenesis of AIDS through its multiple roles in the HIV-1 life cycle and through its effects on the immune system, the strategy of genetically blocking Tat protein function with a humanized anti-Tat sFv intrabody may prove useful for the treatment of HIV-1 infection and AIDS, particularly when used as an adjuvant gene therapy together with highly active antiretroviral therapies that are currently available.

Molecular Analysis of Immunoglobulin Genes in Multiple Myeloma

The study of immunoglobulin genes in multiple myeloma over the last five years has provided important information regarding biology, ontogenetic location, disease evolution, pathogenic consequences and tumor-specific therapeutic intervention with idiotypic vaccination. Detailed analysis of VH genes has revealed clonal relationship between switch variants expressed by the bone marrow plasma cell and myeloma progenitors in the marrow and peripheral blood. VH gene usage is biased against V4–34 (encoding antibodies with cold agglutinin specificity; anti-l/i) explaining the absence of autoimmune phenomena in myeloma compared to other B-cell lymphoproliferative disorders. VH genes accumulate somatic hyper-mutations following a distribution compatible with antigen selection, but with no intraclonal heterogeneity. VL genes indicate a bias in usage of VKI family members and somatic hyper-mutation, in line with antigen selection, of the expressed VK genes is higher than any other B-cell lymphoid disorder. A complementary imprint of antigen selection as evidenced by somatic hypermutation of either the VH or VL clonogenic genes has been observed. The absence of ongoing somatic mutations in either VH or VL genes gives rise to the notion that the cell of origin in myeloma is a post-germinal center memory B-cell. Clinical application of sensitive PCR methods in order to detect clonal immunoglobulin gene rearrangements has made relevant the monitoring and follow-up of minimal residual disease in stem cell autografts and after myeloablative therapy. The fact that surface immunoglobulin VH and VL sequences consitute unique tumor-specific antigenic determinants has stimulated investigators to devise strategies aiming to generate active specific immunity against the idiotype of malignant B-cells in myeloma by constructing vaccines based on expressed single-chain Fv fragments, DNA plasmids carrying VH+VL clonogenic genes for naked DNA vaccination, or dendritic cell-based vaccination armed with the tumor-specific idiotype.

Probing the interaction between a high-affinity single-chain Fv and a pyrimidine (6-4) pyrimidone photodimer by site-directed mutagenesis.

We have used site-directed mutagenesis to uncover the origin of the binding affinity differences exhibited by a series of monoclonal antibodies that recognize pyrimidine (6-4) pyrimidone photoproducts in the context of single- or double-stranded DNA. In this study, we have focused on two antibodies-64M3 and 64M5-that share the same binding specificity but differ in their affinities. We used single-chain Fv (scFv) derivatives for these studies since they can be easily expressed in Escherichia coli. To facilitate this, we also developed a simple, on-column refolding procedure for scFvs that is rapid and does not require high dilution. We took several precautions to ensure that the scFvs faithfully reflected the behavior of the parent monoclonal antibodies. Results obtained from chimeric scFvs constructed from 64M3 and 64M5 suggested that the higher affinity of the 64M5 antibody was mainly due to its VL region. Loop-grafting studies in which VH CDR loops of 64M3 were individually transplanted into 64M5 were consistent with this hypothesis. Since the VL sequences of 64M3 and 64M5 differ at only three positions (L30, L50, and L90), alanine-scanning mutagenesis was used to assess the importance of these three residues in DNA binding by 64M5. These studies highlighted the importance of all three VL CDR loops; furthermore, they suggested that photoproduct binding involved conformational changes within the VL region.

Molecular analyses of VH and VL genes used by 7 cases of Primary Effusion Lymphomas (PEL) disclose the presence of somatic mutations indicative of antigen selection.

