Biomedical subjects

M J Mattes

Publications and source records attributed to M J Mattes.

At least 19 recordsLinked to original sources

PubMed2004

Antibodies to CD20 and MHC class II antigen bound to B-lymphoma cells accumulate in shed cytoplasmic fragments.

Antibodies (Abs) to CD20 and MHC class II antigen were found to exhibit a novel processing pathway after binding to the surface of RL B-lymphoma cells. The Abs were 'excreted' as a part of large cytoplasmic fragments. These fragments formed at cell-cell junctions, but gentle dispersal of the cells, to form a single-cell suspension of high viability, caused the release of most of the fragments. This process also occurred in Raji cells and in three other B-lymphoma cell lines (of seven tested). Six B-lymphoblastoid cell lines tested did not form these objects. Once they were recognised, the fragments could be identified in cell preparations by phase contrast microscopy or after staining with Wright's stain. They were induced by the binding of certain Abs, but not by most Abs bound to the cell surface. The mode of formation, detailed morphology and function of these cytoplasmic fragments remain to be determined. They are similar in many respects to the lymphoglandular bodies that have been described by pathologists for many years, which are characteristic of B-cell lymphoma, but which have not previously been described in cell lines. This type of Ab processing, if it occurs in patients, will have an impact on the therapeutic use of these Abs.

Antibodies, Monoclonal↗

PubMed2001

Single-cell cytotoxicity with radiolabeled antibodies.

Previous studies demonstrated the effective, antigen-specific killing of Raji B-lymphoma cells in vitro by radiolabeled anti-CD74, attributable largely to the high level of uptake, of approximately 10(7) antibody (Ab) molecules/cell/ day. This Ab is rapidly delivered to lysosomes for catabolism, so the radionuclide delivered accumulates primarily in lysosomes. In this study, we have tested Abs that bind to the same target cells in similar amounts, but remain primarily on the cell surface, to compare the potency of radioactivity delivered to the cell surface versus the cytoplasm. The Abs tested were anti-major histocompatibility complex class II and anti-CD20. 111In-labeled conjugates made with these two Abs killed cells very effectively and specifically, with 100% kill of sample of 5 x 10(5) cells. Because these Abs remain primarily on the cell surface, it would be predicted that residualizing radiolabels, which are trapped in lysosomes after Ab catabolism, would not be required, and this was observed, i.e., these two Abs were effective when labeled with either 125I or 131I, using conventional iodination, as well as with the residualizing label 111In-labeled DTPA. These results are in contrast to results obtained with anti-CD74, which required a residualizing radiolabel for effectiveness. The uptake of these radionuclides, in cpm/cell, was monitored, and this allowed estimation of the radiation dose delivered; the cytotoxicity observed was consistent with the estimated radiation dose delivered. To establish the generality of the results, we also demonstrated that 111In-labeled anti-CD74 effectively killed three other B-lymphoma cell lines, in addition to Raji and the adherent melanoma cell line SK-MEL-37. By using more potent radionuclides or conjugates of higher specific activity, this approach might be effective with other, lower density antigens.

Antibodies, Monoclonal↗

PubMed2001

Therapy of disseminated B-cell lymphoma xenografts in severe combined immunodeficient mice with an anti-CD74 antibody conjugated with (111)indium, (67)gallium, or (90)yttrium.

