Biomedical subjects
Publications and source records attributed to S Carrel.
Six patients with neoplastic melanomatous meningitis were studied. The diagnosis of this complication of malignant disease rests on the demonstration of malignant cells within the CSF. The addition of monoclonal antibody immunocytology to conventional techniques significantly improved the diagnostic sensitivity of CSF cytology, allowing for earlier and therefore more effective palliative treatment.
We have previously reported that the amino acid sequence of the common acute lymphoblastic leukemia antigen (CALLA, CD10) translated from a normal human kidney cDNA clone is identical to that of neutral endopeptidase (NEP, EC 3.4.24.11). In this study, we show that by flow cytometry, a monoclonal antibody (135A3) produced against rabbit NEP reacted selectively with leukemia and melanoma cell lines expressing CALLA on their surface. A glycoprotein of apparent Mr 100,000 was immunoprecipitated from surface labeled NALM-1 leukemia or Mel-1477 melanoma cells with monoclonal antibodies to NEP (135A3) or CALLA (44C10). mRNAs hybridizing to a NEP-specific probe were present in CALLA+ leukemia and melanoma cell lines, but absent from CALLA- lines. NEP enzymatic activity was detected on intact cells from CALLA+ lines, but not CALLA- lines. The activity was blocked by two selective inhibitors of NEP, thiorphan and phosphoramidon. CALLA antigen purified from the NALM-6 leukemic cell line by affinity to 44C10-IgG Sepharose retained a peptidase activity that was completely blocked by thiorphan and phosphoramidon. Thus the CALLA antigen present at the surface of leukemia and melanoma cell lines is an enzymatically active neutral endopeptidase.
Pooled F(ab')2 fragments of three MAbs against distinct epitopes of carcinoembryonic antigen (CEA) were used for radioimmunotherapy of nude mice bearing a subcutaneous human colon carcinoma xenograft. 9-10 d after transplantation when tumor nodules were in exponential growth, 36 mice were treated by intravenous injection of different amounts of 131I-labeled MAb F(ab')2. All 14 mice injected with a single dose of 2,200 (n = 10) or 2,800 microCi (n = 4) showed complete tumor remission. 8 of the 10 mice treated with 2,200 microCi survived in good health for 1 yr when they were killed and shown to be tumor free. Four of nine other mice treated with four fractionated doses of 400 microCi showed no tumor relapse for more than 9 mo. In contrast, all 15 mice injected with 1,600-3,000 microCi 131I-control IgG F(ab')2 showed tumor growth retardation of only 1-4 wk, and 15 of 16 mice injected with unlabeled anti-CEA MAb F(ab')2 showed unmodified tumor progression as compared with untreated mice. From tissue radioactivity distributions it was calculated that by an injection of 2,200 microCi 131I-MAb F(ab')2 a mean dose of 8,335 rad was selectively delivered to the tumor, while the tissue-absorbed radiation doses for the normal organs were: peripheral blood, 2,093; stomach, 1,668; kidney, 1,289; lung, 1,185; liver, 617; spleen, 501; small intestine, 427; large intestine, 367; bone, 337; and muscle, 198. These treatments were well tolerated since out of 19 mice with complete tumor remission only 4 required bone marrow transplantation and 17 were in good health for 6-12 mo of observation. The results demonstrate the selective destruction of established human colon carcinoma transplants by intravenous injection of either single or fractionated doses of 131I-MAb F(ab')2.
Human malignant glioma cell lines and clones were incubated with various concentrations of recombinant human TNF-alpha, either alone or in combination with recombinant human IFN-gamma. The surface expression of HLA-ABC (class I) antigens and beta 2-microglobulin, was significantly enhanced by TNF-alpha alone on every cell line and clone tested. After incubation with both TNF-alpha and IFN-gamma, the surface expression of HLA-ABC antigens was only slightly higher than that observed with each cytokine alone. In contrast to IFN-gamma, TNF-alpha had no effect on the surface expression of HLA-DR (class II) antigens. Moreover, the surface expression of HLA-DR induced by IFN-gamma was unaffected by TNF-alpha. The increased expression of HLA-ABC antigens after treatment with TNF-alpha or IFN-gamma correlated with increased levels of HLA-ABC-specific mRNA. In addition, TNF-alpha, like IFN-gamma, selectively enhanced the surface expression of a tumor-associated antigen, Me14-D12, while it had no effect on the expression of various other surface antigens. In the absence of actinomycin D, TNF-alpha exhibited no direct cytotoxic/cytostatic effect on the glioma cell lines tested. These results indicate that TNF-alpha can enhance the surface expression of HLA-ABC antigens on human glioma cells in the absence of a direct cytotoxic/cytostatic effect.
