Development of Anilino-Maytansinoid ADCs that Efficiently Release Cytotoxic Metabolites in Cancer Cells and Induce High Levels of Bystander Killing.

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.

Synthesis and Evaluation of Camptothecin Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) that incorporate the exatecan derivative DXd in their payload are showing promising clinical results in solid tumor indications. The payload has an F-ring that also contains a second chiral center, both of which complicate its synthesis and derivatization. Here we report on new camptothecin-ADCs that do not have an F-ring in their payloads yet behave similarly to DXd-bearing conjugates in vitro and in vivo. This simplification allows easier derivatization of camptothecin A and B rings for structure-activity relationship studies and payload optimization. ADCs having different degrees of bystander killing and the ability to release hydroxyl or thiol-bearing metabolites following peptide linker cleavage were investigated.

Peptide-Cleavable Self-immolative Maytansinoid Antibody-Drug Conjugates Designed To Provide Improved Bystander Killing.

A new type of antibody-drug conjugate (ADC) has been prepared that contains a sulfur-bearing maytansinoid attached to an antibody via a highly stable tripeptide linker. Once internalized by cells, proteases in catabolic vesicles cleave the peptide of the ADC's linker causing self-immolation that releases a thiol-bearing metabolite, which is then S-methylated. Conjugates were prepared with peptide linkers containing only alanyl residues, which were all l isomers or had a single d residue in one of the three positions. A d-alanyl residue in the linker did not significantly impair a conjugate's cytotoxicity or bystander killing unless it was directly attached to the immolative moiety. Increasing the number of methylene units in the maytansinoid side chain of a conjugate did not typically affect an ADC's cytotoxicity to targeted cells but did increase bystander killing activity. ADCs with the highest in vitro bystander killing were then evaluated in vivo in mice, where they displayed improved efficacy compared to previously described types of maytansinoid conjugates.

The murine cytomegalovirus cell death suppressor m38.5 binds Bax and blocks Bax-mediated mitochondrial outer membrane permeabilization.

Apoptosis is increasingly implicated as an early line of defense against viral infections. Viruses have devised numerous strategies to delay apoptosis of infected cells. Many viruses encode cell death suppressors that target mitochondrial apoptotic signaling pathway, indicating the importance of this pathway in the anti-viral response. Human and primate cytomegaloviruses encode the viral mitochondria-localized inhibitor of apoptosis vMIA, but no overt homologue of vMIA was identified in any non-primate cytomegalovirus. Here we report that m38.5 protein encoded by murine cytomegalovirus, which is unrelated to vMIA in its amino acid sequence, delays death receptor ligation-induced cell death, and that m38.5 associates with Bax, recruits it to mitochondria, and blocks Bax-mediated but not Bak-mediated mitochondrial outer membrane permeabilization. Thus, primate and murine cytomegaloviruses have evolved non-homologous but functionally similar cell death suppressors selectively targeting the Bax-mediated branch of the mitochondrial apoptotic signaling pathway, indicating the importance of this branch in the response of diverse host organisms against cytomegalovirus infections.

A New Triglycyl Peptide Linker for Antibody-Drug Conjugates (ADCs) with Improved Targeted Killing of Cancer Cells.

A triglycyl peptide linker (CX) was designed for use in antibody -: drug conjugates (ADC), aiming to provide efficient release and lysosomal efflux of cytotoxic catabolites within targeted cancer cells. ADCs comprising anti-epithelial cell adhesion molecule (anti-EpCAM) and anti-EGFR antibodies with maytansinoid payloads were prepared using CX or a noncleavable SMCC linker (CX and SMCC ADCs). The in vitro cytotoxic activities of CX and SMCC ADCs were similar for several cancer cell lines; however, the CX ADC was more active (5-100-fold lower IC50) than the SMCC ADC in other cell lines, including a multidrug-resistant line. Both CX and SMCC ADCs showed comparable MTDs and pharmacokinetics in CD-1 mice. In Calu-3 tumor xenografts, antitumor efficacy was observed with the anti-EpCAM CX ADC at a 5-fold lower dose than the corresponding SMCC ADC in vivo Similarly, the anti-EGFR CX ADC showed improved antitumor activity over the respective SMCC conjugate in HSC-2 and H1975 tumor models; however, both exhibited similar activity against FaDu xenografts. Mechanistically, in contrast with the charged lysine-linked catabolite of SMCC ADC, a significant fraction of the carboxylic acid catabolite of CX ADC could be uncharged in the acidic lysosomes, and thus diffuse out readily into the cytosol. Upon release from tumor cells, CX catabolites are charged at extracellular pH and do not penetrate and kill neighboring cells, similar to the SMCC catabolite. Overall, these data suggest that CX represents a promising linker option for the development of ADCs with improved therapeutic properties. Mol Cancer Ther; 15(6); 1311-20. ©2016 AACR.

SAR650984, a novel humanized CD38-targeting antibody, demonstrates potent antitumor activity in models of multiple myeloma and other CD38+ hematologic malignancies.

