Anetumab ravtansine: a novel mesothelin-targeting antibody-drug conjugate cures tumors with heterogeneous target expression favored by bystander effect.

Mesothelin is a tumor differentiation antigen frequently overexpressed in tumors such as mesothelioma, ovarian, pancreatic, and lung adenocarcinomas while showing limited expression in nonmalignant tissues. Mesothelin is therefore an attractive target for cancer therapy using antibody-drug conjugates (ADC). This study describes the detailed characterization of anetumab ravtansine, here referred to as BAY 94-9343, a novel ADC consisting of a human anti-mesothelin antibody conjugated to the maytansinoid tubulin inhibitor DM4 via a disulfide-containing linker. Binding properties of the anti-mesothelin antibody were analyzed using surface plasmon resonance, immunohistochemistry, flow cytometry, and fluorescence microscopy. Effects of BAY 94-9343 on cell proliferation were first studied in vitro and subsequently in vivo using subcutaneous, orthotopic, and patient-derived xenograft tumor models. The antibody binds to human mesothelin with high affinity and selectivity, thereby inducing efficient antigen internalization. In vitro, BAY 94-9343 demonstrated potent and selective cytotoxicity of mesothelin-expressing cells with an IC(50) of 0.72 nmol/L, without affecting mesothelin-negative or nonproliferating cells. In vivo, BAY 94-9343 localized specifically to mesothelin-positive tumors and inhibited tumor growth in both subcutaneous and orthotopic xenograft models. In addition, BAY 94-9343 was able to induce a bystander effect on neighboring mesothelin-negative tumor cells. Antitumor efficacy of BAY 94-9343 correlated with the amount of mesothelin expressed and was generally superior to that of standard-of-care regimen resulting in complete tumor eradication in most of the models. BAY 94-9343 is a selective and highly potent ADC, and our data support its development for the treatment of patients with mesothelin-expressing tumors.

Changes in purine specificity in tandem GAF chimeras from cyanobacterial cyaB1 adenylate cyclase and rat phosphodiesterase 2.

The C-terminal catalytic domains of the 11 mammalian phosphodiesterase families (PDEs) are important drug targets. Five of the 11 PDE families contain less well-characterized N-terminal GAF domains. cGMP is the ligand for the GAF domains in PDEs 2, 5, 6 and 11, and cAMP is the ligand for PDE10. Structurally related tandem GAF domains signalling via cAMP are present in the cyanobacterial adenylate cyclases cyaB1 and cyaB2. Because current high-resolution crystal structures of the tandem GAF domains of PDE2 and cyaB2 do not reveal how cNMP specificity is encoded, we generated chimeras between the tandem GAF domains of cyaB1 and PDE2. Both bind the ligand in the GAF B subdomains. Segmental replacements in the highly divergent beta1-beta3 region of the GAF B subdomain of cyaB1 by the corresponding PDE2 regions switched signalling from cAMP to cGMP. Using 10 chimeric constructs, we demonstrated that, for this switch in purine specificity, only 11% of the sequence of the cyanobacterial GAF B needs to be replaced by PDE2 sequences. We were unable, however, to switch the purine specificity of the PDE2 tandem GAF domain from cGMP to cAMP in reverse constructs, i.e. by replacement of PDE2 segments with those from the cyaB1 GAF tandem domain. The data provide a novel view on the structure-function relationships underlying the purine specificity of cNMP-binding GAF domains and indicate that, as potential drug targets, they must be characterized structurally and biochemically one by one.

Characterization of the tandem GAF domain of human phosphodiesterase 5 using a cyanobacterial adenylyl cyclase as a reporter enzyme.

