Therapy of Angiosarcoma with Thalidomide and Lenalidomide.

Angiosarcoma is an uncommon malignancy with a poor prognosis. Systemic therapy options for patients with metastatic disease generally have limited effectiveness. In this case study, a 73-year-old male with metastatic angiosarcoma who previously declined chemotherapy and developed progressive disease after checkpoint inhibitor immunotherapy elected to try thalidomide based on 6 case reports describing its effectiveness. Thalidomide resulted in stable disease for 9 months, but due to severe neuropathy as a side effect, lenalidomide was then substituted for thalidomide. The patient continued to have stable disease on lenalidomide for an additional 16 months and ongoing. This is the first case study to report on effective treatment of angiosarcoma with lenalidomide. Further investigation of lenalidomide in the management of angiosarcoma is warranted.

Therapy of advanced B-lymphoma xenografts with a combination of 90Y-anti-CD22 IgG (epratuzumab) and unlabeled anti-CD20 IgG (veltuzumab).

PURPOSE: Antibodies are effective therapeutic agents in cancer, but cures are rarely if ever obtained. Combination therapies are likely to be more effective than a single agent. In this study, the combination of a new unconjugated humanized anti-CD20 IgG, veltuzumab, with a (90)Y-conjugated humanized antibody to CD22 (epratuzumab) was evaluated for the treatment of B-cell lymphoma in a nude mouse model system. EXPERIMENTAL DESIGN: Nude mice were grafted with the Ramos human B-lymphoma and treatment initiated when tumors were >0.1 cm(3). In most experiments, mice were injected first with unconjugated anti-CD20, then with (90)Y-anti-CD22 1 day later. Additional weekly injections of the unconjugated veltuzumab were administered for 3 weeks. Controls included a single agent only and a nonreactive control radiolabeled antibody. RESULTS: Unconjugated anti-CD20 veltuzumab alone did not have a significant therapeutic effect, even at a total dose of 2.5 mg per mouse. The (90)Y-anti-CD22 epratuzumab alone induced marked regressions of all tumors, but they regrew in a few weeks. The combination of these agents cured approximately 80% of the mice. A nonreactive control antibody labeled with (90)Y, used without veltuzumab, had no therapeutic effect. The therapeutic effect of (90)Y-epratuzumab required the maximum tolerated dose of radioactivity, which was 160 muCi per mouse. CONCLUSIONS: These studies illustrate how combinations of unconjugated and radioconjugated antibodies against different B-cell markers can improve therapeutic outcome, and offer a new therapeutic paradigm for the treatment of B-cell lymphomas.

The mechanism of killing of B-lymphoma cells by 111In-conjugated antibodies.

PURPOSE: To determine the mode of killing of B-lymphoma cell lines by 111In-conjugated antibodies (Ab). MATERIALS AND METHODS: Cells were treated with a range of radiolabeled Ab concentrations to produce a level of killing from 80 - 99.9%, as determined by a long-term proliferation assay. On days 1, 2, 3 and 4, apoptosis was evaluated in individual cells by two assays: (i) Mitochondrial function by JC-1 staining; and (ii) presence of cleaved PARP (poly-ADP-ribose-polymerase) by immunofluorescence. Five cell lines were tested, including two non-Burkitt lines, RL and FSCCL. RESULTS: In preliminary experiments, it was realized that the standard JC-1 assay, which is widely used, does not distinguish between apoptotic and necrotic cells. The JC-1 assay becomes useful if it is combined with a stain for lysed cells, Sytox green, and then is able to distinguish between viable, apoptotic and necrotic cells. Of the 5 cell lines tested, only Ramos had a large population of apoptotic cells detected by the modified JC-1 assay, but all four of the other cell lines showed evidence of cleaved PARP, albeit to markedly different degrees. The caspase inhibitor, Z-VAD (N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone), significantly inhibited the killing of Daudi cells. CONCLUSIONS: Apoptosis was activated in at least four of the five cell lines tested, and participated in the killing of the cells. Staining for cleaved PARP was much more sensitive than the modified JC-1 assay, for most cell lines, probably because it can detect cells that have already lysed, but which died via apoptosis. In addition, the methodological issues discussed may lead to a better understanding of the role of apoptosis in tumor cell cytotoxicity.

CD74: a new candidate target for the immunotherapy of B-cell neoplasms.

