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PubMed2003

Targeting cells that overexpress the epidermal growth factor receptor with polyethylene glycolated BPD verteporfin photosensitizer immunoconjugates.

Photoimmunotherapy was introduced two decades ago but has been studied infrequently in vivo and is virtually untested clinically. Progress has been limited because high-quality, well-characterized photosensitizer immunoconjugates (PICs) have been difficult to make. Here, we describe the development of an innovative conjugation method for producing water-soluble PICs that are free of insoluble aggregates and free of unacceptable amounts of noncovalently associated photosensitizer impurities. The method exploits two procedures previously untried in this research area. First, a small number of antibody lysines (<3 per antibody) are polyethylene glycolated (PEGylated) using a 10 kDa branched polyethylene glycol (PEG), which dramatically enhances PIC solubility and reduces PIC aggregation. Second, a 50% dimethyl sulfoxide-50% aqueous two-solvent system is used to prevent photosensitizer aggregation and noncovalent interactions. These measures allow efficient covalent linkage of the photosensitizer BPD Verteporfin (BPD) to antibody lysines, thorough purification of the resulting PICs (verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), maintenance of PIC antigen-binding activity (verified by cellular binding-uptake assays) and reduction of nonspecific cellular uptake (e.g. macrophage capture) of the PICs. Loading levels could be varied controllably in the range < or = 11 BPD/antibody. PICs of the C225 anti-epidermal growth factor receptor (EGFR) chimeric monoclonal antibody killed EGFR-overexpressing A-431 cells photodynamically but did not significantly affect EGFR-negative NR6 cells. Although fluorescence measurements demonstrated that the PICs were quenched by as much as an order of magnitude compared with free BPD, an impressive 90% reduction in A-431 cell viability was achieved using 20 J/cm2 of 690 nm light after a 40 h incubation with the C225 PICs. The results suggest that PEGylated BPD-C225 PICs merit further investigation in animal models of EGFR-overexpressing cancers.

Animals↗
PubMed2001

A mouse model for calculating the absorbed beta-particle dose from (131)I- and (90)Y-labeled immunoconjugates, including a method for dealing with heterogeneity in kidney and tumor.

Flynn, A. A., Green, A. J., Pedley, R. B., Boxer, G. M., Boden, R. and Begent, R. H. J. A Mouse Model for Calculating the Absorbed Beta-Particle Dose from (131)I- and (90)Y-Labeled Immunoconjugates, Including a Method for Dealing with Heterogeneity in Kidney and Tumor. Radiat. Res. 156, 28-35 (2001). Conventional internal radiation dosimetry methods assume that the beta-particle energy is absorbed uniformly and completely in the source organ and that the radioactivity is distributed uniformly in the source. However, in mice, a considerable proportion of the beta-particle energy can escape the source organ, resulting in large cross-organ doses. Furthermore, the distribution of radioactivity is generally heterogeneous in kidney and tumor. Therefore, a model was developed to account for cross-organ doses and for the effects of heterogeneity in kidney and tumor in mice for two of the most important radionuclides used in therapy, (131)I and (90)Y. Most mouse organs were modeled as single-compartment ellipsoids or cylinders, while heterogeneity in kidney and in tumor was addressed by using two compartments to represent the cortex and the medulla and viable and necrotic cells, respectively. The dimensions of these models were taken from previous studies, with the exception of kidney and tumor, which were defined using radioluminography and mosaics of high-power microscopy images. The absorbed fractions in each compartment were calculated using beta-particle point dose kernels. The self-organ dose was significantly higher for (131)I compared to (90)Y in all compartments, but a considerable amount of beta-particle energy was shown to escape the source organ for both radionuclides, with as much as 85% and 36% escaping the marrow for (90)Y and (131)I, respectively. The cortex was found to occupy a greater proportion of the total kidney volume than the medulla, and consequently the self-dose was higher in the cortex. In addition, the thickness of the viable shell in the tumor increased with tumor size, as did the self-dose fractions in both necrotic and viable areas. This dosimetry model improves dose estimates in mice and gives a conceptual basis for considering dosimetry in humans.