A radiolabeled antibody (Ab) to CD74 (the MHC class II invariant chain, Ii) was shown previously to effectively kill human B-lymphoma cells in vitro. Conjugates with both Auger electron and beta-particle emitters were able to kill cells, but the former displayed less nonspecific toxicity in the in vitro assay used. In this report, we have extended the studies to an in vivo model of tumor growth. The human B-cell lymphoma Raji was injected i.v. into severe combined immunodeficient mice, and radiolabeled Abs were injected at various times after tumor inoculation. The maximum tolerated dose (MTD), as well as lower doses, was tested. Tumor growth was monitored by hind-leg paralysis. With a 3-5-day interval before Ab injection, anti-CD74 conjugated to either (111)In or (67)Ga, at a dose of 240-350 microCi/mouse, produced a strong therapeutic effect, with greatly delayed tumor growth, and many of the treated mice were tumor free for >6 months. Control mice became paralyzed in 16-24 days, uniformly. Treatment at later time points (9-day interval) had little therapeutic effect. The MTD was required for optimal therapy. With the beta-particle emitter (90)Y, the MTD was much less, 25 microCi/mouse, and at this dose there was only a weak therapeutic effect. In conclusion, the data suggest that low-energy electrons are more effective than beta-particles in this model system. These results may be applicable to humans, particularly in the case of micrometastatic disease. This approach may also be effective with other Abs that accrete in large amounts.

Animals↗

PubMed2000

Localization of an antibody to CD74 (MHC class II invariant chain) to human B cell lymphoma xenografts in nude mice.

The tumor-specific localization of an anti-CD74 Ab, LL1, was demonstrated in nude mice bearing xenografts of human B-cell lymphoma. This Ab, conjugated to radionuclides emitting Auger electrons, including 125I and 111In, was previously reported to kill tumor cells in vitro effectively and specifically. The cytotoxic potency of this Ab is due to its uptake and catabolism at a very high level, which also affected the Ab biodistribution experiments. Thus, Ab localization to the tumor was only detected if a "residualizing" radiolabel was used, meaning a label that is trapped within cells, usually within lysosomes, after catabolism of the Ab to which it was conjugated. Similar results were obtained with three different residualizing labels: 111In conjugated via the chelators benzyl diethylenetriaminepentaacetic acid (DTPA) or 1.4,7,10-tetra-azacyclododecane-N, N', N", N"'-tetraacetic acid (DOTA), or 131I-dilactitol-tyramine, a residualizing form of iodine. The Ab protein dose could be high, 0.5 mg/mouse, without causing a decrease in specific tumor uptake, probably reflecting the high capacity for uptake. Moreover, tumors of moderate size were found to cause rapid, specific removal of the Ab from the blood, also a result of catabolic processes. This induced blood clearance naturally affected the Ab localization experiments, but this factor could be circumvented by increasing the Ab protein dose. Using a different Ab, anti-(mature MHC class II), the ability of Ab to penetrate relatively large solid tumors was investigated. Complete saturation of antigenic sites was observed in tumors up to 0.3 g in size, but quite high Ab protein doses were required, 5.0 mg/ mouse. These results provide a rationale for attempting therapy with radiolabeled LL1.

Animals↗

PubMed2000

Limitations in the use of low pH extraction to distinguish internalized from cell surface-bound radiolabeled antibody.

Internalization by cells of radiolabeled protein ligands bound to the cell surface is frequently analyzed by extraction of the cells with low pH buffers. This treatment supposedly strips the ligands from the cell surface, and remaining molecules are considered to be internalized. However, we show herein that: (1) low molecular weight catabolic products that are trapped within lysosomes (residualizing radiolabels) are efficiently extracted by low pH buffers, under the same conditions used to remove cell surface-bound material, and (2) low pH treatment lyses the majority of the cells, as shown with both a nonadherent and an adherent cell line, with the release of most of a (51)Cr label. Still, low pH extraction was effective at demonstrating Ab internalization, as has been demonstrated many times. These effects of low pH treatment may be attributed to the fixative properties of these buffers. Regardless of the mechanism, these data must be taken into consideration in interpreting the results of such experiments.

Acids↗

PubMed2000

Radionuclides linked to a CD74 antibody as therapeutic agents for B-cell lymphoma: comparison of Auger electron emitters with beta-particle emitters.

