Discovery of novel polyamide-pyrrolobenzodiazepine hybrids for antibody-drug conjugates.

Pyrrolobenzodiazepine (PBD) dimers are well-known highly potent antibody drug conjugate (ADC) payloads. The corresponding PBD monomers, in contrast, have received much less attention from the ADC community. We prepared several novel polyamide-linked PBD monomers and evaluated their utility as ADC payloads. The unconjugated polyamide-PBD hybrids exhibited potent antiproliferative activity (IC50 range: 10-11-10-8 M) against a variety of HER2-expressing cancer cell lines. Several peptide-linked variants of the lead compound were prepared and conjugated to trastuzumab to afford ADCs with drug-to-antibody (DAR) ratios ranging from 3 to 5. The ADCs exhibited antigen-dependent cytotoxicity in vitro and potently suppressed tumor xenograft growth in vivo in a target-dependent manner. Moreover, the ADCs were well-tolerated in both mouse and rat. This work demonstrates for the first time that PBD polyamide hybrids can serve as effective ADC payloads.

Dolaflexin: A Novel Antibody-Drug Conjugate Platform Featuring High Drug Loading and a Controlled Bystander Effect.

After significant effort over the last 30 years, antibody-drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement in vitro characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing in vitro, was effective in vivo in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.

The Dolaflexin-based Antibody-Drug Conjugate XMT-1536 Targets the Solid Tumor Lineage Antigen SLC34A2/NaPi2b.

Target selection for antibody-drug conjugates (ADC) frequently focuses on identifying antigens with differential expression in tumor and normal tissue, to mitigate the risk of on-target toxicity. However, this strategy restricts the possible target space. SLC34A2/NaPi2b is a sodium phosphate transporter expressed in a variety of human tumors including lung and ovarian carcinoma, as well as the normal tissues from which these tumors arise. Previous clinical trials with a NaPi2b targeting MMAE-ADCs have shown objective durable responses. However, the protein-based biomarker assay developed for use in that study was unable to discern a statistically significant relationship between NaPi2b protein expression and the probability of response. XMT-1536 is a NaPi2b targeting ADC comprised of a unique humanized antibody conjugated with 10-15 auristatin F- hydroxypropylamide (AF-HPA) payload molecules via the Dolaflexin platform. AF-HPA is a cell-permeable, antimitotic compound that is slowly metabolized intratumorally to an active, very low-permeable metabolite, auristatin F (AF), resulting in controlled bystander killing. We describe the preclinical in vitro and in vivo antitumor effects of XMT-1536 in models of ovarian and lung adenocarcinoma. Pharmacokinetic analysis showed approximately proportional increases in exposure in rat and monkey. Systemic free AF-HPA and AF concentrations were observed to be low in all animal species. Finally, we describe a unique IHC reagent, generated from a chimeric construct of the therapeutic antibody, that was used to derive a target expression and efficacy relationship in a series of ovarian primary xenograft cancer models.

A Polymer-Based Antibody-Vinca Drug Conjugate Platform: Characterization and Preclinical Efficacy.

Antibody-drug conjugates (ADC) are an emerging drug class that uses antibodies to improve cytotoxic drug targeting for cancer treatment. ADCs in current clinical trials achieve a compromise between potency and physicochemical/pharmacokinetic properties by conjugating potent cytotoxins directly to an antibody at a 4:1 or less stoichiometric ratio. Herein, we report a novel, polyacetal polymer-based platform for creating ADC that use poly-1-hydroxymethylethylene hydroxymethyl-formal (PHF), also known as Fleximer. The high hydrophilicity and polyvalency properties of the Fleximer polymer can be used to produce ADC with high drug loading without compromising physicochemical and pharmacokinetic properties. Using trastuzumab and a vinca drug derivative to demonstrate the utility of this platform, a novel Fleximer-based ADC was prepared and characterized in vivo. The ADC prepared had a vinca-antibody ratio of 20:1. It exhibited a high antigen-binding affinity, an excellent pharmacokinetic profile and antigen-dependent efficacy, and tumor accumulation in multiple tumor xenograft models. Our findings illustrate the robust utility of the Fleximer platform as a highly differentiated alternative to the conjugation platforms used to create ADC currently in clinical development.

