Antigen L6 Research Articles

Immunoprofiling of Drosophila Hemocytes by Single-cell Mass Cytometry

Single-cell mass cytometry (SCMC) combines features of traditional flow cytometry (i.e., fluorescence-activated cell sorting) with mass spectrometry, making it possible to measure several parameters at the single-cell level for a complex analysis of biological regulatory mechanisms. In this study, weoptimizedSCMC to analyze hemocytes of the Drosophila innate immune system. We used metal-conjugated antibodies (against cell surface antigens H2, H3, H18, L1, L4, and P1, and intracellular antigens 3A5 and L2) and anti-IgM (against cell surface antigen L6) to detect the levels of antigens, while anti-GFP was used to detect crystal cells in the immune-induced samples. We investigated the antigen expression profile of single cells and hemocyte populations in naive states, in immune-induced states, in tumorous mutants bearing a driver mutation in the Drosophila homologue of Janus kinase (hopTum) and carrying a deficiency of the tumor suppressor gene lethal(3)malignant blood neoplasm-1 [l(3)mbn1], as well as in stem cell maintenance-defective hdcΔ84 mutant larvae. Multidimensional analysis enabled the discrimination of the functionally different major hemocyte subsets for lamellocytes, plasmatocytes, and crystal cells, anddelineated the unique immunophenotype of Drosophila mutants. We have identified subpopulations of L2+/P1+ and L2+/L4+/P1+ transitional phenotype cells in the tumorous strains l(3)mbn1 and hopTum, respectively, and a subpopulation of L4+/P1+ cells upon immune induction. Our results demonstrated for the first time that SCMC, combined with multidimensional bioinformatic analysis, represents a versatile and powerful tool to deeply analyze the regulation of cell-mediated immunity of Drosophila.

A Polypeptide of Tumor-Associated Antigen L6 with Intrinsic Adjuvant Activity Enhances Antitumor Immunity

Peptide vaccines are safe, and aim to elicit and expand tumor-specific immunity so as to eradicate tumors. However, achieving strong and long-lasting anti-tumor immunity with peptide vaccines for the antigen-specific treatment of cancer is challenging, in part because their efficacy depends on strong adjuvants or immunomodulators. We approached this problem by conjugating an epitope-based cancer vaccine with a lipidated sequence (an immunomodulator) to elicit a strong immune response. Lipidated and non-lipidated polyepitope proteins were generated that contained the universal T helper cell epitope (pan-DR), B cell epitopes, and the extended loop sequence of extracellular domain 2 of tumor-associated antigen L6 (TAL6). We show that the lipidated polyepitope cancer vaccine can activate bone marrow-derived dendritic cells, and trigger effective antigen-specific antibody and T helper cell responses, more effectively than the non-lipidated vaccine. Moreover, potent T cell immune responses were elicited in mice inoculated with the lipidated polyepitope cancer vaccine, providing protective antitumor immunity in mice bearing TAL6 tumors. Our study demonstrates that a lipidated polyepitope cancer vaccine could be employed to generate potent anti-tumor immune responses, including humoral and cellular immunity, which could be beneficial in the treatment of TAL6+ cancer.

83P - Synthetic peptide of tumour–associated antigen L6 formulated with polymer-based adjuvant enhances anti-tumour effects in mice

BackgroundTumour associated antigen L6 (TAL6) is a cell surface protein of the transmembrane-4 superfamily (TM4SF) also known as TM4SF1. TAL6 is over-expressed in more than 80 % of human lung, breast, colon and ovarian tumours but not normal tissues. Synthetic peptides are attractive for cancer immunotherapy because of their safety and flexibility. We have identified an B cell epitope of TAL6 using monoclonal antibodies. The synthetic peptide epitope formulated with adjuvants can induce anti-tumour immunity. MethodsThe linear B cell epitope was identified by anti-TAL6 monoclonal antibody using overlapping peptides cover the extracellular domain of TAL6. The synthetic peptide containing B cell epitope was formulated with different adjuvants to immunize mice to determine their immunogenecity. Furthermore, the sera of immunized mice were used to examine the antibody-dependent cellular cytotoxicity (ADCC) against human cancer. Moreover, the anti-tumor effects were evaluated in mouse model. ResultsThe anti-TAL6 recognized a minimal linear region at amino acid 121-129. We formulated synthetic peptide (a.a.114-133) and a pan-DR peptide with different adjuvants to immunize mouse and evaluate their immunogenicity. The peptides formulated with an emulsion type nanoparticle (PELC) adjuvant and a toll-like receptor 9 agonist (CpG ODN) (PELC/CpG) induced the greatest ADCC responses in TAL6-expresssed cell lines. The induced anti-tumor immunity inhibited the growth of TAL6-positive cancer cells in mouse. ConclusionsThese data suggested that a peptide containing B cell epitopes of TAL6 formulated with PELC/CpG adjuvant is feasible for cancer immunotherapy. Legal entity responsible for the studyThe authors. FundingMinistry of Science Technology, Taiwan. DisclosureAll authors have declared no conflicts of interest.

