Radiolabeled MDA2, an oxidation-specific, monoclonal antibody, identifies native atherosclerotic lesions in vivo.

BACKGROUND: Oxidatively modified low-density lipoprotein (LDL) is present in atherosclerotic but not normal arteries and plays a crucial role in the pathogenesis and adverse consequences of atherosclerotic lesions. We previously generated a series of monoclonal antibodies (MoAb) against oxidation-specific neo-epitopes formed during the oxidative modification of LDL. MDA2, a prototype MoAb, recognizes malondialdehyde-lysine epitopes (eg, in malondi-aldehyde-modified LDL) within atherosclerotic lesions. We describe the in vivo characteristics of MDA2 and initial noninvasive imaging studies of atherosclerosis in rabbits. METHODS: To assess the in vivo specificity of MDA2 for atherosclerotic lesions, iodine 125-MDA2 was intravenously injected into 7 LDL-receptor deficient Watanabe heritable hyperlipidemic (WHHL) and 2 normal New Zealand white (NZW) rabbits, and the aortic plaque uptake was evaluated 24 hours later. 125I-Halb, an isotype-matched irrelevant MoAb that binds to human albumin, was injected into 5 WHHL and 2 NZW rabbits as a control. Aortic autoradiography was performed, and the mean uptake of MoAbs was measured as the percent injected dose per gram aortic tissue. Gamma camera imaging was then carried out in 7 WHHL rabbits and 2 NZW rabbits with 99mTc-MDA2. Imaging was carried out at 10 minutes and at 12 or 24 hours. Malondialdehyde-LDL was then injected to clear the blood pool signal, and final images were obtained 2 hours later. RESULTS: Mean uptake of 125I-MDA2 in the entire aorta was 17.4-fold higher in WHHL than in NZW aortas (P < .001), and 2.8-fold higher than 125I-Halb in WHHL aortas. 125I-MDA2 also had higher specificity for lesioned areas than 125I-Halb (plaque/normal ratio 6.3 vs 2.9, P < .001). Autoradiograph of aortas of 125I-MDA2-injected WHHL rabbits revealed uptake in lipid-stained lesions with absence of signal in adjacent normal arterial tissue. Immunostaining of WHHL lesions, which accumulated MDA2 as noted on autoradiography, revealed that uptake was highest in areas with abundant foam cells and in lipid-rich necrotic core areas. Autoradiograph of aortas from NZW rabbits injected with 125I-MDA2 did not yield any visible signal. Planar gamma camera in vivo scintigraphy revealed a visible signal in 4/7 WHHL rabbits, which was confirmed by aortic Sudan staining. CONCLUSION: Radiolabeled MDA2 shows excellent in vivo uptake and specificity for atherosclerotic lesions containing abundant oxidation-specific epitopes. The in vivo imaging studies suggest that noninvasive imaging of oxidation-rich atherosclerotic lesions with radiolabeled MDA2 may be feasible in human beings with optimization of the imaging methods.

Radioimmunotherapy of relapsed B-cell lymphoma with yttrium 90 anti-idiotype monoclonal antibodies.

Tumor-specific anti-idiotype (anti-Id) monoclonal antibodies (MoAbs) to B-cell lymphomas have been administered to patients, resulting in significant clinical responses. However, clinical responses have been limited by the emergence of Id-negative lymphoma. To overcome the problem of tumor heterogeneity, we conducted a pilot evaluation of the safety and effectiveness of yttrium 90 (90Y)-labeled anti-Id and shared Id (sId) MoAbs in non-Hodgkin's B-cell lymphoma. Nine patients with relapsed B-cell lymphoma in whom tumor was successfully targeted with 111In-labeled anti-Id MoAb were treated with 90Y-labeled anti-Id MoAb. A total of 19 courses (one to four per patient) were administered using 1,000 to 2,320 mg unlabeled clearing MoAb and 10 to 54 mCi 90Y MoAb per patient. Two of nine patients had a complete response, one a partial response, three stable disease, and three disease progression. Time to progression varied from 1 to 12 months. Toxicities were predominately hematologic, and only one patient developed infection and required transfusion. At progression, three of five assessable patients had Id-positive lymphoma and two had Id-negative lymphoma. Human antimouse antibodies (HAMA) did not develop in the patients after treatment. 90Y anti-Id MoAbs demonstrated excellent in vivo stability, produced significantly tumor regression in three of nine patients, exhibited acceptable toxicities, and elicited no HAMA formation. Further investigation of repetitive, low-dose 90Y anti-Id and MoAb therapy is warranted; however, the advantages of a pan B MoAb may prove the latter to be the agent of choice for the radio immunotherapy of B-cell lymphoma.

