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PubMed1996

Analysis of monoclonal antibody and immunoconjugate digests by capillary electrophoresis and capillary liquid chromatography.

Comparative peptide mapping of a monoclonal antibody chimeric BR96 and corresponding doxorubicin (DOX) immunoconjugate was performed using capillary electrophoresis (CE) and capillary liquid chromatography (CLC). A unique, highly sensitive and selective approach combined with both UV absorbance and laser-induced fluorescence (LIF) detection has been developed and applied to studies including enzymatic digests of antibody and conjugate and related drug and conjugation linker substances. The analytical methodology has been established based on the unique characteristic of the anticancer drug DOX which yields native fluorescence. With an excitation wavelength of 488 nm from argon-ion laser, DOX conjugated to the monoclonal antibody using a hydrazone linker can be determined with a detection limit at the attomole level. Approaches were developed based on the successful conjugation and analysis of a model peptide conjugate. Enzymatic digests of the monoclonal antibody BR96 and its immunoconjugate were mapped by CE and CLC with on-line UV and LIF detection, which results in a unique fingerprint for structural analysis. With a two-dimensional LC-CE approach, conjugated peptide-DOX species from LC were further analyzed by CE with LIF detection. The drug-containing peptide fragments in the mixture were readily detected, which can be further characterized using other complementary analytical techniques.

Amino Acid Sequence↗
PubMed2000

Equilibrium adsorption of LDL and gold immunoconjugates to affinity membranes containing PEG spacers.

The objective of this study was to provide insight into the effects of spacer chemistry on immunoaffinity separations for the capture of large macromolecules and biological complexes. Immunoaffinity membranes were prepared by immobilization of immunoglobulin G (IgG) to flat sheet microporous membranes. Two different systems were examined: immobilized IgG for the immunoadsorption of human low-density lipoprotein (LDL) and immobilized IgG for the immunoadsorption of gold particle immunoconjugate. The IgG was immobilized either directly to the membrane or via a polyethylene glycol (PEG) spacer. Adsorption of LDL was significantly greater for anti-LDL IgG immobilized via PEG than for IgG immobilized directly to the membrane. With the PEG spacer, the adsorption capacity for LDL matched the theoretical density of a monolayer of LDL particles on the membrane surface. The gold particle immunoconjugate, similar in size to LDL, was examined as a generalized model of restrictions to immunoaffinity adsorption of large (>20 nm) biological complexes. Adsorption of gold particles was greater for IgG immobilized via PEG than for IgG immobilized directly to the membrane. It is postulated that the PEG spacer allows lateral movement of the immobilized IgG and dense monolayer packing of adsorbed particles on the membrane surface. These results are pertinent to the removal of LDL from human plasma and the purification of gene therapy delivery vectors, viral vaccines, and other large biological complexes.

Adsorption↗
PubMed2000

Synthesis and HPLC analysis of enzymatically cleavable linker consisting of poly(ethylene glycol) and dipeptide for the development of immunoconjugate.

A model compound of anti-tumor agent, segment B of duocarmycin derivative DU-86, was conjugated to tumor-specific antibody via a cleavable linker consisting of poly(ethylene glycol) (PEG) and dipeptide, L-alanyl-L-valine (Ala-Val), to confirm the feasibility of the linker for application to immunoconjugate. The release of segment B from the linker was evaluated by HPLC analysis. When segment B was derivatized to have an amino residue and then linked to PEG through a dipeptide, segment B was cleaved at the peptide bond by a particular enzyme, thermolysin (EC 3.4.24.4), but not by plasmin (EC 3.4.2 1.7.), indicating that certain protease specifically expressed at the tumor site would be capable of peptide-specific digestion and release of anti-tumor agent since a thermolysin-like enzyme has been reported to be expressed at many tumor cells. Furthermore, the results showing that cell extract from G361 human melanoma had an ability to digest the linker peptide while the linker was stable in normal human serum suggested the tumor-specific activation of the conjugated agent. Segment B was conjugated via the linker to murine monoclonal antibody KM641 reactive to GD3 ganglioside to form immunoconjugate and the quantitative release of segment B under the treatment with the enzyme was also confirmed. These results indicate the possibility of double targeting based on both the recognition ability of tumor specific antibody and tumor specific activation of the anti-tumor agents to enhance tumor treatment efficacy and to decrease unwanted side effects.

