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PubMed1993

The use of daunomycin-antibody immunoconjugates in managing soft tissue sarcomas: nude mouse xenograft model.

Analysis of human fibrosarcoma cells exposed to radiolabeled monoclonal antibody 19-24, which recognizes sarcoma-associated antigen p102, revealed that over 54% of the cell surface-bound radioactivity was internalized. No modulation of cell surface p102 antigen by monoclonal antibody 19-24 was observed in human fibrosarcoma cells. Monoclonal antibody 19-24 coupled to daunomycin via a dextran bridge was found to be most effective. In different preparations, the daunomycin:total protein molar ratio ranged from 1.9 to 6.1. In vitro cytotoxicity studies using human fibrosarcoma cells showed that, at 10 micrograms/ml concentration, this immunoconjugate was 79.4% as efficient as free daunomycin and, at 1 microgram/ml concentration, 36.8% as efficient. Control nonspecific murine monoclonal antibody P3 immunoconjugates were relatively ineffective. The distribution of 14C-Adriamycin, 125I-labeled monoclonal antibody 19-24, and 125I-labeled 19-24 immunoconjugate was also evaluated over a 24-h period in tumor and normal tissues of athymic mice bearing a human fibrosarcoma xenograft. Poor uptake of radiolabeled Adriamycin by the tumor tissue was observed. The level of 14C radioactivity in the tumor tissue never exceeded 1% of the total injected dose and was 24.8-fold lower than the radioactivity found in the spleen tissue. Tumor tissue uptake of radiolabeled monoclonal antibody 19-24 was characterized by the high tumor tissue:blood ratio of 1.62 +/- 0.28 (SD). However, for monoclonal antibody 19-24 immunoconjugates, this ratio decreased to 0.66 +/- 0.05, which was still higher than normal (liver, 0.48 +/- 0.02; lung, 0.48 +/- 0.07; spleen, 0.28 +/- 0.01) or nonspecific monoclonal antibody P3 immunoconjugates (0.22 +/- 0.03). Thus, it appears that, compared to free daunomycin, monoclonal antibody 19-24 immunoconjugates may be more efficient and less cytotoxic to normal tissues.

Animals↗
PubMed1995

An immunoconjugate of Lys3-bombesin and monoclonal antibody 22 can specifically induce FcgammaRI (CD64)-dependent monocyte- and neutrophil-mediated lysis of small cell carcinoma of the lung cells

Small cell carcinoma of the lung (SCCL) accounts for 25% of all lung cancers and has a very poor prognosis. It is known that SCCL cells produce gastrin-releasing peptide, a peptide which has similar biological actions to that of bombesin, an amphibian counterpart of gastrin-releasing peptide, and express high affinity cell surface bombesin/gastrin-releasing peptide receptors. These receptors can serve as targets for specific immunotherapy. Cell surface receptors for the Fc portion of IgG (FcgammaR) are a family of molecules that can mediate a variety of immune reactions, including tumor cell cytotoxicity. We hypothesized that an immunoconjugate of bombesin and a mAb directed to the high-affinity FcgammaRI (mAb 22) should be able to trigger specific cytotoxicity against SCCL cells. In this article, we report the construction of this immunoconjugate and demonstrate its capacity to redirect immune effector cells toward SCCL cells and elicit lysis of these target cells. The immunoconjugate stained the majority of cells from four SCCL cell lines and reacted with FcgammaRI on activated monocytes and neutrophils. After preincubating monocytes and neutrophils with recombinant gamma interferon to enhance the expression of FcgammaRI on the cell surface, we demonstrated that 60-98% of SCCL cells could be lysed in the presence of the immunoconjugate in a chromium release assay. Tumor cell lysis was observed over a wide range of immunoconjugate concentrations, was dependent on the ratio of E:T cells, and could be blocked by the addition of either parental molecule of the immunoconjugate. Bispecific molecules redirecting immune effector cells to target SCCL cells may have clinical application in the therapy of SCCL.

Journal Article↗
PubMedDate not supplied

Enhanced stability in vitro and in vivo of immunoconjugates prepared with 5-methyl-2-iminothiolane.