Chimeric peptide containing both B and T cells epitope of tumor-associated antigen L6 enhances anti-tumor effects in HLA-A2 transgenic mice

Synthetic peptides are attractive for cancer immunotherapy because of their safety and flexibility. In this report, we identified a new B cell epitope of tumor-associated antigen L6 (TAL6) that could induce antibody-dependent cellular cytotoxicity (ADCC) in vivo. We incorporated the B cell epitope with a cytotoxic T lymphocyte (CTL) and a helper T (Th) epitope to form a chimeric long peptide. We formulated the chimeric peptide with different adjuvants to immunize HLA-A2 transgenic mice and evaluate their immunogenicity. The chimeric peptide formulated with an emulsion type nanoparticle (PELC) adjuvant and a toll-like receptor 9 agonist (CpG ODN) (PELC/CpG) induced the greatest ADCC and CTL responses. The induced anti-tumor immunity inhibited the growth of TAL6-positive cancer cells. Moreover, we observed that immunization with the chimeric peptide inhibited cancer cell migration in vitro and metastasis in vivo. These data suggest that a chimeric peptide containing both B and T cell epitopes of TAL6 formulated with PELC/CpG adjuvant is feasible for cancer immunotherapy.

A HLA-A2-restricted CTL epitope induces anti-tumor effects against human lung cancer in mouse xenograft model.

Cancer immunotherapy is attractive for antigen-specific T cell-mediated anti-tumor therapy, especially in induction of cytotoxic T lymphocytes. In this report, we evaluated human CTL epitope-induced anti-tumor effects in human lung cancer xenograft models. The tumor associated antigen L6 (TAL6) is highly expressed in human lung cancer cell lines and tumor specimens as compared to normal lung tissues. TAL6 derived peptides strongly inhibited tumor growth, cancer metastasis and prolonged survival time in HLA-A2 transgenic mice immunized with a formulation of T-helper (Th) peptide, synthetic CpG ODN, and adjuvant Montanide ISA-51 (ISA-51). Adoptive transfer of peptide-induced CTL cells from HLA-A2 transgenic mice into human tumor xenograft SCID mice significantly inhibited tumor growth. Furthermore, combination of CTL-peptide immunotherapy and gemcitabine additively improved the therapeutic effects. This pre-clinical evaluation model provides a useful platform to develop efficient immunotherapeutic drugs to treat lung cancer and demonstrates a promising strategy with benefit of antitumor immune responses worthy of further development in clinical trials.

A Novel HLA-A2–restricted CTL Epitope of Tumor-associated Antigen L6 can Inhibit Tumor Growth In Vivo