A human CD8+ CTL clone with cytolytic potential was shown to express two serine proteases, a 50-kDa homodimer and a 27-kDa monomer, which were purified from cytoplasmic granules. N-terminal sequencing of the purified proteins revealed that the 50-kDa homodimer is the gene product of the human Hanukah factor cDNA clone and that it represents the human homologue to granzyme A. Similarly, the 27-kDa protein was shown to be the serine esterase encoded by the human lymphocyte protease cDNA clone and corresponds to granzyme B. There was no evidence for the presence of other granzymes, in particular for the human homologues to murine granzymes C, D, E, and F. The substrate best cleaved by granzyme A was Gly-Pro-Arg-amido-4-methyl-coumarin after the Arg residue and the pH optimum was 8 to 8.5. Upon triggering of the TCR-CD3 complex with an anti-CD3 mAb, granzyme A was released into the culture medium. Furthermore, a granule-associated hemolytic activity was detected after salt extraction and partial purification of granule proteins. This suggests that hemolytically active human perforin can be obtained from inactive granules.
Monoclonal antibodies (MAbs) targeted to glioma-associated antigens may allow the selective delivery of imaging and therapeutic agents to brain tumors; the use of MAb fragments may be a strategy to further improve tumor uptake of such agents relative to normal tissues. In this study, we have examined the in vivo localization of radioiodinated MAb Me1-14, a murine immunoglobulin G2a (IgG2a) reactive with gliomas, and its F(ab')2 fragment in s.c. and intracranial xenografts of human glioma cell line D-54 MG in athymic mice. The radiolabeled F(ab')2 fragment of Me1-14 was demonstrated to possess in vitro binding affinity and immunoreactivity comparable to that of whole IgG. Direct comparison of IgG and F(ab')2 biodistribution in s.c. xenograft-bearing mice showed higher tumor: normal tissue ratios of the F(ab')2 fragment compared to IgG. In intracranial tumor-bearing mice paired-label analysis using a nonspecific protein control showed earlier specific tumor localization by the F(ab')2 fragment of Me1-14 compared to IgG. Blood-to-tumor transfer constants (K1) derived for Me1-14 F(ab')2 were significantly greater than those for whole Me1-14 (P = 0.01). Estimated radiation dosimetry revealed that 131I-labeled Me1-14 F(ab')2 would deliver higher radiation doses to tumor than to normal tissues. These studies demonstrate that the F(ab')2 fragment of Me1-14 may be a potential agent for immune-directed brain tumor diagnosis and therapy.
Although it is well known that the CD3/T-cell receptor (TCR) complex modulates from the surface of T cells upon exposure to monoclonal antibodies (mAb) directed against it, the fate of bound mAb has not been yet elucidated. We therefore perform direct binding experiments of 125I-labeled mAb against CD3 or TCR to investigate their fate in Jurkat T cells. We demonstrated that all mAb were progressively internalized and degraded in Jurkat T cells and that this degradation was inhibited by chloroquine, an inhibitor of lysosomal degradation enzymes. The sequestration of anti-CD3 mAb in acid compartments was furthermore shown using cytofluorometry. All together our results show that antibodies against CD3 or against TCR follow the same endocytic pathway.