Substituted 2-iminothiolanes (X2ITs) are new heterobifunctional crosslinking agents designed for the preparation of disulfide-linked conjugates with enhanced resistance to reduction. Based upon 2-IT substituted at the 4 and/or 5 position, these reagents appear to function by sterically protecting the conjugate disulfide bond from attack by thiolate nucleophiles. Here, we have used the X2ITs to prepare and evaluate a series of immunoconjugates (antibody-cytotoxin conjugates) between the murine monoclonal antibody 791/T36, which recognizes a 72-kDa surface antigen present on many human tumor cells, and RTA30, the naturally occurring 30-kDa glycoform of ricin A chain. The X2IT-linked conjugates were also compared to immunoconjugates prepared with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) and 4-[(succinimidyloxy)carbonyl]-alpha-methyl-alpha-(2- pyridyldithio)toluene (SMPT), as well as with methyl- and dimethyl-substituted structural analogs of SPDP. In vitro, 791-(X2IT)-TNB model compounds exhibited a 6000-fold range of stabilities. In contrast, the corresponding 791-(X2IT)-RTA30 immunoconjugates were up to 20-fold more stable than conjugates made with unhindered linkages. These improvements resulted in immunoconjugates with prolonged serum half-lives in animals. Our data indicate that one of the crosslinking agents, 5-methyl-2-iminothiolane (M2IT), has optimal properties for the preparation of disulfide crosslinked immunoconjugates intended for therapeutic use in that (i) it is highly water soluble and reacts rapidly with protein amino groups at neutral pH, preserving the positive charge, (ii) it forms conjugates with RTA30 efficiently, and (iii) its conjugates exhibited enhanced disulfide bond stability in vitro and in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Modifications in synthesis strategy improve the yield and efficacy of geldanamycin-herceptin immunoconjugates.

Geldanamycin (GA) was modified with N-tert-butyloxycarbonyl-1,3-diaminopropane to introduce a latent primary amine. After deprotection, this primary amine provided a site for introduction of a maleimide group that enabled linkage to proteins. This maleimido derivative of geldanamycin (GMB-APA-GA) was linked to the monoclonal antibody Herceptin after the antibody had been modified with Traut's reagent to introduce thiol groups. By this sequence, a new immunoconjugate (H:APA-GA) was generated that showed greater antiproliferative activity than the previously reported analogous immunoconjugate created with a 1,4-diaminobutane spacer derivative of geldanamycin to form an immunoconjugate, H:ABA-GA. Both immunoconjugates inhibited in vitro the growth of MDA-361/DYT2 cells, a cell line overexpressing the HER2 antigen, while Herceptin alone was ineffective. However, H:APA-GA showed better efficacy than H:ABA-GA (IC(50) = 0.2 vs 0.58 mg/mL and cell doubling time >12 vs 6 days, respectively). Results of the in vivo therapy experiments in a xenograft model were consistent with the in vitro findings. Treatment with Herceptin prolonged the survival of the tumor-bearing mice when compared with the control group, but H:ABA-GA and H:APA-GA were each more efficacious than unmodified Herceptin. However, unlike H:ABA-GA, the immunoconjugate H:APA-GA caused stable tumor regression (in 25% of the recipients), showing a qualitative improvement with potential clinical relevance.

Animals↗
PubMed2002

Cellular handling of a dexamethasone-anti-E-selectin immunoconjugate by activated endothelial cells: comparison with free dexamethasone.

PURPOSE: For selective inhibition of endothelial cell activation in chronic inflammation, we have developed a dexamethasone-anti-E-selectin immunoconjugate. The present study was performed to evaluate the cellular handling of this immunoconjugate by activated primary endothelial cells and to compare its drug delivery capacity with free dexamethasone. METHODS: The binding, uptake, and degradation of 125I-radiolabeled dexamethasone-anti-E-selectin immunoconjugate by TNFalpha-activated endothelial cells were studied for different time periods and at different concentrations, as well as in the presence of inhibitors for E-selectin binding and lysosomal degradation. Its drug delivery capacity was compared with the uptake of unconjugated 3H-labeled dexamethasone. RESULTS: The immunoconjugate was internalized by E-selectin expressing activated endothelial cells and degraded in the lysosomal compartment. The receptor-mediated binding and uptake was saturable, implying a maximal attainable intracellular concentration of the drug. In contrast, free dexamethasone entered both resting and activated endothelial cells by passive diffusion. CONCLUSIONS: The dexamethasone-anti-E-selectin immunoconjugate is capable of selective delivering the coupled drug into activated endothelial cells. This targeting concept enables disease-induced drug delivery in which intracellular concentrations can be reached comparable with those obtained after incubation with 3 FM dexamethasone.