Vaccines utilizing cytotoxic T lymphocyte (CTL) epitopes are promising for the treatment of cancer and chronic infectious diseases. Tumor-associated antigen L6 (TAL6) is overexpressed in some epithelial cancer cells. In this report, we detected TAL6 expression in breast cancer tissue using quantitative reverse-transcriptase-polymerase chain reaction. We found that >80% of breast tumor tissue highly expressed TAL6 compared with adjacent normal breast tissue. To identify CTL epitopes from TAL6, we synthesized 18 peptides for HLA-A2-binding assay based on the MHC-binding motif using 4 computer prediction programs. Positive binders identified by ELISA were immunized in HLA-A2 transgenic (A2 Tg) mice. Two peptides, peptide 2 and peptide 5, induced T-cell responses in A2 Tg mice. To confirm whether these peptides could be processed and presented to induce T-cell responses in vivo, A2 Tg mice were immunized with plasmid DNA encoding TAL6. We found that both peptides 2 and 5 stimulated splenocytes from TAL6-immunized mice to secrete interferon-γ. However, only peptide 5 could induce expression of the cytolytic molecule CD107a on CD8+ T cells after immunization. Furthermore, peptide 5-immunized A2 Tg mice could inhibit the growth of TAL6-positive tumors (EL4/TAL6/HLA-A2) in A2 Tg mice but not in wild-type mice. These results demonstrate that the TAL6-derived CTL epitope could induce HLA-A2-restricted immunity against TAL6-expressing tumor cells.

Gene expression analysis after receptor tyrosine kinase activation reveals new potential melanoma proteins

BackgroundMelanoma is an aggressive tumor with increasing incidence. To develop accurate prognostic markers and targeted therapies, changes leading to malignant transformation of melanocytes need to be understood. In the Xiphophorus melanoma model system, a mutated version of the EGF receptor Xmrk (Xiphophorus melanoma receptor kinase) triggers melanomagenesis. Cellular events downstream of Xmrk, such as the activation of Akt, Ras, B-Raf or Stat5, were also shown to play a role in human melanomagenesis. This makes the elucidation of Xmrk downstream targets a useful method for identifying processes involved in melanoma formation.MethodsHere, we analyzed Xmrk-induced gene expression using a microarray approach. Several highly expressed genes were confirmed by realtime PCR, and pathways responsible for their induction were revealed using small molecule inhibitors. The expression of these genes was also monitored in human melanoma cell lines, and the target gene FOSL1 was knocked down by siRNA. Proliferation and migration of siRNA-treated melanoma cell lines were then investigated.ResultsGenes with the strongest upregulation after receptor activation were FOS-like antigen 1 (Fosl1), early growth response 1 (Egr1), osteopontin (Opn), insulin-like growth factor binding protein 3 (Igfbp3), dual-specificity phosphatase 4 (Dusp4), and tumor-associated antigen L6 (Taal6). Interestingly, most genes were blocked in presence of a SRC kinase inhibitor. Importantly, we found that FOSL1, OPN, IGFBP3, DUSP4, and TAAL6 also exhibited increased expression levels in human melanoma cell lines compared to human melanocytes. Knockdown of FOSL1 in human melanoma cell lines reduced their proliferation and migration.ConclusionAltogether, the data show that the receptor tyrosine kinase Xmrk is a useful tool in the identification of target genes that are commonly expressed in Xmrk-transgenic melanocytes and melanoma cell lines. The identified molecules constitute new possible molecular players in melanoma development. Specifically, a role of FOSL1 in melanomagenic processes is demonstrated. These data are the basis for future detailed analyses of the investigated target genes.

Regulation of TM4SF5-mediated tumorigenesis through induction of cell detachment and death by tiarellic acid

mRNA for four-transmembrane L6 family member 5 (TM4SF5), a homolog of tumor antigen L6, was previously shown to be highly expressed in diverse tumors. We recently found that human hepatocarcinoma tissues also overexpressed TM4SF5 protein, in comparison to normal liver tissues. We also found that tiarellic acid (TA) caused cell detachment-related apoptosis in cells expressing endogenous or stably-overexpressing TM4SF5. When cells expressing TM4SF5 were treated with TA, we observed reduced phosphorylation of focal adhesion kinase, paxillin, and p130Cas, but not c-Src. TA treatment also caused focal adhesion loss and reduced cell adhesion, and increased the numbers of floating cells and apoptotic cells. These effects were blocked by overexpression of focal adhesion molecules, suggesting that treatment with TA mediates anoikis of TM4SF5-expressing cells. However, TM4SF5-null cells were not affected by TA, indicating that these effects occur specifically in TM4SF5-positive cells. TA administration reduced tumor formation in nude mice injected with TM4SF5-expressing cells, presumably through increased apoptosis in TM4SF5-positive tumors. These observations indicate that TM4SF5-positive tumorigenesis can be inhibited by TA via induction of cell detachment-related apoptosis, and suggest that TA may be developed as a putative therapeutic reagent against TM4SF5-positive tumorigenesis.

