ABSTRACT
The potential of CD123-targeted therapies in acute lymphoblastic leukemia/lymphoma remains largely unexplored. We examined CD123 expression levels in a large cohort of patients with acute lymphoblastic leukemia/lymphoma and assessed the in vitro impact of IMGN632, a conjugate of CD123-binding antibody with a novel DNA-alkylating payload. CD123 expression on leukemic blasts was surveyed using multicolor/multiparameter flow cytometry. The in vitro effect of IMGN632 was evaluated on B acute lymphoblastic leukemia/lymphoma cell lines and primary B acute lymphoblastic leukemia/lymphoma blasts. The study cohort (n=213) included 183 patients with B acute lymphoblastic leukemia/lymphoma and 30 with T acute lymphoblastic leukemia/lymphoma. CD123 expression was more prevalent in B acute lymphoblastic leukemia/lymphoma than in T acute lymphoblastic leukemia/lymphoma (164/183, 89.6% versus 13/30, 43.3%; P<0.0001), and within B acute lymphoblastic leukemia/lymphoma CD123 expression was more prevalent in Philadelphia chromosome-positive patients than in Philadelphia chromosome-negative patients (96.6% versus 86.3%; P=0.033). In T acute lymphoblastic leukemia/lymphoma, 12/13 (92.3%) patients with CD123-positive blasts had either early T precursor (ETP) or early non-ETP immunophenotype. IMGN632 was highly cytotoxic to B acute lymphoblastic leukemia/lymphoma cell lines, with half maximal inhibitory concentrations (IC50) between 0.6 and 20 pM. In five of eight patients' samples, low picomolar concentrations of IMGN632 eliminated more than 90% of the B acute lymphoblastic leukemia/lymphoma blast population, sparing normal lymphocytes. In conclusion, CD123 expression is prevalent across acute lymphoblastic leukemia/lymphoma subtypes, and the CD123-targeted antibody-drug conjugate IMGN632 demonstrates promising selective activity in preclinical models of B acute lymphoblastic leukemia/lymphoma.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Drug Delivery Systems , Gene Expression Regulation, Leukemic/drug effects , Immunoconjugates/pharmacology , Interleukin-3 Receptor alpha Subunit , Neoplasm Proteins , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Adolescent , Adult , Aged , Aged, 80 and over , Cell Line, Tumor , Child , Child, Preschool , Female , Humans , Infant , Interleukin-3 Receptor alpha Subunit/antagonists & inhibitors , Interleukin-3 Receptor alpha Subunit/biosynthesis , Male , Middle Aged , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/biosynthesis , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathologyABSTRACT
Antibody anilino maytansinoid conjugates (AaMCs) have been prepared in which a maytansinoid bearing an aniline group was linked through the aniline amine to a dipeptide, which in turn was covalently attached to a desired monoclonal antibody. Several such conjugates were prepared utilizing different dipeptides in the linkage including Gly-Gly, l-Val-l-Cit, and all four stereoisomers of the Ala-Ala dipeptide. The properties of AaMCs could be altered by the choice of dipeptide in the linker. Each of the AaMCs, except the AaMC bearing a d-Ala-d-Ala peptide linker, displayed more bystander killing in vitro than maytansinoid ADCs that utilize disulfide linkers. In mouse models, the anti-CanAg AaMC bearing a d-Ala-l-Ala dipeptide in the linker was shown to be more efficacious against heterogeneous HT-29 xenografts than maytansinoid ADCs that utilize disulfide linkers, while both types of the conjugates displayed similar tolerabilities.