CD74 is an integral membrane protein that functions as a MHC class II chaperone. Moreover, it has recently been shown to have a role as an accessory-signaling molecule and has been implicated in malignant B-cell proliferation and survival. These biological functions combined with expression of CD74 on malignant B cells and limited expression on normal tissues implicate CD74 as a potential therapeutic target. The anti-CD74 monoclonal antibody LL1 has been humanized (hLL1 milatuzumab or IMMU-115) and can provide the basis for novel therapeutic approaches to B-cell malignancies, particularly because this antibody shows rapid internalization into CD74+ malignant cells. This article reviews the preclinical evaluations of LL1, its humanized form, and isotope, drug, and toxin conjugates. These studies show that unconjugated hLL1 and conjugates of hLL1 constructs with radioisotopes, doxorubicin, and frog RNase have high antitumor activity in non-Hodgkin's lymphoma and multiple myeloma in vitro and in tumor xenograft models. Single-dose studies of hLL1 in monkeys showed no adverse effects but did decrease circulating B and T lymphocytes and natural killer cells. When evaluated in combination with rituximab, either equivalent or improved efficacy, compared with either antibody alone, was observed. CD74 is a new candidate target for the immunotherapy of neoplasms expressing this antigen, which can be exploited using either a naked antibody or conjugated to isotopes, drugs, or toxins.

A divalent hapten-peptide induces apoptosis in human non hodgkin lymphoma cell lines targeted by anti-CD20xanti-hapten bispecific antibodies.

PURPOSE: Bispecific antibody (bsMAb) pretargeting procedures use divalent hapten-peptides to stabilize the binding of the hapten-peptide on tumor cells by a process known as the affinity enhancement system. The goal of this study was to determine if a divalent hapten-peptide could induce apoptosis by cross-linking bsMAb bound to CD20. METHODS: Three forms of bsMAbs were prepared by coupling the IgG, F(ab')2, or Fab' of a humanized anti-CD20 antibody to a Fab' of a murine antibody directed against the hapten histamine-succinyl-glycine (HSG). A recombinant bsMAb with divalent binding to CD20 and monovalent binding to HSG was also examined. Induction of apoptosis on SU-DHL-6, RL, and Ramos cells was examined by propidium iodide staining, caspase-3 activation, and mitochondrial membrane potential collapse, and compared with induction by cross-linking an anti-CD20 IgG with an antispecies antibody. RESULTS: The various forms of bsMAb had differing baseline levels of apoptosis in the absence of the divalent HSG peptide. The addition of the divalent HSG peptide significantly increased the level of apoptosis seen with the Fab'xFab' bsMAb by 2.2- to 3.9-fold, as well as the F(ab')2xFab', IgGxFab', and the recombinant bsMAbs by approximately 1.5-fold. CONCLUSIONS: The addition of a divalent HSG peptide to various forms of bispecific anti-CD20 MAbs could enhance apoptotic signaling in several lymphoma cells. This effect was more consistently measured when the orientation of the anti-hapten-binding arm of the bsMAb was well defined, such as in the Fab'xFab' and recombinant forms of bsMAb.

Characterization of antibody-containing vesicles shed from B-lymphoma cell lines: exposure of annexin V binding sites.

Antibodies (Abs) to CD20 or HLA-DR, after binding to the B-lymphoma cell line RL following an overnight incubation at 37 degrees C, accumulate in the form of shed vesicles, which develop in the center of the cell clusters that are spontaneously formed by this cell line. These vesicles coalesce into fairly stable large structures, which we refer to as conglomerates of shed vesicles (CSVs). In the present study, we have extended our previous investigations into the nature of this material. Electron microscopy revealed a conglomerate of heterogeneous vesicles, which looked like pinched-off cytoplasmic projections. CSVs developed similarly either with or without Ab, demonstrating that CSV production is a spontaneous process that incorporates bound Abs if they are present. Before delivery to CSVs, the Abs capped on the cell surface. CSVs had high expression of annexin V binding sites, which are phagocytic signals that are exposed on damaged cells. For CSVs that were cell bound, which are frequently observed, the annexin V binding sites were only in the CSVs, and not on the surface of the intact cell. Although all CSVs contained both Abs and annexin V binding sites, the precise distribution of these two ligands was generally different. Annexin V binding sites were present on caps as well as on CSVs, and appear as soon as caps are formed. In cells incubated with anti-HLA-DR, CD20 was delivered to the CSVs together with HLA-DR, suggesting an association between these two molecules. CSVs prepared with anti-HLA-DR, but not CSVs prepared with anti-CD20, contained considerable numbers of nuclear fragments, identified by propidium iodide staining.

