A phase 2 trial of ponatinib in Philadelphia chromosome-positive leukemias.

BACKGROUND: Ponatinib is a potent oral tyrosine kinase inhibitor of unmutated and mutated BCR-ABL, including BCR-ABL with the tyrosine kinase inhibitor-refractory threonine-to-isoleucine mutation at position 315 (T315I). We conducted a phase 2 trial of ponatinib in patients with chronic myeloid leukemia (CML) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-positive ALL). METHODS: We enrolled 449 heavily pretreated patients who had CML or Ph-positive ALL with resistance to or unacceptable side effects from dasatinib or nilotinib or who had the BCR-ABL T315I mutation. Ponatinib was administered at an initial dose of 45 mg once daily. The median follow-up was 15 months. RESULTS: Among 267 patients with chronic-phase CML, 56% had a major cytogenetic response (51% of patients with resistance to or unacceptable side effects from dasatinib or nilotinib and 70% of patients with the T315I mutation), 46% had a complete cytogenetic response (40% and 66% in the two subgroups, respectively), and 34% had a major molecular response (27% and 56% in the two subgroups, respectively). Responses were observed regardless of the baseline BCR-ABL kinase domain mutation status and were durable; the estimated rate of a sustained major cytogenetic response of at least 12 months was 91%. No single BCR-ABL mutation conferring resistance to ponatinib was detected. Among 83 patients with accelerated-phase CML, 55% had a major hematologic response and 39% had a major cytogenetic response. Among 62 patients with blast-phase CML, 31% had a major hematologic response and 23% had a major cytogenetic response. Among 32 patients with Ph-positive ALL, 41% had a major hematologic response and 47% had a major cytogenetic response. Common adverse events were thrombocytopenia (in 37% of patients), rash (in 34%), dry skin (in 32%), and abdominal pain (in 22%). Serious arterial thrombotic events were observed in 9% of patients; these events were considered to be treatment-related in 3%. A total of 12% of patients discontinued treatment because of an adverse event. CONCLUSIONS: Ponatinib had significant antileukemic activity across categories of disease stage and mutation status. (Funded by Ariad Pharmaceuticals and others; PACE ClinicalTrials.gov number, NCT01207440 .).

A humanized system for pharmacologic control of gene expression.

Gene therapy was originally conceived as a medical intervention to replace or correct defective genes in patients with inherited disorders. However, it may have much broader potential as an alternative delivery platform for protein therapeutics, such as cytokines, hormones, antibodies and novel engineered proteins. One key technical barrier to the widespread implementation of this form of therapy is the need for precise control over the level of protein production. A suitable system for pharmacologic control of therapeutic gene expression would permit precise titration of gene product dosage, intermittent or pulsatile treatment, and ready termination of therapy by withdrawal of the activating drug. We set out to design such a system with the following properties: (1) low baseline expression and high induction ratio; (2) positive control by an orally bioavailable small-molecule drug; (3) reduced potential for immune recognition through the exclusive use of human proteins; and (4) modularity to allow the independent optimization of each component using the tools of protein engineering. We report here the properties of this system and demonstrate its use to control circulating levels of human growth hormone in mice implanted with engineered human cells.

Evaluation of a rapamycin-regulated serotype 2 adeno-associated viral vector in macaque parotid glands.

OBJECTIVES: Salivary glands are useful target organs for local and systemic gene therapeutics. For such applications, the regulation of transgene expression is important. Previous studies by us in murine submandibular glands showed that a rapamycin transcriptional regulation system in a single serotype 2, adeno-associated viral (AAV2) vector was effective for this purpose. This study evaluated if such a vector was similarly useful in rhesus macaque parotid glands. METHODS: A recombinant AAV2 vector (AAV-TF-RhEpo-2.3w), encoding rhesus erythropoietin (RhEpo) and a rapamycin-inducible promoter, was constructed. The vector was administered to macaques at either of two doses [1.5 x 10(11) (low dose) or 1.5 x 10(12) (high dose) vector genomes] via cannulation of Stensen's duct. Animals were followed up for 12-14 weeks and treated at intervals with rapamycin (0.1 or 0.5 mg kg(-1)) to induce gene expression. Serum chemistry, hematology, and RhEpo levels were measured at interval. RESULTS: AAV-TF-RhEpo-2.3w administration led to low levels of rapamycin-inducible RhEpo expression in the serum of most macaques. In five animals, no significant changes were seen in serum chemistry and hematology values over the study. One macaque, however, developed pneumonia, became anemic and subsequently required euthanasia. After the onset of anemia, a single administration of rapamycin led to significant RhEpo production in this animal. CONCLUSION: Administration of AAV-TF-RhEpo-2.3w to macaque parotid glands was generally safe, but led only to low levels of serum RhEpo in healthy animals following rapamycin treatment.