Cells, Cultured↗
PubMed1999

The selective cytotoxicity of cobra venom factor immunoconjugate on cultured human nasopharyngeal carcinoma cell line.

The selective cytotoxicity of a CVF immunoconjugate on human nasopharyngeal carcinoma cell line was reported. Cobra venom factor (CVF), a C3b-like glycoprotein, was linked to BAC5, a murine monoclonal antibody directed against a human nasopharyngeal carcinoma-associated membrane antigen, by a disulfide bond. The high affinity to cultured human nasopharyngeal cells (CNE2) and the complement activating potency retained in CVF immunoconjugate. Although the equimolar concentration of BAC5 or CVF alone was harmless to CNE2 cells, the CVF immunoconjugate in the presence of fresh human serum exhibited selective cytotoxicity on CNE2 cells in a concentration- (IC50 3.07 x 10(-7) mol/L) and time-dependent manner. No cytotoxicity occurred on either CNE1 (another human nasopharyngeal carcinoma cell line) or MGC-803 (human gastric carcinoma cell line) cells. Furthermore, direct lytic factor (DLF, cardiotoxin) separated from cobra venom, augmented CVF immunoconjugate-induced cytotoxicity significantly. These results indicate that the CVF immunoconjugate has complement-mediated selective cytotoxicity on CNE2 cells, which can be potentiated by DLF.

Animals↗
PubMed2002

Preparation and comparative evaluation of 99mTc-labeled 2-iminothiolane modified antibodies and CITC-DTPA immunoconjugates of anti-EGF-receptor antibodies.

The use of antibodies as targeting agents for the delivery of radioisotopes to tumors is a promising concept that has received widespread attention since the advent of monoclonal antibody (mAb) technology. The following studies are described in this article: the 99mTc-randiolabeling of 2-iminothiolane (2-IT) modified antibodies and 6-p-isothiocyanatobenzyl- diethylene-triamine penta-acetic acid (CITC-DTPA) immunoconjugates of anti-EGF-receptor antibodies murine ior egf/r3 and humanized h-R3; the analytical methods for quality control of the radiopharmaceutical such as instant thin layer chromatography-silica gel (ITLC-SG); the biological assessment of the radiolabeled molecule using flow cytometry analysis; in vitro stability studies with cysteine and DTPA challenge and the biodistribution studies in 4NMRI xenografted nude mice with U-87 human glioblastoma multiforme and MDA-MB-468 breast cancer cell lines. Labeling efficency of (96.48 +/- 0.70%) (98.42 +/- 0.38%), (94.8 +/- 1.25%) and (96.41 +/- 0.89%) was achieved for 99mTC-2-IT ior efg/r3, 99mTc-CITC-DTPA- ior egf/r3, 99mTc-CITC-DTPA- h-R3 and 99mTc-DIACIM h-R3, respectively. Radiocolloids were less than 2.0% in all cases. The biological activity measured by flow cytometry analysis using the MDA-MB-468 breast cancer cell line showed an immunoreactivity fraction greater than 85% in all concentrations of each immunoconjugate. Challenge studies demonstrated no evidence of transcomplexation of 99mTc to 1.0 mM DTPA for 2-IT modified antibody ior egf/r3 and CITC-DTPA immunoconjugates and only 8.7%, 4.9% and 5.0% of the 99mTc-radiolabeled was transcomplexed to 1.0 mM cysteine after 1 h incubation at 37 degrees C for 2-IT modified antibody ior egf/r3, CITC-DTPA ior egf/r3 and CITC-DTPA h-R3, respectively. Biodistribution studies with 2-IT modified antibodies and CITC-DTPA immunoconjugates indicated high tumor uptake in both cell lines with both immunoconjugates and no accumulation of the radiolabeled antibodies in normal organs.

Adenocarcinoma↗
PubMed1989

The Aspergillus toxin restriction is a suitable cytotoxic agent for generation of immunoconjugates with monoclonal antibodies directed against human carcinoma cells.