A novel melanoma-associated differentiation Ag whose surface expression can be enhanced or induced by IFN-gamma was identified by mAb Me14/D12. Testing of numerous tumor cell lines and tumor tissue sections showed that Me14/D12-defined Ag was present not only on melanoma but also on other tumor lines of neuroectodermal origin such as gliomas and neuroblastomas and on some lymphoblastic B cell lines, on monocytes and macrophages. Immunoprecipitation by mAb Me14/D12 of lysates from [35S]methionine-labeled melanoma cells analyzed by SDS-PAGE revealed two polypeptide chains of 33 and 38 KDa, both under reducing and nonreducing conditions. Cross-linking experiments indicated that the two chains were present at the cell surface as a dimeric structure. Two-dimensional gel electrophoresis showed that the two chains of 33 and 38 KDa had isoelectric points of 6.2 and 5.7, respectively. Treatment of the melanoma cells with tunicamycin, an inhibitor of N-linked glycosylation, resulted in a reduction of the Mr from 33 to 24 KDa and from 38 to 26 KDa. Peptide maps obtained after Staphylococcus aureus V8 protease digestion showed no shared peptides between the two chains. Although biochemical data indicate that Me14/D12 molecules do not correspond to any known MHC class II Ag, their dimeric structure, tissue distribution, and regulation of IFN-gamma suggest that they could represent a new member of the MHC class II family.
Treating human melanoma lines with dibutyryl adenosine 3':5'-cyclic monophosphate (dbc AMP) resulted in morphologic changes associated with the altered expression of cell surface antigens. After treatment, cells developed long cellular projections characteristic of mature melanocytes and showed the presence of an increased number of Stage II premelanosomes. In addition, induction of melanin synthesis, detected as brown perinuclear pigmentation, was observed. The AMP further drastically reduced the growth rate of the five melanoma cell lines that were tested. The influence of dbc AMP was completely reversible 3 days after the agent was removed from the culture medium. The antigenic phenotype of the melanoma lines was compared before and after dbc AMP treatment. This was done with four monoclonal antibodies directed against major histocompatibility complex (MHC) Class I and II antigens and 11 monoclonal antibodies defining eight different melanoma-associated antigenic systems. Treatment with dbc AMP reduced the expression of human leukocyte antigen (HLA)-ABC antigens and beta-2-microglobulin in five of five melanoma lines. In the two HLA-DR-positive cell lines dbc AMP reduced the expression of this antigen in one line and enhanced it in the other. No induction of HLA-DR or HLA-DC antigens was observed in the Class II negative cell lines. Furthermore, dbc-AMP modulated the expression of the majority of the melanoma antigenic systems tested. The expression of a 90-kilodalton (KD) antigen, which has been found to be upregulated by interferon-gamma, was markedly decreased in all the five cell lines. A similar decrease in the expression of the high molecular weight proteoglycan-associated antigen (220-240 KD) was observed. The reduced expression of Class I and II MHC antigens as well as the altered expression of the melanoma-associated antigens studied were shown to be reversible after dbc AMP was removed. Our results collectively show that the monoclonal antibody-defined melanoma-associated molecules are linked to differentiation. They could provide useful tools for monitoring the maturation of melanomas in vivo induced by chemical agents or natural components favoring differentiation.
A mixture of 3 MAbs directed against 3 different CEA epitopes was radiolabelled with 131I and used for the treatment of a human colon carcinoma transplanted s.c. into nude mice. Intact MAbs and F(ab')2 fragments were mixed because it had been shown by autoradiography that these 2 antibody forms can penetrate into different areas of the tumor nodule. Ten days after transplantation of colon tumor T380 a single dose of 600 microCi of 131I MAbs was injected i.v. The tumor grafts were well established (as evidenced by exponential growth in untreated mice) and their size continued to increase up to 6 days after radiolabelled antibody injection. Tumor shrinking was then observed lasting for 4-12 weeks. In a control group injected with 600 microCi of 131I coupled to irrelevant monoclonal IgG, tumor growth was delayed, but no regression was observed. Tumors of mice injected with the corresponding amount of unlabelled antibodies grew like those of untreated mice. Based on measurements of the effective whole-body half-life of injected 131I, the mean radiation dose received by the animals was calculated to be 382 rads for the antibody group and 478 rads for the normal IgG controls. The genetically immunodeficient animals exhibited no increase in mortality, and only limited bone-marrow toxicity was observed. Direct measurement of radioactivity in mice dissected 1, 3 and 7 days after 131I-MAb injection showed that 25, 7.2 and 2.2% of injected dose were recovered per gram of tumor, the mean radiation dose delivered to the tumor being thus more than 5,000 rads. These experiments show that therapeutic doses of radioactivity can be selectively directed to human colon carcinoma by i.v. injection of 131I-labelled anti-CEA MAbs.
