A Novel NAMPT Inhibitor-Based Antibody-Drug Conjugate Payload Class for Cancer Therapy.

Inhibition of intracellular nicotinamide phosphoribosyltransferase (NAMPT) represents a new mode of action for cancer-targeting antibody-drug conjugates (ADCs) with activity also in slowly proliferating cells. To extend the repertoire of available effector chemistries, we have developed a novel structural class of NAMPT inhibitors as ADC payloads. A structure-activity relationship-driven approach supported by protein structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffolds and linker structures led to highly potent effector chemistries which were conjugated to antibodies targeting C4.4a (LYPD3), HER2 (c-erbB2), or B7H3 (CD276) and tested on antigen-positive and -negative cancer cell lines. Pharmacokinetic studies, including metabolite profiling, were performed to optimize the stability and selectivity of the ADCs and to evaluate potential bystander effects. Optimized NAMPTi-ADCs demonstrated potent in vivo antitumor efficacy in target antigen-expressing xenograft mouse models. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile compared with the corresponding isotype control ADCs. Moreover, we demonstrate─to our knowledge for the first time─the generation of NAMPTi payload metabolites from the NAMPTi-ADCs in vitro and in vivo. In conclusion, NAMPTi-ADCs represent an attractive new payload class designed for use in ADCs for the treatment of solid and hematological cancers.

IL3RA-Targeting Antibody-Drug Conjugate BAY-943 with a Kinesin Spindle Protein Inhibitor Payload Shows Efficacy in Preclinical Models of Hematologic Malignancies.

IL3RA (CD123) is the alpha subunit of the interleukin 3 (IL-3) receptor, which regulates the proliferation, survival, and differentiation of hematopoietic cells. IL3RA is frequently expressed in acute myeloid leukemia (AML) and classical Hodgkin lymphoma (HL), presenting an opportunity to treat AML and HL with an IL3RA-directed antibody-drug conjugate (ADC). Here, we describe BAY-943 (IL3RA-ADC), a novel IL3RA-targeting ADC consisting of a humanized anti-IL3RA antibody conjugated to a potent proprietary kinesin spindle protein inhibitor (KSPi). In vitro, IL3RA-ADC showed potent and selective antiproliferative efficacy in a panel of IL3RA-expressing AML and HL cell lines. In vivo, IL3RA-ADC improved survival and reduced tumor burden in IL3RA-positive human AML cell line-derived (MOLM-13 and MV-4-11) as well as in patient-derived xenograft (PDX) models (AM7577 and AML11655) in mice. Furthermore, IL3RA-ADC induced complete tumor remission in 12 out of 13 mice in an IL3RA-positive HL cell line-derived xenograft model (HDLM-2). IL3RA-ADC was well-tolerated and showed no signs of thrombocytopenia, neutropenia, or liver toxicity in rats, or in cynomolgus monkeys when dosed up to 20 mg/kg. Overall, the preclinical results support the further development of BAY-943 as an innovative approach for the treatment of IL3RA-positive hematologic malignancies.

Tailored Linker Chemistries for the Efficient and Selective Activation of ADCs with KSPi Payloads.

Several antibody-drug conjugates (ADCs) have failed to achieve a sufficiently large therapeutic window in patients due to toxicity induced by unspecific payload release in the circulation or ADC uptake into healthy organs. Herein, we describe the successful engineering of ADCs consisting of novel linkers, which are efficiently and selectively cleaved by the tumor-associated protease legumain. ADCs generated via this approach demonstrate high potency and a preferential activation in tumors compared to healthy tissue, thus providing an additional level of safety. A remarkable tolerance of legumain for different linker peptides, including those with just a single asparagine residue, together with a modifier of the physicochemical metabolite profile, proves the broad applicability of this approach for a tailored design of ADCs.

Antibody-Prodrug Conjugates with KSP Inhibitors and Legumain-Mediated Metabolite Formation.