Subject(s)
Aniline Compounds/chemistry , Antineoplastic Agents, Phytogenic/chemistry , Immunoconjugates/chemistry , Maytansine/chemistry , Aniline Compounds/pharmacokinetics , Aniline Compounds/therapeutic use , Animals , Antineoplastic Agents, Phytogenic/pharmacokinetics , Antineoplastic Agents, Phytogenic/therapeutic use , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Humans , Immunoconjugates/pharmacokinetics , Immunoconjugates/therapeutic use , Maytansine/pharmacokinetics , Maytansine/therapeutic use , Mice , Neoplasms/drug therapyABSTRACT
Several antibody-maytansinoid conjugates (AMCs) are in clinical trials for the treatment of various cancers. Each of these conjugates can be metabolized by tumor cells to give cytotoxic maytansinoid metabolites that can kill targeted cells. In preclinical studies in mice, the cytotoxic metabolites initially formed in vivo are further processed in the mouse liver to give several oxidized metabolic species. In this work, the primary AMC metabolites were synthesized and incubated with human liver microsomes (HLMs) to determine if human liver would likely give the same metabolites as those formed in mouse liver. The results of these HLM metabolism studies as well as the subsequent syntheses of the resulting HLM oxidation products are presented. Syntheses of the minor impurities formed during the conjugation of AMCs were also conducted to determine their cytotoxicities and to establish how these impurities would be metabolized by HLM.
Subject(s)
Immunoconjugates/chemistry , Immunoconjugates/metabolism , Maytansine/chemistry , Animals , Cell Line, Tumor , Humans , Magnetic Resonance Spectroscopy , Mice , Microsomes, Liver , Xenograft Model Antitumor AssaysABSTRACT
Antibody-drug conjugates (ADCs) are designed to eradicate cancer cells that express the target antigen on their cell surface. A key component of an ADC is the linker that covalently connects the cytotoxic agent to the antibody. Several antibody-maytansinoid conjugates prepared with disulfide-based linkers such as those targeting the CanAg antigen have been shown to display more activity in preclinical mouse xenograft models than corresponding conjugates prepared with uncleavable thioether-based linkers. To investigate how the linker influences delivery and activation of antibody-maytansinoid conjugates, we isolated and characterized the [(3)H]maytansinoids from CanAg-positive tumor tissues following a single intravenous administration of 300 microg/kg (based on maytansinoid dose) of anti-CanAg antibody (huC242)-(3)H-maytansinoid conjugates prepared with cleavable disulfide linkers and an uncleavable thioether linker. We identified three target-dependent tumor metabolites of the disulfide-linked huC242-SPDB-DM4, namely, lysine-N(epsilon)-SPDB-DM4, DM4, and S-methyl-DM4. We found similar metabolites for the less hindered disulfide-linked huC242-SPP-DM1 conjugate with the exception that no S-methyl-DM1 was detected. The sole metabolite of the uncleavable thioether-linked huC242-SMCC-DM1 was lysine-N(epsilon)-SMCC-DM1. The AUC for the metabolites of huC242-SMCC-DM1 at the tumor over 7 d was about 2-fold greater than the corresponding AUC for the metabolites of the disulfide-linked conjugates. The lipophilic metabolites of the disulfide-linked conjugates were found to be nearly 1000 times more cytotoxic than the more hydrophilic lysine-N(epsilon)-linker-maytansinoids in cell-based viability assays when added extracellularly. The cell killing properties associated with the lipophilic metabolites of the disulfide-linked conjugates (DM4 and S-methyl-DM4, and DM1) provide an explanation for the superior in vivo efficacy that is often observed with antibody-maytansinoid conjugates prepared with disulfide-based linkers in xenograft mouse models.