The protein toxin restriction, isolated from the mould Aspergillus restrictus, inactivates protein synthesis in eukaryotic cells by blocking the ribosome elongation cycle. This protein acts as a specific nuclease that cuts off a small fragment from the 28-S rRNA. Biochemical and biological characterization of this toxin indicated that it is a non-glycosylated polypeptide of Mr 16836, exhibiting in cell-free systems a protein synthesis inhibition capacity similar to that of the ricin A chain. This polypeptide seemed unable to penetrate most of the cancer cell lines tested, as measured by its low in vitro cytotoxicity. In addition in vivo studies in BALB/c mice demonstrated that restriction toxicity was very low and that in rabbits, after intravenous injection 15% of the toxin was still present in the blood stream 24 h later. After derivatization with N-succinimidyl 3-(2-pyridyldithio)propionate and reduction by dithiothreitol, the restrictocin maintained its protein synthesis inhibitory activity, as assayed in a cell-free system. This derivatized toxin was then coupled to monoclonal antibodies (MBr1, MLuC1, MLuC2, MOv17, MOv18, MOv19) which exhibited a restricted spectrum of reactivity against human carcinomas. The biochemical and biological characterization of the immunoconjugates indicated that (a) when restrictocin was coupled to monoclonal antibodies with an average molar ratio of about 2, the immunoconjugates maintained the binding activity of the antibody and protein synthesis inhibition activity of the toxin; (b) four immunoconjugates were tested for cytotoxicity and three of them obtained with the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited a good level of cytotoxicity for relevant target cells and low or no toxicity for the irrelevant cell lines. The MLuC2 monoclonal antibody which gave rise to a completely ineffective immunoconjugate, induced internalization of less than one tenth of the antigenic sites whereas the MBr1, MLuC1 and MOv17 monoclonal antibodies exhibited about one third of the antigenic sites interanalized. From these data it is concluded that, providing an appropriate target antigen and coupling procedure are selected, restrictocin can be considered a suitable toxin for immunoconjugate generation.

Allergens↗
PubMed1992

Efficacy of an anti-CD7-ricin A chain immunoconjugate in a novel murine model of human T-cell leukemia.

In vivo efficacy testing of monoclonal antibody-based drugs specific for human leukemias is hampered by the paucity of suitable animal models, due in part to the inability of many anti-human monoclonal antibodies to cross-react with antigens expressed in animal tissues or cells. Moreover, human leukemic cells have proven difficult to establish in immunosuppressed mice except as solid tumors. We report here the establishment of a murine model for human leukemia displaying features of human disease, such as growth of malignant cells and localization of such cells to lymphoid compartments, and the effective depletion of leukemic cells from these mice by an immunoconjugate. Human T-leukemia cells (CEM) injected into cyclophosphamide-pretreated NIH-III mice engrafted in all mice (n = 41), with CEM cells detected in the bone marrow, spleen, and blood 4 weeks after injection. There was no evidence of solid tumors. Treatment of CEM-engrafted mice with 4A2-RTA30, an immunoconjugate of an anti-CD7 monoclonal antibody and ricin A chain (RTA30), resulted in a 100- to 200-fold overall depletion of CEM cells from the spleen and the bone marrow (P less than 0.02). This depletion was specific and toxin-dependent, as a control immunoconjugate had no demonstrable effect (P greater than 0.5). Depletion of CEM cells was also observed after treatment with unconjugated anti-CD7 mAb, but this effect was not significantly different from controls (P greater than 0.1). Therefore, significant depletion of CEM cells required the presence of the ricin A chain moiety. Further investigations revealed that CEM cells recovered from NIH-III mice expressed less CD7 antigen, but remained sensitive to subsequent in vitro exposure to 4A2-RTA30. In conclusion, we have established a model for studying the efficacy of immunoconjugates and have successfully depleted human T-leukemic cells from lymphoid tissues in immunodeficient mice by treatment with an anti-CD7-RTA30 immunoconjugate.

Animals↗
PubMed1991

Phase I trial of H65-RTA immunoconjugate in patients with cutaneous T-cell lymphoma.