Focal adhesion and actin organization by a cross-talk of TM4SF5 with integrin α2 are regulated by serum treatment

The biological functions of transmembrane 4 L6 family member 5 (TM4SF5) homologues to a tumor-associated antigen L6 are unknown, although it is over-expressed in certain forms of cancer. In the present study, the ectopic expression of TM4SF5 in Cos7 cells reduced integrin signaling under serum-containing conditions, but increased integrin signaling upon serum-free replating on substrates. TM4SF5 regulated actin organization and focal contact dynamics via the serum treatment-dependent differential regulation of FAK Tyr925 and paxillin Tyr118 phosphorylations and their localizations on peripheral cell boundaries. Y925F FAK mutation abolished the TM4SF5 effects. TM4SF5 associated with integrin α2 subunit, and this association was abolished by serum treatment. Furthermore, functional blocking anti-integrin α2 antibody abolished TM4SF5-enhanced signaling activity and caused membrane blebbing with abnormal actin organization. TM4SF5 increased chemotactic but decreased haptotactic migration. Altogether, this study reveals the functions of TM4SF5 collaborative with integrin signaling to alter focal contact dynamics, actin reorganization, and migration. Furthermore, this study suggests a mechanism of cross-talk between TM4SF5 and integrin which is further regulated by growth factor signaling.

CD13 (aminopeptidase N) can associate with tumor‐associated antigen L6 and enhance the motility of human lung cancer cells

Cancer metastasis is a multiple-step process that involves the regulated interaction of diverse cellular proteins. We recently reported that the expression of tumor-associated antigen L6 (TAL6) promoted the invasiveness of lung cancer cells and was inversely correlated with disease-free survival of squamous lung carcinoma patients. We now report that CD13 (aminopeptidase N) can associate with TAL6 and can enhance cancer cell migration. CD13 was shown by coimmunoprecipitation to associate in vitro with TAL6 on several cancer cell lines and to associate in vivo by antibody-mediated copatching immunofluorescence. CD13 was selectively expressed on highly invasive CL1-5 lung cancer cells as compared to poorly invasive CL1-0 lung cancer cells. The role of CD13 aminopeptidase activity in regulating cell motility was investigated with chemical inhibitors, specific antibodies and a catalytically inactive CD13 protein. Inhibition of CD13 aminopeptidase activity by nontoxic concentrations of leuhistin modestly decreased the migration of CL1-5 cells. In contrast, binding of CD13 by specific antibodies significantly reduced both the migration and the invasion of CL1-5 cells. Poorly invasive CL1-0 cells that stably expressed CD13 displayed significantly (p < or = 0.0005) enhanced cell migration (300% of control). Expression of an enzymatically inactive CD13 mutant on CL1-0 cells also significantly (p < or = 0.0005) enhanced cell migration (200% of control). Our results show that TAL6 and CD13 can form a complex on lung cancer cells, that these molecules can modulate cell migration and invasion and that the influence of CD13 on cell motility did not strictly depend on its aminopeptidase activity.

P-330 Tumor-associated antigen L6 (TAL6) is involved in the invasion of human lung cancer cells

P-330 Tumor-associated antigen L6 (TAL6) is involved in the invasion of human lung cancer cells

Diagnosis and monitoring of colorectal cancer by L6 blood serum polymerase chain reaction is superior to carcinoembryonic antigen-enzyme-linked immunosorbent assay.