Therapy of small subcutaneous B-lymphoma xenografts with antibodies conjugated to radionuclides emitting low-energy electrons.

PURPOSE: Radionuclides emitting low-energy electrons (Auger and conversion electrons of <50 keV) are potentially useful for cancer therapy when conjugated to an antibody, because they can irradiate the cell to which they bind while producing relatively little irradiation of surrounding cells and tissues. We showed previously the ability of such antibody conjugates to treat micrometastatic, disseminated human B-lymphoma in a severe combined immunodeficient mouse model using an anti-CD74 antibody. In this study, we have evaluated the ability of such conjugates to treat s.c. tumors. EXPERIMENTAL DESIGN: Severe combined immunodeficient mice were injected s.c. with Raji, Daudi, or RL B-lymphoma human tumor cells. Antibodies to CD74, CD20, or HLA-DR were radiolabeled with (111)In or (125)I and injected i.v. at various times starting at day 5, and tumor growth was monitored. Controls included the testing of unlabeled antibodies, labeled nonreactive antibodies, and a combination of the two. RESULTS: Therapy of s.c. B-lymphoma was more difficult than therapy of tumor cells that had been injected i.v. Although large, macroscopic tumors were not effectively treated, therapy was effective on s.c. Daudi tumors on day 36 after injection of this slowly growing tumor, with an (111)In anti-CD74 antibody given in two doses. An anti-CD20 antibody labeled with either (111)In or (125)I was able to effectively treat s.c. RL tumors when given as late as day 16 after tumor inoculation. The largest tumors that were effectively treated were macroscopic thin discs (<2 mm in diameter) growing on the mesentery. CONCLUSION: These results extend previous evidence that antibody conjugates with emitters of low-energy electrons can be effective therapeutic agents for micrometastatic cancer.

Improved iodine radiolabels for monoclonal antibody therapy.

A major disadvantage of (131)iodine (I)-labeled monoclonal antibodies (MAbs) for radioimmunotherapy has been the rapid diffusion of iodotyrosine from target cells after internalization and catabolism of the radioiodinated MAbs. We recently reported that a radioiodinated, diethylenetriaminepentaacetic acid-appended peptide, designated immunomedics' residualizing peptide 1 (IMP-R1), was a residualizing iodine label that overcame many of the limitations that had impeded the development of residualizing iodine for clinical use. To determine the factors governing the therapeutic index of the labeled MAb, as well as the factors required for production of radioiodinated MAb in high yield and with high specific activity, variations in the peptide structure of IMP-R1 were evaluated. A series of radioiodinated, diethylenetriaminepentaacetic acid-appended peptide moieties (IMP-R1 through IMP-R8) that differed in overall hydrophilicity and charge were compared. Radioiodinations of the peptides followed by conjugations to disulfide-reduced RS7 (an anti-epithelial glycoprotein-1 MAb) furnished radioimmunoconjugates in good overall incorporations, with immunoreactivities comparable to that of directly radioiodinated RS7. Specific activities of up to 8 mCi/mg and yields > 80% have been achieved. In vitro processing experiments showed marked increases in radioiodine retention with all of the adducts; radioiodine retention at 45 h was up to 86% greater in cells than with directly iodinated RS7. Each of the (125)I-peptide-RS7 conjugates was compared with (131)I-RS7 (labeled by the chloramine-T method) in paired-label biodistribution studies in nude mice bearing human lung tumor xenografts. All of the residualizing substrates exhibited significantly enhanced retention in tumor in comparison to directly radioiodinated RS7, but the nontarget uptakes differed significantly among the residualizing labels. The best labels were IMP-R4 and IMP-R8, showing superior tumor-to-non-tumor ratios by virtue of high tumor uptake and retention and low normal organ uptake, as well as superior radiochemical properties. The therapeutic efficacy of (131)I-IMP-R4-RS7 was compared with that of conventionally (131)I-labeled RS7 and (90)yttrium-RS7 in the nude mice lung cancer model. The therapeutic efficacy of (131)I-IMP-R4-RS7 and (90)yttrium-RS7 were equivalent, and both agents yielded significantly improved control of tumor growth compared with conventional (131)I-labeled RS7.

