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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.

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.

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.

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.

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.

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.

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.

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.

The optimal use of radioiodinated internalizing monoclonal antibodies (mAbs) for radioimmunotherapy necessitates the development of practical methods for increasing the level of retention of 131I in the tumor. Lysosomally trapped ("residualizing") iodine radiolabels that have been previously designed are based mostly on carbohydrate-tyramine adducts, but these methods have drawbacks of low overall yields and/or high levels of mAb aggregation. We have developed a method using thiol-reactive diethylenetriaminepentaacetic acid (DTPA)-peptide adducts wherein the peptides are assembled with one or more D-amino acids, including D-tyrosine. Two such substrates, R-Gly-D-Tyr-D-Lys[1-(p-thiocarbonylaminobenzyl)DTPA], referred to as IMP-R1, and [R-D-Ala-D-Tyr-D-Tyr-D-Lys]2(CA-DTPA), referred to as IMP-R2, wherein R is 4-(N-maleimidomethyl)cyclohexane-1-carbonyl, were synthesized by preparing functional group-protected peptides on a solid phase, selectively derivatizing the lysine side chain with 1-(p-isothiocyanatobenzyl)DTPA or DTPA dianhydride (CA-DTPA), deprotecting other functional groups, and finally derivatizing the peptide's N-terminus so it contained a maleimide group. Radioiodinations of the peptides followed by conjugations to disulfide-reduced mAbs, carried out as a one-vial procedure, resulted in 32-89% overall yields, at specific activities of 1.8-11. 1 mCi/mg, with less than 2% aggregation. Two internalizing mAbs, LL2 (anti-CD 22 B-cell lymphoma mAb) and RS7 (an anti-adenocarcinoma mAb which targets EGP-1 antigen), labeled with this procedure exhibited a 2-3-fold better cellular retention in Ramos and Calu-3 tumor cell lines, in vitro, respectively, compared to the same mAbs radioiodinated with the chloramine-T method. The rationale for the new approach, syntheses, radiochemistry and in vitro data are presented.

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.

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.

Unlabelled: We have attempted to improve the therapeutic index of radioimmunotherapy by manipulating the blood clearance rate and the catabolism of the radiolabel. The general strategy is to allow the antibody (Ab) to circulate in the blood for 2-3 days, then to clear it rapidly by a method that delivers the Ab to hepatocytes. In addition, the radiolabel selected has two key properties: it is a residualizing label (which is lysosomally trapped after catabolism), so it is retained well by tumor cells, but is excreted rapidly by hepatocytes into bile., Methods: In initial experiments, three residualizing radiolabels were tested for their rate of excretion after specific delivery in vivo to either hepatocytes, via galactosylated Ab, or Kupffer cells, via immune complexes. A label showing rapid biliary excretion only after delivery to hepatocytes, (111)In-benzyl-diethylenetriamine tetraacetic acid, was then used for radioimmunodetection in a protocol of delayed rapid blood clearance in which clearance was by hepatocytes. This was achieved by using galactosylated Ab, combined with temporary inhibition of the asialo-glycoprotein receptor on hepatocytes. Ab RS11 and the lung adenocarcinoma Calu-3 xenograft in nude mice were used. Control experiments were performed with a conventional 125I label and with 125I-dilactitol-tyramine., Results: Indium-benzyl-diethylenetriamine tetraacetic acid was identified as a label that was excreted more rapidly from hepatocytes than from Kupffer cells, by biliary excretion. Using this radiolabel with delayed rapid blood clearance, very high tumor/blood ratios were obtained, 166:1 at day 3, but tumor/normal tissue ratios for other tissues were not as high. There appeared to be some uptake of the radiolabel by all normal tissues tested, including the lungs and muscle. Dosimetry calculations suggested that the therapeutic index was no better than with a simple Ab injection., Conclusion: Antibody catabolism can be directed towards either hepatocytes or Kupffer cells, and this difference can strongly affect the excretion rate of radiolabels, since only hepatocytes can excrete degradation products into bile. Processing will also depend on the particular radiolabel. These factors are particularly important for protocols involving delayed rapid blood clearance, since liver uptake is so rapid. The methods described should stimulate other approaches of manipulating Ab blood clearance and radiolabel catabolism to achieve improved therapeutic results.