Monoclonal antibodies (MoAb) that react with the T-lymphocyte markers called cluster of differentiation CD5 and CD2 were labeled with iodine 131 (131I) and were injected intravenously in nude mice bearing solid subcutaneous xenografts derived from the human T-cell leukemia line Ichikawa. Both MoAb anti-CD5 and anti-CD2 yielded favorable mean tumor to whole-body ratios of 3.8 and 5.1, respectively. These ratios were further increased up to 10.0 for MoAb anti-CD5 and 15.5 for MoAb anti-CD2 by using their F(ab')2 fragments. The tumors could be imaged clearly by external scanning after injection of F(ab')2 fragments from both MoAb. F(ab')2 fragments from MoAb anti-CD2 and of a third MoAb recognizing the clonotypic determinant (Ti) of the antigen receptor expressed by the human T-cell line Jurkat were injected in mice bearing intrasplenic Jurkat xenografts. A selective localization of both fragments in tumor tissue was demonstrated with mean tumor to whole-body ratios of 7.5 and 4.1 for MoAb anti-CD2 and anti-Ti, respectively. These in vivo experimental results may provide useful information for the potential use of radiolabeled MoAb and fragments in the diagnosis and treatment of patients with T-cell lymphoma and different other forms of T-cell malignancies.
There is experimental evidence that the CD4 molecule participates in the antigen-driven activation of T cells expressing this surface glycoprotein. Whether CD4, a member of the immunoglobulin supergene family, acts as a ligand-binding molecule and/or is directly involved in the activation pathway has yet to be established. In this study, we show that human CD4+ lymphocytes can be activated by exposure to the anti-CD4 monoclonal antibody (mAb) B66. Normal peripheral blood CD4+ cells were induced to proliferate and to synthesize interleukin 2 (IL 2) by the antibody. The specificity of the antibody stimulatory activity was tested by using IL 2-producing clones bearing either CD4 or CD8 on their surface. IL 2 production was induced by mAb B66 in CD4+, but not CD8+, clones, whereas both types of clones responded to stimulation by the anti-CD3 mAb Leu-4. Despite its unique stimulatory activity, mAb B66 shared with other anti-CD4 antibodies the ability to inhibit the specific cytolytic activity of CD4+ effector cells. These results clearly indicate that cross-linking of surface CD4 molecules with appropriate antibodies can fully activate CD4+ lymphocytes. Whether the natural ligand for CD4 can trigger this activation pathway remains to be defined.
A new heterodimeric structure, Tp135-145, which can mediate interleukin 2 (IL2) production and Ca2+ mobilization by Jurkat cells is described. This structure was identified by a monoclonal antibody, MX24, on the surface of either T3/TcR+ or T3/TcR- human T cell lines as well as on B cell lines. Biochemical studies showed that antibody MX24 precipitated two polypeptide chains of 135 and 145 kDa, respectively, in lysates from 125I-labeled T cells. After reduction the 135-kDa polypeptide chain shifted to 140 kDa, whereas the molecular mass of the other polypeptide remained unchanged. The apparent molecular masses of the desialylated polypeptides differed by 5 kDa. No common peptide fragments between the two polypeptide chains were found after limited proteolysis by Staphylococcus aureus V8 protease. The expression of Tp135-145 was independent of the expression of the T3/TcR molecular complex. Incubation of Jurkat cells with anti-TcR or anti-T3 monoclonal antibody induced complete modulation only of the T3/TcR complex but not of Tp135-145. Conversely complete modulation of Tp135-145 was observed after incubation of these cells with MX24 antibody. Functional studies showed that anti-Tp135-145 antibody MX24 induced high levels of IL2 production in Jurkat cells. In addition, incubation of these cells with MX24 resulted in Ca2+ mobilization from internal stores. In peripheral blood, Tp135-145 was found to be expressed by 39%-76% of resting T cells in individual donors. Two-color flow microfluorimetry showed that the Tp135-145+ cells were equally distributed on the CD4+ and CD8+ subsets. Incubation of peripheral blood T cells with antibody MX24 resulted in IL2 production and cell proliferation. Taken together these results suggest that Tp135-145 is a novel surface molecule involved in antigen-independent pathway of T cell activation.
