The L49 monoclonal antibody against the p97 antigen on melanomas and carcinomas was chemically conjugated to E. coli beta-galactosidase (beta-gal), forming a largely monomeric conjugate with preserved enzymatic activity. The resulting L49-beta-gal conjugate was used to activate (N-[(4"R,S)-4"-hexyloxy-4"-(1'''-O-beta-D-galactopyranosyl)butyl]daunorubicin) (1), a derivative of daunorubicin that has low cytotoxicity and high chemical stability. Addition of the conjugate to the prodrug resulted in an increase in cytotoxicity of approximately 10(5)-fold, a level of activation that is higher than any mAb-enzyme/prodrug combination yet described. Furthermore, the released drug had an IC(50) value of approximately 10 pM, making it significantly more potent than the vast majority of clinically approved anticancer drugs. The potential of this enzyme/prodrug combination for cancer therapy is discussed.
Abstract The activity of monoclonal antibodies (mAbs) can be enhanced by a number of chemical and genetic strategies. We describe a novel strategy for enhancing antibody-dependent cellular cytotoxicity (ADCC) through modification of the mAb carbohydrate during expression, designated Sugar Engineered Antibody (SEA) technology. A series of small molecule fucose analogs were added to mAb-expressing Chinese hamster ovary (CHO) cells, with the resulting mAbs showing a significant reduction in their carbohydrate fucosylation. We demonstrate that these mAbs show a substantial increase in ADCC activity and an improvement in CD16 binding. We find that the mechanism of action is inhibition of GDP-mannose dehydratase (GMD), the first enzyme in de novo synthesis of GDP-fucose, leading to a depletion of substrate for the fucosyltransferase responsible for mAb carbohydrate fucosylation. We also demonstrate that this strategy yields mAbs with significantly reduced fucosylation in large scale CHO cell culture as well as in additional expression systems such as hybridomas and transient transfectants. Since genetic modification of the mAb-producing cell line is not required, SEA technology can be readily applied from the mAb screening to manufacturing stage to generate effector function enhanced therapeutic antibodies. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B133.
Seven different monoclonal antibodies of theIgG class that are reactive with four different antigens on human lymphoid cells were utilized to form immunotoxins with the ribosome-inactivating proteins gelonin and the three known pokeweed antiviral proteins.Thirteen different immunotoxin combinations were prepared.The ribosome-inactivating proteins were modified with 2-iminothiolane.The sulfhydryl groups so introduced were reacted with maleimido groups or with dithiopyridyl groups that had been introduced into the antibodies.The toxin-antibody conjugates so formed were purified by affinity chromatography on protein A-Sepharose CL-4B, ion exchange chromatography, and by gel filtration and were characterized by polyacrylamide-dodecyl sulfate gel electrophoresis.The purified immunotoxins were free of nonconjugated monomeric proteins and aggregates of very high molecular weight.All the immunotoxins~showed the specific binding of the component antibody as measured by indirect immunofluorescence binding assays.The activities of the ribosome-inactivating proteins were unaffected by conjugation where the cross-link to the antibody contained a disulfide bond and when assayed after reductive cleavage of the linker.Disulfide-linked immunotoxins with six of the antibodies were highly cytotoxic for the target cells.However, immunotoxins containing an anti-B1 antibody showed no cytotoxicity.The possible use of antibodies to target pharmacologic agents, such as toxins, was first proposed by Ehrlich (1).Research to exploit this idea has developed rapidly in the last decade, owing much to the ability to produce pure highly specific monoclonal antibodies using the hybridoma technology ( 2 ) .Recently, monoclonal antibodies have been developed that recognize tumor-associated antigens (3, 4, 11, 12), and it is the hope that such antibodies can be exploited to deliver toxic agents to particular types of tumor cells in order to kill them selectively.The ribosome-inactivating proteins (5, 6) seem to be ideal toxic agents for this purpose.Most effort has been directed toward using ricin (extracted from castor beans, Ricinus communis) which consists of two nonidentical sub-
Proc Amer Assoc Cancer Res, Volume 46, 2005
6181
The class III receptor tyrosine kinase FLT3 is an attractive therapeutic target as it is overexpressed in blasts of ∼90% of acute myelogenous leukemia (AML) and the majority of B-lymphoid leukemia patients. Internal tandem duplications (ITDs) in the juxtamembrane region and point mutations in the kinase domain of FLT3 are found in ∼37% of AML patients and are associated with a poor prognosis. We have recently developed a fully human, high affinity monoclonal antibody (EB10) which is readily internalized upon binding to FLT3 receptor on human leukemia cells. In the present study, a novel auristatin conjugate of the anti-FLT3 antibody (EB10-MMAF) was prepared using a dipeptide linker that allows for drug release inside the lysosomes of antigen-positive cells. The MMAF conjugates were stable in buffers and plasma. EB10-MMAF (drug/antibody ratio = 8) retained the binding affinity and internalizing capability of EB10. The conjugate was highly potent, and selectively inhibited the growth of FLT3-expressing leukemia cells with an IC50 of 0.19 nM and 0.08 nM for MV4;11 and BaF3-ITD cells (both positive for FLT3-ITD), 1.11 nM, 6.18 nM and 1.82 nM for REH, EOL-1, EM3 cells (all three positive for wild-type FLT3), and 135 nM for JM1 (negative for FLT3). An MMAF conjugate with a control antibody was not active in these cell lines (IC50s > 5.9 uM). Flow cytometric analysis with annexin V indicated that EB10-MMAF treatment induced apoptosis of leukemia cells in vitro . In vivo treatment with EB10-MMAF strongly inhibited leukemia growth and prolonged survival of mice in both EOL-1 (mean survival time ∼81.0 +/− 46.8 days for the 3 mg/kg group compared to 41.5+/−15 days for the untreated group) and BaF3-ITD (mean survival time ∼91.5 +/− 39.5 days for the 5 mg/kg group compared to 32.8 +/− 5.2 days for the untreated group) leukemia models. In summary, immunoconjugates composed of a fully human anti-FLT3 antibody and a potent auristatin drug may provide a valuable therapeutic approach for AML and other FLT3-positive leukemias. In summary, immunoconjugates composed of a fully human anti-FLT3 antibody and a potent auristatin drug may provide a valuable therapeutic approach for AML and other types of FLT3-positive leukemia.
