Costimulation of T-cell-mediated tumor immunity.
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Animals↗
Biomedical subjects
I Hellström
Publications and source records attributed to I Hellström.
MR of carcinoma-specific monoclonal antibody conjugated to monocrystalline iron oxide nanoparticles: the potential for noninvasive diagnosis.
PURPOSE: To determine if tumor-specific monoclonal antibodies conjugated to superparamagnetic monocrystalline iron oxide nanoparticles can be used to yield specific diagnoses with the use of MR imaging. METHODS: Monoclonal antibodies conjugated to monocrystalline iron oxide nanoparticles were given to nude rats with intracranial tumors either by intravenous injection, intraarterial injection with osmotic blood-brain barrier disruption, or direct intratumoral inoculation. Either L6, a tumor-specific antibody, or P-1.17, a control isotype-matched antibody, was used. Coronal T1-weighted, T2-weighted, and spoiled gradient-recalled acquisition in the steady state images were obtained before, 30 minutes after, 6 hours after, and 24 hours after injection. RESULTS: Intravenous injection of greater than 2 mg of the tumor-specific antibody showed a specific pattern of enhancement of the tumors with the largest concentration of antibody in the area with the greatest density of tumor cells. The control antibody showed nonspecific changes. After intraarterial injection with barrier disruption to increase delivery globally or direct inoculation to increase delivery focally, no specific enhancement pattern was seen. CONCLUSION: Monoclonal antibodies conjugated with monocrystalline iron oxide particles may provide a method to obtain specific diagnoses with the use of MR imaging.
Animals↗
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)
Amino Acid Sequence↗
Regressions and cures of melanoma xenografts following treatment with monoclonal antibody beta-lactamase conjugates in combination with anticancer prodrugs.
Cephalosporin doxorubicin (C-Dox) and 7-(4-carboxybutanamido)-cephalosporin mustard (CCM) are prodrugs that are catalytically converted by Enterobacter cloacae beta-lactamase (bL) to the active anticancer agents doxorubicin and phenylenediamine mustard, respectively. Both prodrugs were less cytotoxic to the 3677 human melanoma line than their respective drugs and were activated in an immunologically specific manner by 96.5-bL, a mAb-bL conjugate that binds to 3677 cell surface antigens. Similar results were obtained using the CCM prodrug on SK-MEL 28 human melanoma cells. Experiments in mice with established s.c. 3677 tumors demonstrated that although no tumors were cured in mice receiving the 96.5-bL/C-Dox combination, the activities were greater than those obtained from systemic doxorubicin treatment or from administration of the nonbinding conjugate P1.17-bL in combination with C-Dox. In contrast, when CCM was used as a prodrug, cures of established 3677 tumors were obtained in 80% of the 96.5-bL treated animals. This combination was also able to induce regressions of large 3677 tumor masses (800 mm3) without any apparent toxic side effects. We conclude that 96.5-bL in combination with C-Dox or CCM has greater antitumor activity than systemic treatment with the corresponding drugs and that CCM is a more effective prodrug than C-Dox for treating human 3677 melanoma xenografts.
Animals↗
Affinity maturation of the BR96 anti-carcinoma antibody by codon-based mutagenesis.
We have increased up to 65-fold the avidity of BR96, a mAb recognizing Lewis Y (Le(y))-related Ags expressed on the surface of many human carcinomas. Libraries of mutations in the complementarity-determining regions (CDRs) of BR96 were constructed in an M13 phage Fab expression vector by codon-based mutagenesis, a method that efficiently introduces large numbers and potentially all combinations of amino acid substitutions. Two mutants that improved the affinity of BR96 to tumor Ag were identified by screening the libraries on carcinoma cell lines. One mutant, M1, at position 97 (Asp to Ala) in CDR3 of the heavy chain, resulted in an 8- to 10-fold improvement in Ag binding, as assessed by ELISA. A second mutant, M2, at position 53 (Gly to Asp) in CDR2 of VH increased binding three- to fivefold. When these mutations were combined, the resulting Fab M3 was improved approximately 30-fold. An additional library was constructed in CDR1 of M1. M4, a mutation with three amino acid substitutions in CDR1, was isolated by screening the library with an enzyme conjugate of synthetic Le(y) tetrasaccharide (sLe(y)). This mutant improved BR96 Fab affinity to sLe(y) an estimated 15- to 20-fold by ELISA, and 14-fold as measured by surface plasmon resonance. The M4 IgG had 65-fold improved avidity to sLe(y) relative to the BR96 IgG. The mutants will be useful for comparison of the efficacy of Abs with different affinities for delivery of cytotoxic agents to tumor cells.
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HER4 expression correlates with cytotoxicity directed by a heregulin-toxin fusion protein.