Restriction fragment analysis has been applied to genomic DNA extracted from human tumor cell lines. Polymorphic restriction fragments encompassing the HLA-DQ alpha gene were observed upon digestion with Bgl II, Eco RI, and Hind III. Analysis of these polymorphic fragments (or allogenotopes) showed that for each restriction enzyme a series of three differently sized allogenotopes existed. Clusters of cosegregating allogenotopes belonging to the different allelic series defined three different allogenotypes. Each allogenotype exhibited a distinctive restriction map generated by digestion with five restriction enzymes. Comparison of these restriction maps showed that generation of the polymorphisms observed at the HLA-DQ alpha region in these sets of cell lines is not caused by a single event. In some B- and T-lymphoma cell lines a fourth allogenotype was found. The restriction site map of genomic DNA from these cell lines suggested that the latter distribution of restriction enzyme sites was most probably generated by recombination between two of the previously observed allogenotypes at a crossover site(s) adjacent to the HLA-DQ alpha gene.
A great number of techniques are currently available for the conservative treatment of uveal melanomas: ocular applicators emitting gamma rays 60Co, 125I) or high-energy beta rays (106Ru/106Rh), light photocoagulation, surgical excision, and accelerated proton beam irradiation. Life expectancy following conservative treatment is equal to or better than that following enucleation. This is demonstrated by nonrandomized comparative studies, and by the authors' own long-term results following the conservative treatment of melanomas by 60Co applicators: mortality due to metastases of small melanomas was 3% (V = smaller than 10 x 10 x 3 mm), with medium-size melanomas it was 12% (V = 10 x 10 x 3-15 x 15 x 5 mm), and with large melanomas 21% (V = larger than 15 x 15 x 5 mm). Accelerated proton beam irradiation of uveal melanomas is currently the method of choice for the conservative treatment of uveal melanomas. The sharp boundaries of the irradiated zone, the uniformly distributed irradiation dose, and beam-splitting are the main advantages of this technique. During the last three years, 310 cases of uveal melanoma have been treated in Switzerland with an accelerated proton beam. Of these, 214 were followed up for more than one year. Eight patients (3.9%) died of metastases. Visual acuity was identical or superior to initial visual acuity in 60.3% of the cases, while 39.6% exhibited a deterioration of vision or a functional loss. Favorable results achieved by conservative treatment of uveal melanomas considerably limited the indications for enucleation, which is now only performed in exceptional situations.
The reactive center of C1-inhibitor, a plasma protease inhibitor that belongs to the serpin superfamily, is located on a peptide loop which is highly susceptible to proteolytic cleavage. With plasma kallikrein, C1s and beta-Factor XIIa, this cleavage occurs at the reactive site residue P1 (Arg444); with neutrophil elastase, it takes place near P1, probably at residue P3 (Val442). After these cleavages, C1-inhibitor is inactivated and its conformation is modified. Moreover, in vivo, cleaved C1-inhibitor is removed from the blood stream more rapidly than the intact serpin, which suggests that proteolysis unmasks sites responsible for cellular recognition and the uptake of the cleaved inhibitor. In the study reported here, we show, using an MAb, that an identical neoepitope is created on C1-inhibitor after the cleavage of its exposed loop by plasma kallikrein, C1s, beta-Factor XIIa, and by neutrophil elastase.
F(ab')2-fragments of the anti-melanoma monoclonal antibody MeI-14 were labelled with 123I for external scanning and with 125I for tissue measurement of radioactivity and injected intravenously into patients scheduled for surgical resection of a glioma. The paired-label study was performed by injecting simultaneously 131I-labelled control (F(ab')2-fragments. The patients were scanned by computerised tomoscintigraphy. After surgery, the activities of 125I and 131I were counted in tumour and normal tissues. The results indicate that there was a low but definite uptake of the antibody in the tumour due to its specificity. The external detection was difficult because of accumulation of antibody fragments in the skull.
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