Many antibody-drug conjugates (ADCs) have failed to achieve a sufficient therapeutic window in clinical studies either due to target-mediated or off-target toxicities. To achieve an additional safety level, a new class of antibody-prodrug conjugates (APDCs) directed against different targets in solid tumors is here described. The tumor-associated lysosomal endopeptidase legumain with a unique cleavage sequence was utilized for APDC metabolism. Legumain-activatable APDCs were as potent as their cathepsin B-activatable analogues. The peptide sequence susceptible to legumain cleavage was optimized for further discrimination of the formation of active metabolites within tumor cells versus healthy tissues, leveraging different tissue-specific legumain activities. Optimized APDCs with slow legumain-mediated conversion reduced preclinically the levels of active metabolite in healthy organs while retaining high activity against different TWEAKR- and B7H3-expressing tumors.

Antibody-Drug Conjugates with Pyrrole-Based KSP Inhibitors as the Payload Class.

The number of cytotoxic payload classes successfully employed in antibody-drug conjugates (ADCs) is still rather limited. The identification of ADC payloads with a novel mode of action will increase therapeutic options and potentially increase the therapeutic window. Herein, we describe the utilization of kinesin spindle protein inhibitors (KSPi) as a novel payload class providing highly potent ADCs against different targets, for instance HER-2 or TWEAKR/Fn14. Aspects of technical optimization include the development of different linker attachment sites, the stabilization of ADC linkage to avoid payload deconjugation and finally, the tailor-made design of active metabolites with a long lasting intracellular exposure in the tumor matching the mode of action of KSP inhibition. These KSPi-ADCs are highly potent and selective in vitro and demonstrate in vivo efficacy in a broad panel of tumor models including complete regressions in a patient-derived urothelial cancer model.

Preclinical Efficacy of the Auristatin-Based Antibody-Drug Conjugate BAY 1187982 for the Treatment of FGFR2-Positive Solid Tumors.

The fibroblast growth factor receptor FGFR2 is overexpressed in a variety of solid tumors, including breast, gastric, and ovarian tumors, where it offers a potential therapeutic target. In this study, we present evidence of the preclinical efficacy of BAY 1187982, a novel antibody-drug conjugate (ADC). It consists of a fully human FGFR2 monoclonal antibody (mAb BAY 1179470), which binds to the FGFR2 isoforms FGFR2-IIIb and FGFR2-IIIc, conjugated through a noncleavable linker to a novel derivative of the microtubule-disrupting cytotoxic drug auristatin (FGFR2-ADC). In FGFR2-expressing cancer cell lines, this FGFR2-ADC exhibited potency in the low nanomolar to subnanomolar range and was more than 100-fold selective against FGFR2-negative cell lines. High expression levels of FGFR2 in cells correlated with efficient internalization, efficacy, and cytotoxic effects in vitro Pharmacokinetic analyses in mice bearing FGFR2-positive NCI-H716 tumors indicated that the toxophore metabolite of FGFR2-ADC was enriched more than 30-fold in tumors compared with healthy tissues. Efficacy studies demonstrated that FGFR2-ADC treatment leads to a significant tumor growth inhibition or tumor regression of cell line-based or patient-derived xenograft models of human gastric or breast cancer. Furthermore, FGFR2 amplification or mRNA overexpression predicted high efficacy in both of these types of in vivo model systems. Taken together, our results strongly support the clinical evaluation of BAY 1187982 in cancer patients and a phase I study (NCT02368951) has been initiated. Cancer Res; 76(21); 6331-9. ©2016 AACR.

In vitro sortagging of an antibody fab fragment: overcoming unproductive reactions of sortase with water and lysine side chains.

