ABSTRACT
Camptothecins exist in a pH-dependent equilibrium between the active, closed lactone and the inactive open-carboxylate forms. Several previous reports underscore the need for lactone stabilization in generating improved camptothecins, and indeed, such designs have been incorporated into antibody-drug conjugates containing this drug. Here, we demonstrate that lactone stabilization is not necessary for camptothecin-based ADC efficacy. We synthesized and evaluated camptothecin SN-38 drug linkers that differed with respect to lactone stability and released SN-38 or the hydrolyzed open-lactone form upon cleavage from the antibody carrier. An α-hydroxy lactone-linked SN-38 drug linker preserved the closed-lactone ring structure, while the phenol-linked version allowed conversion between the closed-lactone and open-carboxylate structures. The in vitro cytotoxicity, pharmacokinetic properties, and in vivo efficacy in the L540cy Hodgkin's lymphoma model of the corresponding ADCs were found to be indistinguishable, leading us to conclude that camptothecin-based antibody-drug conjugates possess pronounced activity regardless of the lactone state of the bound drug. This is most likely a result of ADC processing within acidic intracellular vesicles, delivering camptothecin in its active closed-lactone form.
Subject(s)
Antibodies/chemistry , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacokinetics , Camptothecin/chemistry , Camptothecin/pharmacokinetics , Lactones/chemistry , Animals , Antineoplastic Agents, Phytogenic/therapeutic use , Camptothecin/therapeutic use , Cell Line, Tumor , Hodgkin Disease/drug therapy , Humans , Hydrogen-Ion Concentration , Irinotecan/chemistry , Irinotecan/pharmacokinetics , Irinotecan/therapeutic use , Mice , Mice, SCID , PharmacokineticsABSTRACT
A strategy for the preparation of homogeneous antibody-drug conjugates (ADCs) containing multiple payloads has been developed. This approach utilizes sequential unmasking of cysteine residues with orthogonal protection to enable site-specific conjugation of each drug. In addition, because the approach utilizes conjugation to native antibody cysteine residues, it is widely applicable and enables high drug loading for improved ADC potency. To highlight the benefits of ADC dual drug delivery, this strategy was applied to the preparation of ADCs containing two classes of auristatin drug-linkers that have differing physiochemical properties and exert complementary anti-cancer activities. Dual-auristatin ADCs imparted activity in cell line and xenograft models that are refractory to ADCs comprised of the individual auristatin components. This work presents a facile method for construction of potent dual-drug ADCs and demonstrates how delivery of multiple cytotoxic warheads can lead to improved ADC activities. Lastly, we anticipate that the conditions utilized herein for orthogonal cysteine unmasking are not restricted to ADCs and can be broadly utilized for site-specific protein modification.
Subject(s)
Aminobenzoates/chemistry , Antibodies, Monoclonal/chemistry , Antineoplastic Agents/chemistry , Cysteine/chemistry , Immunoconjugates/chemistry , Oligopeptides/chemistry , Drug Delivery Systems , Molecular ConformationABSTRACT
A strategy for the conjugation of alcohol-containing payloads to antibodies has been developed and involves the methylene alkoxy carbamate (MAC) self-immolative unit. A series of MAC ß-glucuronide model constructs were prepared to evaluate stability and enzymatic release, and the results demonstrated high stability at physiological pH in a substitution-dependent manner. All the MAC model compounds efficiently released alcohol drug surrogates under the action of ß-glucuronidase. To assess the MAC technology for ADCs, the potent microtubule-disrupting agent auristatinâ E (AE) was incorporated through the norephedrine alcohol. Conjugation of the MAC ß-glucuronide AE drug linker to the anti-CD30 antibody cAC10, and an IgG control antibody, gave potent and immunologically specific activities inâ vitro and inâ vivo. These studies validate the MAC self-immolative unit for alcohol-containing payloads within ADCs, a class that has not been widely exploited.