We analyzed cGMP signaling by the human phosphodiesterase 5 (hPDE5) tandem GAF domain based on a functional activation assay. The C-terminal catalytic domain of the cyanobacterial adenylyl cyclase (AC) cyaB1 was used as a reporter enzyme. We demonstrate functional coupling between the hPDE5 GAF ensemble and the AC resulting in a chimera stimulated 10-fold by cGMP. The hPDE5 GAF domain has an inhibitory effect on AC activity, which is released upon cGMP activation. Removal of 109 amino acids from the N terminus resulted in partial disengagement of the GAF domain and AC, i.e. in a 10-fold increase in basal activity, and affected cGMP affinity. The Ser-102 phosphorylation site of hPDE5 increased cGMP affinity, as shown by a 5-fold lower K(D) for cGMP in a S102D mutant, which mimicked complete modification. The function of the NKFDE motif, which is a signature of all GAF domains with known cyclic nucleotide binding capacity, was elucidated by targeted mutations. Data with either single and double mutants in either GAF A or GAF B or a quadruple mutant affecting both subdomains simultaneously indicated that it is impossible to functionally assign cGMP binding and intramolecular signaling to either GAF A or B of hPDE5. Both subdomains are structurally and functionally interdependent and act in concert in regulating cycaB1 AC and, most likely, also hPDE5.

The cyanobacterial tandem GAF domains from the cyaB2 adenylyl cyclase signal via both cAMP-binding sites.

The tandem GAF domains from the cyanobacterium Anabaena PCC7120 cyaB2 adenylyl cyclase form an antiparallel dimer with cAMP bound to all four binding sites. cAMP binding causes highly cooperative allosteric enzyme activation (>500-fold; EC(50) = 1 microM; Hill coefficient >2.0). The cyaB2 GAF domains, like those of the cyclic nucleotide phosphodiesterases (PDEs), contain conserved NKFDE motifs that when mutated in the PDEs abrogate cyclic nucleotide binding. We mutated the aspartic acids within this motif in cyaB2 to determine which domains were required for signaling. Constructs containing an Asp/Ala mutation in either GAF domain still showed positive cooperative cAMP stimulation but with reduced Hill coefficients. The cyaB2 GAF domain NKFDE motifs contain inserts of 14 (GAF-A) and 19 (GAF-B) amino acids not present in PDE2 or cyaB1. Constructs having these inserts deleted could still be activated by cAMP (23- to 100-fold) but lost all positive cooperative activation, suggesting that the inserts play an important role in domain interaction and/or stabilization of the cAMP-binding pockets. In the shortened constructs, even those with a single Asp/Ala mutation in the NKFDE motifs could still be activated by cAMP. However, in a double Asp/Ala mutant of the shortened construct, stimulation by cAMP was almost completely lost, and the EC(50) shifted far to the right. Overall, the data suggest that in GAF domains without these inserts, only the canonical lysine:aspartate salt bridge keeps the alpha4-helix and the alpha4-beta5 linker that close over the cyclic nucleotide properly oriented, thereby stabilizing the binding pocket. The cyaB2 GAF ensemble appears to be an evolutionary intermediate where both GAF domains still participate in allosteric activation by cAMP.

Crystal structure of the tandem GAF domains from a cyanobacterial adenylyl cyclase: modes of ligand binding and dimerization.

In several species, GAF domains, which are widely expressed small-molecule-binding domains that regulate enzyme activity, are known to bind cyclic nucleotides. However, the molecular mechanism by which cyclic nucleotide binding affects enzyme activity is not known for any GAF domain. In the cyanobacterium, Anabaena, the cyaB1 and cyaB2 genes encode adenylyl cyclases that are stimulated by binding of cAMP to their N-terminal GAF domains. Replacement of the tandem GAF-A/B domains in cyaB1 with the mammalian phosphodiesterase 2A GAF-A/B tandem domains allows regulation of the chimeric protein by cGMP, suggesting a highly conserved mechanism of activation. Here, we describe the 1.9-A crystal structure of the tandem GAF-A/B domains of cyaB2 with bound cAMP and compare it to the previously reported structure of the PDE2A GAF-A/B. Unexpectedly, the cyaB2 GAF-A/B dimer is antiparallel, unlike the parallel dimer of PDE2A. Moreover, there is clear electron density for cAMP in both GAF-A and -B, whereas in PDE2A, cGMP is found only in GAF-B. Phosphate and ribose group contacts are similar to those in PDE2A. However, the purine-binding pockets appear very different from that in PDE2A GAF-B. Differences in the beta2-beta3 loop suggest that this loop confers much of the ligand specificity in this and perhaps in many other GAF domains. Finally, a conserved asparagine appears to be a new addition to the signature NKFDE motif, and a mechanism for this motif to stabilize the cNMP-binding pocket is proposed.