The aim of this study was to compare carcinoembryonic antigen levels with detection of messenger ribonucleic acid coding for the tumor-associated antigen L6 in patients with colorectal cancer. Not only are carcinoembryonic antigens expressed by the corresponding tumor cell, but the messenger ribonucleic acid of tumor-associated antigens, in contrast, is produced exclusively by viable tumor cells. L6 messenger ribonucleic acid was determined by reverse-transcription polymerase chain reaction. Carcinoembryonic antigen was measured by the enzyme-linked immunosorbent assay technique, with a cutoff value of 40 microg/l. Blood serum was sampled from 187 patients with colorectal cancer. Statistical significance was calculated with the McNemar chi-squared test. Preoperatively, 79 percent of patients in all stages were positive for L6 messenger ribonucleic acid, whereas only 35 percent had elevated carcinoembryonic antigen titers (P < 0.001). In Dukes A tumors, 84.9 percent of patients were positive for L6 messenger ribonucleic acid, whereas carcinoembryonic antigen was elevated in only 16.9 percent of patients. Only in Dukes D tumors did the enzyme-linked immunosorbent assay for carcinoembryonic antigen exhibit the same sensitivity as reverse-transcription polymerase chain reaction for L6 messenger ribonucleic acid. Recurrence was detected significantly earlier by reverse-transcription polymerase chain reaction for L6 messenger ribonucleic acid than by enzyme-linked immunosorbent assay for carcinoembryonic antigen. L6 is more sensitive and precise than carcinoembryonic antigen in diagnosing and monitoring colorectal cancer.

Differential Expression of the L-Plastin Gene in Human Colorectal Cancer Progression and Metastasis

To identify molecular alterations in the progression of colorectal carcinoma, we analyzed gene expression profiles of colon cancer cell lines derived from primary and metastatic tumors from a single patient. Of 2280 cDNAs investigated using our in-house microarray, the expression of 6 genes (tumor-associated antigen L6, L-plastin, the human homologue of yeast ribosomal protein S28, the B-cell translocation gene, mitochondrial aspartate-aminotransferase, and HLA-A) increased, while that of 2 genes (keratin 5 and phosphoglucomutase) decreased in metastatic-tumor-derived cells compared with primary-tumor-derived cells. Of these genes, we assessed the L-plastin gene, an actin-bundling protein, at the protein level using a tissue microarray consisting of 58 clinically stratified colorectal cancer specimens. Consistent with our microarray results, the expression of L-plastin was significantly correlated with the progression of cancer staging. Therefore, our results suggest that the L-plastin gene is a potential metastatic marker. In addition, combining cDNA microarrays and tissue arrays, as shown here, is thought to facilitate the rapid characterization of candidate biomarkers.

Isolation and characterization of differentially expressed genes in invasive and non-invasive immortalized murine male germ cells in vitro

In an attempt to elucidate the potential of premeiotic male germ cells to malignant transformation both the invasiveness and the differential gene expression of several putative tumor markers of the spermatogonia-derived cell line GC-1spg and the spermatocyte-derived cell line GC-4spc were analyzed. Studies, using RT-PCR analysis, of the expression pattern of the alkaline phosphatase isoenzymes which serve as markers for testicular germ cell tumors demonstrated that the expression of the endogenous mouse embryonic alkaline phosphatase (EAP) is upregulated in the GC-1spg cell line. Additionally, after transfection of GC-1spg cells and GC-4spc cells with a GCAP-CAT construct, an increased promoter activity of the human germ cell alkaline phosphatase (GCAP), the equivalent human isoenzyme of EAP, was shown in GC-1spg. Furthermore, an in vitro Matrigel invasion assay revealed a significant higher invasive potential of GC-1spg cells as compared to GC-4spc cells. Finally, a suppression subtractive hybridization on RNA of invasive GC-1spg cells and non-invasive GC-4spc cells was performed. In total, 31 cDNA sequences were isolated and further analyzed. Among these, 18 known sequences and 13 unknown sequences were determined. Northern blot analysis revealed that one unknown gene and eight known genes, namely integrin alpha 6, L6 antigen, annexin VIII, BVL-1 retrotransposon, protective protein, replacement variant histone 3.3, alpha-catenin and LPS-binding protein, are over-expressed in invasive GC-1spg cells. Taken together, both the enhanced invasive activity of GC-1spg cells and the upregulated expression of genes involved in the process of tumor progression suggest that the immortalized spermatogonia-derived cell line GC-1spg does have a higher potential to malignant transformation than the immortalized spermatocyte-derived cell line GC-4spc.

The carboxy-terminal cysteine of the tetraspanin L6 antigen is required for its interaction with SITAC, a novel PDZ protein.