H65-RTA is an immunoconjugate that consists of the A chain of ricin (RTA), a ribosomal-inhibiting protein, coupled to a murine monoclonal antibody (H65) directed against the pan-T-cell antigen CD5. The CD5 antigen is heterogeneously expressed on cutaneous T-cell lymphoma tumor cells, but is not expressed on normal cells except lymphocytes. A phase I trial was therefore conducted in which 14 patients with cutaneous T-cell lymphoma progressive on other therapies were treated with up to three cycles of H65-RTA. The maximal tolerated dose (MTD) of H65-RTA was 0.33 mg/kg/d administered intravenously for 10 days as defined by dyspnea at rest at higher doses. Other reversible side effects included myalgia, mild hypoalbuminemia with weight gain, pedal edema, fatigue, fevers, and chills. Six patients received more than one cycle of H65-RTA without increased side effects compared with the first cycle. Pharmacokinetic analysis showed that peak serum drug levels were dose-dependent, and ranged from 1.13 to 5.56 micrograms/mL, with a terminal half-life ranging from 1.0 to 2.9 hours. The development of antibodies against the immunoconjugate was associated with a lower peak drug level, but not with enhanced side effects. Partial responses lasting from 3 to 8 months were documented in four patients. Three of the responding patients received more than one cycle of H65-RTA in the presence of anti-immunoconjugate antibodies. The results from this phase I trial suggest that H65-RTA is an active drug in the treatment of cutaneous T-cell lymphoma. The immunoconjugate may be safely administered repeatedly, even in the presence of anti-immunoconjugate antibodies, with responses noted. Additional studies at the MTD are needed to define the response rate in this disease.

Adult↗
PubMed1990

Evaluation in vitro of adriamycin immunoconjugates synthesized using an acid-sensitive hydrazone linker.

A novel method for linking Adriamycin (ADM) to monoclonal antibodies is described in which the 13-keto position of the anthracycline is used as the attachment site to the linker arm. A new ADM acylhydrazone derivative, Adriamycin 13-[3-(2-pyridyldithio)propionyl]hydrazone hydrochloride, which contains a pyridyl-protected disulfide, was synthesized and used for conjugation to monoclonal antibodies (MAbs) that were thiolated with N-succinimidyl 3-(pyridyldithiol)propionate or 2-iminothiolane. This resulted in formation of a linker between MAb and drug that contained a disulfide bond. Conjugation conditions were optimized to yield conjugates with high ADM:MAb molar ratios. The final immunoconjugate yields were found to decrease as the ADM:MAb molar ratio of the conjugates increased. Stability studies indicated that ADM was released from the immunoconjugates at mildly acidic pHs ranging from 4.5-6.5. Treatment of immunoconjugates with mild reducing agent dithiothreitol resulted in release of an acylhydrazone derivative of ADM. Flow-cytometric studies showed that the binding activity of various MAbs following conjugation to ADM was preserved at ADM:MAb molar ratios up to 10. Antibody-directed cytotoxicity was demonstrated under several assay conditions using combinations of antigen-positive and antigen-negative cells and binding and nonbinding immunoconjugates. In several experiments, ADM immunoconjugates were more potent than equivalent amounts of unconjugated ADM.

Antibodies, Monoclonal↗
PubMed2005

Analysis of the composition of immunoconjugates using size-exclusion chromatography coupled to mass spectrometry.

Recombinant monoclonal antibody drug products play an increasingly important role in the treatment of various diseases. Antibodies are large, multi-chain proteins and antibody preparations often contain several molecular variants, which renders them heterogeneous. The heterogeneity is further increased in immunoconjugates prepared by covalently linking several drug molecules per antibody molecule. As part of the product characterization, the molecular weights of the antibodies or their drug conjugates need to be measured. Electrospray ionization mass spectrometry (ESI-MS) is well suited for the analysis of recombinant antibodies and immunoconjugates. Sample preparation is an important element of ESI-MS analysis, in particular samples need to be freed of interfering charged species, such as salts and buffer components. In this paper, Amicon centrifugal filters, reversed-phase high-performance liquid chromatography (HPLC), and size-exclusion HPLC were evaluated for sample desalting. Size-exclusion HPLC, using aqueous acetonitrile as the mobile phase, directly coupled to ESI-MS provided the best performance and was optimized for the study of immunoconjugates. The results showed that antibodies carrying covalently linked maytansinoid molecules generated charge envelope profiles that differ from those of the non-conjugated antibody. For the determination of the distribution of the various conjugate species in an immunoconjugate sample prepared by randomly linking in the average 3.6 drug molecules per antibody molecule, the experimental conditions needed to be carefully selected to allow acquisition of the whole spectrum containing the charge envelopes of all species.

Chromatography, Gel↗
PubMed1996

Preparation and functional evaluation of new doxorubicin immunoconjugates containing an acid-sensitive linker on small-cell lung cancer cells.