99 Background: PELs are an uncommon subset of AIDS related lymphomas that grow mainly in the body cavities as lymphomatous effusions. Because of the presence of a clonal rearrangement of the Ig genes these lymphomas are classified of B lineage origin although they lack of surface B cell antigens. To date no data are available on the sequences of these rearranged genes. Methods: We have sequenced the VH and VL genes used by 4 cell lines (HBL6, BC1, BC2, and BC3) derived from PEL and from 4 biopsy samples. HBL6 and BC1 were derived from the same patient though at different times. Results: VH gene usage among the samples was the following: 3 cases used a VH3 family gene (two the 3-23 gene and one the 3-73 gene), 2 cases employed a VH4 gene (both 4-39), 1 case used a VH1 gene (1-03) and 1 a VH5 gene (5-51). The malignant cells expressed a JH5 (2 cases), JH6 (4 cases) segments, and quite strikingly JH4 (the most common JH) was expressed only by one case. The D segments were not clearly assignable except than in two cases in which stretches deriving from the D2-15/D and from the D5-18 genes were identified. Indeed in these two cases the CDR3 length was longer than in the others (16 and 20 aa respectively against a general average of 13 aa). A productive rearrangement of a Lambda light chain was found in 6 out of 7 samples. The only sample having a productive rearrangement was the cell line HBL6 (and BC1). Among the Lambda genes the VL3 family was used predominantly (4 out of six samples). In all instances but one (BC3 cell line), the comparison of the VH and VL sequences with a database discloses significant nucleotide differences with the presumptive germline. We analyzed the pattern of these mutations using the classical R:S ratio and using a statistical analysis proposed by Chang and Casali. Both these analyses disclosed that the mutations present in the antigen receptors were compatible with antigen selection either at VH or VL level in all the samples. Conclusions: The presence and the pattern of the point mutations at VH and VL level in the cases reported suggest that these were "experienced" cells that had undergone stimulation/selection by antigen(s). Overall these data suggest that (i) PEL frequently derives from memory cells possibly of GC- or post-GC B cell and (li) antigen induced stimulation and selection may be part of the pathogenic origin of these lymphomas.

Monoclonal anti-idiotypic antibodies mimicking the immunodominant epitope of influenza virus haemagglutinin elicit biologically significant immune responses.

The MAb IIB4 recognizes an immunodominant epitope on influenza virus haemagglutinin (HA) which is shared by different strains of the human subtype H3. This epitope includes amino acids 198, 199 and 201, as determined by selection of IIB4 escape mutants, and is involved in haemagglutination-inhibition (HI) and virus-neutralization (VN). We have developed anti-idiotypic MAbs (Ab2) that mimic the IIB4 epitope. Two Ab2, 78 and 464, completely inhibited binding of MAb IIB4 to the virus. Nucleotide sequences of VL- and VH-encoding regions were determined for IIB4 and both Ab2. VH of IIB4 and 78 belong to the same IgG family (VII) and show high nucleotide identity (89%). Conversely, VH and VL sequences of both internal image-bearing Ab2 revealed lower degrees of identity (61 and 50%, respectively). Ab2 were used for syngeneic immunization to elicit polyclonal Ab3 responses. Like Ab1, Ab3 immunoprecipitated viral HA and displayed HI and VN activity. The different VN activity of anti-78 and anti-464 in vitro correlated with the affinities of their corresponding Ab2 to IIB4. In vivo immunization with either Ab2 protected 15-37% of mice against lethal influenza infection or delayed dying. In contrast to VN activity of Ab3 in vitro, there was no significant difference between the protection of mice induced by Ab2 78 and 464. We demonstrate, for the influenza model, that active immunization with a single influenza virus HA epitope in the form of its internal image leads to partial protection in vivo.

Cardiolipin binding a light chain from lupus-prone mice.

Autoantibodies in systemic lupus erythematosus react with multiple epitopes on highly conserved molecules such as nucleic acids, cytoskeletal proteins, phospholipids, and phospholipid-binding proteins. Analysis of the heavy- and light-chain variable sequences (VH and VL) has shown that a restricted set of V genes gives rise to these autoantibodies. Several monoclonal antibodies were developed from a strain of mouse prone to lupus (F1 male NZW x BXSB). Two of these antibodies, A1.72 and A1.84, reacted directly with cardiolipin and their VH and VL sequences were analyzed. Surprisingly, these two antibodies had identical light-chain variable sequences despite having substantially different heavy-chain variable sequences. This VL sequence, VL 72/84 was 97% identical with the germ-line sequences with only four single nucleotide substitutions. When this VL sequence was shuffled with the VH sequence of other monoclonal antibodies and expressed as single chain variable fragment (scFv) in Escherichia coli, it imparted cardiolipin-binding activity to the hybrids. Furthermore, the VL 72/84 sequence, when expressed alone without any VH sequence, also bound to cardiolipin. The antibodies and their recombinant fragments were immunoaffinity-purified on cardiolipin liposomes. The dissociation constant of the light chain for cardiolipin was similar to the intact molecule (21 +/- 0.01 vs 20 +/- 0.03 nM). These studies demonstrate that the VL sequence alone, in the absence of any other immunoglobulin domains, can mediate cardiolipin binding, raising the possibility that antigen specificity of certain antibodies may exclusively reside in their light-chain sequences.