Antibodies, Monoclonal↗
PubMed2002

Synthesis of an immunoconjugate of camptothecin.

The first immunoconjugate of camptothecin has been synthesized wherein the drug is attached to the tumor-recognizing antibody BR96 via a Cathepsin B cleavable linker. Endocytosis of the immunoconjugate upon binding to the tumor cell followed by enzymatic cleavage of the linker inside the endosome ensures tumor-specific release of the drug. In this way, it is hoped that the dose-limiting side effects associated with camptothecin can be eliminated while the antitumor activity is preserved.

Camptothecin↗
PubMed2002

Syntheses of dendritic linkers containing chlorambucil residues for the preparation of antibody-multidrug immunoconjugates.

A novel dendritic molecule with nine chlorambucil (CBL) residues on the surface and a maleimide moiety at the core terminus was synthesized using a convergent synthetic methodology. This molecule is ready for attachment to single-chain Fv antibodies (scFvs) to form antibody-multidrug immunoconjugates in an effort to study the relevance of drug/antibody molar ratio and the potency of these drug-antibody immunoconjugates. A monomer and a trimer with a similar structural motif were also prepared for comparative purposes.

Antibodies↗
PubMedDate not supplied

A facile, water-soluble method for modification of proteins with DOTA. Use of elevated temperature and optimized pH to achieve high specific activity and high chelate stability in radiolabeled immunoconjugates.

We have developed a method for attachment of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane N,N',N",N"'-tetraacetic acid (DOTA) to proteins by activation of a single carboxyl group with N-hydroxysulfosuccinimide (sulfo-NHS). The sulfo-NHS active ester of DOTA was prepared in a single step using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), and DOTA conjugates of cytochrome c and the anti-carcinoembryonic antigen chimeric monoclonal antibody cT84.66 were prepared by adding the DOTA active ester reaction mixture to the proteins at pH 8.5-9.0. Mass spectrometry of the cytochrome c conjugates showed that as the molar ratio of DOTA active ester to protein in the reaction mixture was increased from 10:1 to 100:1, the average number of chelators attached to the protein molecule increased from 2.64 to 8.79. When DOTA active ester reacted with the antibody at a molar ratio of 100:1, the conjugate averaged 3.8 chelates per antibody. Immunoreactivity of the antibody conjugate radiolabeled with 111In(III) and 90Y(III) remained quantitative. Variation of the DOTA:sulfo-NHS:EDC activation stoichiometry from 2:2:1 to 10:10:1 revealed that the kinetic stability of the radioconjugates increased as the molar ratio of carbodiimide, relative to DOTA and sulfo-NHS, was decreased. Radiolabeling of the protein conjugates with 111In(III) and 90Y(III) proved to be sensitive to pH, buffer, and temperature effects. The optimum pH for the labeling reaction was different for each protein and may be related to the isoelectric point of the protein. Radiometal incorporation at high specific activity was accomplished in acetate and Tris buffers, but the presence of citrate inhibited the labeling reaction. Increasing the temperature of the radiolabeling reaction from 25 to 43 degrees C greatly increased both the efficiency of radiometal incorporation and the kinetic stability of the radioconjugates. Stability studies of the conjugates in human serum and in the presence of a 5000- to 250,000-fold excess of diethylenetriaminepentaacetic acid (DTPA) demonstrated that the radiolabeled proteins are kinetically inert under physiological conditions. In serum, the 111In(III)-labeled antibody showed a rate of radiometal loss of approximately 0.08% per day. In the presence of excess DTPA, both conjugates lost 111In(III) at a rate of about 0.3% per day. No loss of 90Y(III) from the conjugates was observed in serum, but in excess DTPA, both 90Y(III) labeled proteins showed a rate of radiometal loss of approximately 0.2% per day. Therefore, kinetic analysis of metal loss from a radiolabeled immunoconjugate in the presence of a vast excess of DTPA may provide a better indication of the in vivo stability of that immunoconjugate than serum stability studies.