Pretargeting methodologies can produce high tumor:blood ratios, but their role in cancer radioimmunotherapy (RAIT) is uncertain. A pretargeting method was developed using a streptavidin (StAv) conjugate of MN-14 IgG, an anti-carcinoembryonic antigen (CEA) murine monoclonal antibody (mab) as the primary targeting agent, an anti-idiotype antibody (WI2 IgG) as a clearing agent, and DTPA- or DOTA-conjugated biotin as the radiolabeled targeting agent. A variety of reagents and conditions were examined to optimize this method. At 3 h, 111In-DTPA-peptide-biotin tumor uptake was 3.9 +/- 0.8% per gram and tumor:blood ratios were > 11:1. By 24 h, this ratio was 178:1, but tumor accretion declined in accordance with the gradual loss of StAv-MN-14 from the tumor. Tissue retention was highest in the liver and kidneys, but their tumor:organ ratios were > 2:1. Dosimetry predicted that radiolabeled MN-14 alone would deliver higher tumor doses than this pretargeting method. Increasing the specific activity and using DOTA-biotin in place of DTPA increased tumor uptake nearly 2-fold, but analysis of StAv-MN-14's biotin-binding capacity indicated over 90% of the initial biotin-binding sites were blocked within 24 h. Animals fed a biotin-deficient diet had 2-fold higher 111In-DOTA-biotin uptake in the tumor, but higher uptake also was observed in all normal tissues. Although exceptionally adept at achieving high tumor:blood ratios rapidly, the tumor uptake of radiolabeled biotin with this pretargeting method is significantly (p < 0.0001) lower than that with a radiolabeled antibody. Endogenous biotin and enhanced liver and kidney uptake may limit the application of this method to RAIT, especially when evaluating the method in animals, but with strategies to overcome these limitations, this pretargeting method could be an effective therapeutic alternative.

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.

LL2 is an anti-CD22 pan-B-cell monoclonal antibody which, when radiolabeled, has a high sensitivity for detecting B-cell, non-Hodgkin's lymphoma (NHL), as well as an antitumor efficacy in therapeutic applications. The aim of this study was to determine whether intracellularly retained radiolabels have an advantage in the diagnosis and therapy of lymphoma with LL2. In vitro studies showed that iodinated LL2 is intracellularly catabolized, with a rapid release of the radioiodine from the cell. In contrast, residualizing radiolabels, such as radioactive metals, are retained intracellularly for substantially longer. In vivo studies were performed using LL2-labeled with radioiodine by a non-residualizing (chloramine-T) or a residualizing method (dilactitol-tyramine, DLT), or with a radioactive metal (111In). The biodistribution of a mixture of 125I (non-residualizing chloramine-T compared to residualizing DLT), 111In-labeled LL2 murine IgG2a or its fragments [F(ab')2, Fab'], as well as its humanized, CDR-grafted form, was studied in nude mice bearing the RL human B-cell NHL cell line. Radiation doses were calculated from the biodistribution data according to the Medical International Radiation Dose scheme to assess the potential advantage for therapeutic applications. At all assay times, tumor uptake was higher with the residualizing labels (i.e., 111In and DLT-125I) than with the non-residualizing iodine label. For example, tumor/blood ratios of 111In-labeled IgG were 3.2-, 3.5- and 2.8-fold higher than for non-residualizing iodinated IgG on days 3, 7 and 14, respectively. Similar results were obtained for DLT-labeled IgG and fragments with residualized radiolabels. Tumor/organ ratios also were higher with residualizing labels. No significant differences in tumor, blood and organ uptake were observed between murine and humanized LL2. The conventionally iodinated anti-CD20 antibody, 1F5, had tumor uptake values comparable to those of iodinated LL2, the uptake of both antibodies being strongly dependent on tumor size. These data suggest that, with internalizing antibodies such as LL2, labeling with intracellularly retained isotopes has an advantage over released ones, which justifies further clinical trials with residualizing 111In-labeled LL2 for diagnosis, and residualizing 131I and 90Y labels for therapy.