Animals↗
PubMed2001

Immunoconjugates of soybean Bowman-Birk protease inhibitor as targeted antitumor polymeric agents.

To enhance the antitumor potential of soybean Bowman-Birk inhibitor (BBI), the conjugate of BBI with an antibody via a macromolecular carrier was prepared. Clinical dextran (D) was used as a biocompatible biodegradable carrier for co-immobilization of BBI and antibody. A model immunoglobulin isolated from sheep serum (sIgG), raised against human IgM was utilized to develop the procedure of immunoconjugate synthesis. The molar ratio of the ingredients in the conjugate was the following BBI:D:sIgG=9:1:1. Comparison of the dose response curves for the native sIgG and the BBI-D-sIgG conjugate indicated that sIgG completely retained its specific activity (>90%) after modification with dextran. The determination of the Ki values for chymotrypsin interaction with the native BBI and the BBI-D-sIgG conjugate indicated high anti-chymotrypsin activity. In the next step, the monoclonal antibody (ICO 25 MAb) against the mucin-like human epithelial membrane antigen was used for conjugation as it is the most universal vector for targeting different agents to human tumors of epithelial origin. The influence of conjugation on the specificity of the Mab reaction with its antigen was studied. The conjugated MAb reacted with tumor cells of different epithelial genesis (breast, lung, gastric, ovarian and uterus tumors), but did not react with tumor cells of non-epithelial origin. It was shown that BBI-D-ICO 25 MAb conjugate has almost the same immunohistochemical activity as non-conjugated MAb. These results demonstrated the feasibility of exploiting the activities of covalently bound BBI and ICO 25 MAb for anticarcinogenic agent targeting.

Animals↗
PubMedDate not supplied

Targeted therapy of cancer: new prospects for antibodies and immunoconjugates.

Immunotherapy of cancer has been explored for over a century, but it is only in the last decade that various antibody-based products have been introduced into the management of patients with diverse cancers. At present, this is one of the most active areas of clinical research, with eight therapeutic products already approved in oncology. Antibodies against tumor-associated markers have been a part of medical practice in immunohistology and in vitro immunoassays for several decades, have even been used as radioconjugates in diagnostic imaging, and are now becoming increasingly recognized as important biological agents for the detection and treatment of cancer. Molecular engineering has improved the prospects for such antibody-based therapeutics, resulting in different constructs and humanized/human antibodies that can be administered frequently. Consequently, a renewed interest in the development of antibodies conjugated with radionuclides, drugs, and toxins has emerged. We review how antibodies and immunoconjugates have influenced cancer detection and therapy, and also describe promising new developments and challenges for broader applications.

Angiogenesis Inhibitors↗
PubMed2003

Meeting report on the Ninth Conference on Cancer Therapy with Antibodies and Immunoconjugates.

This conference, held biennially for the past 12 years, provides a forum where investigators from throughout the world can gather and discuss how monoclonal antibodies can be used to improve the treatment of cancer. As in the past, this meeting focused primarily on the use of radiolabeled antibodies in cancer treatment, but this year there were many additional contributions on the use of unconjugated "naked" antibodies for the treatment of cancer, reflecting a growing understanding that antibodies not only can be used to direct isotopes and drugs to tumors but can also be effective agents in themselves. Preclinical studies using immunoconjugates prepared with toxins, drugs, or other agents were also reported to be highly effective therapeutic agents. Some of these are now showing efficacy in clinical trials. In addition, presentations focused on a variety of approaches, including pretargeting, regional delivery, and combinations with other standard treatment regimens designed to optimize antibody-targeted treatment strategies. Although the most efficacious treatments were reported in a variety of hematological malignancies, there were a number of presentations, primarily in early preclinical development, that provided evidence for potential future improvements in the treatment of solid tumors.

Animals↗
PubMed1995

Immunologic assessment during treatment of rheumatoid arthritis with anti-CD5 immunoconjugate.