Antibodies, Monoclonal↗
PubMedDate not supplied

Monoclonal antibody conjugates of doxorubicin prepared with branched linkers: A novel method for increasing the potency of doxorubicin immunoconjugates.

Immunoconjugates of monoclonal antibody BR96 and Doxorubicin have been prepared using a novel series of branched hydrazone linkers. Since each linker bound to the mAb carries two DOX molecules, the DOX/mAb molar ratios of these conjugates were approximately 16, twice that of those previously prepared with single-chain hydrazone linkers. The conjugates were stable at a physiological pH of 7, but released DOX rapidly at lysosomal pH 5. The branched series of BR96 conjugates demonstrated antigen-specific cytotoxicity, and were more potent in vitro than the single-chain conjugate on both a DOX (4-14-fold) and mAb (7-23-fold) basis. The results suggest that, by using the branched linker methodology, it is possible to significantly reduce the amount of mAb required to achieve antigen-specific cytotoxic activity. In this paper, the synthesis and in vitro biology of branched chain immunoconjugates are described.

Animals↗
PubMed2003

Targeting of human squamous carcinomas by SPA470-doxorubicin immunoconjugates.

In a quest to identify a favorable target for head and neck cancers and squamous cell carcinoma, we sought to determine if Hsp47/CBP2 could be used as a target and whether the expression of this target was influenced by hypoxia. Moreover, we determined if doxorubicin (DOX) immunoconjugates directed against Hsp47/CBP2 that linked monoclonal antibodies (MAbs) to the 13-keto position of the drug possessed high cytotoxic drug activity and antibody-directed killing of antigen bearing tumor target cells. Experiments were performed using established cell lines of human oral squamous carcinoma cells (SCCs) (SCC-4, -9, -15 and -25) obtained from American Type Culture Collection (ATCC) (Manassas, VA). In addition, the UMB2 cell line is a spontaneous mutant of SCC-9 that does not express Hsp47/CBP2 was also used. Synthesis of the immunoconjugates was accomplished by thiolating the MAbs with 2 IT and reacting the MAbs with the DOX-hydrazone. The binding of MAb-DOX conjugates to SCC cells was determined by indirect immunofluorescence and analyzed using a Becton Dickinson FACS scan with Cell Quest software. Comparison of the cytotoxicity of DOX, MAb-DOX conjugates and MAb+DOX were determined using a limited dilution assay and colony survival assays during normoxia and hypoxia. These studies revealed that SCC cells treated with the SPA470-DOX conjugate for 2 h retained the original binding activity for targeted SCC cells and was significantly more potent that unconjugated DOX, DOX-hydrazone or equivalent MAb protein+DOX. Also, SPA47-DOX produced equal to and at lower concentrations greater cell killing than equivalent dose of free DOX. During hypoxia cells treated with SPA470-DOX demonstrated a small increase in colony survival and a diminishment in cytotoxicity. SPA470-DOX conjugates target SCC cells that express Hsp47/CBP2. The demonstration that SPA470-DOX is effective during hypoxia or conditions that mimic hypoxia presumes the further utility of SPA470-DOX in treating head and neck cancers.

Antibodies, Monoclonal↗
PubMed1995

Cytotoxicity of 213Bi- and 225Ac-immunoconjugates.