Insight into Burkitt's Lymphoma from Immunoglobulin Variable Region Gene Analysis

Analysis of usage of VH and VL genes, and the degree and pattern of somatic mutation, has been used to investigate the cell of origin and clonal history in cases of Burkitt's lymphoma (BL). Tumor cell lines and biopsy material from patients with endemic, sporadic and AIDS-associated BL have been compared. VH genes were most commonly derived from the VH3 (52%) and VH4 (39%) families. This shows a similar gene usage of the VH3 family to that seen in the normal peripheral blood repertoire (55%), but a biased usage of the VH4 family (22% in normal). There was no restriction in VL gene usage. This overall distribution was similar in all subsets of BL. In all categories, there was significant somatic mutation in both VH and VL sequences. There was no evidence for accumulation of mutations in cell lines cultured in vitro indicating that all mutations in BL-derived cell lines have accumulated in vivo, The mean percentage level of mutation ± ± standard deviation was greater in endemic BL (VH = 7.7 ± 4.0, VL = 5.3 ± 2.2) and AIDS-associated BL (VH = 7.5 ± 3.6, VL = 3.9 ± 1.9) than in sporadic BL (VH = 4.0 ± 2.5, VL = 2.2 ± 1.2). The pattern of somatic hypermutation was similar in VH and VL sequences of the different types of BL although the light chain genes were less mutated. Mutational patterns in the VH genes did not reveal a conventional role for antigen in selection of tumor cell sequences in 23/25 VH genes analysed. In contrast, patterns in VL sequences were consistent with a role for antigen in 8/13 sBL + eBL cases and 8/17 cases overall.The presence of EBV did not seem to influence the quantity or pattern of somatic mutations. Evidence for intraclonal variation was seen in uncloned cell lines from cases of eBL and AIDS-associated BL and confirmed in biopsy material in some, but not all cases of eBL, sBL and AIDS-associated BL examined. These common features indicate that the B-cells involved in all types of BL are derived from cells that have traversed the germinal centre, and that the somatic mutation mechanism may still be operative following neoplastic transformation. Overall, in 10/30 cases, there was evidence of significant clustering of replacement amino acids, in CDRs, particularly in VL, indicating that the B-cell of origin is likely to have been selected by antigen.

Phage surface expression for analysis of recognition sites of human autoantibodies: Comparison of single chain Fv and Fab

Human autoantibodies can mediate the pathology of autoimmune disease. To plan therapeutic intervention based on inhibition of binding to autoantigen, it is desirable to map the amino acid residues involved in recognition. Bacterial expression provides a simple and rapid system for production and mutagenesis of recombinant antibody fragments, but there are problems with reproducible folding and dispersity of soluble products. Expression at the surface of phage particles avoids both these problems, and allows variable region gene sequences to be expressed as functional monomers. To apply this technology, we have expressed a human anti-DNA monoclonal antibody, derived from a hybridoma, at the surface of the phage. The presence of the phage particle allowed highly sensitive detection of antibody activity by ELISA, so that minimal levels of expression were required. By measuring the amount of expressed antibody protein, it was possible to analyze the antibody activity/microgram. We expressed the antibody as FAB or single chain (sc) Fv, and found binding activities to be closely similar. The parental anti-DNA antibody utilizes the human V4-34 gene and displays the associated conformation-dependent 9G4 idiotope. Both the Fab and scFv expressed the idiotope, confirming similar molecular folding. This technology can be applied rapidly and efficiently to investigations of human autoantibody structure, with the option for expression as either Fab or scFv, and the only requirement being knowledge of VH and VL sequences.

Construction, expression, and activities of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation.

The L49 (IgG1) monoclonal antibody binds to p97 (melanotransferrin), a tumor-selective antigen that is expressed on human melanomas and carcinomas. A recombinant fusion protein, L49-sFv-bL, that contains the antibody binding regions of L49 fused to the Enterobacter cloacae r2-1 beta-lactamase (bL) was constructed, expressed, and purified to homogeneity in an Escherichia coli soluble expression system. The variable regions of L49 were cloned by reverse transcription-polymerase chain reaction from L49 hybridoma mRNA using signal sequence and constant region primers. Construction of the gene encoding L49-sFv-bL was accomplished by hybridization insertion of VH, VL, and sFv linker sequences onto a pET phagemid template containing the bL gene fused to the pelB leader sequence. Optimal soluble expression of L49-sFv-bL in E. coli was found to take place at 23 degrees C with 50 microM isopropyl beta-D-thiogalactopyranoside induction and the use of the nonionic detergent Nonidet P-40 for isolation from the bacteria. Construction and expression of a soluble form of the p97 antigen in Chinese hamster ovary cells allowed affinity-based methods for analysis and purification of the fusion protein. Surface plasmon resonance, fluorescent activated cell sorting, and Michaelis-Menten kinetic analyses showed that L49-sFv-bL retained the antigen binding capability of monovalent L49 as well as the enzymatic activity of bL. In vitro experiments demonstrated that L49-sFv-bL bound to 3677 melanoma cells expressing the p97 antigen and effected the activation of 7-(4-carboxybutanamido)cephalosporin mustard (CCM), a cephalosporin nitrogen mustard prodrug. On the basis of these results, L49-sFv-bL was injected into nude mice with subcutaneous 3677 tumors, and localization was determined by measuring bL activity. Tumor to blood conjugate ratios of 13 and 150 were obtained 4 and 48 h post conjugate administration, respectively, and the tumor to liver, spleen, and kidney ratios were even higher. A chemically produced L49-Fab'-bL conjugate yielded a much lower tumor to blood ratio (5.6 at 72 h post administration) than L49-sFv-bL. Therapy experiments established that well-tolerated doses of L49-sFv-bL/CCM combinations resulted in cures of 3677 tumors in nude mice. The favorable pharmacokinetic properties of L49-sFv-bL allowed prodrug treatment to be initiated 12 h after the conjugate was administered. Thus, L49-sFv-bL appears to have promising characteristics for site-selective anticancer prodrug activation.