UNLABELLED: We demonstrated previously that human B-cell lymphomas were effectively and specifically killed in vitro by an antibody to CD74 (LL1) linked to (111)In or other Auger electron emitters. This study was intended to more accurately compare the potency and specificity of 3Auger electron emitters, (111)In, 67Ga, and 125I, and to evaluate beta-particle emitters, 131I and 90Y. The unique property of LL1 is its high level of intracellular uptake. METHODS: Raji B-lymphoma cells were incubated with serial dilutions of the radiolabeled Abs for 2 d and then monitored for cell growth by 2 assays: a cell counting assay and a clonogenic assay. The uptake of radioactivity per cell was monitored at various time points, and the radiation dose was calculated using published S values for radioactivity located in the cytoplasm. Both specific and nonspecific toxicity were evaluated. RESULTS: The beta-particle emitters had considerably higher levels of nonspecific toxicity than the Auger electron emitters, but both 131I and 90Y, and particularly 131I, still had high levels of specificity. Both of these results were consistent with dosimetry calculations. Relative to the delivered disintegrations per cell, 131I and 67Ga were the most potent of the radionuclides tested, with 125I and (111)In being significantly weaker and 90Y being intermediate. The high potency of 67Ga, together with its low nonspecific toxicity, caused this radionuclide to have the highest specificity index. CONCLUSION: When delivered by Ab LL1, both Auger electron and beta-particle emitters can produce specific and effective toxicity. The choice of the optimal radionuclide for therapy may depend on the ease and efficiency of labeling, the specific activity obtained, the nature of the tumor being targeted, and other factors, but the high specificity indices of the Auger electron emitters may be an advantage.

Antibodies↗

PubMed1999

Cytotoxicity with Auger electron-emitting radionuclides delivered by antibodies.

We investigated the in vitro cytotoxic potential of Auger electron-emitting radionuclides delivered to the cytoplasm or, more specifically, to lysosomes, via antibodies. The antibody (Ab) used was LL1, which is specific for CD74, an epitope of the major histocompatibility complex (MHC) class II antigen invariant chain, Ii, present on the cell surface. It is taken up in large amounts, approximately 10(7) Ab molecules per cell per day, and delivered to lysosomes. The radioisotopes tested included (111)In, 99mTc and 125I. With sufficient specific activity, approximately 10 mCi/mg Ab, all of these isotopes were potent cytotoxic agents. 125I was active only if a "residualizing" form was used, meaning a form that is trapped within cells after catabolism of the Ab to which it was conjugated (conventional oxidative iodination produces a non-residualizing label). The conjugates of (111)In and 99mTc used are known to be residualizing. One hundred percent cell kill in vitro was obtained with (111)In and 125I, under conditions in which a non-reactive control Ab, conjugated in the same way, produced no significant toxicity. 99mTc was also potent and specific, but appeared somewhat less active than the other isotopes under the conditions evaluated. Although few Abs are accreted by cells at the same rate as LL1, it may be possible to use other Abs to deliver similar amounts of radioactivity, if Abs with higher specific activity can be produced. Such conjugated radioisotopes may be useful for attacking tumor cells in vivo, particularly for single cells or micrometastases.

Antibodies↗

PubMed1999

Processing of antibodies to the MHC class II antigen by B-cell lymphomas: release of Fab-like fragments into the medium.

Lym-1, an anti-MHC class II Ab, displayed a unique processing pathway after binding to the surface of Raji B-lymphoma cells, in which Fab-like fragments were gradually released into the medium. The fragments had reduced interchain disulfide bonds. Fragmentation was markedly reduced by inhibitors of intracellular catabolism, namely ammonium chloride, chloroquine and leupeptin. The capacity of the process was high, and fragmentation of approximately 5x10(6) Ab molecules per cell per day was measured directly, in what can be considered to be a minimum estimate. Five other Abs to the MHC class II antigen were tested similarly on Raji and on three other B-cell lymphomas: none showed the same high level of fragmentation seen with Lym-1 binding to Raji, but significant fragmentation did occur with some of the Abs, particularly EDU-1 and L243. The level of fragmentation depended on the cell line as well as on the particular Ab. The other 5 Abs were all catabolized, to low molecular weight material, much more extensively than Lym-1. Part of the difference between Abs can probably be attributed to the fortuitous, preferential labeling of Lym-1 on the light chain, since the data suggest that the Fc fragment is fully degraded while the Fab-like fragment is released into the supernatant. This pathway of Ab processing is likely to be related to the physiology of the MHC class II antigen, which recycles into a mildly proteolytic intracellular compartment.