A hot spot of binding energy in a hormone-receptor interface.

The x-ray crystal structure of the complex between human growth hormone (hGH) and the extracellular domian of its first bound receptor (hGHbp) shows that about 30 side chains from each protein make contact. Individual replacement of contact residues in the hGHbp with alanine showed that a central hydrophobic region, dominated by two tryptophan residues, accounts for more than three-quarters of the binding free energy. This "functional epitope" is surrounded by less important contact residues that are generally hydrophilic and partially hydrated, so that the interface resembles a cross section through a globular protein. The functionally important residues on the hGHbp directly contact those on hGH. Thus, only a small and complementary set of contact residues maintains binding affinity, a property that may be general to protein-protein interfaces.

Regulation of protein secretion through controlled aggregation in the endoplasmic reticulum.

A system for direct pharmacologic control of protein secretion was developed to allow rapid and pulsatile delivery of therapeutic proteins. A protein was engineered so that it accumulated as aggregates in the endoplasmic reticulum. Secretion was then stimulated by a synthetic small-molecule drug that induces protein disaggregation. Rapid and transient secretion of growth hormone and insulin was achieved in vitro and in vivo. A regulated pulse of insulin secretion resulted in a transient correction of serum glucose concentrations in a mouse model of hyperglycemia. This approach may make gene therapy a viable method for delivery of polypeptides that require rapid and regulated delivery.

Redesigning small molecule-protein interfaces.

Several recent reports have described the rational redesign of small molecule-protein interfaces, generating modified ligands that interact only with suitably mutated proteins. These studies provide powerful reagents for specifically manipulating engineered proteins inside cells, as well as general insights into the factors underlying the binding affinity and specificity of small molecules in general. Progress this year includes the development of allele-specific inhibitors of Src-family tyrosine kinases and the crystal structure determination of a remodeled FKBP-ligand interface.

Controlling mammalian gene expression with small molecules.

Several systems are now available that enable transcription of a gene introduced into mammalian cells to be controlled using small molecules. Among the potential applications are the analysis of gene function through creation of inducible alleles in cell culture and transgenic animals, and, ultimately, the pharmacological control of therapeutic protein production in vivo in the context of gene therapy. Highlights of the past year include several demonstrations of regulated protein production in animal models of gene and cell therapy, and the development of a new approach to transcriptional regulation using chemical inducers of dimerization.

Structural and functional analysis of the 1:1 growth hormone:receptor complex reveals the molecular basis for receptor affinity.

The designed G120R mutant of human growth hormone (hGH) is an antagonist and can bind only one molecule of the growth hormone receptor. We have determined the crystal structure of the 1:1 complex between this mutant and the receptor extracellular domain (hGHbp) at 2.6 A resolution, and used it to guide a detailed survey of the structural and functional basis for hormone-receptor recognition. The overall structure of the complex is very similar to the equivalent portion of the 1:2 complex, showing that formation of the active complex does not involve major conformational changes. However, a segment involved in receptor-receptor interactions in the 1:2 complex is disordered in this structure, suggesting that its productive conformation is stabilized by receptor dimerization. The hormone binding site of the receptor comprises a central hydrophobic patch dominated by Trp104 and Trp169, surrounded by a hydrophilic periphery containing several well-ordered water molecules. Previous alanine scanning showed that the hydrophobic "hot spot" confers most of the binding energy. The new structural data, coupled with binding and kinetic analysis of further mutants, indicate that the hot spot is assembled cooperatively and that many residues contribute indirectly to binding. Several hydrophobic residues serve to orient the key tryptophan residues; kinetic analysis suggests that Pro106 locks the Trp104 main-chain into a required conformation. The electrostatic contacts of Arg43 to hGH are less important than the intramolecular packing of its alkyl chain with Trp169. The true functional epitope that directly contributes binding energy may therefore comprise as few as six side-chains, participating mostly in alkyl-aromatic stacking interactions. Outside the functional epitope, multiple mutation of residues to alanine resulted in non-additive increases in affinity: up to tenfold for a hepta-alanine mutant. Contacts in the epitope periphery can therefore attenuate the affinity of the central hot spot, perhaps reflecting a role in conferring specificity to the interaction.

In vitro selection from protein and peptide libraries.

In vitro selection from molecular libraries has rapidly come of age as a protein-engineering tool. Dramatic increases in protein affinity can be engineered using phage-display libraries, and specific antibodies can be selected directly from a single 'naïve' library of their genes. Repertoires of small molecules are a potentially valuable resource for drug discovery. Libraries of linear peptides provide ligands for proteins that recognize continuous epitopes, and low-affinity mimics of some small molecules, but generally do not contain mimics of large molecular interfaces. Switching to constrained peptide formats, and deploying more diverse, non-peptide chemical libraries, may bring greater success.