Sortase A from Staphylococcus aureus attracts growing interest for its use in biotechnological protein modification. This enzyme binds to a short signal sequence at the C terminus of a target protein, cleaves it by formation of an acyl-enzyme intermediate, and subsequently attaches an oligoglycine with a peptide bond. In this work, we explored its usability for the modification of the L19 Fab fragment (specific for fibronectin ED-B), a promising candidate for antibody-based cancer therapy. The Fab fragment was expressed with a sortase signal sequence attached to its light chain, and was successfully modified with a fluorescent oligoglycine probe in good yield. Our interest focused on performance under conditions of limited oligoglycine concentrations. Two unproductive side reactions of sortase were observed. The first was hydrolysis of the acyl-enzyme intermediate; in the second, sortase accepted the ε-amino group of lysine as substrate, thereby resulting in polypeptide crosslinking. In case of the L19 Fab fragment, it led to the covalent connection of the heavy and light chains. Both side reactions were effectively suppressed by sufficient concentrations of the oligoglycine probe.

Stereospecific enzymatic transformation of alpha-ketoglutarate to (2S,3R)-3-methyl glutamate during acidic lipopeptide biosynthesis.

The acidic lipopeptides, including the calcium-dependent antibiotics (CDA), daptomycin, and A54145, are important macrocyclic peptide natural products produced by Streptomyces species. All three compounds contain a 3-methyl glutamate (3-MeGlu) as the penultimate C-terminal residue, which is important for bioactivity. Here, biochemical in vitro reconstitution of the 3-MeGlu biosynthetic pathway is presented, using exclusively enzymes from the CDA producer Streptomyces coelicolor. It is shown that the predicted 3-MeGlu methyltransferase GlmT and its homologues DptI from the daptomycin producer Streptomyces roseosporus and LptI from the A54145 producer Streptomyces fradiae do not methylate free glutamic acid, PCP-bound glutamate, or Glu-containing CDA in vitro. Instead, GlmT, DptI, and LptI are S-adenosyl methionine (SAM)-dependent alpha-ketoglutarate methyltransferases that catalyze the stereospecific methylation of alpha-ketoglutarate (alphaKG) leading to (3R)-3-methyl-2-oxoglutarate. Subsequent enzyme screening identified the branched chain amino acid transaminase IlvE (SCO5523) as an efficient catalyst for the transformation of (3R)-3-methyl-2-oxoglutarate into (2S,3R)-3-MeGlu. Comparison of reversed-phase HPLC retention time of dabsylated 3-MeGlu generated by the coupled enzymatic reaction with dabsylated synthetic standards confirmed complete stereocontrol during enzymatic catalysis. This stereospecific two-step conversion of alphaKG to (2S,3R)-3-MeGlu completes our understanding of the biosynthesis and incorporation of beta-methylated amino acids into the nonribosomal lipopeptides. Finally, understanding this pathway may provide new possibilities for the production of modified peptides in engineered microbes.

Mechanistic and structural basis of stereospecific Cbeta-hydroxylation in calcium-dependent antibiotic, a daptomycin-type lipopeptide.

Non-ribosomally synthesized lipopeptide antibiotics of the daptomycin type are known to contain unnatural beta-modified amino acids, which are essential for bioactivity. Here we present the biochemical and structural basis for the incorporation of 3-hydroxyasparagine at position 9 in the 11-residue acidic lipopeptide lactone calcium-dependent antibiotic (CDA). Direct hydroxylation of l-asparagine by AsnO, a non-heme Fe(2+)/alpha-ketoglutarate-dependent oxygenase encoded by the CDA biosynthesis gene cluster, was validated by Fmoc derivatization of the reaction product and LC/MS analysis. The 1.45, 1.92, and 1.66 A crystal structures of AsnO as apoprotein, Fe(2+) complex, and product complex, respectively, with (2S,3S)-3-hydroxyasparagine and succinate revealed the stereoselectivity and substrate specificity of AsnO. The comparison of native and product-complex structures of AsnO showed a lid-like region (residues F208-E223) that seals the active site upon substrate binding and shields it from sterically demanding peptide substrates. Accordingly, beta-hydroxylated asparagine is synthesized prior to its incorporation into the growing CDA peptide. The AsnO structure could serve as a template for engineering novel enzymes for the synthesis of beta-hydroxylated amino acids.

The iterative gramicidin s thioesterase catalyzes peptide ligation and cyclization.