Animals↗

PubMed1999

Cell surface expression and metabolism of major histocompatibility complex class II invariant chain (CD74) by diverse cell lines.

We previously described the processing of antibodies to CD74 (the major histocompatibility complex class II-associated invariant chain, Ii), by B-cell lymphoma cell lines. These cells expressed relatively low levels of Ii on the surface, but the molecules were rapidly internalized and replaced by new molecules, so that approximately 8 x 10(6) antibody molecules per cell were taken up per day. We herein report the results of similar studies with other cell types, namely a melanoma, a colon carcinoma, a T-cell lymphoma and B-lymphoblastoid cell lines. The melanoma and the carcinoma were treated with interferon-gamma to induce high levels of the antigen. The T-cell lymphoma, HUT 78, was selected specifically because it was previously reported to lack cell surface Ii, while expressing the molecule intracellularly. However, HUT 78 displayed Ii on the cell surface, as did the other cell lines tested, and catabolism of the antibody was very fast on all of the cell lines. The capacity of four of the cell lines for cumulative antibody uptake was evaluated, using 'residualizing' radiolabels, which are trapped within the cell after catabolism of the antibody to which they were conjugated. A high level of uptake was observed in all cases, although there was significant variation between the cell lines. With melanoma SK-MEL-37, the total LL1 uptake in 24 hr was nearly 10(7) molecules per cell and the average turnover time for Ii on the cell surface was 4 min; with carcinoma HT-29, the total LL1 uptake in 24 hr was approximately 10(6) molecules per cell, and the average turnover time for Ii on the cell surface was 27 min. Based on the cell content of mature class II antigens (alphabeta), these data suggest that a large fraction, or all, of immature class II molecules (alphabetaIi) reach the cell surface before entering the peptide-loading compartment, independent of the particular cell type.

Antigen-Antibody Reactions↗

PubMed1999

Carcinoembryonic antigen as a target for radioimmunotherapy of human medullary thyroid carcinoma: antibody processing, targeting, and experimental therapy with 131I and 90Y labeled MAbs.

The poor prognosis of patients with advanced medullary thyroid carcinoma (MTC) has prompted a search for new treatment modalities. In this report we explore the characteristics of carcinoembyronic antigen (CEA) as a target for radioimmunotherapy (RAIT) of MTC, with respect to antibody processing, targeting, and experimental therapy. In vitro studies showed a high level of CEA expression on the cell surface of the MTC cell line TT. MAbs bound to the cell were predominantly retained for several days, although there was also a significant level of internalization and catabolism. Immunohistology of frozen sections of tumor xenografts demonstrated that approximately half of the cells were darkly stained, however, some cells expressed little or no CEA. In biodistribution studies in nude mice bearing TT tumors, the mean percent injected dose per gram of tumor observed at three days post injection (time of maximum uptake) of 125I-MN-14 was 19.7%. When the MAb was labeled with 88Y, a residualizing label, a much higher accretion, 50.5%, was observed at the time of maximum uptake (7 days). Significant anti-tumor effects were seen at the maximum tolerated doses of 131I- and 90Y-MN-14, compared with relatively rapid tumor growth in untreated animals or those treated with the same dose of control MAbs. Importantly, it was observed that 90Y-MN-14 yielded significantly improved therapeutic efficacy in comparison to 131I-MN-14, which may have important implications for design and conduct of future clinical trials for the treatment of MTC.