In vitro cytotoxicity of carcinoma cells with 111In-labeled antibodies to HER-2.

Antibodies conjugated to radionuclides emitting low-energy electrons, which include Auger electrons and some conversion electrons, were recently shown to efficiently kill cells bearing a high density of the antigen recognized. The primary purpose of this study was to determine if such killing could be obtained with anti-HER-2 antibodies conjugated to (111)In, using the chelator benzyl-diethylenetriaminepentaacetic acid, or (125)I. Target cells were the breast carcinoma SK-BR-3 and the ovarian carcinoma SK-OV-3.ip1. In preliminary experiments, antibody accumulation and catabolism during a 2- to 3-day incubation with antibody was investigated. The level of antibody uptake, in terms of molecules per cell, was high enough such that killing seemed feasible. With an (125)I label, but not an (111)In label, increasing the antibody concentration past a certain point caused a decrease in total antibody accumulation, which might be attributed to effects of antibody binding. To test for cytotoxicity, cells were incubated for 2 days with the labeled antibody, then assayed for colony-forming units with a limiting dilution assay. SK-BR-3 cells were strongly killed ( approximately 3 logs) by antibody 21.1, and 100% kill was obtained by combining two noncompeting antibodies to HER-2 (21.1 and 4D5). SK-OV-3.ip.1 cells were more resistant to killing, but use of the two-antibody mixture produced a surviving fraction of approximately 0.002. (111)In-labeled antibodies to other high-density antigens, epithelial glycoprotein-1 and epithelial glycoprotein-2, also killed these target cells. In contrast, unlabeled antibodies or a nonreactive-labeled antibody produced much less cytotoxicity. The same experiment with an (131)I label (a beta-particle emitter) resulted in much greater levels of nonspecific cytotoxicity and essentially no specific cytotoxicity. This approach may be effective for therapy of micrometastases.

177Lu-antibody conjugates for single-cell kill of B-lymphoma cells in vitro and for therapy of micrometastases in vivo.

Antibodies (Abs) conjugated to 177Lu, a relatively low-energy beta-particle emitter, were evaluated in vitro for their cytotoxic activity and in vivo for their therapeutic activity against disseminated B-cell lymphoma xenografts in SCID mice. 177Lu was compared with other beta-particle emitters ((131)I and 90Y), and also with emitters of low-energy electrons (LEEs, meaning Auger and conversion electrons of < 50 keV). The Abs used reacted with CD20, CD74 or HLA-DR, and the target cell was the Raji B lymphoma. Like the other beta-particle emitters, 177Lu was a potent and specific toxic agent in vitro, when conjugated to Abs recognizing high-density antigens. It appeared to be slightly less potent than (131)I per decay, but this difference was relatively small, and would not be a major factor in the selection of the optimal radionuclide for clinical use. The nonspecific toxicity from 177Lu was less than from 90Y, but 177Lu still produced greater nonspecific toxicity in vitro than LEE emitters. The maximum tolerated dose (MTD) of 177Lu-anti-CD74 in SCID mice was 1.81 MBq (49 microCi)/mouse. When this dose was administered on day 5 after tumor inoculation, significant protection was obtained, but considerably less than the protection obtained in previous experiments with LEE emitters (111)In and 67Ga. In conclusion, 177Lu has advantages over other available beta-particle emitters as a therapeutic agent, but its efficacy in the treatment of micrometastases seems to be less than that of LEE emitters, due to greater nonspecific toxicity. This conclusion, however, may not apply to therapy of macroscopic tumors.

Deferoxamine as a chelator for 67Ga in the preparation of antibody conjugates.