Subject(s)
Antibodies/metabolism , Disulfides/chemistry , Immunoconjugates/metabolism , Immunoconjugates/therapeutic use , Maytansine/metabolism , Neoplasms/metabolism , Sulfides/chemistry , Animals , Antibodies/chemistry , Antibodies/immunology , Antibodies/therapeutic use , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Immunoconjugates/chemistry , Immunoconjugates/immunology , Maytansine/chemistry , Maytansine/immunology , Maytansine/therapeutic use , Mice , Mice, SCID , Neoplasms/drug therapy , Neoplasms/immunology , Xenograft Model Antitumor AssaysABSTRACT
Conjugates of the anti-CanAg humanized monoclonal antibody huC242 with the microtubule-formation inhibitor DM1 (a maytansinoid), or with the DNA alkylator DC1 (a CC1065 analogue), have been evaluated for their ability to eradicate mixed cell populations formed from CanAg-positive and CanAg-negative cells in culture and in xenograft tumors in mice. We found that in culture, conjugates of either drug killed not only the target antigen-positive cells but also the neighboring antigen-negative cells. Furthermore, we showed that, in vivo, these conjugates were effective in eradicating tumors containing both antigen-positive and antigen-negative cells. The presence of antigen-positive cells was required for this killing of bystander cells. This target cell-activated killing of bystander cells was dependent on the nature of the linker between the antibody and the drug. Conjugates linked via a reducible disulfide bond were capable of exerting the bystander effect whereas equally potent conjugates linked via a nonreducible thioether bond were not. Our data offer a rationale for developing optimally constructed antibody-drug conjugates for treating tumors that express the target antigen either in a homogeneous or heterogeneous manner.
Subject(s)
Colonic Neoplasms/drug therapy , Immunoconjugates/pharmacology , Maytansine/analogs & derivatives , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antigens, Neoplasm/immunology , Cell Line, Tumor , Colonic Neoplasms/immunology , Cross-Linking Reagents/chemistry , Female , HT29 Cells , Humans , Immunoconjugates/chemistry , Immunoconjugates/immunology , Maytansine/chemistry , Maytansine/pharmacology , Mice , Mice, SCID , Xenograft Model Antitumor AssaysABSTRACT
The outlook for patients with refractory/relapsed acute myeloid leukemia (AML) remains poor, with conventional chemotherapeutic treatments often associated with unacceptable toxicities, including severe infections due to profound myelosuppression. Thus there exists an urgent need for more effective agents to treat AML that confer high therapeutic indices and favorable tolerability profiles. Because of its high expression on leukemic blast and stem cells compared with normal hematopoietic stem cells and progenitors, CD123 has emerged as a rational candidate for molecularly targeted therapeutic approaches in this disease. Here we describe the development and preclinical characterization of a CD123-targeting antibody-drug conjugate (ADC), IMGN632, that comprises a novel humanized anti-CD123 antibody G4723A linked to a recently reported DNA mono-alkylating payload of the indolinobenzodiazepine pseudodimer (IGN) class of cytotoxic compounds. The activity of IMGN632 was compared with X-ADC, the ADC utilizing the G4723A antibody linked to a DNA crosslinking IGN payload. With low picomolar potency, both ADCs reduced viability in AML cell lines and patient-derived samples in culture, irrespective of their multidrug resistance or disease status. However, X-ADC exposure was >40-fold more cytotoxic to the normal myeloid progenitors than IMGN632. Of particular note, IMGN632 demonstrated potent activity in all AML samples at concentrations well below levels that impacted normal bone marrow progenitors, suggesting the potential for efficacy in AML patients in the absence of or with limited myelosuppression. Furthermore, IMGN632 demonstrated robust antitumor efficacy in multiple AML xenograft models. Overall, these findings identify IMGN632 as a promising candidate for evaluation as a novel therapy in AML.
Subject(s)
Immunoconjugates/therapeutic use , Interleukin-3 Receptor alpha Subunit/immunology , Leukemia, Myeloid, Acute/drug therapy , Animals , Cell Line, Tumor , Cell Survival/drug effects , Hematopoietic Stem Cells/drug effects , Heterografts , Humans , Immunoconjugates/immunology , Mice , Neoplastic Stem Cells/drug effects , Tumor Cells, CulturedABSTRACT
The promise of tumor-selective delivery of cytotoxic agents in the form of antibody-drug conjugates (ADC) has now been realized, evidenced by the approval of two ADCs, both of which incorporate highly cytotoxic tubulin-interacting agents, for cancer therapy. An ongoing challenge remains in identifying potent agents with alternative mechanisms of cell killing that can provide ADCs with high therapeutic indices and favorable tolerability. Here, we describe the development of a new class of potent DNA alkylating agents that meets these objectives. Through chemical design, we changed the mechanism of action of our novel DNA cross-linking agent to a monofunctional DNA alkylator. This modification, coupled with linker optimization, generated ADCs that were well tolerated in mice and demonstrated robust antitumor activity in multiple tumor models at doses 1.5% to 3.5% of maximally tolerated levels. These properties underscore the considerable potential of these purpose-created, unique DNA-interacting conjugates for broadening the clinical application of ADC technology. Mol Cancer Ther; 15(8); 1870-8. ©2016 AACR.
Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Immunoconjugates/pharmacology , Animals , Antineoplastic Agents, Alkylating/chemistry , Bystander Effect , Cell Line, Tumor , Cell Survival/drug effects , DNA/chemistry , DNA/metabolism , DNA Adducts , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Humans , Immunoconjugates/chemistry , Mice , Molecular Structure , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacokinetics , Maytansine/pharmacokinetics , Neoplasms/metabolism , Adult , Aged , Antibodies, Monoclonal , Antineoplastic Agents, Phytogenic/pharmacology , Dose-Response Relationship, Drug , Female , Half-Life , Humans , Immunoconjugates , Infusions, Intravenous , Male , Maximum Tolerated Dose , Maytansine/pharmacology , Middle Aged , Mucin-1/immunologyABSTRACT
The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4-6 × 10(7) copies). Efflux of 3[H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of 3 [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death.
Subject(s)
Maytansine/metabolism , Tubulin/metabolism , Cell Line, Tumor , Dose-Response Relationship, Drug , Humans , Liposomes , Maytansine/pharmacology , Microtubules/drug effects , Microtubules/metabolism , Models, Biological , Protein BindingABSTRACT
Lorvotuzumab mertansine (LM) is an antibody-drug conjugate composed of a humanized anti-CD56 antibody, lorvotuzumab, linked via a cleavable disulfide linker to the tubulin-binding maytansinoid DM1. CD56 is expressed on most small cell lung cancers (SCLC), providing a promising therapeutic target for treatment of this aggressive cancer, which has a poor five-year survival rate of only 5-10%. We performed immunohistochemical staining on SCLC tumor microarrays, which confirmed that CD56 is expressed at high levels on most (~74%) SCLC tumors. Conjugation of lorvotuzumab with DM1 did not alter its specific binding to cells and LM demonstrated potent target-dependent cytotoxicity against CD56-positive SCLC cells in vitro. The anti-tumor activity of LM was evaluated against SCLC xenograft models in mice, both as monotherapy and in combination with platinum/etoposide and paclitaxel/carboplatin. Dose-dependent and antigen-specific anti-tumor activity of LM monotherapy was demonstrated at doses as low as 3 mg/kg. LM was highly active in combination with standard-of-care platinum/etoposide therapies, even in relatively resistant xenograft models. LM demonstrated outstanding anti-tumor activity in combination with carboplatin/etoposide, with superior activity over chemotherapy alone when LM was used in combinations at significantly reduced doses (6-fold below the minimally efficacious dose for LM monotherapy). The combination of LM with carboplatin/paclitaxel was also highly active. This study provides the rationale for clinical evaluation of LM as a promising novel targeted therapy for SCLC, both as monotherapy and in combination with chemotherapy.