Subject(s)
Aminobenzoates/chemistry , Carbamates/chemistry , Immunoconjugates/chemistry , Oligopeptides/chemistry , Phenylpropanolamine/analogs & derivatives , Tubulin Modulators/chemistry , Aminobenzoates/administration & dosage , Aminobenzoates/therapeutic use , Animals , Antineoplastic Agents, Immunological/administration & dosage , Antineoplastic Agents, Immunological/chemistry , Antineoplastic Agents, Immunological/therapeutic use , Cell Line, Tumor , Drug Delivery Systems , Hodgkin Disease/drug therapy , Humans , Immunoconjugates/administration & dosage , Immunoconjugates/therapeutic use , Mice , Neoplasms/drug therapy , Oligopeptides/administration & dosage , Oligopeptides/therapeutic use , Tubulin Modulators/administration & dosage , Tubulin Modulators/therapeutic useABSTRACT
Tubulysins have emerged in recent years as a compelling drug class for delivery to tumor cells via antibodies. The ability of this drug class to exert bystander activity while retaining potency against multidrug-resistant cell lines differentiates them from other microtubule-disrupting agents. Tubulysinâ M, a synthetic analogue, has proven to be active and well tolerated as an antibody-drug conjugate (ADC) payload, but has the liability of being susceptible to acetate hydrolysis at the C11 position, leading to attenuated potency. In this work, we examine the ability of the drug-linker and conjugation site to preserve acetate stability. Our findings show that, in contrast to a more conventional protease-cleavable dipeptide linker, the ß-glucuronidase-cleavable glucuronide linker protects against acetate hydrolysis and improves ADC activity inâ vivo. In addition, site-specific conjugation can positively impact both acetate stability and inâ vivo activity. Together, these findings provide the basis for a highly optimized delivery strategy for tubulysinâ M.
Subject(s)
Immunoconjugates/chemistry , Oligopeptides/chemistry , Animals , Humans , Immunoconjugates/therapeutic use , Mice , Molecular Structure , Oligopeptides/therapeutic use , Rats , Rats, Sprague-Dawley , Xenograft Model Antitumor AssaysABSTRACT
Auristatins, a class of clinically validated anti-tubulin agents utilized as payloads in antibody-drug conjugates, are generally classified by their membrane permeability and the extent of cytotoxic bystander activity on neighboring cells after targeted delivery. The drugs typically fall within two categories: membrane permeable monomethyl auristatin E-type molecules with high bystander activities and susceptibility to efflux pumps, or charged and less permeable monomethyl auristatin F (MMAF) analogs with low bystander activities and resistance to efflux pumps. Herein, we report the development of novel auristatins that combine the attributes of each class by having both bystander activity and cytotoxicity on multidrug-resistant (MDR+) cell lines. Structure-based design focused on the hydrophobic functionalization of the N-terminal N-methylvaline of the MMAF scaffold to increase cell permeability. The resulting structure-activity relationships of the new auristatins demonstrate that optimization of hydrophobicity and structure can lead to highly active free drugs and antibody-drug conjugates with in vivo bystander activities.
Subject(s)
Aminobenzoates/therapeutic use , Oligopeptides/therapeutic use , Aminobenzoates/pharmacology , Animals , Cell Line, Tumor , Disease Models, Animal , Humans , Mice , Oligopeptides/pharmacology , Rats , Structure-Activity RelationshipABSTRACT
We have developed a highly active and well-tolerated camptothecin (CPT) drug-linker designed for antibody-mediated drug delivery in which the lead molecule consists of a 7-aminomethyl-10,11-methylenedioxy CPT (CPT1) derivative payload attached to a novel hydrophilic protease-cleavable valine-lysine-glycine tripeptide linker. A defined polyethylene glycol stretcher was included to improve the properties of the drug-linker, facilitating high antibody-drug conjugate (ADC) drug loading, while reducing the propensity for aggregation. A CPT1 ADC with 8 drug-linkers/mAb displayed a pharmacokinetic profile coincident with parental unconjugated antibody and had high serum stability. The ADCs were broadly active against cancer cells in vitro and in mouse xenograft models, giving tumor regressions and complete responses at low (≤3 mg/kg, single administration) doses. Pronounced activities were obtained in both solid and hematologic tumor models and in models of bystander killing activity and multidrug resistance. Payload release studies demonstrated that two CPTs, CPT1 and the corresponding glycine analog (CPT2), were released from a cAC10 ADC by tumor cells. An ADC containing this drug-linker was well tolerated in rats at 60 mg/kg, given weekly four times. Thus, ADCs comprised of this valine-lysine-glycine linker with CPT drug payloads have promise in targeted drug delivery.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents, Phytogenic/therapeutic use , Camptothecin/therapeutic use , Animals , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Camptothecin/pharmacology , Disease Models, Animal , Female , Humans , Mice , Rats , Rats, Sprague-DawleyABSTRACT
Although antibody-drug conjugates (ADCs) find increasing applications in cancer treatment, de novo or treatment-emergent resistance mechanisms may impair clinical benefit. Two resistance mechanisms that emerge under prolonged exposure include upregulation of transporter proteins that confer multidrug resistance (MDR+) and loss of cognate antigen expression. New technologies that circumvent these resistance mechanisms may serve to extend the utility of next-generation ADCs. Recently, we developed the quaternary ammonium linker system to expand the scope of conjugatable payloads to include tertiary amines and applied the linker to tubulysins, a highly potent class of tubulin binders that maintain activity in MDR+ cell lines. In this work, tubulysin M, which contains an unstable acetate susceptible to enzymatic hydrolysis, and two stabilized tubulysin analogues were prepared as quaternary ammonium-linked glucuronide-linkers and assessed as ADC payloads in preclinical models. The conjugates were potent across a panel of cancer cell lines and active in tumor xenografts, including those displaying the MDR+ phenotype. The ADCs also demonstrated potent bystander activity in a coculture model comprised of a mixture of antigen-positive and -negative cell lines, and in an antigen-heterogeneous tumor model. Thus, the glucuronide-tubulysin drug-linkers represent a promising ADC payload class, combining conjugate potency in the presence of the MDR+ phenotype and robust activity in models of tumor heterogeneity in a structure-dependent manner. Mol Cancer Ther; 17(8); 1752-60. ©2018 AACR.