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.

Unlabelled: Attachment of 131I to MAbs through adducts such as dilactitol-tyramine (DLT), which remain lysosomally trapped after catabolism of the labeled MAb, can greatly increase the residence time of the radiolabel at the tumor site. Our previous studies demonstrated a marked increase in accretion of 131I in a human lung-cancer xenograft model, using 131I-DLT in comparison to 131I linked to MAb by the conventional chloramine-T methodology., Methods: In this study, biodistribution experiments were performed to evaluate the effect of protein dose on the accretion of 131I-DLT-labeled MAb RS11 in tumor and nontumor tissues, and in vivo radioimmunotherapy experiments compared the effect of single injections of 131I-DLT-labeled MAb RS11 to conventional 131I-labeled RS11 and an untreated control group., Results: Dosimetry calculations based on the biodistribution data indicate only small changes in absorbed dose-to-tumor and nontumor tissues with increasing protein dose up to 100 micrograms, with a predicted absorbed dose to tumor of from 21,000 to 25,000 cGy/mCi. A single dose of 100 microCi of 131I-DLT-RS11 was found to cause tumor regression. At 7 wk postinjection of the radiolabeled MAbs, tumor volume in 73% of the animals administered 131I-DLT-labeled RS11 remained smaller than at the time of MAb injection. This is compared to 14% of the tumors in the conventionally labeled 131I-RS11 group and none in the untreated group., Conclusion: The use of the residualizing radiolabel DLT provides a therapeutic advantage in comparison to conventional 131I-labeled RS11.

The fate of antibody (Ab) LL1, which reacts with the invariant chain (Ii) subunit of the immature MHC class-II antigen (CD74) after binding to the surface of B-cell lymphomas was investigated. This Ab was internalized and catabolized very rapidly, much faster than other Abs that are considered to be rapidly internalized, such as CD19, CD22 and anti-(transferrin receptor). Such internalization did not depend on Ab cross-linking. The capacity of this uptake process was determined in long-term experiments by increasing the Ab concentration: in 1 day, approx. 8 x 10(5) Ab molecules per cell were catabolized. This analysis was facilitated by the use of radiolabels that are trapped within cells after catabolism of the Abs to which they were conjugated. If the Ab is a reliable marker for the Ii antigen, which is likely, we can conclude that Ii directed to the cell surface appears to be sufficient, indeed more than sufficient, to account for the cell content of mature class-II molecules.

A new cell line derived from a human adenocarcinoma of the colon, GS-7, was propagated as a s.c. tumor in nude mice. This tumor histologically is a mucinous adenocarcinoma (also designated mucoid or colloid) with characteristic large mucin pools that are not lined by an epithelial layer but may contain scattered, randomly distributed cancer cells. Ten to 20% of human colorectal adenocarcinomas are of this histological type, but rapidly growing xenografts with this histology have been rarely used experimentally. This tumor, therefore, constitutes a useful model for similar human tumors. The mucin pools contain large amounts of carcinoembryonic antigen and tumor-associated glycoprotein 72, and the cells express epithelial glycoprotein 2 on their surface. The ability of antibodies injected i.v. to penetrate this tumor was investigated, using both biotinylated and radioiodinated antibodies (Abs). The results demonstrate that Abs can effectively penetrate the mucin pools, and that large amounts of Ab can localize there. This tumor type may have advantages as a target for certain forms of experimental immunotherapy.