Subject(s)
Antibodies, Monoclonal/metabolism , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , CD56 Antigen/immunology , Immunotherapy/methods , Lung Neoplasms/therapy , Maytansine/analogs & derivatives , Maytansine/metabolism , Small Cell Lung Carcinoma/therapy , Tubulin Modulators/metabolism , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Cell Line, Tumor , Cytotoxicity, Immunologic , Drug Synergism , Drug Therapy, Combination , Female , Humans , Lung Neoplasms/immunology , Maytansine/chemistry , Maytansine/immunology , Mice , Mice, SCID , Small Cell Lung Carcinoma/immunology , Tubulin Modulators/chemistry , Xenograft Model Antitumor AssaysABSTRACT
Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate consisting of the anti-HER2 antibody trastuzumab linked via a nonreducible thioether linker to the maytansinoid antitubulin agent DM1. T-DM1 has shown favorable safety and efficacy in patients with HER2-positive metastatic breast cancer. In previous animal studies, T-DM1 exhibited better pharmacokinetics (PK) and slightly more efficacy than several disulfide-linked versions. The efficacy findings are unique, as other disulfide-linked antibody-drug conjugates (ADC) have shown greater efficacy than thioether-linked designs. To explore this further, the in vitro and in vivo activity, PK, and target cell activation of T-DM1 and the disulfide-linked T-SPP-DM1 were examined. Both ADCs showed high in vitro potency, with T-DM1 displaying greater potency in two of four breast cancer cell lines. In vitro target cell processing of T-DM1 and T-SPP-DM1 produced lysine-N(ε)-MCC-DM1, and lysine-N(ε)-SPP-DM1 and DM1, respectively; in vivo studies confirmed these results. The in vitro processing rates for the two conjugate to their respective catabolites were similar. In vivo, the potencies of the conjugates were similar, and T-SPP-DM1 had a faster plasma clearance than T-DM1. Slower T-DM1 clearance translated to higher overall tumor concentrations (conjugate plus catabolites), but unexpectedly, similar levels of tumor catabolite. These results indicate that, although the ADC linker can have clear impact on the PK and the chemical nature of the catabolites formed, both linkers seem to offer the same payload delivery to the tumor.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Maytansine/analogs & derivatives , Ado-Trastuzumab Emtansine , Animals , Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Monoclonal, Humanized/metabolism , Antibodies, Monoclonal, Humanized/pharmacokinetics , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Cell Survival/drug effects , Female , Humans , Maytansine/chemistry , Maytansine/metabolism , Maytansine/pharmacokinetics , Maytansine/pharmacology , Mice , Mice, Nude , Receptor, ErbB-2/metabolism , Trastuzumab , Xenograft Model Antitumor AssaysABSTRACT
The synthesis and biological evaluation of hydrophilic heterobifunctional cross-linkers for conjugation of antibodies with highly cytotoxic agents are described. These linkers contain either a negatively charged sulfonate group or a hydrophilic, noncharged PEG group in addition to an amine-reactive N-hydroxysuccinimide (NHS) ester and sulfhydryl reactive termini. These hydrophilic linkers enable conjugation of hydrophobic organic molecule drugs, such as a maytansinoid, at a higher drug/antibody ratio (DAR) than hydrophobic SPDB and SMCC linkers used earlier without triggering aggregation or loss of affinity of the resulting conjugate. Antibody-maytansinoid conjugates (AMCs) bearing these sulfonate- or PEG-containing hydrophilic linkers were, depending on the nature of the targeted cells, equally to more cytotoxic to antigen-positive cells and equally to less cytotoxic to antigen-negative cells than conjugates made with SPDB or SMCC linkers and thus typically displayed a wider selectivity window, particularly against multidrug resistant (MDR) cancer cell lines in vitro and tumor xenograft models in vivo.