Pharmacokinetics of an indium-labeled IgG monoclonal antibody over a prolonged period.

This study was directed toward determining the pharmacokinetic fate of an IgG2a monoclonal antibody (MoAb). The 96.5 anti-melanoma MoAb was labeled with indium-111 and indium-114m and administered to BALB/c mice. The mice receiving 111In MoAb were sacrificed at 4 and 72 h, while those receiving 114mIn 96.5 MoAb (50-day physical half-life) were sacrificed at 4 h and 3, 15, and 30 days. Multiple tissues were counted against a standard of the injectate and the data expressed as percent injected dose per organ and percent total dose excreted in the urine and feces. The 111In- and 114mIn-labeled MoAbs had nearly identical distribution through 72 h. Over the 30-day period 25% of the 114mIn label was excreted in the urine and 50% eliminated in the feces. All of the tissues studied showed a decrease in 114mIn in the 30-day period. We conclude that the metabolic products of indium-labeled MoAbs, the indium itself, or a combination of both are eliminated from the tissues over a period of several weeks and do not accumulate to a significant extent in any single site.

Pharmacokinetics of 111In-labelled monoclonal antibody ZCE-025 and fragments in tumour-bearing mice.

Radiolabelled anti-tumour antibodies, their fragments and derivatives hold promise for imaging and therapeutics in oncology. A better understanding of the pharmacokinetics of these entities is therefore important for clinical applications and management. In the present study, the in vivo behaviour of 111Indium-labelled monoclonal anti-CEA antibody ZCE-025 and its F(ab')2 and Fab' fragments and a Fab' derivative are compared in the nude mouse-human tumour model. The object of the derivative was to improve the tumour uptake of the fragment yet reduce its high renal uptake while continuing to achieve desirable kinetics in the normal tissues. Uptake of the derivative in the tumour was comparable to that of the intact antibody and exceeded that of the underivatized fragments. Moreover, uptake in non-target tissues was lower with the derivative than with the intact entity. The renal uptake of the derivative was dramatically lower than for the fragments. The modelling data strongly suggest that the derivatives will be advantageous for clinical use compared with the underivatized whole antibodies or their fragments.

Human anti-mouse antibody response in cancer patients following single low-dose injections of radiolabeled murine monoclonal antibodies.

We examined the human anti-mouse antibody (HAMA) response in 61 cancer patients following a single, diagnostic injection of any one of ten 111In conjugated murine monoclonal antibodies. Between 1 and 22 mg of antibody containing 1-5 mCi 111In was administered. The populations studied included 30 patients with colorectal carcinoma (four different antibodies), 22 with malignant melanoma (four antibodies), and nine with prostate cancer (two antibodies). Forty-one percent of the patients developed HAMA within 14 days. Three patients (5%) developed an IgM response, five patients (8%) developed an IgG response, and 17 patients (28%) developed both IgM and IgG. Only 27% of the patients with colon cancer developed HAMA, compared to 55% of the melanoma patients and 56% of the prostate cancer patients. There were no correlations among injected dose, various clinical parameters, and HAMA response. There were variations in the HAMA response to different monoclonal antibodies, but population samples were too small to infer significance. Most of the HAMA responses had a significant proportion of idiotypic or isotypic specificity. Only 1/6 patients who were HAMA negative after the first infusion developed HAMA following subsequent infusions of the same monoclonal antibody. Our data demonstrate that a significant percent of cancer patients develop HAMA following a single, low-dose injection of a radiolabeled monoclonal antibody for diagnostic purposes. This may have important implications for the future therapeutic use of monoclonal antibodies in such patients.

Chromatofocusing studies involving a monoclonal Fab'.