(67)Ga antibodies (Abs) have been shown to be effective agents for single-cell killing due to the Auger electrons emitted, but their specific activities have not been as high as desired. We therefore evaluated deferoxamine (DFO) as a chelator, as opposed to the cyclic chelator NOTA, which was used previously. Use of DFO for Ab conjugation to (67)Ga was reported previously by several laboratories. DFO was conjugated to Abs by two methods, one using Ablysine conjugation and another using mild reduction of Abs to generate thiols in the hinge region. Labeling with (67)Ga was efficient, and the specific activities obtained under nonoptimized conditions were twice as high as those achieved previously. However, analysis of these conjugates revealed two problems that appear to prevent their further development. First, the stability was inadequate for the 3-day half-life of the nuclide. Second, the labels were poorly retained within cells after Ab internalization and catabolism. Also, it was found that stability was significantly affected by the incubation buffer used: buffers lacking physiological concentrations of divalent cations Ca and Mg resulted in much lower stability than buffers including them. In conclusion, DFO does not seem to be a suitable chelator for (67)Ga conjugation for our purposes.

Intracellular accumulation of the anti-CD20 antibody 1F5 in B-lymphoma cells.

In previous experiments, 125I-labeled 1F5 (anti-CD20)was found to kill B-lymphoma cells efficiently and specifically.Unexpectedly, the number of antibody (Ab) molecules taken up per cell was much larger than the number of antigen sites on the cell surface. The present studies were designed to explain this apparent discrepancy. Incubation with fluorophore-conjugated 1F5, using the Raji cell line, demonstrated that the Ab accumulated in large amounts in a juxtanuclear spot. Double labeling showed that the same spot was labeled by transferrin, but the transferrin labeling was much faster (45 min versus 18 h). Experiments with brefeldin A demonstrated that the spot stained was distinct from the Golgi cisternae; thus, it appears to be the endocytic recycling compartment. A fluorescent Fab fragment of 1F5 produced much weaker, barely detectable staining of the juxtanuclear spot. Experiments with three other B-lymphoma cell lines demonstrated marked heterogeneity among them. With Ramos cells, 1F5 and transferrin localized to multiple smaller intracellular spots, rather than a single large spot. There were also major differences between different Abs to CD20, as tested on Raji cells. Rituximab showed some staining of the juxtanuclear spot, but not as homogeneously as the staining with 1F5. B1 and L27 were not tested as thoroughly but did not appear to stain the juxtanuclear spot. Such internalization may have a major impact on the therapy of this tumor type with conjugates of anti-CD20 Abs. However, internalization did not correlate with sensitivity to specific killing by 125I-labeled 1F5.

Antibody localization to B-cell lymphoma xenografts in immunodeficient mice: importance of using residualizing radiolabels.

Antibody (Ab) localization to Raji B-cell lymphoma xenografts in severe combined immunodeficient (SCID) mice was investigated using three Abs: anti-CD20; anti-CD147; and anti-MHC class II. These antigens are all high-density cell surface antigens, and the Abs are all considered to be slowly internalized and catabolized, with catabolism primarily due to the basal turnover rate of cell surface constituents. Unexpectedly, specific Ab uptake was demonstrated only when residualizing labels were used. The residualizing labels tested were 111In-benzyl-diethylenetriaminepentaacetic acid and [125I]iodo-dilactitol-tyramine, whereas the nonresidualizing label was a conventional iodine label. In contrast, in vitro experiments demonstrated very slow catabolism of the same Abs. These data strongly suggest that Ab catabolism is much more rapid in vivo than in vitro and has a strong impact on Ab accumulation in the tumor. If autologous human tumors are similar to these xenografts, then there should be a large advantage in the use of residualizing radiolabels for radioimmunotherapy.

In vitro toxicity of A-431 carcinoma cells with antibodies to epidermal growth factor receptor and epithelial glycoprotein-1 conjugated to radionuclides emitting low-energy electrons.