Animals↗

PubMed1999

Enhancement of tumor-to-nontumor localization ratios by hepatocyte-directed blood clearance of antibodies labeled with certain residualizing radiolabels.

UNLABELLED: To increase tumor-to-nontumor localization ratios of injected radiolabeled antibodies (Abs), several interrelated methods were used. METHODS: The model systems used were two human carcinoma xenografts grown in nude mice, targeted by antibodies RS11 (antiepithelial glycoprotein-2) or MN-14 (anticarcinoembryonic antigen). The Abs were conjugated with biotin and 111In-benzyl diethylenetriamine pentaacetic acid, and, at various times after injection, were cleared by intraperitoneal injection of galactosylated streptavidin, which delivers the complexes to hepatocytes. The radiolabel used was selected because it is retained within tumors after catabolism of the Ab by the tumor cell but is quite rapidly excreted from hepatocytes into bile. RESULTS: With blood clearance induced at 24 h, and dissection 5 h later, high tumor-to-nontumor ratios were attained. Depending on the model used, tumor-to-blood ratios were 16:1 to 31:1, and tumor-to-nontumor ratios for the kidney, lungs and bone were also high and greatly increased by the clearance regimen. Despite clearance into the liver, tumor-to-liver ratios remained >1, due to fairly rapid biliary excretion of the label. The absolute antibody uptake by the tumors was also high, because 24 h was allowed for the Ab to penetrate and bind to cells within the subcutaneous tumors. CONCLUSION: The method described produced high tumor-to-nontumor ratios at 1 d after injection and may be advantageous for tumor imaging with antibodies. Radiation dosimetry calculations indicate that there is only a slight advantage with this approach for radioimmunotherapy.

Animals↗

PubMed1999

Targeting human cancer xenografts with monoclonal antibodies labeled using radioiodinated, diethylenetriaminepentaacetic acid-appended peptides.

A new nonmetabolizable peptide approach to the production of residualizing radioiodine was evaluated in nude mice bearing xenografts of human lung adenocarcinoma (Calu-3) and B-cell lymphoma (Ramos). Monoclonal antibodies (MAbs) RS7 (anti-epithelial glycoprotein-1) and LL2 (anti-CD22) were radioiodinated using the thiol-reactive diethylenetriaminepentaacetic acid-D-peptide adducts IMP-R1 and IMP-R2. 125I-IMP-R1- and 125I-IMP-R2-labeled MAbs were compared to the MAbs iodinated by the conventional chloramine-T approach, (111)In, and 131I-dilactitoltyramine (DLT). In vivo biodistribution studies demonstrated a significant improvement in the tumor accretion of radiolabel using the 125I-IMP-R1 labeled MAbs compared with the conventionally iodinated antibodies. For example, at day 7, the percentage of injected dose per gram of tissue in Calu-3 was 7.9 +/- 4.1% and 18.1 +/- 7.9% (P < 0.05) for the conventional 131I- and 125I-IMP-R1-RS7, respectively, and tumor:nontumor ratios were 2.6-4.5-fold higher with the 125I-IMP-R1-RS7. It is estimated that 131I-IMP-R1-RS7 would deliver a dose to tumor (at the estimated maximum tolerated dose) 3.9 times greater than conventional 131I-labeled RS7, 1.4 times greater than 90Y-labeled RS7, and 0.7 times that of 131I-DLT-labeled RS7. Tumor accretion of 125I-IMP-R2-RS7 was also improved compared with conventionally iodinated antibody. However, this label also caused a large increase in kidney accretion. Similar improvements in tumor accretion and tumor:nontumor ratios were observed when 125I-IMP-R1-LL2 was used in the Ramos model. IMP-R1 offers a practical and useful residualizing radioiodine label because labeling efficiency is at least 10 times greater than that of the residualizing label DLT, without MAb aggregation. Structural modifications can be envisioned for further improvements in radioiodine incorporation, specific activity, and tumor dosimetry, and efforts along these lines are under way.