We have constructed, expressed, and purified a fusion protein, HAR-TX beta 2, consisting of heregulin-beta 2 fused to a binding-defective form of Pseudomonas exotoxin A, PE40. The fusion protein was found to induce receptor tyrosine phosphorylation in CEM cells transfected with HER4 alone or in combination with HER2 but not in cells transfected with HER2 or HER1 alone. The phosphorylation of receptor tyrosines was both dose-dependent and saturable in amounts similar to those shown to be active for native heregulin. HAR-TX beta 2 was specifically cytotoxic toward a variety of carcinoma cell lines in the ng/ml range. However, some tumor cell lines were found to be insensitive to the cytotoxic action of the fusion protein even at > 2 micrograms/ml. Relative amounts of HER4, HER3, and HER2 were determined on seven cell lines sensitive and four cell lines insensitive to HAR-TX beta 2. All lines that express HER4 were killed by HAR-TX beta 2, while none lacking HER4 were affected. HAR-TX beta 2 was able to bind to and signal via tyrosine phosphorylation in cell lines that co-express HER2 and HER3 in the absence of HER4 without inducing cytotoxicity. Thus HAR-TX beta 2 may prove to be a useful reagent for the targeting and elimination of HER4-positive tumor cells.
ADP Ribose Transferases↗
Antitumor immunity elicited by tumor cells transfected with B7-2, a second ligand for CD28/CTLA-4 costimulatory molecules.
We have examined the role of the B7-2 costimulatory molecule, a second ligand for CD28/CTLA-4 counter-receptors, in the induction of antitumor immunity. A plasmid containing murine B7-2 cDNA was transfected into the immunogenic mouse mastocytoma P815 of DBA/2 origin. In contrast to the lethal growth of the wild-type (wt) P815 tumor, B7-2-positive (B7-2+) P815 cells inoculated into syngeneic mice regressed, and immunization of mice with such tumor cells protected them against the challenge of wt P815 tumor. Depletion of CD8+, but not of CD4+, lymphocytes in vivo by specific Abs abolished the regression of B7-2+ P815 tumors. CD8+ cytolytic T cells could be generated from mice immunized with B7-2+ P815. They were found to be MHC class I-restricted and specific for the P815 tumor. In contrast, transfection of the B7-2 gene into the nonimmunogenic MCA102 fibrosarcoma of C57BL/6 origin induced neither tumor regression nor protective immunity. Co-expression on MCA102 cells of B7-2 together with the related costimulator B7-1 also failed to induce immunity to MCA102 tumor. Our results indicate that transfection of B7-2 into tumor cells can improve host response to some tumors, and that the effects seen are similar to those previously observed for B7-1.
Abatacept↗
[An unethical and unscientific survey on environmental health effects].
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Environmental Health↗
Immunoconjugates and immunotoxins for therapy of carcinomas.
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Animals↗
The x-ray structure of an anti-tumour antibody in complex with antigen.
The crystal structures of the murine BR96 Fab and its human chimera have been determined in complex with the nonoate methyl ester derivative of Lewis Y (nLey) at 2.8 A and 2.5 A resolution, respectively. BR96 binds the carbohydrate in a large pocket which is formed by residues of all CDR loops except L2. The binding of the carbohydrate is mediated predominantly by aromatic residues in BR96. Analysis of the structure suggests that BR96 is capable of recognizing a structure larger than the Le(y) tetrasaccharide, providing a possible explanation for its high tumour selectivity. The structure provides a rationale for mutagenesis experiments that have resulted in BR96 CDR loop mutants with increased affinity for nLey and/or tumour cells.
Animals↗
Can co-stimulated tumor immunity be therapeutically efficacious?
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Animals↗
Antitumour activity of a melanoma-specific immunotoxin, ME20-LysPE40.
An immunotoxin conjugate has been prepared by linking an internalizing antibody with melanoma selectivity, ME20, with a binding-defective form of Pseudomonas exotoxin A, LysPE40. ME20-LysPE40 binds to a 105,000 Da cell-surface antigen present on melanoma cells (ME20-M) within twofold of unmodified ME20 and was cytotoxic to two human melanoma cell lines, H3606 and MALME-3M, with EC50 values of 100 and 200 pM, respectively. Immunotoxin treatment, initiated 1 day following subcutaneous implantation of H3606 melanoma cells into mice, prevented outgrowth of tumour xenografts in > 50% of the mice. In contrast, only a modest inhibition in tumour growth was observed if the immunotoxin was administered 5 days after implantation of in vivo passaged H3606 tumour fragments in mice. This study shows that the internalizing monoclonal antibody ME20 IgG can be used for targeting a toxin toward melanoma cells displaying the ME20-M antigen.
Animals↗
B7-1/CD80-transduced tumor cells elicit better systemic immunity than wild-type tumor cells admixed with Corynebacterium parvum.