Investigating protein-ligand interactions with a mutant FKBP possessing a designed specificity pocket.

Using structure-based design and protein mutagenesis we have remodeled the FKBP12 ligand binding site to include a sizable, hydrophobic specificity pocket. This mutant (F36V-FKBP) is capable of binding, with low or subnanomolar affinities, novel synthetic ligands possessing designed substituents that sterically prevent binding to the wild-type protein. Using binding and structural analysis of bumped compounds, we show here that the pocket is highly promiscuous-capable of binding a range of hydrophobic alkyl and aryl moieties with comparable affinity. Ligand affinity therefore appears largely insensitive to the degree of occupancy or quality of packing of the pocket. NMR spectroscopic analysis indicates that similar ligands can adopt radically different binding modes, thus complicating the interpretation of structure-activity relationships.

Intravenous safety and pharmacokinetics of a novel dimerizer drug, AP1903, in healthy volunteers.

AP1903 is a novel gene-targeted drug that is being developed for use in drug-regulated cell therapies. An intravenous, single-blind, placebo- and saline-controlled, ascending-dose study was performed to evaluate the safety, tolerability, and pharmacokinetics of AP1903. Twenty-eight normal healthy male volunteers were randomized into five dosage groups of AP1903 (0.01, 0.05, 0.1, 0.5, and 1 mg/kg). Within each group, 4 volunteers received a single dose of AP1903, 1 volunteer received an equal volume of placebo, and 1 received an equal volume of normal saline. The only exception was in the 0.5 mg/kg group, in which 4 volunteers were dosed: 3 received AP1903 and 1 received normal saline. All dosages were administered as intravenous infusions over 2 hours. Clinical safety parameters were monitored, and serial blood and urine samples were collected for analysis of AP1903. No drug-related adverse events were observed at any of the dose levels with the possible exception of facial flushing in 1 volunteer at the 1.0 mg/kg dose level. AP1903 plasma levels were directly proportional to the administered dose, with mean Cmax values ranging from approximately 10 to 1,275 ng/mL over the 0.01 to 1.0 mg/kg dose range. Following the infusion period, blood concentrations revealed a rapid distribution phase, with plasma levels being reduced to approximately 18%, 7%, and 1% of the maximal concentration at 0.5, 2, and 10 hours postdose, respectively. AP1903 was shown to be safe and well tolerated at all dose levels and demonstrated a favorable pharmacokinetic profile at doses well above the anticipated therapeutic dose.

By-passing immunization: building high affinity human antibodies by chain shuffling.

Diverse antibody libraries can be displayed on the surface of filamentous bacteriophage, and selected by panning of the phage with antigen. This allows human antibodies to be made directly in vitro without prior immunization, thus mimicking the primary immune response. Here we have improved the affinity of one such "primary" antibody by sequentially replacing the heavy and light chain variable (V) region genes with repertoires of V-genes (chain shuffling) obtained from unimmunized donors. For a human phage antibody for the hapten 2-phenyloxazol-5-one (phOx) (Kd = 3.2 x 10(-7) M), we shuffled the light chains and isolated an antibody with a 20 fold improved affinity. By shuffling the first two hypervariable loops of the heavy chain, we isolated an antibody with a further 15-fold improved affinity. The reshuffled antibody differed in five of the six hypervariable loops from the original antibody and the affinity for phOx (Kd = 1.1 x 10(-9) M) was comparable to that of mouse hybridomas from the tertiary immune response. Reshuffling offers an alternative to random point mutation for affinity maturation of human antibodies in vitro.

Rapamycin control of transgene expression from a single AAV vector in mouse salivary glands.

Salivary glands (SGs) appear to be a useful target site for gene therapeutics. The ability to control transgene expression is essential for clinical application. Previously, in a proof-of-concept study, we have shown that the rapamycin-inducible transcriptional regulation system can regulate protein expression after adenoviral-mediated gene transfer to SGs. To evaluate the potential ability to utilize this regulatory system for long-term control of transgene expression in this tissue, we employed a 'third generation', single adenoassociated serotype 2 viral (AAV2) vector encoding human erythropoietin (hEPO) under the control of a rapamycin-inducible promoter. The vector, rAAV-TF2.3-hEPO (10(10) particles/animal), was delivered to mouse SGs. No detectable increase in serum hEPO or hematocrit levels was observed in the absence of rapamycin administration. However, rapamycin induced elevation of serum hEPO levels, as well as concomitant hematocrit changes, that were dose-dependent, completely reversible, and relatively stable over the course of this study (6 months), with no appreciable change in rapamycin responsiveness. Our results suggest that the rapamycin transcriptional regulation system delivered in a single AAV2 vector to SGs may be valuable for systemic protein replacement applications.