Transferrin as a model system for method development to study structure, dynamics and interactions of metalloproteins using mass spectrometry.

BACKGROUND: Transferrin (Tf) is a paradigmatic metalloprotein, which has been extensively studied in the past and still is a focal point of numerous investigation efforts owing to its unique role in iron homeostasis and enormous promise as a component of a wide range of therapies. SCOPE OF REVIEW: Electrospray ionization mass spectrometry (ESI MS) is a potent analytical tool that has been used successfully to study various properties of Tf and Tf-based products, ranging from covalent structure and metal binding to conformation and interaction with their physiological partners. MAJOR CONCLUSIONS: Various ESI MS-based techniques produce unique information on Tf properties and behavior that is highly complementary to information provided by other experimental techniques. GENERAL SIGNIFICANCE: The experimental ESI MS-based techniques developed for Tf studies are not only useful for understanding of fundamental aspects of the iron-binding properties of this protein and optimizing Tf-based therapeutic products, but can also be applied to study a range of other metalloproteins. This article is part of a Special Issue entitled Transferrins: Molecular mechanisms of iron transport and disorders.

Towards high-throughput metabolomics using ultrahigh-field Fourier transform ion cyclotron resonance mass spectrometry.

With unmatched mass resolution, mass accuracy, and exceptional detection sensitivity, Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) has the potential to be a powerful new technique for high-throughput metabolomic analysis. In this study, we examine the properties of an ultrahigh-field 12-Tesla (12T) FTICR-MS for the identification and absolute quantitation of human plasma metabolites, and for the untargeted metabolic fingerprinting of inbred-strain mouse serum by direct infusion (DI). Using internal mass calibration (mass error ≤1 ppm), we determined the rational elemental compositions (incorporating unlimited C, H, N and O, and a maximum of two S, three P, two Na, and one K per formula) of approximately 250 out of 570 metabolite features detected in a 3-min infusion analysis of aqueous extract of human plasma, and were able to identify more than 100 metabolites. Using isotopically-labeled internal standards, we were able to obtain excellent calibration curves for the absolute quantitation of choline with sub-pmol sensitivity, using 500 times less sample than previous LC/MS analyses. Under optimized serum dilution conditions, chemical compounds spiked into mouse serum as metabolite mimics showed a linear response over a 600-fold concentration range. DI/FTICR-MS analysis of serum from 26 mice from 2 inbred strains, with and without acute trichloroethylene (TCE) treatment, gave a relative standard deviation (RSD) of 4.5%. Finally, we extended this method to the metabolomic fingerprinting of serum samples from 49 mice from 5 inbred strains involved in an acute alcohol toxicity study, using both positive and negative electrospray ionization (ESI). Using these samples, we demonstrated the utility of this method for high-throughput metabolomics, with more than 400 metabolites profiled in only 24 h. Our experiments demonstrate that DI/FTICR-MS is well-suited for high-throughput metabolomic analysis.

Indirect detection of protein-metal binding: interaction of serum transferrin with In3+ and Bi3+.