PDZ domains are protein modules that mediate protein-protein interactions. Here, we present the identification and characterization of a protein similar to the recently identified PDZ-containing protein TACIP18, which we have named SITAC (similar to TACIP18). SITAC is preferentially expressed in cells of the digestive tract, associated with intracellular membranes. Despite the high degree of sequence identity between the PDZ domains of TACIP18 and those of SITAC, none of the known ligands of the former shows interaction with the latter, as judged by two-hybrid analysis. SITAC interacts with peptides containing bulky hydrophobic amino acids two positions upstream of the C-terminal residue. Surprisingly, SITAC also shows interaction with peptides ending in C, a previously unacknowledged ability of PDZ domains. The sequence -Y-X-C-COOH, bound in vitro by SITAC, is present in the member of the tetraspanin superfamily, the L6 antigen. Coimmunoprecipitation experiments show that SITAC interacts with L6A, but not with an L6A C-terminal mutant, confirming the capacity of SITAC to interact with proteins ending in C. Confocal analysis shows that the interaction between L6A and SITAC is necessary for the precise colocalization of both molecules in the same subcellular compartment. In summary, the characterization of the protein SITAC has unveiled novel sequences recognized by PDZ domains, and it suggests that L6A is a natural ligand of this PDZ protein.

Identification of a new tumour-associated antigen TM4SF5 and its expression in human cancer.

In a previous large-scale screening for differentially expressed genes in pancreatic cancer, a gene was identified that was highly overexpressed in pancreatic cancer encoding a novel putative tetraspan transmembrane protein highly homologous to the tumour-associated antigen L6. Using a radiation hybrid panel the identified human gene named TM4SF5 (transmembrane 4 superfamily member 5) was localized to chromosome 17 in the region 17p13.3. The cloned TM4SF5 cDNA has a 32bp 5'-untranslated region (UTR), a 591bp open reading frame (ORF) and a 85bp 3'UTR. The predicted TM4SF5 protein with 197 amino acids contains three NH2-terminal hydrophobic transmembrane regions, followed by an extracellular hydrophilic domain containing two potential N-linked glycosylation sites and a COOH-terminal hydrophobic transmembrane region. These structural features are shared by the L6 antigen and a number of related cell surface proteins associated with cell growth. TM4SF5 was overexpressed in pancreatic cancer tissues as compared to both normal pancreas and chronic pancreatitis tissues, and was detected at high levels in other tumour tissues. Although the precise function of TM4SF5 remains to be elucidated it may be useful in a clinical setting for tumour diagnosis and/or therapy. This hypothesis is supported by the strong homology to the L6 antigen, which has proved promising in immunological, therapeutic and diagnostic approaches.

Costimulation by CD28 sFv expressed on the tumor cell surface or as a soluble bispecific molecule targeted to the L6 carcinoma antigen.

Interaction of the CD80 (B7-1) and CD86 (B7-2) molecules on antigen presenting cells with the receptors CD28 and CTLA-4 on T cells generates signals important in the regulation of immune responses. Because this receptor system involves multiple receptor-ligand interactions, determining the function for individual receptors has been difficult. One approach is the use of antibodies and their derivatives with singular specificity as substitute ligands to explore the activities of these molecules. We have constructed recombinant mono- and bi-specific sFv molecules specific for the CD28 receptor that are capable of binding and generating costimulatory signals to activate T cells. We demonstrate that these soluble molecules are capable of higher levels of costimulation than soluble CD80Ig at equivalent concentrations. We also constructed artificial adhesion receptors on the cell surface using two different CD28-specific sFvIgs fused to the CD80 cytoplasmic and transmembrane domains. In this report, we compared costimulation by a soluble bispecific (alpha CD28-alpha L6) single chain sFvIg fusion protein to that generated by L6 antigen positive (L6+) H3347 tumor cells transduced with cell surface expressed forms of alpha CD28 sFv's. We show that the bispecific protein can target potent CD28 costimulatory activity to L6+ tumor cells in vitro. We also show that transfection of the cell surface forms of the two different CD28 sFvIgs into H3347 tumor cells allows them to generate significant costimulatory signals to activated T cells. Finally, we demonstrate that tumor cell presentation of either the soluble bispecific or transduced cell surface sFv generate similar costimulatory effects resulting in T cell activation.

Cloning of the murine counterpart of the tumor-associated antigen H-L6: epitope mapping of the human and murine L6 antigens.