Regulated gene expression systems.

Most gene therapy research to date has focused on solving the delivery problem--getting genes efficiently and stably into target cells and tissues. Those working on systems for regulating the expression of genes once delivered have often been accused of trying to run before they can walk. Yet regulation is likely to be essential if gene therapy is to realize its full potential as a mainstream clinical option for delivering proteins. Dramatic progress has been made in designing and testing systems in which expression is controlled by orally active drugs. The next few years should see the first clinical trials of drug-regulated gene therapies.

'Sticky feet'-directed mutagenesis and its application to swapping antibody domains.

We describe a novel technique for precisely cutting and pasting two DNA sequences without using restriction sites. The method is based on site-directed mutagenesis and uses a long primer, generated by the polymerase chain reaction (PCR), to transfer large segments of DNA into a single-stranded template. The primer anneals to the template by virtue of 'sticky feet' sequences (complementary to the template) which are introduced at the ends of the primer by the PCR. Yields of desired recombinants were high (approximately 36%) and the transplanted sequences (approximately 400bp) free of errors. We have used this technique to swap CH2 domains between two mouse antibodies, and find that this domain can carry features critical for triggering complement lysis, in addition to the C1q binding motif.

The antagonistic effects of acetate and lactate upon the trichomonacidal activity of metronidazole.

Lactate and acetate at 5 mM were found to be antagonistic to the killing action of metronidazole on Trichomonas vaginalis under aerobic but not anaerobic conditions. These compounds had no effect on the growth of the parasite, or the rate of metronidazole reduction by, and the activities of NADH oxidase and NADPH oxidase in, parasite homogenates. The phenomenon reported could be one reason why metronidazole is not always effective in the treatment of trichomoniasis.

Antitrichomonal activity of compounds that affect DNA and its repair.

The trichomonacidal activity of dimethyl sulphate (DMS) was increased a 100-fold by administration in combination with benzamide and aminobenzamides, compounds thought to interfere with DNA repair in mammalian cells. The same benzamides did not enhance the trichomonacidal activities of metronidazole or a thiosemicarbazone. The results suggest that Trichomonas vaginalis is able to repair damaged DNA and that neither metronidazole nor the thiosemicarbazone affect trichomonal DNA in the same way as DMS. They do not rule out the possibility that the drugs have some other action on DNA.

Making antibody fragments using phage display libraries.

To by-pass hybridoma technology and animal immunization, we are trying to build antibodies in bacteria by mimicking features of immune selection. Recently we used fd phage to display antibody fragments fused to a minor coat protein, allowing enrichment of phage with antigen. Using a random combinatorial library of the rearranged heavy (VH) and kappa (V kappa) light chains from mice immune to the hapten 2-phenyloxazol-5-one (phOx), we have now displayed diverse libraries of antibody fragments on the surface of fd phage. After a single pass over a hapten affinity column, fd phage with a range of phOx binding activities were detected, at least one with high affinity (dissociation constant, Kd = 10(-8) M). A second pass enriched for the strong binders at the expense of the weak. The binders were encoded by V genes similar to those found in anti-phOx hybridomas but in promiscuous combinations (where the same V gene is found with several different partners). By combining a promiscuous VH or V kappa gene with diverse repertoires of partners to create hierarchical libraries, we elicited many more pairings with strong binding activities. Phage display offers new ways of making antibodies from V-gene libraries, altering V-domain pairings and selecting for antibodies with good affinities.

Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector.

Small molecule-regulated transcription has broad utility and would benefit from an easily delivered self-contained regulatory cassette capable of robust, tightly controlled target gene expression. We describe the delivery of a modified dimerizer-regulated gene expression system to cells on a single retrovirus. A transcription factor cassette responsive to the natural product dimerizer rapamycin was optimized for retroviral delivery by fusing a highly potent chimeric activation domain to the rapamycin-binding domain of FKBP-rapamycin-associated protein (FRAP). This improvement led to an increase in both the potency and maximal levels of gene expression induced by rapamycin, or nonimmunosuppressive rapamycin analogs. The modified transcription factor cassette was incorporated along with a target gene into a single rapamycin-responsive retrovirus. Cell pools stably transduced with the single virus system displayed negligible basal expression and gave induction ratios of at least three orders of magnitude in the presence of rapamycin or a nonimmunosuppressive rapamycin analog. Levels of induced gene expression were comparable to those obtained with the constitutive retroviral long terminal repeat and the single virus system performed well in four different mammalian cell lines. Regulation with the dimerizer-responsive retrovirus was tight enough to allow the generation of cell lines displaying inducible expression of the highly toxic diphtheria toxin A chain gene. The ability to deliver the tightly inducible rapamycin system in a single retrovirus should facilitate its use in the study of gene function in a broad range of cell types.