Isoelectric focusing (IEF) of the Fab' derivative of murine monoclonal antibody ZCE-025 is known to detect at least six bands having isoelectric points (pI) ranging from 5.4 to 7.8. Chromatofocusing was employed to separate these bands. Electrophoresis of the starting materials under nonreducing conditions indicated all of the materials to migrate as Fab'. The electrophoresis of urine samples obtained from Balb/c and nude mice 8 hr after the i.v. injection of the various 125I bands revealed the low pI bands to migrate approximately as a 125I-Fab'. The higher pI band activity was located in lower molecular weight regions. Serum samples taken at 8 hr postinjection from the above mice revealed a series of what appeared to be high molecular weight complexes and some low molecular weight species. Biodistribution studies in comparison Balb/c mice and nude mice revealed that the low pI 125I-Fab' bands gave an organ and tumor uptake at 8 hr very similar to Fab', while the high pI 125I-Fab' bands were rapidly excreted into the urine and feces and did not concentrate in the tumor. The data suggest that the population of molecules making up the Fab' of this antibody is heterogeneous and variably stable. Theoretically, some of the entities observed could be counter productive to successful radioimmunoimaging. It is also possible that some of the labeled molecules are associating in vivo with endogenous proteins that might, in some Mabs, affect the biodistribution of the radiopharmaceutical.

Detection of occult tumor using indium 111-labeled anticarcinoembryonic antigen antibodies.

Even with the advancement of radiologic techniques, metastatic cancers can still be difficult to detect. In this study, 48 patients suspected of having occult metastases were studied by radioimmunodetection following the administration of 92.5 to 181.3 MBq of indium 111-labeled monoclonal anticarcinoembryonic antigen antibody. All but seven patients were thought to have metastatic colorectal carcinoma. In the majority of cases, physical examinations and computed tomographic scans had failed to detect a lesion. At least one lesion that was later proved to exist was detected in 34 of the 50 studies performed on these patients. Seven of eight patients with normal radioimmunodetection scans remain free of disease. One hundred one sites were detected overall; 60 were considered true-positive sites and 27 false-positive sites. Fourteen sites remained in question. Nineteen false-negative sites occurred. Radioimmunoimaging appears valuable for the detection of occult cancer where standard, noninterventional techniques have failed to detect the suspected disease.

Alterations in an indium-111 Fab' under conditions of utilization.

This study was conducted to investigate alterations that occur in an indium/111 Fab' of a monoclonal antibody following its in vivo administration. Patients were infused with 111In-Fab' of the monoclonal antibody ZCE-025. Serum and urine specimens were collected from these patients. Starting materials, serum, urine and controls samples were studied by electrophoresis. Animal distribution studies were performed in normal Balb/c mice and, in some cases, nude mice bearing a carcinoembryonic antigen (CEA)/producing human colon tumour since the antibody targets CEA. The studies indicated that the molecule circulated almost totally intact for at least 4 h and to a considerable extent for 24 h, with some evidence for in vivo fragmentation by 24 h. Evidence was also obtained suggesting the formation of a high molecular weight species in some patients. Shortly after infusion, some of the 111In in the urine appeared as the intact Fab', but within hours the majority migrated electro-phoretically as low molecular weight species. We conclude that while the majority of the 111In-Fab' of this particular antibody remains intact and immunoreactive following its administration, the molecule is structurally changed to some degree shortly after its infusion into humans. Since each monoclonal antibody is unique, the degree and rapidity of degradation of its Fab' in vivo could vary markedly from the above and possibly adversely effect its utility as a radiopharmaceutical.

"CORT-EX:" a program for quantitative analysis of brain SPECT data.

A program was developed to extract from brain SPECT data global as well as regional concentrations of a radiopharmaceutical while allowing for improved subjective evaluation of its distribution. This program was used to process the data obtained from 17 normal subjects, 20 min, 2 hr, and 4 hr after the injection of iodine-labeled iodoamphetamines. The mean absolute cortical uptake at these three time periods was 0.921 (+/- 0.185), 0.803 (+/- 0.107), and 0.748 (+/- 0.103) in arbitrary units (+/- s.d.), respectively. The regional distribution of the tracer became more uniform with time due to an uneven washout rate. The cerebellum was noted to have a very high variability in its uptake and a high washout rate, making it unsuitable as an internal standard for relative quantification. Finally, a repeat study was performed on 10 subjects. No significant difference could be demonstrated in the mean uptake of the group at 2 and 4 hr, however the difference observed in the 20 min uptake values was significant at the p = 0.05 level.