Primary structure and functional scFv antibody expression of an antibody against the human protooncogen c-myc.

The immunoglobulin heavy- and light-chain variable region (Vh and Vl) genes were isolated from Myc1-9E10 hybridoma cells, which secreted monoclonal antibody against human oncogen c-myc. The expression vector pOPE52-c-myc was constructed for the recombinant production in E. coli. A 30 kDa single chain fragment (scFv) expression product was found in the periplasmic space by SDS-PAGE and immunoblotting. A significant fraction was processed correctly as demonstrated with an antiserum recognizing the processed aminoterminus only. The specific binding of the scFv fragment to the peptide epitope of the maternal monoclonal antibody was demonstrated and the primary sequence of the variable regions was determined. Sequence comparison with previously published partial Vh and Vl sequences from this hybridoma cell line revealed a genetic heterogeneity for the light chain variable region. The potential use of this scFv as a new tool for detection and purification of tagged proteins, for adding costimulatory signals to the surface of cancer cells as well as for analyzing c-myc function in the living cell by cytoplasmic expression is discussed.

Myeloma VL and VH Gene Sequences Reveal a Complementary Imprint of Antigen Selection in Tumor Cells

In multiple myeloma, sequence studies of VH genes used to encode clonal Ig in neoplastic plasma cells have shown a common pattern of extensive somatic hypermutation. A further consistent feature of these VH sequences is a complete lack of intraclonal variation. These findings indicate that the malignant cell arises at a mature, postfollicular stage of B-cell development. However, only a minority of cases have a distribution of somatic mutations in VH consistent with a prior role for antigen in selecting the B cell of origin. To complement these studies, and to take further the investigation of a role for antigen in the clonal history of myeloma, we have investigated tumor-derived VL sequences from bone marrows of 15 patients. All sequences (9Vκ and 6Vλ) were potentially functional and 5 of 15 had evidence for N-region additions. All had undergone extensive somatic hypermutation, and showed no intraclonal variation. In 4 of 15 cases, the distribution of mutations revealed a significant (P < .05) clustering of replacement mutations in the CDR sequences, indicating a role for VL in selection by antigen. Comparison with the VH sequences used by the same tumor cells showed that, if significant clustering was present, it was in either VH or VL, but not both. Altogether, 10 of 15 V-regions showed evidence for antigen selection, suggesting that the B cell of origin has behaved as a normal germinal center B cell. Deductions concerning a role for antigen selection may require both VH and VL sequences for validation.

Myeloma VL and VH Gene Sequences Reveal a Complementary Imprint of Antigen Selection in Tumor Cells

AbstractIn multiple myeloma, sequence studies of VH genes used to encode clonal Ig in neoplastic plasma cells have shown a common pattern of extensive somatic hypermutation. A further consistent feature of these VH sequences is a complete lack of intraclonal variation. These findings indicate that the malignant cell arises at a mature, postfollicular stage of B-cell development. However, only a minority of cases have a distribution of somatic mutations in VH consistent with a prior role for antigen in selecting the B cell of origin. To complement these studies, and to take further the investigation of a role for antigen in the clonal history of myeloma, we have investigated tumor-derived VL sequences from bone marrows of 15 patients. All sequences (9Vκ and 6Vλ) were potentially functional and 5 of 15 had evidence for N-region additions. All had undergone extensive somatic hypermutation, and showed no intraclonal variation. In 4 of 15 cases, the distribution of mutations revealed a significant (P < .05) clustering of replacement mutations in the CDR sequences, indicating a role for VL in selection by antigen. Comparison with the VH sequences used by the same tumor cells showed that, if significant clustering was present, it was in either VH or VL,but not both. Altogether, 10 of 15 V-regions showed evidence for antigen selection, suggesting that the B cell of origin has behaved as a normal germinal center B cell. Deductions concerning a role for antigen selection may require both VH and VL sequences for validation.