The murine monoclonal antibody (mAb) L6 was raised against human lung carcinoma cells and found to recognize an antigen which is highly expressed on lung, breast, colon, and ovarian carcinomas. Promising results in phase 1 clinical studies with this antibody or its chimerized counterpart suggest the antigen recognized by mAb L6 (H-L6) is an attractive target for monoclonal antibody-based cancer therapy. Further development of L6 as an anti-tumor-targeting agent would benefit from the development of a murine model. However, initial attempts to develop such a model were hampered by our inability to generate antibodies against the murine homologue of the L6 antigen, M-L6. Here we describe the preparation of the mAb 12A8, which was raised against murine thymic epithelial cells, the tissue distribution of the murine antigen recognized by 12A8, the cloning of a cDNA encoding the 12A8 target antigen, and the demonstration that this antigen is M-L6. Using H-L6/M-L6 chimeric proteins, we show that the region of the M-L6 protein recognized by mAb 12A8 corresponds to the region of H-L6 recognized by mAb L6. There are five amino acid differences in the regions of the H-L6 and M-L6 proteins recognized by L6 and 12A8, respectively. We further mapped the protein epitope recognized by L6 by individually exchanging each of these residues in H-L6 with the corresponding residue found in M-L6. Substitution of the single H-L6 residue Leu122 with Ser resulted in the H-L6 mutant HL6-L122S which failed to bind L6. The HL6-L122S mutant also failed to bind 12A8.(ABSTRACT TRUNCATED AT 250 WORDS)

Sulfated etoposide and nitrogen mustard prodrugs and their activation bystreptomycesarylsulfatase

AbstractEtoposide sulfate (ES) and p-di-2-chloroethylaminophenyl sulfate (CAPS) were designed as nontoxic anticancer prodrugs of etoposide and p-di-2-chloroethylaminophenol (CAP) that could be activated on tumor cell surfaces by monoclonal antibody (mAb)-arylsulfatase conjugates. In vitro assays indicated that CAPS and ES were nontoxic to the H2981 human lung adenocarcinoma cell line, while etoposide and CAP had IC50 values of 1–2 μM. Several commercially available arylsulfatases, as well as the arylsulfatases from human liver and urine, were either unable or poorly able to effect the hydrolysis of ES and CAPS. In contrast, arylsulfatase from Streptomyces (SAS) hydrolyzed ES and CAPS with specific activities of 3.0 and 4.8 μmol/min/ mg, respectively. SAS was conjugated to the L6 mono-clonal antibody and was able to activate ES in an immunologically specific manner on H2981 cells (L6 antigen positive). ES and CAPS were stable in mouse serum and were at least 9–13 times less toxic to mice on a molar basis t...

Identification of residues in the monoclonal antitumor antibody L6 important for binding to its tumor antigen.

L6 is a monoclonal antitumor antibody which recognizes an epitope located in a 42-residue extracellular domain of a tumor-associated approximately 22 kDa glycoprotein antigen. The L6 mAb localizes to solid tumors in vivo and triggers complement activation and antibody-dependent cellular cytotoxicity. It has been the subject of phase I clinical trials. Previously, we had reported the derivation and analysis of a three-dimensional model of the L6 Fv. The model suggests that L6 displays a generally aromatic CDR surface. We aim at improving the affinity for tumor antigen of L6 by in vitro mutagenesis. As the first step toward this end, we have attempted to identify residues critical for the binding of L6 to tumor antigen. On the basis of the model, seven residues were selected which we thought may be critical for L6 antigen binding. Criteria for the selection of these residues were their accessibility and central position on the CDR surface and the residue character. Large polar or charged residues such as arginine, asparagine, and tyrosine were preferred. Nine site-specific single and double mutants were generated using oligonucleotide-directed mutagenesis in an M13 expression vector encoding the L6 Fab. The binding of these mutant Fabs to the L6 tumor antigen and a set of three anti-idiotypic antibodies was quantified in an ELISA. In eight out of nine mutants, binding to L6 tumor antigen was either abolished or substantially reduced. In contrast, the binding of the mutants to the anti-idiotypic antibodies was largely unaffected, suggesting that no significant structural perturbations were introduced as a consequence of these mutations.