Effect of acetylation on monoclonal antibody ZCE-025 Fab': distribution in normal and tumor-bearing mice.

Studies were performed to determine in vitro and in vivo effects of acetylation on Fab' fragments of ZCE-025, a monoclonal anti-CEA antibody. Isoelectric focusing revealed a drop in isoelectric point of 1.7 pI units following acetylation. Biodistribution studies of acetylated and nonacetylated [111In]Fab' were performed in normal BALB/c mice and in nude mice bearing the T-380 CEA-producing human colon tumor. The acetylated fragments remained in the vascular compartment longer and had significantly diminished renal uptake of 111In compared to controls. While acetylation itself effected a 50% drop in immunoreactivity, tumor uptake of the acetylated and nonacetylated 111In-labeled Fab' fragments was comparable, with the exception of one data point, through 72 h.

Radioimmunotherapy of human B-cell lymphoma with 90Y-conjugated antiidiotype monoclonal antibody.

We report the first case of 90Y-conjugated monoclonal antibody (MoAb) administration for human radioimmunotherapy. Ten mCi 90Y-labeled antiidiotype (anti-Id) MoAb were administered to a patient with B-cell lymphoma whose tumor successfully imaged with 111In-labeled anti-Id MoAb. No significant toxicities were observed. More than 2 g of unlabeled anti-Id MoAb were administered while clearing the circulating IgM idiotype prior to administration of the 90Y-MoAb. Transient partial regression of disease was observed. Serial fine needle aspirations of a malignant lymph node documented in vivo anti-Id penetration into a site that did not image by radioimmunoscintigraphy. The radiosensitivity of B-cell lymphoma, the tumor specificity of anti-Id, the antitumor activity of anti-Id alone, and the safe administration of 10 mCi 90Y-labeled anti-Id MoAb in this report suggest further investigation of this radioimmunoconjugate for therapy of B-cell lymphoma is warranted.

Distribution of radiolabeled human and mouse monoclonal IgM antibodies in murine models.

The distribution and kinetics of six human and one murine monoclonal IgM antibodies (MoAb) were studied in BALB/c mice. Labeling was with 111In, 75Se, and 125I. The monomers and pentamers of certain MoAbs were studied. Human distribution studies were also performed. The serum containing [111In]MoAb was obtained from one of the patients 24 hr after administration and injected into mice which were then killed and assayed for 111In distribution. In general, the [75Se] and [111In]MoAbs had distribution and kinetic patterns that were similar while the 125I-labeled MoAbs dehalogenated after 4 hr. Monomers and pentamers had highly similar distributions suggesting that the distribution of IgMs may be based on factors other than molecular size. The murine IgM showed a somewhat different distribution in mice than did human IgMs. Serum from the patient containing [111In]MoAb had a distribution in mice similar to that of the patient with high liver and gastrointestinal uptake. The human imaging indicates that it is possible to target tumor with human IgM MoAbs, but significant problems remain in regard to their clinical use.

Scintigraphy with In-111-labeled monoclonal antitumor antibodies: kinetics, biodistribution, and tumor detection.

The purpose of this study was to evaluate and compare the kinetics, biodistribution, and tumor-depicting properties of three intact indium-111-labeled murine monoclonal antibodies (MoAb) and to determine if use of In-111-labeled F(ab')2 fragments of one of them had advantages over its intact counterpart for immunoscintigraphy. Ten patients with prostate cancer were studied with an anti-prostatic acid phosphatase MoAb (PAY-276), with a resultant tumor detection rate of 15%. Twenty-eight patients with melanoma were studied with ZME-018, a MoAb that targets the KD-240 melanoma antigen. Forty-three percent of the known lesions were detected. Forty patients with carcinoembryonic antigen (CEA)-producing tumors were studied, 24 with intact ZCE-025, and anti-CEA MoAb, and 16 with its F(ab')2 fragment. With use of intact ZCE-025, 34% of known lesions were detected versus 83% with its F(ab')2 fragment. The distribution of each MoAb appears unique unto itself with regard to kinetics, normal tissue distribution, and response to MoAb mass.

Toxicities associated with monoclonal antibody infusions in cancer patients.