OBJECTIVE: To determine if sustained immunologic effects occurred after treatment of patients with rheumatoid arthritis (RA) with an immunoconjugate of murine anti-CD5 monoclonal antibody with ricin A chain (anti-CD5). METHODS: We measured lymphocyte populations, mitogen induced peripheral blood mononuclear cell (PBMC) stimulation, cytokine levels, immunoglobulin levels, in vivo immune function, and clinical outcomes in 9 patients with RA treated with anti-CD5. RESULTS: The treatment of patients with RA with anti-CD5 was associated with marked acute depletion of peripheral blood lymphocytes (p < 0.01) during and immediately after treatment. A sustained decrease in the number of CD3, CD4, CD5, and CD8 bearing lymphocytes persisted for 2 months after treatment (p < 0.05). After 3 months a mild decrease in the number of these lymphocyte populations persisted, but when compared to baseline values, the differences were not found to be statistically significant. Phytohemagglutinin induced PBMC proliferation was decreased at the 3-month followup (p < 0.05). Evaluations of mitogen induced cytokine and immunoglobulin production, immunoglobulin level, autoantibody, and in vivo antibody response to tetanus toxoid did not show any consistent change from baseline. CONCLUSION: Anti-CD5 treatment of RA appears to be associated with a decrease in the population of cells bearing CD5, but does not appear to induce any persistent immunologic abnormalities.

Adolescent↗
PubMedDate not supplied

Cytotoxic effect of immunoconjugate composed of glucose-oxidase coupled to an anti-ganglioside (GD2) antibody on spheroids.

As a new treatment protocol for neuroblastoma, the chimeric (human/mouse) antiganglioside GD2 antibody chl4.18 is being clinically tested. To improve the therapeutic effect of the antibody alone, we are currently investigating the cytotoxicity of glucose-oxidase coupled to the antibody chl4.18 on spheroids of the neuroblastoma cell line SK-N-LO. The cytotoxic effect of glucose-oxidase is achieved by the production of hydrogenperoxide (H2O2) and probably by the following reaction of H2O2 with iron to form hydrogen radicals (OH.). The cytotoxicity of glucose-oxidase was measured by two viability tests (MTT and WST 1). After a 4 hour treatment of the spheroids with the immunoconjugate, a reduction of viability to 50% (MTT-test) and 25% (WST 1-test), respectively, was obtained. The difference between the results of these two tests, might be explained by the different measurement protocols.

Antibodies, Monoclonal↗
PubMed1997

Overcoming the nephrotoxicity of radiometal-labeled immunoconjugates: improved cancer therapy administered to a nude mouse model in relation to the internal radiation dosimetry.