PURPOSE: The ability of antibodies (Abs) conjugated to radionuclides emitting low-energy electrons to specifically kill nonadherent lymphoma target cells in vitro was demonstrated previously. This study extends this work to adherent carcinoma cells. The fact that these cells are spread out on plastic can potentially make it more difficult to deliver radiation to the nucleus from decays in the cytoplasm or on the cell surface. EXPERIMENTAL DESIGN: The Abs tested were anti-epidermal growth factor receptor and anti-epithelial glycoprotein-1, conjugated to indium-111 or iodine-125, which emit low-energy Auger and conversion electrons. Conjugates of the beta-particle emitter, iodine-131, also were tested, for comparison. Abs were incubated with the cells for 2 days, and then the treated cells were assayed for colony-forming units. The radiation dose delivered to the nucleus was calculated from the cumulative decays per cell. RESULTS: With conjugates of (111)In, very potent killing was obtained with both of the Abs, with 100% kill (approximately 4-5 logs) even at subsaturating Ab concentrations. Lower levels of kill were obtained with (125)I or (131)I conjugates. Conjugates with (131)I, a beta-particle emitter, produced greater nonspecific toxicity. The greater potency of (111)In could be attributed to the higher specific activity that was obtained routinely with this radiolabel, up to 70 mCi/mg. Uptake of radioactivity peaked at approximately 200 cpm per cell. Dosimetry calculations, using subcellular S values, demonstrated that the toxicity observed was consistent with the amount of radiation delivered to the nucleus. CONCLUSIONS: These results are similar to previous results obtained with B lymphoma cells and indicate that this approach is applicable to a wide range of tumor types. Radionuclides emitting low-energy electrons are effective at killing target cells with relatively little nonspecific toxicity, if sufficient activity is delivered to the cell. Most Abs to high-density cell surface antigens would probably be effective.

Cure of SCID mice bearing human B-lymphoma xenografts by an anti-CD74 antibody-anthracycline drug conjugate.

PURPOSE: The purpose of this research was to test the therapeutic efficacy of an anthracycline-antibody conjugate for the treatment of human B-cell lymphoma in a preclinical animal model. EXPERIMENTAL DESIGN: Doxorubicin (dox) conjugates of the murine and humanized versions of the anti B-cell antibody LL1, targeting CD74, were prepared, along with a nonspecific control dox-antibody conjugate, targeting carcinoembryonic antigen. Antibody conjugates carried approximately 8-10 drug molecules attached site-specifically at thiols of reduced interchain disulfide bonds. Conjugates were tested, initially in vitro, and then for therapeutic efficacy in a systemic model, using a lethal i.v. dose of Raji cells in SCID mice. RESULTS: Dox-LL1 conjugates were shown to be stable and 3-fold more effective in vitro against the human B-cell Burkitt's lymphoma line, Raji, compared with the nonspecific control conjugate that did not target CD74 or B cells. When SCID mice were given an i.v. dose of 2.5 million Raji cells, they would die of disseminated disease within 15-25 days postinjection. A single dose of dox-LL1 conjugate, 117-350 micro g, given 5 days to 14 (advanced disease) days after injection of the Raji cells resulted in cure of most animals out to 180 days after injection of the cells, whereas animals in treatment control groups were not cured. The dose of dox-LL1 found useful in this work corresponds with a significantly lower drug dose than reported previously with other drug-antibody conjugates CONCLUSION: CD74 appears to be a uniquely useful target antigen for delivery of drugs, effecting cures of animals with single, low doses of conjugate.

A comparison of 4 radionuclides conjugated to antibodies for single-cell kill.

UNLABELLED: We previously found that (67)Ga was more potent and more specific in single-cell kill than other Auger electron emitters and beta-particle emitters, using an anti-CD74 antibody (Ab) (major histocompatibility complex [MHC] class II invariant chain). Because anti-CD74 Abs follow an unusual processing pathway, with rapid delivery in very large amounts to lysosomes, it was important to determine if similar results would be obtained with other, more typical Abs. METHODS: Target cells were Raji B-lymphoma cells, and the Abs tested were antimature MHC class II antigen (lacking the invariant chain) and anti-CD20, both of which react with high-density antigens. Labeling was with (125)I or (131)I, by conventional iodination; with (111)In using the chelator benzyl-diethylenetriaminepentaacetic acid; or with (67)Ga using the chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid. Abs were incubated with the cells for 2 d, uptake of radioactivity was assayed at various times, and toxicity was assayed primarily by a clonogenic assay. The fraction surviving was plotted versus cumulative disintegrations per cell to determine relative potency. RESULTS: The ranking of the radionuclides for potency was (131)I > (67)Ga > (125)I > (111)In. (67)Ga was approximately 2- to 3-fold more potent than (111)In. This was very similar to previous results with anti-CD74. Dosimetry calculations were generally consistent with the level of toxicity observed. In previous studies of nonspecific toxicity, the order of ranking was the same. CONCLUSION: The subcellular location of the bound Ab (whether on the cell surface or the cytoplasm) does not appear to be an important variable in the choice of radionuclide for single-cell kill. (67)Ga is a promising radionuclide for killing micrometastases, for high-density target antigens, but methods for achieving higher specific activity are required to fully exploit this approach. Each of the 4 radionuclides tested has certain advantages, and further studies are required to select the optimal radionuclide for a particular purpose.