Tumor cells genetically modified by transduction of B7 (B7-1/CD80), a natural ligand for the T-cell costimulatory molecules CD28 and CTLA-4, can elicit potent tumor immunity, and they can be effective for treatment of established cancers in animal models. In this study, three tumor lines, the EL4 lymphoma, the P815 mastocytoma, and the MCA102 sarcoma were transduced with recombinant retrovirus containing the murine B7 gene, and their potency to induce systemic immunity protective against challenge with wild-type tumor was compared to that of the same tumor cells admixed with the commonly used adjuvant Corynebacterium parvum. While admixture of tumor cells with C. parvum resulted in complete regression of tumors in syngeneic mice, it did not induce protective immunity against a subsequent challenge of wild-type cells from any of the 3 tumors tested. In contrast, B7-transduced EL4 and P815 tumors regressed locally and induced a potent systemic immunity to wild-type tumors and a higher level of cytotoxic T-cell activity than did tumor cells admixed with C. parvum. No systemic immunity was induced by B7-transduced nonimmunogenic MCA102 sarcoma cells. Our results demonstrate that immunogenic tumor cells transduced with the B7 gene are superior to tumor cells mixed with C. parvum for the induction of systemic tumor immunity.
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Costimulation of tumor-reactive CD4+ and CD8+ T lymphocytes by B7, a natural ligand for CD28, can be used to treat established mouse melanoma.
Interactions between the costimulatory molecule B7 on APC and its counter-receptor CD28 on T lymphocytes play a key role in the induction of cell-mediated immune responses. We studied the role of costimulation of tumor-reactive T cells by B7 in the immune destruction of the K1735-M2 mouse melanoma into which the gene encoding the human melanoma-associated Ag, p97, had been transfected. Previous work has demonstrated that the p97 transfectant cl62 is immunogenic but still grows progressively in immunocompetent C3H/HeN mice and that adoptive transfer of p97-specific CD4+ T cells can induce the regression of small established cl62 tumors metastatic to the lungs. We have now shown that expression of B7 in cl62 after retroviral-mediated gene transfer eliminated its ability to grow in immunocompetent mice but not in T cell-deficient nude mice. Mice immunized with B7-transduced p97+ cells had an increased activity of both CD4+ T cells, which could proliferate in response to the p97 Ag, and CD8+ CTL, which could lyse a broad spectrum of cultured syngeneic p97+ and p97- tumor lines but not allogeneic tumor lines or syngeneic lymphoblasts. Both CD4+ and CD8+ T cell subsets were required for tumor rejection, and the depletion of CD4+ T cells in vivo decreased the tumoricidal activity of CD8+ CTL. Treatment of mice bearing an 8-day established s.c. cl62 melanoma by i.p. injection of B7+ cells from 2A, a highly immunogenic p97 transfectant, resulted in complete tumor regression and cure, injection of B7- 2A cells did not. The therapeutic effect was specific for the cl62 tumor. Our results demonstrate that costimulation by B7 can amplify both CD4+ and CD8+ T cell responses against small tumors toward therapeutic benefit.
Animals↗
Differential processing and secretion of the melanoma-associated ME20 antigen.
A murine monoclonal antibody, ME20, with high selectivity for melanomas, has been utilized to isolate a unique membrane-bound (designated ME20-M) and secreted (designated ME20-S) antigen from H3606 human melanoma cells. ME20-M was purified from the cell lysate and ME20-S from the conditioned medium of H3606 cells by immunoaffinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weights were 105,000 and 76,000, respectively. Analyses of ME20-M and ME20-S by amino acid sequencing identified the processing sites. Signal peptide cleavage occurs at Thr-24 of pro-ME20 antigen, yielding ME20-M (25 to 661). In addition, proteolytic processing of the precursor at Val-467 yields ME20-S (25 to 467). We report the characterization of Asn-linked glycosylation sites in ME20-M and ME20-S to determine the involvement of oligosaccharides in the proteolytic processing of pro-ME20 antigen. Tryptic peptide maps of ME20-M and ME20-S were prepared and the glycosylation sites identified by sequence analyses. Oligosaccharides were enzymatically released and characterized by high-performance anion-exchange chromatography. We found high-mannose-type structures at Asn-57, Asn-82, and Asn-87 of ME20-M, whereas ME20-S contained 73% complex-type and 27% high-mannose-type oligosaccharides at the same sites. To assess the role of oligosaccharides in the processing of the ME20 antigen, we tested the effect of the oligosaccharide processing modifier deoxymannojirimycin, a compound that inhibits synthesis of hybrid- and complex-type oligosaccharides. Deoxymannojirimycin had no effect on the synthesis and relative rate of synthesis of ME20-M, but markedly reduced the synthesis of ME20-S without affecting the rate of secretion. The reported results suggest that carbohydrate maturation of the ME20 antigen may be important for processing and secretion.