This paper describes in vitro cytotoxicity experiments with 213Bi- and 225Ac-immunoconjugates on the human epidermoid tumour cell line A431 using a blood group A-reactive murine IgG (2D11) as the specific antibody and MOPC 21 as the control antibody. With both radionuclides, specific cell-killing was achieved. The observed cytotoxicity of 213Bi (T1/2 - 47 min) indicates that this radionuclide is a useful alternative for the alpha-emitter 212Bi in the treatment of blood-borne malignancies. 225Ac-immunoconjugates (T1/2 of 225Ac is 10 days) may be applicable for the treatment of solid tumours, since the daughter radionuclides of 225Ac contribute to the cytotoxic efficacy by a field effect (i.e. toxicity in an area distal from the antibody-binding site). The lack of an adequate chelator for 225Ac is a major drawback.

Actinium↗
PubMed2005

Structural characterization of the maytansinoid-monoclonal antibody immunoconjugate, huN901-DM1, by mass spectrometry.

Immunoconjugates are being explored as novel cancer therapies with the promise of target-specific drug delivery. The immunoconjugate, huN901-DM1, composed of the humanized monoclonal IgG1 antibody, huN901, and the maytansinoid drug, DM1, is being tested in clinical trials to treat small cell lung carcinoma (SCLC). huN901-DM1 contains an average of three to four DM1 drug molecules per huN901 antibody molecule. The drug molecules are linked to huN901 through random modification of huN901 at epsilon-amino groups of lysine residues, thus yielding a heterogeneous population of conjugate species. We studied the drug distribution profile of huN901-DM1 by electrospray time-of-flight mass spectrometry(ESI-TOFMS), which showed that one to six DM1 drug molecules were attached to an antibody molecule. Both light and heavy chains contained linked drugs. The conjugation sites in both chains were determined by peptide mapping using trypsin and Asp-N protease digestion. Trypsin digestion identified modified lysine residues, since these residues were no longer susceptible to enzymatic cleavage after conjugation with the drug. With respect to Asp-N digestion, modified peptides were identified by observing a mass increase corresponding to the modification. The two digestion methods provided consistent results, leading to the identification of 20 modified lysine residues in both light and heavy chains. Each lysine residue was only partially modified. No conjugation sites were found in complementarity determining regions (CDRs). Using structural models of human IgG1, it was found that modified lysine residues were on the surface in areas of structural flexibility and had large solvent accessibility.

Amino Acid Sequence↗
PubMed2006

Eukaryotic expression vectors and immunoconjugates for cancer therapy.

This review considers ways to address specificity to therapeutic targeted anticancer agents. These include transcriptional activation of tissue- and tumor-specific promoters in eukaryotic expression vectors and use of antitumor-directed immunoconjugates. The review deals with analysis of strategies used for selection of targeted promoters and examples of antibody fusion proteins exhibiting antitumor activity. A new direction in antitumor treatment pooling together methods of gene therapy and antibody therapy has appeared. This direction is based on the development of vectors encoding secreted forms of immunoconjugates. After vector introduction into a cell, the latter is capable of synthesizing and secreting antibody fusion protein composed of a therapeutic anticancer agent and antibody specifically targeted to cancer cells.

Animals↗
PubMed2004

Herceptin-geldanamycin immunoconjugates: pharmacokinetics, biodistribution, and enhanced antitumor activity.

The efficacy of monoclonal antibodies (mAbs) as single agents in targeted cancer therapy has proven to be limited. Arming mAbs with a potent toxic drug could enhance their activity. Here we report that conjugating geldanamycin (GA) to the anti-HER2 mAb Herceptin improved the activity of Herceptin. The IC(50)s of the immunoconjugate H-GA were 10-200-fold lower than that of Herceptin in antiproliferative assays, depending on the cell line. The H-GA mode of action involved HER2 degradation, which was partially lactacystin sensitive and thus proteasome dependent. The linkage between GA and Herceptin remained stable in the circulation, as suggested by the pharmacokinetics of Herceptin and conjugated GA, which were almost identical and significantly different from that of free GA. Tumor uptake of Herceptin and H-GA were similar (52 +/- 7 and 43 +/- 7% of the initial injected dose per gram tissue, respectively; P = 0.077), indicating no apparent damage attributable to conjugation. Therapy experiments in xenograft-bearing mice consisted of weekly i.p. doses, 4 mg/kg for 4 months. H-GA showed a greater antitumor effect than Herceptin because it induced tumor regression in 69% of the recipients compared with 7% by Herceptin alone. Median survival time was 145 days as opposed to 78 days, and 31% of the recipients remained tumor free 2 months after therapy was terminated versus 0% in the Herceptin group. Enhancement of Herceptin activity could be of significant clinical value. In addition, the chemical linkage and the considerations in therapeutic regimen described here could be applied to other immunoconjugates for targeted therapy of a broad spectrum of cancers.