Here, we present a comprehensive in vitro characterization of the excised iterative, bimodular PCP-TE of the gramicidin S synthetase GrsB, which is able to act both as a ligation and a cyclization catalyst. Using the native pentapeptidyl-thioester substrates, GrsB PCP-TE catalyzes the dimerization and subsequent formation of the decapeptide lactam gramicidin S. Interestingly, the detection of linear decapeptidyl-SNAC as an enzyme-dependent intermediate supports the iterative mechanism in vivo, in which two pentapeptides, one bound as an ester to the active site serine of the TE domain and the second bound as a thioester to the adjacent pan-PCP, are ligated to a decapeptidyl-pan-PCP that subsequently transferred to the adjacent TE domain and cyclized. Moreover, GrsB PCP-TE can handle different substrates length, leading not only to dimerization, but also to trimerization and the formation of different ring sizes.

Peptide macrocyclization: the reductase of the nostocyclopeptide synthetase triggers the self-assembly of a macrocyclic imine.

Many biologically active natural products have macrocyclic structures. In nonribosomal peptides macrocyclization is commonly achieved via the formation of intramolecular ester or amide bond catalyzed by thioesterase domains during biosynthesis. A unique and so far unknown type of peptide cyclization occurs in the nostocyclopeptide, a macrocyclic imine produced by the terrestrial cyanobacterium Nostoc sp. ATCC53789. In this work we show that a C-terminal reductase domain of the nostocyclopeptide nonribosomal peptide synthetase catalyzes the reductive release of a linear peptide aldehyde and thereby triggers the spontaneous formation of a stable imino head-to-tail linkage. This type of molecular self-assembly induced by the reductive release of reactive aldehydes may be more commonplace in other complex nonribosomal peptides than originally thought.

Chemoenzymatic design of acidic lipopeptide hybrids: new insights into the structure-activity relationship of daptomycin and A54145.

The acidic lipopeptides, including the clinically approved antibiotic daptomycin, constitute a class of structurally related branched cyclic peptidolactones and peptidolactams synthesized by nonribosomal peptide synthetases (NRPSs). In this study, the excised peptide cyclases from A54145 and daptomycin NRPSs were shown to be able to catalyze the macrocyclization of peptide thioester substrates, which were chemically produced by solid phase peptide synthesis. Applying this chemoenzymatic strategy, we generated derivatives of A54145 and daptomycin as well as hybrid molecules of both compounds. Bioactivity determination of the derived cyclic molecules revealed new insights into the structure-activity relationship of the acidic lipopeptide family. The general importance of several amino acid positions, including two conserved aspartic acid residues, was confirmed to be substantial for antibiotic potency. As a robust macrocyclization catalyst, the peptide cyclase excised from A54145 synthetase is the first cyclase of a branched cyclic lipopeptide, which catalyzes both macrolactonization and macrolactamization. The results presented herein illustrate the advantages of combining organic synthesis with natural product biosynthetic enzymes to explore the interplay between structural features and biological activity.

Fluorescence resonance energy transfer as a probe of peptide cyclization catalyzed by nonribosomal thioesterase domains.

Macrocyclization of synthetic peptides by thioesterase (TE) domains excised from nonribosomal peptide synthetases (NRPS) has been limited to peptides that contain TE-specific recognition elements. To alter substrate specificity of these enzymes by evolution efforts, macrocyclization has to be detected under high-throughput conditions. Here we describe a method to selectively detect cyclic peptides by fluorescence resonance energy transfer (FRET). Using this method, picomolar detection limits were easily realized, providing novel entry for kinetic studies of catalyzed macrocyclization. Application of this method also provides an ideal tool to track TE-mediated peptide cyclization in real time. The general utility of FRET-assisted detection of cyclopeptides was demonstrated for two cyclases, namely tyrocidine (Tyc) TE and calcium-dependent antibiotic (CDA) TE. For the latter cyclase, this approach was combined with site-directed affinity labeling, opening the possibility for high-throughput enzymatic screening.

Chemoenzymatic approach to enantiopure streptogramin B variants: characterization of stereoselective pristinamycin I cyclase from Streptomyces pristinaespiralis.