Experimental therapy of disseminated B-Cell lymphoma xenografts with 213Bi-labeled anti-CD74.

A (213)Bi-labeled antibody to CD74 was evaluated as a therapeutic agent for B-cell lymphoma. The alpha-particle emission, with a half-life of 46 min, is appropriate for therapy of micrometastases. The labeled Ab retained full immunoreactivity, and was potent at single-cell kill of the Raji B-lymphoma cell line. Approximately 30 decays of cell-bound (213)Bi was required for a cell kill of 99%, and dosimetry calculations suggested that the cGy dose delivered was sufficient to produce the level of toxicity observed. A non-reactive control Ab, labeled similarly, also produced toxicity, due to decays occurring in the medium, but was approximately 3-fold less potent than the reactive Ab. In a SCID mouse xenograft micrometastatic model, Ab injection at day 2 or day 5 after tumor inoculation resulted in strong, specific suppression of tumor growth, with some apparent cures.

Use of galactosylated-streptavidin as a clearing agent with 111In-labeled, biotinylated antibodies to enhance tumor/non-tumor localization ratios.

Optimal tumor imaging using radiolabeled antibodies (Abs) depends on obtaining the highest possible tumor/non-tumor localization ratios. To increase this ratio, in a mouse xenograft model system, we induced rapid blood clearance of the Ab after extensive penetration of a solid tumor, at 24 hr after Ab injection. By using galactosylated streptavidin (gal-SA) as a clearing agent for biotinylated Abs, and by using an 111In-DTPA (diethylenetriaminepentaacetic acid) label, clearance was directed to hepatocytes (as opposed to Kupffer cells), and the radiolabel was excreted by the hepatocytes into bile, thereby reducing accumulation in the liver. In this study, we directly compared this approach with the use of 99mTc-F(ab)2 fragments, using the same Ab to carcinoembryonic antigen (CEA), with a colon carcinoma xenograft. The gal-SA clearance method produced substantially higher tumor/non-tumor localization ratios for all tissues except the liver, and even for the liver the disadvantage of the gal-SA clearance method was small. We also tested the gal-SA clearance method with a xenograft model of human B-cell lymphoma, using anti-CD22. High tumor/non-tumor ratios were obtained, as previously described with carcinomas of the lung and colon. Therefore, this approach appears to be a generally applicable strategy to obtain relatively high tumor/non-tumor ratios.

Induction of apoptosis by cross-linking antibodies bound to human B-lymphoma cells: expression of Annexin V binding sites on the antibody cap.

UNLABELLED: There are many reports that cross-linking antibodies (Abs) bound to the surface of B-lymphoma cells can induce apoptosis and/or cell death, especially with anti-CD20 Abs. This study was intended to extend our understanding of these effects. To determine if CD20 is a unique target in this respect, or whether Abs to other antigens would have similar effects, six Abs were tested, with and without cross-linking with a secondary Ab, on three target cell lines. We utilized assays that distinguish between apoptotic, dead, and viable cells. Two assays were used: Annexin V plus propidium iodide, and JC-1 plus SYTOX green (Molecular Probes, Eugene, OR). Most of the Abs tested induced a low level of apoptosis and cell death in Ramos cells, but not in the other two cell lines (Raji and RL). In general, the level of toxicity was correlated with the level of antigen expression, with Abs to high-density antigens having the strongest effects. However, since the majority of Ramos cells continued to multiply, it is questionable whether toxicity at this level can provide a significant clinical benefit. Unexpectedly, there was also a population of cells that stained weakly with Annexin V. These cells were distinct from classical apoptotic cells, and appeared to belong to the viable cell population. In these cells, Annexin V stained the region of the Ab cap, in contrast to the ringed staining of classical apoptotic cells. IN CONCLUSION: 1) Low-level induction of apoptosis was not unique for anti-CD20 Abs, but occurred similarly with other Abs, and 2) results of Annexin V staining experiments may need to be reevaluated. Further studies are required to explain why Annexin V binding sites are exposed in the region of an Ab cap.