Abstract Antibody-drug conjugates (ADCs) enable targeted delivery of therapeutics to cancer cells and offer potential for more selective therapy. Several ADCs are demonstrating promising clinical efficacy, however due to the complexity of human cancer, tumors become refractory to most drug treatments. We hypothesized that cultured tumor cells chronically treated with an ADC would acquire mechanisms of resistance unique to ADC-based therapy. Human breast cancer cell lines were exposed to multiple cycles of an anti-Her2 trastuzumab-maytansinoid conjugate (TM) at IC80 concentrations for 3 days followed by ∼1 week without treatment to simulate a maximally tolerated dose followed by recovery. After ∼2 months, significant resistance developed in JIMT1 and MDA-MB-361 cell lines. The potency of TM conjugate on drug-selected cell lines was reduced to the activity observed on Her2-negative cells (>20 & >270X in JIMT1 & 361 cell models, respectively). Flow cytometry revealed 58% and 25% decreases in Her2 receptor number in JIMT-TM & 361-TM, respectively. Proteomic profiling of surface proteins in JIMT-TM cells demonstrated significant increases in proteins involved in post-translational modification (e.g., ubiquitinating enzymes, kinases, and phosphatases), as well as elevated levels of endosomal and vesicle proteins (e.g., RAB family members), and proteins mediating microtubule and actin dynamics. Notably, ABC drug transporters were not altered in JIMT-TM cells. In 361-TM cells, an increase in ABCC1 (MRP1) was observed, but no changes in ABCB1 (MDR1) which typically effluxes tubulin inhibitors. These data suggest the acquisition of complex resistance mechanisms upon ADC treatment. The cross-resistance profile of these ADC refractory models was evaluated. Minimal or no resistance (1 - 6X) was observed to free drugs, including maytansine or other standard-of-care tubulin or DNA targeted therapeutics. In JIMT-TM cells, cross-resistance was observed to other trastuzumab-based ADCs, including those containing either non-cleavable or cleavable linkers, and delivering payloads with various biological mechanisms of action. In contrast, 361-TM cells (which were made resistant to an ADC delivering a tubulin inhibitor via a non-cleavable linker) retained significant sensitivity to ADCs containing cleavable linkers, even those containing other tubulin inhibitor-based payloads. Moreover, both 361-TM and JIMT-TM resistant cell lines retained sensitivity to ADCs delivering payloads with alternative (non-tubulin) mechanisms of action. Therefore, despite the reduction in antigen levels observed in both cell lines, modification of the linker and/or the payload was able to overcome resistance mediated by the initial ADC therapy. These data offer the potential to treat refractory tumors with ADCs containing the same antibody vehicle, but delivering alternative linkers or payloads. Citation Format: Xingzhi Tan, Guixian Jin, Jeremy Myers, Veronica Diesl, Max Follettie, My-Hanh Lam, Sylvia Musto, Kiran Khandke, Manoj Charati, Edmund Graziani, Andreas Maderna, Chakrapani Subramanyam, Frank Koehn, Russell Dushin, Kim Arndt, Christopher J. O'Donnell, Hans-Peter Gerber, Frank Loganzo. Tumor cells selected for resistance to an antibody-drug conjugate retain sensitivity to ADCs with modified linkers and payloads. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4629. doi:10.1158/1538-7445.AM2013-4629
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
(trans)-2-(3-Methoxy-5-methylsulfonyl-4-propoxyphenyl)-5-(3,4,5- trimethoxyphenyl)tetrahydrofuran (L-659,989) is a potent and orally active platelet-activating factor (PAF)-specific and competitive receptor antagonist. The 2,5-tritium-labeled L-659,989 ([3H]L-659,989) specifically binds to rabbit platelet membranes with an equilibrium dissociation constant (KD) of 1.60 +/- 0.20 nM in 10 mM MgCl2. Several selected PAF analogs and PAF receptor antagonists show equilibrium inhibition constants roughly similar to those found in the specific [3H]PAF binding assay. Other pharmacological agents with no PAF antagonistic activities do not inhibit the specific binding of [3H]L-659,989. K+ and divalent cations such as Mg2+, Ca2+, and Mn2+ potentiate the specific [3H]L-659,989 binding. Na+ and Li+ also enhance but GTP shows no effect on the specific binding of [3H]L-659,989. However, Ni2+ inhibits the specific binding. Scatchard analysis demonstrates that the potentiating effect of these cations is due to an increase in the detectable receptor number for L-659,989. In 10 mM MgCl2 [3H]L-659,989 shows higher receptor number than [3H]PAF. Under various ionic conditions with or without GTP, in which [3H] L-659,989 binding remains approximately the same, C16-PAF shows different potencies in competing against the specific [3H] L-659,989 binding. These results demonstrate the existence of multiple conformational states of the PAF-specific receptor. The variation in the detectable receptor number under different conditions is due to the coexistence of the high and low affinity states and the fact that the low affinity state(s) of the receptor with KD value(s) possibly in the micromolar range cannot be detected in the Scatchard analysis with the radioligand at nanomolar concentrations. In the presence of 150 mM NaCl and 1 mM GTP, receptors exist in a single conformational state with an equilibrium dissociation constant (KB) of 0.931 microM for PAF.
Abstract Antibody drug conjugates (ADCs) are designed to deliver cytotoxics to tumor cells via binding to surface antigen followed by internalization and intracellular drug release. ADC linkers are typically categorized as non-cleavable or cleavable; a cleavable linker example is Y_mcValCitPABC_X, with antibody Y, a dipeptide sequence with self-immolative PABC spacer, and payload X. This linker is known to be cleaved by endosomal/lysosomal proteases such as cathepsins, releasing attached payload. In addition to intracellular processing of this linker, we report that conditioned media of cultured tumor cell lines is sufficient to promote extracellular cleavage of ADCs with peptide-linked payloads. Cultured cell lines N87 (gastric) and U87 (glioblastoma), and patient-derived xenograft PA0165 (pancreatic) adapted to in vitro culture, were plated either in standard 2D culture or in 3D Cultrex embedded culture. After 3 - 7 days, conditioned media from cells was transferred onto MDA-MB-468 or HT29 cells, and then ADCs (Y_mcValCitPABC_Aur) were added to cultures. ADCs were non-targeting IgG conjugated via cleavable dipeptide-PABC linker to auristatin tubulin inhibitor. Minimal cytotoxicity was observed with ADC alone on 468 or HT29 cells. However, in the presence of conditioned media from N87, U87, or PA0165 cells plus the ADC, cytotoxicity was observed in the recipient cells (up to 31, 22, 56% growth inhibition respectively at 100 nM ADC). Moreover, in all cases, the magnitude of the response was greatest when cells providing conditioned media were grown in 3D culture (up to 56, 48, 70%, respectively). In contrast, minimal response was observed using conditioned media from other cancer cell lines (ie HCC2429, 1 - 17%). Additional analyses were conducted by incubating conditioned media from these cells with a dipeptide-based cleavable substrate with fluorescent probe and measuring released product in a plate-based assay. Conditioned media promoted fluorescence, suggesting proteolytic enzymes secreted by cells. An ELISA confirmed the presence of cathepsins in conditioned media. Complementing these studies, proteolytic activity was detected in the interstitial fluid derived from tumors grown in athymic mice. Fluid extracted from xenograft tumors (cultured cancer lines and patient-derived tumors) was analyzed for proteolytic activity using cleavable-fluorescent linker-probe in a plate assay. The majority of samples demonstrated proteolytic activity. These data are consistent with reported secretion of cathepsins by cancer cells and we now show that these proteases may mediate extracellular release of cytotoxic payloads from ADCs containing peptide-based cleavable linkers. This activity is magnified when cells are grown in 3D culture and is observed in tumor xenografts grown in vivo. This response may provide a beneficial bystander effect of ADCs on antigen negative cells in a heterogenous tumor population. Citation Format: My-Hanh Lam, Judy Lucas, Andreas Maderna, Hallie Wald, Megan Wojciechowicz, Russell Dushin, Bryan Peano, Fang Wang, Jeremy Myers, Xingzhi Tan, Sylvia Musto, Manoj Charati, Hans-Peter Gerber, Frank Loganzo. Extracellular proteolytic cleavage of peptide-linked antibody-drug conjugates promotes bystander killing of cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4837. doi:10.1158/1538-7445.AM2014-4837