Streptogramin B antibiotics are cyclic peptide natural products produced by Streptomyces species. In combination with the synergistic group A component, they are "last line of defense" antimicrobial agents against multiresistant cocci. The racemization sensitivity of the phenylglycine (Phg(7)) ester is a complex challenge in total chemical synthesis of streptogramin B molecules. To provide fast and easy access to novel streptogramin antibiotics, we introduce a novel chemoenzymatic strategy in which diversity is generated by standard solid phase protocols and stereoselectivity by subsequent enzymatic cyclization. For this approach, we cloned, overproduced, and biochemically characterized the recombinant thioesterase domain SnbDE TE of the pristinamycin I nonribosomal peptide synthetase from Streptomyces pristinaespiralis. SnbDE TE catalyzes regioselective ring closure of linear peptide thioester analogues of pristinamycin I as well as stereoselective cyclization out of complex in situ racemizing substrate mixtures, enabling synthesis of Streptogramin B variants via a dynamic kinetic resolution assay. A remarkable substrate tolerance was detected for the enzymatic cyclization including all the seven positions of the peptide backbone. Interestingly, SnbDE TE was observed to be the first cyclase from a macrolactone forming NRPS which is additionally able to catalyze macrolactamization of peptide thioester substrates. An N-methylated peptide bond between positions 4 and 5 is mandatory for a high substrate turnover. The presented strategy is potent to screen for analogues with improved activity and guides our understanding of structure--activity relationships in the important class of streptogramin antibiotics.

Synthesis of linear gramicidin requires the cooperation of two independent reductases.

The linear pentadecapeptide gramicidin has been reported to be assembled by four large multimodular nonribosomal peptide synthetases (NRPSs), LgrABCD, that comprise 16 modules. During biosynthesis, the N-formylated 16mer peptide is bound to the peptidyl carrier protein (PCP) of the terminal module via a thioester bond to the carboxyl group of the last amino acid glycine(16). In a first reaction the peptide is released from the protein template in an NAD(P)H-dependent reduction step catalyzed by the adjacent reductase forming an aldehyde intermediate. Here we present the biochemical proof that this aldehyde intermediate is further reduced by an aldoreductase, LgrE, in an NADPH-dependent manner to form the final product gramicidin A, N-formyl-pentadecapeptide-ethanolamine. To determine the potential use of the two reductases in the construction of hybrid NRPSs, we have tested their ability to accept a variety of different substrates in vitro. The results obtained give way to a broad spectrum of possible use.

Synthesis and derivatization of daptomycin: a chemoenzymatic route to acidic lipopeptide antibiotics.

Daptomycin is a branched cyclic nonribosomally assembled acidic lipopeptide, which is the first clinically approved antibiotic of this class. Here we show that the recombinant cyclization domain of the Streptomyces coelicolor calcium-dependent antibiotic (CDA) nonribosomal peptide synthetase (NRPS) is a versatile tool for the chemoenzymatic generation of daptomycin derivatives. Linear CDA undecapeptide thioesters with single exchanges at six daptomycin-specific residues were successfully cyclized by CDA cyclase. Simultaneous incorporation of all six of these residues into the peptide backbone and elongation of the N-terminus of CDA by two residues yielded a daptomycin derivative that lacked only the beta-methyl group of l-3-methylglutamate. Bioactivity studies with several substrate analogues revealed a significant role of nonproteinogenic constituents for antibacterial potency. In accordance with acidic lipopeptides, the bioactivity of the chemoenzymatic assembled daptomycin analogue is dependent on the concentration of calcium ions. Single deletions of the four acidic residues in the peptide backbone suggest that only two aspartic acid residues are essential for antimicrobial potency. These two residues are strictly conserved among other nonribosomal acidic lipopeptides and the EF-motif of ribosomally assembled calmodulin. Based on these findings CDA cyclase is a versatile catalyst that can be used to generate novel daptomycin derivatives that are otherwise difficult to obtain by chemical modification of the parental tridecapeptide to improve further its therapeutic activity.