Animals↗
PubMed2003

Antibody-targeted chemotherapy with CMC-544: a CD22-targeted immunoconjugate of calicheamicin for the treatment of B-lymphoid malignancies.

Antibody-targeted chemotherapy with gemtuzumab ozogamicin (CMA-676, a CD33-targeted immunoconjugate of N-acetyl-gamma-calicheamicin dimethyl hydrazide [CalichDMH], a potent DNA-binding cytotoxic antitumor antibiotic) is a clinically validated therapeutic option for patients with acute myeloid leukemia (AML). Here, we describe the preclinical profile of another immunoconjugate of CalichDMH, CMC-544, targeted to CD22 expressed by B-lymphoid malignancies. CMC-544 comprises a humanized IgG4 anti-CD22 monoclonal antibody (mAb), G5/44, covalently linked to CalichDMH via an acid-labile 4-(4'-acetylphenoxy) butanoic acid (AcBut) linker. Both CMC-544 and unconjugated G5/44 bound human CD22 with subnanomolar affinity. CMC-544, but not unconjugated G5/44, exerted potent cytotoxicity against CD22+ B-cell lymphoma (BCL) cell lines (inhibitory concentration of 50%: 6-600 pM CalichDMH). CMC-544 caused a potent inhibition of growth of small but established BCL xenografts leading to cures (therapeutic index > 10). CMC-544 prevented the establishment of BCL xenografts and also caused regression of large BCLs (> 1.5 g tumor mass). In contrast, unconjugated CalichDMH, unconjugated G5/44, and an isotype-matched control conjugate, CMA-676, were ineffective against these BCL xenografts. Thus, CD22-targeted delivery of CalichDMH is a potent and effective preclinical therapeutic strategy for BCLs. The strong antitumor profile of CMC-544 supports its clinical evaluation as a treatment option for B-lymphoid malignancies.

Animals↗
PubMed2003

Cantuzumab mertansine, a maytansinoid immunoconjugate directed to the CanAg antigen: a phase I, pharmacokinetic, and biologic correlative study.

PURPOSE: To determine the maximum tolerated dose and pharmacokinetics of cantuzumab mertansine, an immunoconjugate of the potent maytansine derivative (DM1) and the humanized monoclonal antibody (huC242) directed to CanAg, intravenously (i.v.) once every 3 weeks and to seek evidence of antitumor activity. PATIENTS AND METHODS: Patients with CanAg-expressing solid malignancies were treated with escalating doses of cantuzumab mertansine administered i.v. every 3 weeks. The pharmacokinetic parameters of cantuzumab mertansine, the presence of plasma-shed CanAg, and the development of both human antihuman and human anti-DM1 conjugate antibodies also were characterized. RESULTS: Thirty-seven patients received 110 courses of cantuzumab mertansine at doses ranging from 22 to 295 mg/m2. Acute, transient, and reversible elevations of hepatic transaminases were the principal toxic effects. Nausea, vomiting, fatigue, and diarrhea were common but rarely severe at the highest dose levels. Dose, peak concentration, and area under the concentration-time curve correlated with the severity of transaminase elevation. The mean (+/- SD) clearance and terminal elimination half-life values for cantuzumab mertansine averaged 39.5 (+/-13.1) mL/h/m2 and 41.1 (+/-16.1) hours, respectively. Strong expression (3+) of CanAg was documented in 68% of patients. Two patients with chemotherapy-refractory colorectal carcinoma had minor regressions, and four patients had persistently stable disease for more than six courses. CONCLUSION: The recommended dose for cantuzumab mertansine is 235 mg/m2 i.v. every 3 weeks. The absence of severe hematologic toxic effects, preliminary evidence of cantuzumab mertansine tumor localization, and encouraging biologic activity in chemotherapy-refractory patients warrant further broad clinical development of this immunoconjugate in CanAg-expressing tumors.