Animals↗

PubMed1998

Rapid blood clearance of mouse IgG2a and human IgG1 in many nude and nu/+ mouse strains is due to low IgG2a serum concentrations.

We reported previously that the blood clearance of injected mouse IgG2a was extremely rapid in many strains of nude and nu/+ mice. In an attempt to determine the cause of this phenomenon, the levels of endogenous IgG2a in the blood of these mice was assayed. It was found that the serum level of IgG2a was extremely low in many of these mice, below 50 microg/ml, which is 20-100 times lower than the expected normal value. Great heterogeneity between individual mice was observed in their blood level of IgG2a, and there was an excellent correlation between low blood IgG2a levels and rapid clearance of injected IgG2a. Thus, the blood IgG2a levels are so low that a novel, previously undescribed effect occurs, namely the rapid clearance of small amounts of injected IgG2a. The clearance is due primarily to binding sites in the spleen and liver. The low level of endogenous IgG2a is not due to the lack of a thymus, since it occurs in nu/+ as well as nude mice, but can probably be attributed to the very clean environment in which these mice are raised. In assays of sera from approximately 50 mouse strains, low IgG2a levels were found in all nude colonies and also in some normal mouse strains. Some nude mice displayed relatively normal IgG2a clearance rates despite having low levels of endogenous IgG2a. In repeated bleedings of individual mice, IgG2a levels were found to fluctuate greatly. A similar clearance effect was observed with a human IgG1 Ab injected into mice. This rapid clearance of injected IgG, of certain subclasses, represents a practical problem for many experiments in which antibodies are used for diagnosis or therapy, and several methods of circumventing the problem are discussed.

Animals↗

PubMed1998

Intracellular processing of 99Tcm-antibody conjugates.

The catabolism of 99Tcm-antibody conjugates after internalization by B-cell lymphomas was investigated, using antibody LL1, an antibody to the MHC class II invariant chain which is internalized and catabolized very rapidly. Intact IgG antibodies were labelled with 99Tcm after mild reduction. The 99Tcm label was strongly retained within cells, similar to 'residualizing' labels such as 111In-diethylenetriamine pentaacetate (111In-DTPA), but different from a conventional iodine label. Unlike 111In-DTPA, 99Tcm was not retained in a low molecular weight form, but instead was found to be bound to a large number of different cellular proteins, and was retained in the cytoplasm rather than in lysosomes. Therefore, this form of 99Tcm represents a new paradigm of intracellular retention of a radiolabel.

Antibodies↗

PubMed1997

Processing of antibodies bound to B-cell lymphomas and lymphoblastoid cell lines.

BACKGROUND: Previous experiments demonstrated that some human B-cell lymphoma cell lines were unusual in that antibodies bound to the cell surface dissociated at high levels. This did not occur with non-B-cell hematologic tumors or with carcinomas. In this study, additional B-cell lymphoma and lymphoblastoid (Epstein-Barr virus-transformed) cell lines were tested. METHODS: The antibodies selected for most experiments, MA103 and anti-CD45, react with relatively high avidity to the cell surface. Antibodies to CD19, CD20, and CD22 also were tested on certain cell lines. The antibodies were labeled with 125I. After binding to the surface of viable cells, unbound antibody was washed away, and the fate of the bound antibody was investigated for 2-3 days. RESULTS: Of the eight B-cell lymphomas tested, three had high levels of dissociation, two had low levels of dissociation, and three had intermediate levels of dissociation. The six lymphoblastoid cell lines had only slightly elevated levels of dissociation, relative to non-B cell lines. Sublines of Raji and Ramos cells were identified that varied greatly in the level of antibody dissociation. The level of dissociation from lymphomas was correlated with the tendency of the cell lines to cluster, with single cells displaying less dissociation than clustered cells. However, some exceptions to this correlation were noted. Cell lines such as Ramos, which showed little dissociation of anti-CD20, displayed relatively rapid catabolism of this antibody. CONCLUSIONS: The level of antibody dissociation as well as the rate of antibody catabolism will affect the results of radioimmunotherapy strongly because these factors affect the time interval for which the cells are in contact with the radioisotope. Different B-cell lines display markedly different levels of dissociation. There is some evidence suggesting that antibody dissociation is high with fresh human tumor cells, but further investigation of this point is required.