PURPOSE: The CD38 cell surface antigen is expressed in diverse hematologic malignancies including multiple myeloma, B-cell non-Hodgkin lymphoma (NHL), B-cell chronic lymphocytic leukemia, B-cell acute lymphoblastic leukemia (ALL), and T-cell ALL. Here, we assessed the antitumor activity of the anti-CD38 antibody SAR650984. EXPERIMENTAL DESIGN: Activity of SAR650984 was examined on lymphoma, leukemia and multiple myeloma cell lines, primary multiple myeloma samples, and multiple myeloma xenograft models in immunodeficient mice. RESULTS: We identified a humanized anti-CD38 antibody with strong proapoptotic activity independent of cross-linking agents, and potent effector functions including complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity, and antibody-dependent cellular phagocytosis (ADCP), equivalent in vitro to rituximab in CD20+ and CD38+ models. This unique antibody, termed SAR650984, inhibited the ADP-ribosyl cyclase activity of CD38, likely through an allosteric antagonism as suggested by 3D structure analysis of the complex. In vivo, SAR650984 was active in diverse NHL, ALL, and multiple myeloma CD38+ tumor xenograft models. SAR650984 demonstrated single-agent activity comparable with rituximab or cyclophosphamide in Daudi or SU-DHL-8 lymphoma xenograft models with induction of the proapoptotic marker cleaved capase-7. In addition, SAR650984 had more potent antitumor activity than bortezomib in NCI-H929 and Molp-8 multiple myeloma xenograft studies. Consistent with its mode of action, SAR650984 demonstrated potent proapoptotic activity against CD38+ human primary multiple myeloma cells. CONCLUSION: These results validate CD38 as a therapeutic target and support the current evaluation of this unique CD38-targeting functional antibody in phase I clinical trials in patients with CD38+ B-cell malignancies.

Differential function and expression of the viral inhibitor of caspase 8-induced apoptosis (vICA) and the viral mitochondria-localized inhibitor of apoptosis (vMIA) cell death suppressors conserved in primate and rodent cytomegaloviruses.

Human cytomegalovirus (CMV) genes UL36 and UL37 encode viral inhibitor of caspase-8-induced apoptosis (vICA) and viral mitochondria inhibitor of apoptosis (vMIA), respectively. Rhesus macaque CMV homologues, denoted Rh-vICA and Rh-vMIA, were identified and found to suppress apoptosis. One of these functions was conserved in MCMV, encoded by the M36 gene and denoted M-vICA. Conserved regions were compared to domains important to vICA- and vMIA-mediated cell death suppression. The conserved sequences of primate CMV vMIA homologues overlapped with the two known functional domains, providing further evidence supporting a crucial role of vMIA in cell death suppression. RNA blot analyses revealed that expression of murine and rhesus macaque CMV UL36 and UL37 homologues started early and continued through late times of infection. Murine CMV homologues were expressed with alpha (immediate early) kinetics, like human CMV UL36 and UL37, whereas rhesus macaque CMV homologues exhibited beta (delayed early) kinetics. Despite differences in organization and transcriptional regulation, this region appears to carry out a conserved role in cell death suppression. When viewed in light of sequence conservation, a functional vMIA homologue appears to be encoded by every primate CMV, whereas a functional vICA homologue appears to be encoded by all cytomegaloviruses for which sequence data are available.

Cytomegalovirus cell death suppressor vMIA blocks Bax- but not Bak-mediated apoptosis by binding and sequestering Bax at mitochondria.

We report that the cytomegalovirus-encoded cell death suppressor vMIA binds Bax and prevents Bax-mediated mitochondrial membrane permeabilization by sequestering Bax at mitochondria in the form of a vMIA-Bax complex. vMIA mutants with a defective mitochondria-targeting domain retain their Bax-binding function but not their ability to suppress mitochondrial membrane permeabilization or cell death. vMIA does not seem to either specifically associate with Bak or suppress Bak-mediated mitochondrial membrane permeabilization. Recent evidence suggests that the contribution of Bax and Bak in the mitochondrial apoptotic signaling pathway depends on the distinct phenotypes of cells, and it appears from our data that vMIA is capable of suppressing apoptosis in cells in which this pathway is dominated by Bax, but not in cells where Bak also plays a role.

An anti-apoptotic viral protein that recruits Bax to mitochondria.

The viral mitochondria-localized inhibitor of apoptosis (vMIA), encoded by the UL37 gene of human cytomegalovirus, inhibits apoptosis-associated mitochondrial membrane permeabilization by a mechanism different from that of Bcl-2. Here we show that vMIA induces several changes in Bax that resemble those found in apoptotic cells yet take place in unstimulated, non-apoptotic vMIA-expressing cells. These changes include the constitutive localization of Bax at mitochondria, where it associates tightly with the mitochondrial membrane, forming high molecular weight aggregates that contain vMIA. vMIA recruits Bax to mitochondria but delays relocation of caspase-8-activated truncated Bid-green fluorescent protein (GFP) (t-Bid-GFP) to mitochondria. The ability of vMIA and its deletion mutants to associate with Bax and to induce relocation of Bax to mitochondria correlates with their anti-apoptotic activity and with their ability to suppress mitochondrial membrane permeabilization. Taken together, our data indicate that vMIA blocks apoptosis via its interaction with Bax. vMIA neutralizes Bax by recruiting it to mitochondria and "freezing" its pro-apoptotic activity. These data unravel a novel strategy of subverting an intrinsic pathway of apoptotic signaling.