Nicotinamide phosphoribosyltransferase (NAMPT) regulates the biosynthesis of NAD from nicotinamide via a salvage biosynthetic pathway. Inhibition of NAMPT depletes cellular NAD levels leading to disruption of energy metabolism and cell death. Non-targeted small molecule NAMPT inhibitors have demonstrated poor tolerability in clinical trials and in preclinical models, including cardiac and retinal toxicities in rats. In an effort to improve the therapeutic window of this drug class, we pursued a targeted-delivery approach using antibody-drug conjugates. Through a medicinal chemistry effort, we identified novel NAMPT inhibitors that incorporate chemical functionality in the solvent-exposed terminus to allow construction of enzyme-cleavable drug linkers. Additionally, we applied a pyridinium-based linker strategy that allows for traceless linker attachment through a conserved nicotinamide-mimetic moiety of NAMPT inhibitors. Candidate molecules were evaluated for NAMPT binding affinity and cellular cytotoxicity as free drugs, and for cellular cytotoxicity as ADCs with the alternate linker strategies. Comparisons across inhibitors and linker strategies provide insight into optimal design of cleavable drug linkers for this class of drugs. In vitro, the ADCs deplete NAD and lead to downstream ATP depletion in a time-dependent manner. In vivo evaluation using human tumor xenografts shows translation of the pharmacodynamic effect resulting in tumor regression in models of Hodgkin lymphoma, non-Hodgkin lymphoma, and acute myeloid leukemia. Toxicology studies in Sprague Dawley rats demonstrate excellent tolerability at active doses, with no observable cardiac or retinal toxicities at the highest tested doses in single- and multi-dose regimens. These findings detail the development of a novel payload class and optimized linker strategy for use with antibody-drug conjugates, and demonstrate a preclinical efficacy and safety profile to support continued efforts toward clinical therapeutics.Citation Format: Chris Neumann, Kathleen C. Olivas, Kung Pern Wang, Andrew B. Waight, David W. Meyer, Luke V. Loftus, Margo C. Zaval, Martha E. Anderson, Steven Jin, Julia H. Cochran, Jessica K. Simmons, Paul G. Pittman, Fu Li, Michelle L. Ulrich, Abbie Wong, Weiping Zeng, Robert P. Lyon, Peter D. Senter. Antibody-drug conjugates of NAMPT inhibitors: Discovery, optimization, and preclinical characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 983.
Abstract The EphA2 receptor tyrosine kinase is selectively expressed on the surface of many different human tumors. We have previously shown that tumor cells can be targeted by EphA2 monoclonal antibodies and that these antibodies function, in part, by inducing EphA2 internalization and degradation. In this report, we describe the isolation and characterization of a fully human monoclonal antibody (1C1) that selectively binds both the human and rodent EphA2 receptor. After cell binding, the antibody induces rapid tyrosine phosphorylation, internalization, and degradation of the EphA2 receptor. Because monoclonal antibodies that selectively bind tumor cells and internalize provide a vehicle for targeted delivery of cytotoxics, 1C1 was conjugated to the microtubule inhibitor monomethylauristatin phenylalanine using a stable maleimidocaproyl linker. The anti-EphA2 antibody-drug conjugate [1C1–maleimidocaproyl-MMAF (mcMMAF)] stimulated the activation of caspase-3/caspase-7 and the death of EphA2-expressing cells with IC50 values as low as 3 ng/mL. Similarly, the conjugate induced degradation of the EphA2 receptor and inhibited tumor growth in vivo. Administration of 1C1-mcMMAF at doses as low as 1 mg/kg once weekly resulted in significant growth inhibition of EphA2-expressing tumors without any observable adverse effects in mouse xenograft and rat syngeneic tumor models. Our data support the use of an antibody-drug conjugate approach to selectively target and inhibit the growth of EphA2-expressing tumors. [Cancer Res 2008;68(22):9367–74]