Antibodies↗

PubMed1997

The advantage of residualizing radiolabels for targeting B-cell lymphomas with a radiolabeled anti-CD22 monoclonal antibody.

CD22 antibodies (Abs) bound to B-cell lymphomas are known to be internalized and catabolized rapidly. Therefore, it would be expected that use of CD22 as a target for radioimmunotherapy should be enhanced by the use of "residualizing" radiolabels, which are trapped within the cell after catabolism of the Ab to which they had been conjugated. Our study was intended to evaluate this hypothesis using Ab LL2. In initial experiments, we found that LL2 binding was strongly temperature dependent, with approximately 15-fold greater binding at 37 degrees C than at 0 degrees C. A series of experiments suggested that this difference is due to a conformational change in the antigen at low temperature, so that the LL2 epitope is partially blocked. In vitro, residualizing labels-including 125I-dilactitol tyramine and 111In-DTPA-were retained by cells much longer than a conventional iodine label. In vivo, residualizing labels also showed a marked advantage in terms of uptake by Ramos B-cell lymphoma xenografts in nude mice. However, the absolute Ab uptake by xenografts was quite low, in comparison with results obtained with many carcinoma xenografts, which appears to be due in part to vascular properties of the B-cell lymphoma xenografts.

Animals↗

PubMed1997

Binding parameters of antibodies reacting with multivalent antigens: functional affinity or pseudo-affinity.

For antibodies binding bivalently to multivalent antigens, including the cell surface, it has been common to determine the 'functional affinity', even though it is clear that the equations describing true affinity only apply to monovalent interactions. We here summarize evidence that 'functional affinities' are not valid, because they do not accurately describe the interactions that occur. A basic discrepancy is that dissociation, in practice, is generally not a first-order reaction. A more useful approach to compare antibodies is to determine the kinetics of dissociation over a long time period, until near-complete dissociation has occurred.

Animals↗

PubMed1997

The Oka blood group antigen is a marker for the M6 leukocyte activation antigen, the human homolog of OX-47 antigen, basigin and neurothelin, an immunoglobulin superfamily molecule that is widely expressed in human cells and tissues.

The high-frequency blood group antigen Ok(a) is carried on a red cell membrane glycoprotein (gp) of 35-69 kDa that is widely distributed on malignant cells of different origins. Immunostaining of hemopoietic cells and a range of normal human tissues demonstrated a wide distribution of the Ok(a) gp that appears to be nonlineage-restricted, although certain tissues show differentiation-related expression. Ok(a) gp was purified from red cell membranes by immunoaffinity chromatography using mAb A103 and amino acid sequence analysis was performed. The N-terminal 30 amino acids are identical to the predicted sequence of M6 leukocyte activation antigen (M6), a member of the Ig superfamily (IgSF) with two IgSF domains. There are homologs in rat (MRC OX-47 or CE9), in mouse (basigin or gp42), and in chicken (HT7 or neurothelin). The molecular basis of the Ok(a) mutation was established by sequencing M6 cDNA derived from normal and Ok(a-) EBV-transformed B cell lines. A point mutation in the translated portion of M6 cDNA, G331AG-->AAG gives rise to a predicted E92-->K amino acid change in the first Ig-like domain of the Ok(a-) form of the protein. Transfection of mouse NS-0 cells with normal or Ok(a-) cDNA confirmed the identity of the protein and only the Ok(a-) transfectants failed to react with monoclonal anti-Ok(a) Ab.

ABO Blood-Group System↗