Transferrins comprise a class of monomeric glycoproteins found in all vertebrates, whose function is iron sequestration and transport. In addition to iron, serum transferrin also binds a variety of other metals and is believed to provide a route for the in vivo delivery of such metals to cells. In the present study, ESI MS is used to investigate interactions between human serum transferrin and two nonferrous metals, indium (a commonly used imaging agent) and bismuth (a component of many antiulcer drugs). While the UV-Vis absorption spectroscopy measurements clearly indicate that both metals bind strongly to transferrin in solution, the metal-protein complex can be detected by ESI MS only for indium, but not for bismuth. Despite the apparently low stability of the transferrin-bismuth complex in the gas phase, presence of such complex in solution can be established by ESI MS indirectly. This is done by monitoring the evolution of charge state distributions of transferrin ions upon acid-induced protein unfolding in the presence and in the absence of the metal in solution. The anomalous instability of the transferrin-bismuth complex in the gas phase is rationalized in terms of conformational differences between this form of transferrin and the holo-forms of this protein produced by binding of metals with smaller ionic radii (e.g., Fe3+ and In3+). The large size of Bi3+ ion is likely to prevent formation of a closed conformation (canonical structure of the holo-protein), resulting in a non-native metal coordination. It is suggested that transferrin retains the open conformation (characteristic of the apo-form) upon binding Bi3+, with only two ligands in the metal coordination sphere provided by the protein itself. This suggestion is corroborated by the results of circular dichroism measurements in the near-UV range. Since the cellular consumption of metals in the transferrin cycle critically depends upon recognition of the holo-protein complex by the transferrin receptor, the noncanonical conformation of the transferrin-bismuth complex may explain very inefficient delivery of bismuth to cells even when a high dosage of bismuth-containing drugs is administered for prolonged periods of time.

Interlobe communication in human serum transferrin: metal binding and conformational dynamics investigated by electrospray ionization mass spectrometry.

Human serum transferrin (hTF) is an iron transport protein, comprising two lobes (N and C), each containing a single metal-binding center. Despite substantial structural similarity between the two lobes, studies have demonstrated the existence of significant differences in their metal-binding properties. The nature of these differences has been elucidated through the use of electrospray ionization mass spectrometry to study both metal retention and conformational properties of hTF under a variety of conditions. In the absence of chelating agents or nonsynergistic anions, the diferric form of hTF remains intact until the pH is lowered to 4.5. The monoferric form of hTF retains the compact conformation until the pH is lowered to 4.0, whereas the apoprotein becomes partially unfolded at pH as high as 5.5. Selective (lobe-specific) modulation of the iron-binding properties of hTF using recombinant forms of the protein (in which the pH-sensitive elements in each lobe were mutated) verifies that the N-lobe of the protein has a lower affinity for ferric ion. Surprisingly, the apo-N-lobe is significantly less flexible compared to the apo-C-lobe. Furthermore, the conformation of the iron-free N-lobe is stabilized when the C-lobe contains iron, confirming the existence of an interlobe interaction within the protein. The experimental results provide strong support for the earlier suggestion that hTF interacts with its receptor (TFR) primarily through the C-lobe both at the cell surface and inside the endosome.

Differential effect of a his tag at the N- and C-termini: functional studies with recombinant human serum transferrin.

Attachment of a cleavable hexa His tag is a common strategy for the production of recombinant proteins. Production of two recombinant nonglycosylated human serum transferrins (hTF-NG), containing a factor Xa cleavage site and a hexa His tag at the carboxyl terminus, has been described [Mason et al. (2001) Prot. Exp. Purif 23, 142-150]. More recently, hTF-NG with an amino-terminal His tag and a factor Xa cleavage site has been expressed (>30 mg/L) in baby hamster kidney cells and purified from the tissue culture medium. Although it is frequently assumed that addition of a His tag has little or no effect on function, this is not always confirmed experimentally. In the present study, in vitro quantitative data clearly shows that the presence of the C-terminal His tag has an effect on the release of iron from recombinant hTF at pH 7.4 and 5.6. Measurement of the rate of release from both the N- and C-lobes is reduced 2-4-fold. These findings provide further compelling evidence that the two lobes communicate with each other and highlight the importance of the C-terminal portion of the C-terminal lobe in this interaction. In contrast to these results, we demonstrate that the presence of a His tag at the N-terminus of hTF has no effect on the rate of iron release from either lobe. In a competition experiment, both unlabeled N- and C-terminal His-tagged constructs were equally effective at inhibiting the binding of radio-iodinated diferric glycosylated hTF from a commercial source to receptors on HeLa cells as the unlabeled recombinant diferric hTF-NG control. Thus, the presence of a His tag at either the N- or C-terminus of hTF-NG has no apparent effect on the ability of these hTF species to bind to transferrin receptors.