Adult↗
PubMed2004

Antitumor effects of the molecule-downsized immunoconjugate composed of lidamycin and Fab' fragment of monoclonal antibody directed against type IV collagenase.

Type IV collagenase plays an important role in tumor invasion and metastasis through cleaving type IV collagen in the basement membrane and extracellular matrix. In this study a molecule-downsized immunoconjugate (Fab'-LDM) was constructed by linking lidamycin (LDM), a highly potent antitumor antibiotic, to the Fab' fragment of a monoclonal antibody directed against type IV collagenase and its antitumor effect was investigated. As assayed in 10% SDS-PAGE gel, the molecular weight of Fab'-LDM conjugate was 65 kD with a 1 : 1 molecular ratio of Fab' and LDM. The Fab'-LDM conjugate maintained most part of the immunoreactivity of Fab' fragment to both type IV collagense and mouse hepatoma 22 cells by ELISA. By MTT assay, Fab'-LDM conjugate showed more potent cytotoxicity to hepatoma 22 cells than that of LDM. Administered intravenously, Fab'-LDM conjugate proved to be more effective against the growth of subcutaneously transplanted hepatoma 22 in mice than free LDM in two experiment settings. In Experiment I, the drugs were given intravenously on day 1 and day 8. Fab'-LDM at the doses of 0.025 mg/kg, 0.05 mg/kg and 0.1 mg/kg inhibited tumor growth by 76.7%, 93.3% and 94.8%, while free LDM at 0.05 mg/kg inhibited tumor growth by 76.1%, respectively. In experiment II, the drugs were given intravenously on day 4 and day 11, Fab'-LDM at the doses of 0.025 mg/kg and 0.05 mg/kg inhibited tumor growth by 74.2%, 80.9%, while free LDM at 0.05 mg/kg inhibited tumor growth by 60.5%, respectively. In terms of survival time, Fab'-LDM was more effective than free LDM. The results suggest that the molecule-downsized immunoconjugate directed against type IV collagenase is of high efficacy in experimental cancer therapy.

Aminoglycosides↗
PubMed1998

In vivo toxicity and pharmacokinetic features of B43(Anti-CD19)-Genistein immunoconjugate.

B43(anti-CD19)-Genistein immunoconjugate targets genistein, a naturally occurring protein tyrosine kinase inhibitory isoflavone to the membrane-associated anti-apoptotic CD19-LYN complexes and triggers apoptotic cell death. In this preclinical study, the toxicity profiles of B43-Genistein as well as unconjugated genistein were evaluated in mice. B43-Genistein and genistein were administered either as single bolus injections or daily injections for 10 consecutive days via the intraperitoneal route to mice. Genistein was not toxic to mice at the highest dose of 40 mg/kg and no test article-related histopathological lesions were found in any of the 64 genistein-treated mice. B43-Genistein had a significantly longer elimination half-life and slower plasma and tissue clearance than unconjugated genistein. B43-Genistein was not toxic to mice at the highest single dose of 40 mg/kg or highest cumulative dose of 100 mg/kg and no test article-related histopathological lesions were found in any of the 108 mice treated with B43-genistein. To our knowledge, this is the first preclinical toxicity and pharmacokinetic study of a tyrosine kinase inhibitor-containing immunoconjugate.

Animals↗
PubMed1999

Selective ablation of acute myeloid leukemia using antibody-targeted chemotherapy: a phase I study of an anti-CD33 calicheamicin immunoconjugate.