Mammalian macrophages contain a transport system that binds and internalizes glycoproteins with exposed mannose residues. This system and analogous systems on other types of cells require substrates to bear multiple nonreducing terminal residues of the appropriate sugar for effective uptake. Small multivalent synthetic glycopeptides with mannose residues covalently linked through a spacer arm to the alpha- and epsilon-amino groups of lysine, dilysine, and trilysine are competitive inhibitors of rat alveolar macrophage uptake of the neoglycoprotein mannosyl-bovine serum albumin with inhibition constants in the microM range. Various compounds could be covalently attached to the alpha-carboxyl group of these glycopeptides with substantial retention of inhibitory potency. This uptake system does not recognize galactose residues, and the galactosyl analog of an inhibitory mannosylpeptide did not inhibit uptake of mannosyl-bovine serum albumin. The trimannosyldilysine ligand is not only an inhibitor but also a substrate for specific uptake by macrophage, as shown with an 125I-labeled derivative. Macrophages bound 6.4 x 10(5) molecules per cell at 0 degrees C with a dissociation constant of 2 microM. At 21 degrees C the cells could internalize the labeled conjugate with an apparent Michaelis constant of 6 microM and a maximal velocity of 1.7 x 10(5) molecules per min per cel. The dissociation constant and Michaelis constant are similar to the inhibition constant of 9 microM determined at 21 degrees C for inhibition by this conjugate at mannosyl-bovine serum albumin uptake. These synthetic substrates may be useful in targeting pharmacologic agents to macrophages, and analogous compounds may target such agents to other types of cell.
Dystrophic (mdx) mice were subjected to a 15 week exercise programme consisting of endurance swimming. Single fibres from the extensor digitorum longus (EDL, fast-twitch) and soleus (SOL, mixed fast- and slow-twitch) muscles were attached to a sensitive force-recording apparatus, and activated in Ca(2+)- and Sr(2+)-buffered solutions. In addition to the normal well-defined fibre types in these muscles, a small number of fibres were also sampled from the soleus of both experimental groups, which were 'Intermediate' to the other two SOL fibre types. Type IIB fibres from the EDL and type IIA fibres from the soleus of the Swim group were significantly less sensitive to Ca2+ and Sr2+ compared with those fibres sampled from the sedentary (Sedent) group, suggesting that endurance exercise was able to modify Ca(2+)- and Sr(2+)-activated contractile characteristics. The swim-trained (Swim) group's increased incidence of SOL fibres with characteristics intermediate to those of the fast- and slow-twitch fibre types suggests a possible exercise-induced fibre type transformation as an adaptation to the functional demand.
Auristatins, synthetic analogues of the antineoplastic natural product Dolastatin 10, are ultrapotent cytotoxic microtubule inhibitors that are clinically used as payloads in antibody-drug conjugates (ADCs). The design and synthesis of several new auristatin analogues with N-terminal modifications that include amino acids with α,α-disubstituted carbon atoms are described, including the discovery of our lead auristatin, PF-06380101. This modification of the peptide structure is unprecedented and led to analogues with excellent potencies in tumor cell proliferation assays and differential ADME properties when compared to other synthetic auristatin analogues that are used in the preparation of ADCs. In addition, auristatin cocrystal structures with tubulin are being presented that allow for the detailed examination of their binding modes. A surprising finding is that all analyzed analogues have a cis-configuration at the Val-Dil amide bond in their functionally relevant tubulin bound state, whereas in solution this bond is exclusively in the trans-configuration. This remarkable observation shines light onto the preferred binding mode of auristatins and serves as a valuable tool for structure-based drug design.
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
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