BACKGROUND: Elevated renal uptake and extended retention of radiolabeled antibody fragments and peptides is a problem in the therapeutic application of such agents. However, cationic amino acids have been shown to reduce renal accretion. The aims of the current study were to evaluate whether this methodology would benefit therapy with yttrium 90 (90Y)-labeled antibody fragments (Fab, F(ab)2), to establish the relationship between radiation dosimetry and observed biologic effects, and to compare the antitumor efficacy of antibody fragments with that of whole immunoglobulin (Ig)G. METHODS: The maximum tolerated dose (MTD) and the dose-limiting organ toxicity of 90Y-labeled anti-carcinoembryonic antigen (CEA) MN-14 monoclonal antibodies (Fab, F(ab)2, and IgG) were determined in nude mice bearing GW-39 human colon carcinoma xenografts. The mice were treated with or without kidney protection by administration of D-lysine, with or without bone marrow transplantation (BMT), or with combinations of each. Toxicity and tumor growth were monitored at weekly intervals after radioimmunotherapy. Dosimetry was calculated from biodistribution studies using 88Y-labeled antibody. Three different dosimetric models were examined: 1) taking solely self-to-self doses into account, using S factors for 90Y in spheroids from 0.1 to 1 g; 2) correcting for cross-organ radiation; and 3) using actual mouse anatomy as represented by nuclear magnetic resonance imaging with a three-dimensional internal dosimetry package (3D-ID). RESULTS: The kidney was the first dose-limiting organ with the use of Fab fragments. Acute radiation nephritis occurred at injected activities > or = 325 microCi, and chronic nephrosis at doses > or = 250 microCi. Activities of 200 microCi were tolerated by 100% of the animals (i.e., the MTD). Application of lysine decreased the renal dose by approximately fivefold, facilitating a 25% increase in the MTD (to 250 microCi), because myelotoxicity became dose-limiting despite red marrow doses of less than 5 gray (Gy). By using BMT and lysine, the MTD could be doubled from 200 to 400 microCi, where no biochemical or histologic evidence of renal damage was observed (kidney dose, < or = 40 Gy). With injected activities of > or = 325 microCi without kidney protection, and with a hepatic self-to-self dose of only 4 Gy, rising liver enzymes were observed, which could be explained only by cross-organ radiation from radioactivity in the kidneys (in the immediate neighborhood of the right kidney up to > or = 150 Gy). The MTD of F(ab)2 fragments could be elevated only by a combination of BMT and lysine. With IgG, the bone marrow alone was dose-limiting. Tumor dosimetry correlated well with antitumor effects; Fab was more effective than F(ab)2, which was consistent with its more favorable dosimetry, and it may also be more effective than IgG due to its higher dose rate and more homogenous distribution. Dosimetry Model 1 was insufficient for predicting biologic effects. Model 2 seemed to be more accurate, accounting for interorgan crossfire. However, Model 3 showed an additional substantial contribution to the red bone marrow dose due to crossfire from the abdominal organs. CONCLUSIONS: These data show that radiation nephrotoxicity is an important effect of cancer therapy with radiometal-conjugated antibody fragments or peptides. However, this effect can be overcome successfully with the application of cationic amino acids, which substantially increase the anti-tumor efficacy of radiometal-labeled immunoconjugates. For understanding the biologic effects (e.g., liver toxicity) of 90Y in a mouse model, accounting for cross-organ radiation is essential. Further studies with radiometal-conjugated monoclonal antibody fragments and peptides are necessary to determine the MTD, dose-limiting organs, antitumor effectiveness, and nephroprotective effects of cationic amino acids in humans.

Animals↗
PubMed1993

Immunoscintigraphy in patients with colorectal, ovarian, and prostate cancer. Results with site-specific immunoconjugates.

Application of monoclonal antibody (MoAb) technology to cancer management is discussed by reviewing the development and clinical evaluation of two MoAb-based immunoscintigraphic agents (111In-satumomab pendetide [OncoScint CR/OV-In] and 111In-CYT-356; Cytogen Corporation, Princeton, NJ). Both agents were prepared using a site-specific MoAb modification method that preserves the immunoreactivity of the radiolabeled immunoconjugate. 111In-satumomab pendetide is an 111In-labeled conjugate of the murine MoAb B72.3, which is directed to TAG-72, an antigen expressed by the majority of adenocarcinomas. By providing information that complements the results of standard radiographic diagnostic modalities, this imaging agent can aid in the treatment of patients with colorectal or ovarian cancer. Immunoscintigraphy with 111In-satumomab pendetide has been shown to assist in medical-surgical management by detecting occult extrahepatic lesions, clarifying equivocal results of other diagnostic imaging tests, and evaluating the extent and resectability of known tumor lesions. 111In-CYT-356 is an 111In-labeled conjugate of the murine MoAb 7E11-C5.3, which is reactive with prostatic carcinoma, benign prostatic hypertrophy, and, to a lesser extent, normal prostatic tissue. Results of preliminary clinical investigations suggest that 111In-CYT-356 immunoscintigraphy can be useful for the presurgical staging of prostatic carcinoma and for the detection of occult distant disease in patients with negative or equivocal results on standard imaging tests. Results with these site-specifically radiolabeled immunconjugates demonstrate the clinical utility of MoAb-based imaging agents in the treatment of patients with solid tumors.

Antibodies, Monoclonal↗
PubMed1994

Biodistribution and preclinical radioimmunotherapy studies using radiolanthanide-labeled immunoconjugates.