The anthracycline doxorubicin (DOX) is one of the most effective drugs for the treatment of small-cell lung cancer (SCLC), but its clinical application is limited by unspecific side-effects like cardiotoxicity. In the present study doxorubicin was conjugated to the monoclonal antibodies (mAb) SEN7, MOC31, and SWA11 via a novel acid-sensitive hydrazone linker. These mAb recognize SCLC-associated antigens of cluster 1 (NCAM), cluster 2 (EGP-2/GA733-2), and cluster 4 (CD24) respectively. To assess their potential therapeutic use against SCLC, the antigen-binding activities, the rates of internalization and the cytotoxic effects of the immunoconjugates were examined on tumour cell lines. The preparation procedure preserved the antigen-binding activities of the mAb and yielded immunoconjugates with average drug:mAb ratios of 7:1. The hydrazone linker was found to be stable at neutral pH but to release doxorubicin under acidic conditions. In contrast to SEN7-DOX, MOC31-DOX and SWA11-DOX were rapidly internalized into SCLC target cells upon binding to their specific cell-surface antigens. Accordingly, both immunoconjugates proved to be highly cytotoxic agents, inhibiting thymidine incorporation by 50% at concentrations between 0.5 microM and 1 microM and were 100-fold more selective than free doxorubicin. The results suggest that binding to selective cell-surface antigens, rapid internalization and efficient release of doxorubicin from the mAb by acid hydrolysis are required for the selective and potent function of the immunoconjugates. In particular, the use of MOC31-DOX for targeted cytotoxic therapy might be promising because of the limited cross-reactivity of the mAb with normal human tissues and its recently demonstrated tumour localization potential in SCLC patients.

Antibiotics, Antineoplastic↗
PubMed1995

Cytolysis of human erythrocytes by a covalent antibody-selenium immunoconjugate.

A selenium (Se)-containing immunoconjugate of a human anti-erythrocyte membrane antibody (Ab-Se) has been synthesized via oxidation of the carbohydrate moieties of the antibody and covalent coupling with selenocystamine. The isolated Ab-Se immunoconjugate is shown to be more hemolytic than is selenocystamine when expressed on equivalent selenium basis. Native antibody preincubated with the human erythrocytes prevented hemolysis induced by the Ab-Se immunoconjugate. As observed microscopically, the Ab-Se immunoconjugate caused extensive damage to the erythrocyte membrane and lysis. The cytotoxicity of Se toward the human erythrocytes is believed to be caused initially by the localized generation of superoxide (O2.-) within the cell membrane. This is the first demonstration of site-directed immunoselectivity of Se cytotoxicity and demonstrates the potential for a free radical pharmacology based on localized Se-generated O2.-.

Cystamine↗
PubMed2005

Arming antibodies: prospects and challenges for immunoconjugates.

Immunoconjugates--monoclonal antibodies (mAbs) coupled to highly toxic agents, including radioisotopes and toxic drugs (ineffective when administered systemically alone)--are becoming a significant component of anticancer treatments. By combining the exquisite targeting specificity of mAbs with the enhanced tumor-killing power of toxic effector molecules, immunoconjugates permit sensitive discrimination between target and normal tissue, resulting in fewer toxic side effects than most conventional chemotherapeutic drugs. Two radioimmunoconjugates, ibritumomab tiuxetan (Zevalin) and tositumomab-131I (Bexxar), and one drug conjugate, gemtuzumab ozogamicin (Mylotarg), are now on the market. For the next generation of immunoconjugates, advances in protein engineering will permit greater control of mAb targeting, clearance and pharmacokinetics, resulting in significantly improved delivery to tumors of radioisotopes and potent anticancer drugs. Pre-targeting strategies, which separate the two functions of antibody-based localization and delivery or generation of the toxic agent into two steps, also promise to afford superior tumor targeting and therapeutic efficacy. Several challenges in optimizing immunoconjugates remain, however, including poor intratumoral mAb uptake, normal tissue conjugate exposure and issues surrounding drug potency and conditional release from mAb carriers. Nonetheless, highly promising results from preclinical models will continue to drive the clinical development of this therapeutic class.

Aminoglycosides↗
PubMed2000

Immunoconjugates of geldanamycin and anti-HER2 monoclonal antibodies: antiproliferative activity on human breast carcinoma cell lines.