1-Deoxynojirimycin↗
Membrane topology of the L6 antigen and identification of the protein epitope recognized by the L6 monoclonal antibody.
The murine monoclonal antibody (mAb) L6 recognizes an integral membrane glycoprotein that is highly expressed on lung, breast, colon, and ovarian carcinomas and is referred to as the L6 antigen. This antigen is an attractive target for therapeutic intervention due to its high level expression on malignant cells. We have previously reported the isolation of a cDNA encoding the human L6 antigen (H-L6). Here, we report the isolation of a cDNA clone encoding the murine L6 antigen (M-L6). This cDNA contains one long open reading frame, which encodes a 220-amino acid polypeptide that is 78% homologous to H-L6. This protein contains short NH2- and COOH-terminal hydrophilic domains and four hydrophobic regions, each long enough to span the plasma membrane. Each of these hydrophobic domains is separated by a hydrophilic domain, the longest of which contains one possible N-linked glycosylation site and is located between the third and fourth hydrophobic domains. We have previously demonstrated that the murine L6 mAb recognizes a protein epitope expressed on human tumor-derived cell lines. Now, using chimeric cDNA constructs encoding human-murine L6 antigen hybrids in conjunction with monoclonal antibody binding experiments, we show that the 42-residue hydrophilic domain of the L6 antigen, located between the third and fourth hydrophobic domains, is outside the cell and that residues in the NH2-terminal region of this domain are critical for the binding of the murine L6 mAb to H-L6.
Amino Acid Sequence↗
In vitro and in vivo characterization of BR96 sFv-PE40. A single-chain immunotoxin fusion protein that cures human breast carcinoma xenografts in athymic mice and rats.
BR96 sFv-PE40 is a single-chain immunotoxin fusion protein targeted to the Ley Ag, which is expressed in many different human carcinomas as well as in normal gastrointestinal epithelium of humans and certain animals, including athymic rats but not mice. In vitro binding analysis determined that BR96 sFv-PE40 was similar in affinity to BR96 Fab. BR96 sFv-PE40 internalizes rapidly, similar to BR96 IgG. H3396 cells, derived from metastatic human breast carcinoma, have been established as tumor xenografts in estradiol-supplemented athymic mice and rats. H3396 tumor xenografts established in athymic mice (up to 350 mm3) and rats (up to 100 mm3) completely regressed after i.v. administration of BR96 sFv-PE40, given as 0.625 mg/kg (1.975 mg/m2) every 4th day for a total of five doses (mice) or 0.25 mg/kg (1.475 mg/m2) every 4th day for a total of four doses (rats). The tumors remained regressed for the duration of the study (> 85 days post-implant), which represents > 10 doubling times, indicating that the animals were cured. There was no toxicity in rats receiving a curative dose of 0.25 mg/kg, although liver and lung toxicity could be detected at a 16 times higher dose, 4 mg/kg or 23.6 mg/m2. We conclude, therefore, that BR96 sFv-PE40 can cure tumor xenografts at well tolerated doses and also in the presence of Ley expression in normal tissues.
ADP Ribose Transferases↗
Tumor immunogenicity determines the effect of B7 costimulation on T cell-mediated tumor immunity.
A costimulatory signal through B7 to its counter-receptor CD28 on T cells enhances T cell activation. We have generated recombinant retroviruses containing cDNA for murine B7 and transduced a panel of murine tumor lines with varying immunogenicity to study the effect of B7 costimulation on antitumor immunity. In contrast to the progressive outgrowth of all wild-type (B7-) tumors in unimmunized syngeneic mice, four immunogenic tumors, lymphoma RMA, EL4, mastocytoma P815, and melanoma E6B2, regressed completely when transduced with the B7 gene. In contrast, four nonimmunogenic tumors, sarcomas MCA101, MCA102, and Ag104, and melanoma B16, remained tumorigenic after transduction of the B7 gene. Immunization with B7-transduced immunogenic tumors enhanced protective immunity and increased specific cytotoxic T lymphocyte (CTL) activity against the respective wild-type tumors as compared to immunization with nontransduced or mock-transduced tumors. Moreover, cocultivation of CTL with B7-transduced EL4 cells augmented the specificity of tumor-reactive CTL in long-term cultures. Treatment by injection of B7-transduced tumor cells cured 60% of mice with established wild-type EL4 lymphoma. In contrast, immunization with nonimmunogenic tumors transduced with B7 did not provide protective immunity and did not increase specific CTL activity. Our results show that tumor immunogenicity is critical to the outcome of costimulation of T cell-mediated tumor immunity by B7.
Animals↗