Lutetium-177 (177Lu), samarium-153 (153Sm), and yttrium-90 (90Y) are members of the family of elements known as lanthanides or rare earths. Monoclonal antibody CC49, a murine immunoglobulin (Ig) G1, which is reactive with the tumor-associated antigen TAG-72, previously has been shown to react with a wide range of human carcinomas. The authors review here the comparative biodistributions of CC49 IgG and F(ab')2 fragments labeled with 177Lu, 153Sm, and 90Y using the bifunctional chelating agent PA-DOTA. The authors also review the results of a biodistribution study comparing iodine-125-labeled and 177Lu-labeled CC49 sFv, and the use of 177Lu-CC+9 IgG in an experimental therapy model. Chelation and conjugations gave similar yields, and the labeled proteins showed similar retention of immunoreactivity regardless of the isotope used for both IgG and F(ab')2. Biodistribution data obtained in athymic mice bearing LS-174T human colon carcinoma xenografts likewise showed no differences among the three radioisotopes for both IgG and F(ab')2. Femur uptake of radioactivity was lower than previously reported for other radiolanthanide immunoconjugates. Different metabolic patterns were observed for radioiodinated versus radiometal-labeled sFv, particularly in the kidney, where localization of the latter was increased dramatically. 177Lu-CC49 was found to delay the growth of established LS-174T human colon carcinomas in athymic mice at a single dose of 50 microCi. Elimination of established tumors was demonstrated over the observation period (77 days) using single administrations of 200 or 350 microCi. Dose fractionation experiments revealed that the mice tolerated 750 microCi (3 x 250 microCi, given weekly), whereas > 50% of the mice died after receiving a single administration of approximately 500 microCi. In isotype-matched control experiments, a large differential in the therapeutic effects was observed between 177Lu-labeled control antibody and CC49.

Aniline Compounds↗
PubMed1993

Effects of administration of an anti-CD5 plus immunoconjugate in rheumatoid arthritis. Results of two phase II studies. The CD5 Plus Rheumatoid Arthritis Investigators Group.

OBJECTIVE: To evaluate the safety and activity of an immunoconjugate of ricin A chain and anti-CD5 monoclonal antibody (anti-CD5 IC), with and without concomitant methotrexate and/or azathioprine, in the treatment of rheumatoid arthritis (RA). METHODS: Seventy-nine patients with active RA were enrolled in 2 prospective open-label protocols. RESULTS: Using composite criteria, response rates were 50-68% at 1 month and 22-25% at 6 months. Transient depletion of CD3/CD5 T cells was observed on days 2 and 5 of treatment, with reconstitution on day 15 or day 29. Treatment-associated adverse effects were common but resolved rapidly without sequelae. CONCLUSION: These findings suggest activity of anti-CD5 IC in active RA and warrant confirmation in a multicenter randomized study (currently underway).

Adult↗
PubMed2004

Effect of vitamin D analog (1alpha hydroxy D5) immunoconjugated to Her-2 antibody on breast cancer.

We previously showed that a new vitamin D analog, 1alpha(OH)D5 (D5), induced differentiation and inhibited the growth of breast cancer cells. In this report, we examined whether D5 specifically delivered to breast cancer cells could have any therapeutic effect. D5 was linked to Her-2 antibody using sulfosuccinimidyl 6-4 azido nitrophenylamido hexanode (SANPAH) as a linker. The Her-2 antibody selected in our study had no significant effect on the in vitro or in vivo growth of breast cancer cells; however, it had cell-differentiating action. In vitro, D5-Her-2 antibody conjugate (IMC) showed the ability to specifically bind to Her-2-expressing cells, to compete with Her-2 antibody for surface receptor and to cause internalization. IMC (equivalent to 5 microg Her-2 antibody given intraperitoneally once weekly for 6 weeks) significantly inhibited the growth of BT-474 cells transplanted into athymic mice. The in vivo growth-inhibitory effect of IMC treatment was similar to that observed in animals receiving D5 continuously as a dietary supplement. These results show that the targeted delivery of D5 by immunoconjugation to cell surface receptor antibodies may be of potential therapeutic value for the treatment of Her-2 positive breast cancer.