Subject(s)
Glucuronides/metabolism , Immunoconjugates/metabolism , Animals , Humans , Mice , Mice, SCID , Xenograft Model Antitumor AssaysABSTRACT
Antibody-drug conjugates (ADC) comprise targeting antibodies armed with potent small-molecule payloads. ADCs demonstrate specific cell killing in clinic, but the basis of their antitumor activity is not fully understood. In this study, we investigated the degree to which payload release predicts ADC activity in vitro and in vivo ADCs were generated to target different receptors on the anaplastic large cell lymphoma line L-82, but delivered the same cytotoxic payload (monomethyl auristatin E, MMAE), and we found that the intracellular concentration of released MMAE correlated with in vitro ADC-mediated cytotoxicity independent of target expression or drug:antibody ratios. Intratumoral MMAE concentrations consistently correlated with the extent of tumor growth inhibition in tumor xenograft models. In addition, we developed a robust admixed tumor model consisting of CD30(+) and CD30(-) cancer cells to study how heterogeneity of target antigen expression, a phenomenon often observed in cancer specimens, affects the treatment response. CD30-targeting ADC delivering membrane permeable MMAE or pyrrolobenzodiazepine dimers demonstrated potent bystander killing of neighboring CD30(-) cells. In contrast, a less membrane permeable payload, MMAF, failed to mediate bystander killing in vivo, suggesting local diffusion and distribution of released payloads represents a potential mechanism of ADC-mediated bystander killing. Collectively, our findings establish that the biophysical properties and amount of released payloads are chief factors determining the overall ADC potency and bystander killing. Cancer Res; 76(9); 2710-9. ©2016 AACR.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Immunoconjugates/pharmacology , Oligopeptides/pharmacology , Animals , Cell Line, Tumor , Chromatography, Liquid , Drug Delivery Systems/methods , Flow Cytometry , Humans , Immunohistochemistry , Lymphoma/pathology , Mass Spectrometry , Mice , Mice, SCID , Xenograft Model Antitumor AssaysABSTRACT
A quaternary ammonium-based drug-linker has been developed to expand the scope of antibody-drug conjugate (ADC) payloads to include tertiary amines, a functional group commonly present in biologically active compounds. The linker strategy was exemplified with a ß-glucuronidase-cleavable auristatin E construct. The drug-linker was found to efficiently release free auristatin E (AE) in the presence of ß-glucuronidase and provide ADCs that were highly stable in plasma. Anti-CD30 conjugates comprised of the glucuronide-AE linker were potent and immunologically specific in vitro and in vivo, displaying pharmacologic properties comparable with a carbamate-linked glucuronide-monomethylauristatin E control. The quaternary ammonium linker was then applied to a tubulysin antimitotic drug that contained an N-terminal tertiary amine that was important for activity. A glucuronide-tubulysin quaternary ammonium linker was synthesized and evaluated as an ADC payload, in which the resulting conjugates were found to be potent and immunologically specific in vitro, and displayed a high level of activity in a Hodgkin lymphoma xenograft. Furthermore, the results were superior to those obtained with a related tubulysin derivative containing a secondary amine N-terminus for conjugation using previously known linker technology. The quaternary ammonium linker represents a significant advance in linker technology, enabling stable conjugation of payloads with tertiary amine residues. Mol Cancer Ther; 15(5); 938-45. ©2016 AACR.