Subject(s)
Antibodies/chemistry , Immunoconjugates/chemistry , Maytansine/chemistry , Animals , Chemistry, Pharmaceutical/methods , Drug Design , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Humans , Mice , Models, Chemical , Neoplasm Transplantation , Polyethylene Glycols/chemistry , Succinimides/chemistry , Sulfones/chemistryABSTRACT
Maytansine and its analogues (maytansinoids) are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis. Antibody-maytansinoid conjugates consisting of maytansinoids (DM1 and DM4) attached to tumor-specific antibodies have shown promising clinical results. To determine the mechanism by which the antibody-DM1 conjugates inhibit cell proliferation, we examined the effects of the cleavable anti-EpCAM-SPP-DM1 and uncleavable anti-EpCAM-SMCC-DM1 conjugates on MCF7 human breast tumor cells. We also examined the effects of the free maytansinoids, maytansine and S-methyl DM1 (a version of DM1 that is stable in cell culture medium), for comparison. Both the conjugates and free maytansinoids potently inhibited MCF7 cell proliferation at nanomolar and subnanomolar concentrations, respectively, by arresting the cells in mitotic prometaphase/metaphase. Arrest occurred in concert with the internalization and intracellular processing of both conjugates under conditions that induced abnormal spindle organization and suppressed microtubule dynamic instability. Microtubule depolymerization occurred only at significantly higher drug concentrations. The results indicate that free maytansinoids, antibody-maytansinoid conjugates, and their metabolites exert their potent antimitotic effects through a common mechanism involving suppression of microtubule dynamic instability.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Immunoconjugates/pharmacology , Maytansine/pharmacology , Microtubules/drug effects , Mitosis/drug effects , Antineoplastic Agents, Phytogenic/immunology , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Chromatography, High Pressure Liquid , Humans , Immunoconjugates/immunology , Maytansine/immunology , Microscopy, Fluorescence , Mitosis/immunologyABSTRACT
Conjugation of cytotoxic compounds to antibodies that bind to cancer-specific antigens makes these drugs selective in killing cancer cells. However, many of the compounds used in such antibody-drug conjugates (ADC) are substrates for the multidrug transporter MDR1. To evade the MDR1-mediated resistance, we conjugated the highly cytotoxic maytansinoid DM1 to antibodies via the maleimidyl-based hydrophilic linker PEG(4)Mal. Following uptake into target cells, conjugates made with the PEG(4)Mal linker were processed to a cytotoxic metabolite that was retained by MDR1-expressing cells better than a metabolite of similar conjugates prepared with the nonpolar linker N-succinimidyl-4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). In accord, PEG(4)Mal-linked conjugates were more potent in killing MDR1-expressing cells in culture. In addition, PEG(4)Mal-linked conjugates were markedly more effective in eradicating MDR1-expressing human xenograft tumors than SMCC-linked conjugates while being tolerated similarly, thus showing an improved therapeutic index. This study points the way to the development of ADCs that bypass multidrug resistance.
Subject(s)
Immunotoxins/pharmacology , Maytansine/analogs & derivatives , ATP Binding Cassette Transporter, Subfamily B , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Adenocarcinoma/drug therapy , Adenocarcinoma/metabolism , Animals , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/immunology , Carcinoma, Renal Cell/drug therapy , Carcinoma, Renal Cell/metabolism , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/immunology , Cell Line, Tumor , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Epithelial Cell Adhesion Molecule , Female , Humans , Immunotoxins/chemistry , Immunotoxins/pharmacokinetics , Kidney Neoplasms/drug therapy , Kidney Neoplasms/metabolism , Maleimides/chemistry , Maytansine/chemistry , Maytansine/pharmacokinetics , Maytansine/pharmacology , Mice , Mice, SCID , Polyethylene Glycols/chemistryABSTRACT
The humanized monoclonal antibody maytansinoid conjugate, cantuzumab mertansine (huC242-DM1) that contains on average three to four linked drug molecules per antibody molecule was evaluated in CD-1 mice for its pharmacokinetic behavior and tissue distribution, and the results were compared with those of the free antibody huC242. The pharmacokinetics in blood were similar for (125)I-labeled conjugate and antibody with terminal half-lives of 154 and 156 h, respectively. Pharmacokinetic analysis using an enzyme-linked immunosorbent assay (ELISA) method, which measures intact conjugate in plasma samples revealed a faster clearance for the conjugate corresponding to a half-life of 42.2 h. This faster clearance is explained as the result of clearance from circulation and concomitant clearance of drug from circulating conjugate through linker cleavage. An antibody-specific ELISA allowed the determination of the clearance rate of the antibody component from circulation. The drug clearance rate from circulating conjugate was then calculated as the difference between the clearance of the conjugate and the clearance of the antibody component and found to be about three times that of the antibody component. The above results were confirmed with a conjugate, huC242-[(3)H]DM1, where the linked DM1 drugs carried a stable tritium label. Tissue distribution studies with (125)I-labeled conjugate and antibody showed antibody-like behavior for the conjugate; the antibody of the conjugate did not distribute or bind significantly to any solid tissue.