Animals↗
PubMed1990

A fluorouridine-anti-CEA immunoconjugate is therapeutically effective in a human colonic cancer xenograft model.

5-fluorouridine (FUR), an antineoplastic agent, was site-specifically conjugated to the carbohydrate moiety of a anticarcinoembryonic antigen (CEA) monoclonal antibody (MAb) by using amino-dextran as the intermediate carrier. The final immunoconjugate contains approximately 30-35 molecules of FUR per molecule of immunoglobulin, has immunoreactivity retained as examined by flow cytometry, and is cytotoxic to the target cells as examined by 75selenomethionine incorporation studies. In the GW-39/nude mouse model, the conjugate remained efficient in targeting the human colonic tumor and possessed greater inhibitory growth effects on the subcutaneous tumor than free FUR or an irrelevant antibody conjugate. In addition, the reduced host toxicity of the conjugate may permit the use of this agent in a high-dose therapy of this tumor system.

Animals↗
PubMed1993

Preclinical investigation of the antitumour effects of anti-CD19-idarubicin immunoconjugates.

Idarubicin (Ida), an analogue of daunomycin, was linked to a mouse monoclonal antibody against the B cell differentiation antigen CD19. Determination of the activity of both the antibody and drug moieties was carried out in vitro using a pre-B cell human acute lymphoblastic leukaemia cell line (NALM-6). A 23% loss in antibody binding and a 20-fold loss in drug activity were observed upon conjugation. Selective cytotoxicity for CD19+ cells, however, was obtained. Measurement of the cytotoxicity, antibody activity and release of the breakdown product, 14-OH-Ida, showed that the conjugates remained stable for more than 100 days after lyophilization and storage at -20 degrees C. In vivo studies were performed in irradiated nu/nu mice bearing NALM-6 tumours. Initial dose response studies with free idarubicin demonstrated that three i.p. doses (every other day) of 10 micrograms resulted in little antitumour activity, but the death of all the animals by day 23. The same treatment regime using 15 micrograms Ida-anti-CD19 conjugate caused the disappearance of four out of five tumours with three complete cures and no evidence of toxicity as assessed by weight loss. Administration of a conjugate of idarubicin with an irrelevant antibody at this dose led to no significant antitumour response. The administration of free drug at a dose of 6 micrograms resulted in a minor antitumour response but high toxicity, whereas injection of Ida-anti-CD19 conjugate at this dose caused no toxicity and a substantial antitumour effect with eradication of two out of five tumours. These results clearly demonstrate that the administration of Ida-anti-CD19 conjugates can result in complete tumour regression in an experimental model. Idarubicin-containing immunoconjugates should be useful for the treatment of patients with non-Hodgkin's lymphoma.

Animals↗
PubMed1989

Preparation and analysis of bifunctional immunoconjugates containing monoclonal antibodies OKT3 and BABR1.

OKT3 and BABR1 [anti-(breast tumour) antibody] were conjugated using N-succinimidyl-3-(2-pyridy]dithio)propionate (SPDP). The procedure employed mild reducing conditions for SPDP-BABR1 and short conjugation incubations at 37 degrees C. The bifunctional immunoconjugates thus produced were isolated by HPLC gel filtration on a preparative TSK 3000 SW column. Both intact IgG and F(ab')2 fragments have been conjugated. The ratio of SPDP to IgG for the optimal yield of dimeric OKT3-BABR1 heteroconjugates was determined to be 2 for OKT3 and 1-2 for BABR1. The OKT3-BABR1 dimers were shown to be bifunctional heteroconjugates by polyacrylamide gel electrophoresis, isoelectric focusing, radial immunodiffusion, and flow cytometry. The binding specificities of the bifunctional heteroconjugates were identical to the specificities of both parent antibodies.