BACKGROUND: HER2 is a membrane receptor whose overexpression is strongly associated with poor prognosis in breast carcinomas. Inhibition of HER2 activity can reduce tumor growth, which led to the development of Herceptin, an anti-HER2 monoclonal antibody (MAb) that is already in clinical use. However, the objective response rate to Herceptin monotherapy is quite low. HER2 activity can also be inhibited by the highly cytotoxic antibiotic geldanamycin (GA). However, GA is not used clinically because of its adverse toxicity. Our purpose was to enhance the inhibitory activity of anti-HER2 MAb by coupling it to GA. METHODS: We synthesized 17-(3-aminopropylamino)GA (17-APA-GA) and conjugated it to the anti-HER2 MAb e21, to form e21 : GA. The noninternalizing anti-HER2 MAb AE1 was used as a control. Internalization assays and western blot analyses were used to determine whether the anti-HER2 MAbs and their immunoconjugates were internalized into HER2-expressing cells and reduced HER2 levels. All statistical tests were two-sided. RESULTS: The immunoconjugate e21 : GA inhibited the proliferation of HER2-overexpressing cell lines better than unconjugated e21 (concentration required for 50% inhibition = 40 versus 1650 microg/mL, respectively). At 15 microg/mL, e21 : GA reduced HER2 levels by 86% within 16 hours, whereas unconjugated e21, 17-APA-GA, or AE1 : GA reduced HER2 levels by only 20%. These effects were not caused by release of 17-APA-GA from the immunoconjugate because immunoconjugates containing [(3)H]GA were stable in serum at 37 degrees C. Furthermore, e21 : GA did not significantly inhibit proliferation of the adult T-cell leukemia cell line HuT102, which is HER2 negative yet highly sensitive to GA. CONCLUSIONS: Our findings suggest that conjugating GA to internalizing MAbs enhances the inhibitory effect of the MAbs. This approach might also be applied in cellular targeting via growth factors and may be of clinical interest.

Animals↗
PubMed2004

Tumour-targeted chemotherapy with immunoconjugates of calicheamicin.

Antibody-targeted chemotherapy is a therapeutic strategy in cancer therapy that involves a monoclonal antibody specific for a tumour-associated antigen, covalently linked via a suitable linker to a potent cytotoxic agent. Tumour-targeted delivery of a cytotoxic agent in the form of an immunoconjugate is expected to improve its antitumour activity and safety. Calicheamicin is a cytotoxic natural product isolated from Micromonospora echinospora that is at least 1000-fold more potent than conventional cytotoxic chemotherapeutics. Calicheamicin binds DNA in the minor groove and causes double-strand DNA breaks, leading to cell death. Immunoconjugates of calicheamicin targeted against tumour-associated antigens exhibit tumour-specific cytotoxic effects and cause regression of established human tumour xenografts in nude mice. Gemtuzumab ozogamicin is the first clinically validated cytotoxic immunoconjugate in which a humanised anti-CD33 antibody is linked to a derivative of calicheamicin. Gemtuzumab ozogamicin is indicated for the treatment of elderly patients with relapsed acute myeloid leukaemia. A similar conjugate, inotuzumab ozogamicin, is being evaluated at present in Phase I clinical trials in patients with non-Hodgkin's lymphoma. A number of tumour-targeted immunoconjugates of calicheamicin are being explored preclinically at present for their therapeutic applications.

Aged↗
PubMed1993

The comparative effects of IL-1 and TNF on AMN-anti-Ly 2.1 immunoconjugate therapy.

The administration of mTNF alpha and hIL-1 alpha was investigated for their potential to increase the anti-tumor activity of AMN-anti-Ly-2.1 against the Ly-2.1+ murine thymoma ITT(1) 75 NS E3. Dose response studies using mTNF alpha alone demonstrated a single 10 micrograms iv injection produced 30% inhibition in tumor size while 3 doses of 1 microgram administered on alternative days produced 70% tumor inhibition. By contrast, hIL-1 alpha was unable to significantly reduce E3 tumor size using single doses up to 10 micrograms or a total of 30 micrograms administered in 3 doses (iv or ip). However, intratumor injection of hIL-1 alpha (20 micrograms injected in 2 doses) produced 20% inhibition in tumor size. Combination therapy using AMN immunoconjugates with mTNF alpha showed enhanced antitumor activity compared to each agent alone. Biodistribution studies revealed that anti-tumor activity, was due to increased localization (2-3 fold) of AMN immunoconjugates in the presence of mTNF-alpha whereas huIL-1 alpha was without effect unless accompanying toxicity was seen. Clearly for this tumor, mTNF alpha potentiated the effects of AMN immunoconjugates. Despite the shared biological properties of these cytokines, mTNF alpha is superior to hIL-alpha for augmenting drug immunoconjugate.

Aminopterin↗