The toxicity during and following 291 infusions of 19 murine and three human monoclonal antibodies (MoAB) in 177 cancer patients with 10 different malignancies was assessed. Doses ranged from 0.5 to 500 mg administered over 0.25 to 24 hours. Various reactions in varying degrees were observed in 45 (28%) patients during their first MoAb infusion. Nine additional patients experienced toxicity following a subsequent antibody infusion. Antibodies that reacted with circulating cells were associated with toxicity in 20 of 28 (71%) of the first infusions, compared to 24 of 127 (19%) for patients receiving antibodies that did not react with circulating cells. Fevers, rigors, chills, and diaphoresis were observed in 10% to 12% of the patients and were associated with binding to circulating cells. Presumed hypersensitivity reactions, including urticaria, pruritus, bronchospasm, and anaphylaxis occurred in 20 patients (11%). There were five episodes of bronchospasm and a single episode of anaphylaxis. Liver transaminases were elevated in 14%. There was no correlation between dose or infusion rate and toxicity. Murine monoclonal antibodies that are not conjugated to cytotoxic agents can be given with an acceptable frequency of side effects and serious allergic reactions. There is a small risk of anaphylaxis, and one should avoid rapid infusion of high antibody doses in the presence of circulating target cells and/or circulating free antigen.

Clinical efficacy of intravenous morphine administration in hepatobiliary imaging for acute cholecystitis.

The most urgent diagnosis addressed by cholescintigraphy is acute cholecystitis. By administering low-dose intravenous morphine sulfate to patients undergoing cholescintigraphy (who demonstrate visualization of both the common bile duct and intestine and nonvisualization of the gallbladder), the time required to complete the study has been reduced to a maximum of 90 minutes. One hundred twenty-eight patients underwent cholescintigraphy for clinically suspected acute cholecystitis. Forty patients received intravenous morphine sulfate during the procedure. In patients who received morphine sulfate during the examination, the sensitivity of cholescintigraphy for the diagnosis of acute cholecystitis was 100%; the specificity was 85%.

Problems associated with radioimmunodetection and possibilities for future solutions.

It is the opinion of the authors that the molecule of the future for radioimmunodetection (and hopefully radioimmunotherapy and delivery of drugs) will have the following characteristics: It will be an altered fragment, rather than an intact molecule or fragment. It will be small, perhaps 60,000 molecular weight, yet remain in the vascular compartment for a comparatively long period of time, then be eliminated via the kidney. It will be of human origin or a murine molecule altered to mask its immunogenic properties. It will be 111In- or 99mTc-labeled or bifunctionally chelated to another metal ion. It will target a cell surface antigen, but one that does not circulate. It will be used in combination with derivatives of other MoAbs that target other antigens on the cell. It would be a hapten-type device that follows the administration of a "bifunctional MoAb." Whatever the final molecule, it will be administered to a patient who has been "prepped" with an antigen-enhancing substance or one that "unmasks" a gene and allows a repressed marker to be expressed by the tumor cell to which the MoAb was developed. It may be a MoAb that attaches to a white cell, which then chemotactically seeks the tumor that has been previously induced to produce a substance that the white cell recognizes.

In vitro and in vivo tumor models for studies of distribution of radiolabelled monoclonal antibodies and fragments.

Colon carcinoma multicellular spheroids were incubated in vitro with radiolabelled MAbs. The more rapid penetration of fragments as compared to intact MAbs was clearly demonstrated. For the study of antibody localization in tumors in vivo, the model of nude mice with ligated kidneys was used. Although very artificial, this model allowed to demonstrate that, without urinary excretion, Fab fragments accumulated more rapidly into the tumor than intact MAbs and disappeared faster from the blood. This difference was less striking for F(ab')2 fragments. In the liver a decreased accumulation of both types of fragments as compared to intact MAbs was observed. Concerning radioimmunotherapy we think that Fab fragments are not useful because of their too short half-life in the circulation and in tumor and because they will probably be too toxic for the kidneys. Intact MAbs and F(ab')2 fragments have each their advantages. Intact MAbs show highest tumor accumulation in mice without ligated kidney, however, they remain mostly on the periphery of tumor nodules, as shown by autoradiography. F(ab')2 fragments have been found to penetrate deeper into the tumor and to accumulate less in the liver. It might be therefore an advantage to combine intact MAbs with F(ab')2 fragments, so that in the tumor two different regions could be attacked whereas in normal tissues toxicity could be distributed to different organs such as to the liver with intact MAbs and to the kidney with F(ab')2 fragments.