Antibodies, Monoclonal↗
PubMed1990

Effects of methotrexate-carcinoembryonic-antigen-antibody immunoconjugates on GW-39 human tumors in nude mice.

Methotrexate (MTX) was conjugated to an anti-carcinoembryonic antigen monoclonal antibody (NP2) by using amino-dextran as an intermediate carrier. The drug was chemically linked to amino-dextran (average Mr = 40,000), and the resulting MTX-dextran was then site-specifically attached to the carbohydrate moiety of the antibody. Athymic nude mice that carried human colonic GW-39 tumors (s.c.) were treated with the immunoconjugate. In this study, the specific conjugate caused a greater inhibition of the tumor growth than either free MTX or its conjugate with dextran and an irrelevant antibody. The intermediate MTX-dextran and the unlinked mixture of MTX-dextran with NP2 were both relatively ineffective in inhibiting tumor growth. The greatly reduced host toxicity permitted the use of the MTX-dextran-NP2 in a high-dose therapy of this tumor system.

Animals↗
PubMed1992

The specific cytotoxicity of immunoconjugates containing blocked ricin is dependent on the residual binding capacity of blocked ricin: evidence that the membrane binding and A-chain translocation activities of ricin cannot be separated.

Recently we have developed blocked ricin, a derivative of native ricin in which the galactose-binding sites of the B-chain are blocked by covalent modification with affinity ligands. This modification impedes the binding function of the B-chain, while sparing its ability to facilitate the entry of the toxic subunit of ricin, the A-chain, into the cytoplasm. Immunotoxins prepared with blocked ricin approach the cytotoxic potency of native ricin with antibody-dependent specificity. Here we report that the high cytotoxic potency of these immunoconjugates, which is attributed to the preserved translocation function of the ricin B-chain, is dependent on the minimal residual lectin activity of blocked ricin. Our findings support the notion that two functions of ricin, membrane binding and translocation, cannot be separated.

Antigen-Antibody Complex↗
PubMed1992

Long-term reduction of vasopressin excretion induced by the central injection of an immunoconjugate (antibody to vasopressin linked to ricin A chain).

We have previously demonstrated that vasopressin-producing neurons are the target of monoclonal antibodies to vasopressin microinjected into the brain tissue. At the same time, this central microinjection of vasopressin-monoclonal antibody into the supraoptic nuclei produced hydro-osmotic disorders mimicking the effects of a central diabetes insipidus. In order to investigate the increase in both duration and amplitude of the biological effects seen after the injection of vasopressin-monoclonal antibody, an immunoconjugate was constructed with the vasopressin-monoclonal antibody IgG1k isotype and the cytotoxic part of the ricin molecule, the ricin A chain. The biological parameters, such as diuresis and urine osmolality which are directly regulated by vasopressin, and vasopressin excretion, were measured after the central injection of this immunotoxin/immunoconjugate. The consequences of immunotoxin injection were also studied when immunotoxin was co-injected with monensin (50 nM) which has been shown to decrease the intracellular degradation of immunotoxin, and plasma complement, which has been shown to increase the neuronal uptake of immunotoxin. Single injection of immunotoxin near the hypothalamic supraoptic nuclei significantly increased diuresis and decreased vasopressin excretion. However, these effects were only transient and disappeared 24 h later. Four successive injections of immunotoxin (one per day) with monensin induced a decrease of vasopressin excretion which was still observed after a resting period of four days after the fourth injection. The long-term reduction of vasopressin excretion was induced in rats receiving four successive injections of a mixture consisting of immunotoxin with monensin and plasma complement. In such experiments, the vasopressin content of urine remained low (55% under the baseline value), two weeks after the fourth injection of immunotoxin. At the same time, the diuresis was increased (80% above the baseline value) and urine osmolality lowered (45% under the baseline value). When non-specific IgG replaced specific antibody, vasopressin excretion, diuresis as well as urine osmolality were unchanged. The results of this study demonstrated that the use of a specific immunotoxin results in a local interference with the vasopressinergic neurons and induces a long-term reduction of vasopressin secretion.

Animals↗