Leukemic blast cells express the CD33 antigen in most patients with acute myeloid leukemia (AML), but this antigen is not expressed by hematopoietic stem cells. We conducted a study to determine whether normal hematopoiesis could be restored in patients with AML by selective ablation of cells expressing the CD33 antigen. In a dose escalation study, 40 patients with relapsed or refractory CD33(+) AML were treated with an immunoconjugate (CMA-676) consisting of humanized anti-CD33 antibody linked to the potent antitumor antibiotic calicheamicin. The capacity of leukemic cells to efflux 3, 3'-diethyloxacarbocyanine iodide (DiOC2) was used to estimate pretreatment functional drug resistance. Leukemia was eliminated from the blood and marrow of 8 (20%) of the 40 patients; blood counts returned to normal in three (8%) patients. A high rate of clinical response was observed in leukemias characterized by low dye efflux in vitro. Infusions of CMA-676 were generally well tolerated, and a postinfusion syndrome of fever and chills was the most common toxic effect. Two patients who were treated at the highest dose level (9 mg/m2) were neutropenic >5 weeks after the last dose of CMA-676. These results show that an immunoconjugate targeted to CD33 can selectively ablate malignant hematopoiesis in some patients with AML.

Adult↗
PubMed2000

Interferon-alpha-2b immunoconjugate for improving immunoscintigraphy and immunotherapy.

UNLABELLED: A pretreatment with a single dose of an immunoconjugate (IC) that promises to enhance tumor uptake and decrease liver uptake of radiolabeled monoclonal antibodies (MAbs) might be of use in radioimmunodetection and radioimmunotherapy (RIT). We have shown previously that an interferon (IFN)-MAb (1:1) immunoconjugate (IC) enhances tumor uptake by a factor of 2 or more and reduces liver uptake by 50% in nude mice bearing human tumors. The aim of this study was to determine whether IFN modulates antigenic expression and to ascertain the most effective route of its administration, the optimal quantity to be administered, and the optimal duration of time to lapse between the administration of IC and the radiolabeled MAb. METHODS: IFN-alpha-2b and anticarcinoembryonic antigen-F6 (IgG2a) MAb were conjugated (1:1), and F(ab')2 of the MAb was labeled with 99mTc. Human colorectal tumors were grown in nude mice by implanting 5 x 10(6) LS174T confluent cells grown in culture. Mice, 5 in each group, received 20 x 10(3) IU intravenously, intramuscularly, or intraperitoneally and 40 x 10(3), 60 x 10(3), and 80 x 10(3) IU intravenously 30 min before the intravenous administration of 25.9 MBq 99mTc/20 microg F(ab')2. Mice in the control groups received 99mTc-F(ab')2 but not the conjugate. Twenty-four hours later mice were killed and imaged, and tissues were removed for quantitative (percentage injected dose/g [% ID/g]) distribution of 99mTc. RESULTS: In all conjugate-receiving mice, the tumor uptake was higher and the liver uptake was lower (P < 0.01) than that in the control mice with the exception of liver uptake, which was not significantly different in mice receiving 80 x 10(3) IU conjugate. The optimal results were apparent in mice pretreated with 40 x 10(3) IU conjugate in which tumor uptake was enhanced by a factor of 2.3 (4.8 +/- 0.5 %ID/g versus 11 +/- 0.7 %ID/g; P < 0.01). The renal uptake remained unchanged, and the tumor-to-muscle ratios increased from 11.5 +/- 6.8 to 14.6 +/- 3.9, and the tumor-to-blood ratios increased from 4.4 +/- 1.8 to 8.3 +/- 2.4. The liver uptake decreased from 9.5% +/- 1% to 5% +/- 1.6%. Results were attributed to enhanced tumor blood flow, increased antigenic expression, and blocking of hepatic nonspecific Fc receptors. CONCLUSION: A pretreatment with IFN-MAb conjugate is a worthwhile approach to consider in radioimmunoscintigraphy and RIT.

Animals↗