Jennifer R. Allen

Preparation and evaluation of unimolecular pentavalent and hexavalent antigenic constructs targeting prostate and breast cancer: a synthetic route to anticancer vaccine candidates.

Several novel, fully synthetic, carbohydrate-based antitumor vaccines have been assembled. Each construct consists of multiple cancer-related antigens displayed on a single polypeptide backbone. Recent advances in synthetic methodology have allowed for the incorporation of a complex oligosaccharide terminating in a sialic acid residue (i.e., GM2) as one of the carbohydrate antigens. Details of the vaccine synthesis as well as the results of preliminary immunological investigations are described herein.

On the power of chemical synthesis: Immunological evaluation of models for multiantigenic carbohydrate-based cancer vaccines

Synthetic carbohydrate cancer vaccines have been shown to stimulate antibody-based immune responses in both preclinical and clinical settings. The antibodies have been observed to react in vitro with the corresponding natural carbohydrate antigens expressed on the surface of tumor cells, and are able to mediate complement-dependent and/or antibody-dependent cell-mediated cytotoxicity. Furthermore, these vaccines have proven to be safe when administered to cancer patients. Until recently, only monovalent antigen constructs had been prepared and evaluated. Advances in total synthesis have now enabled the preparation of multivalent vaccine constructs, which contain several different tumor-associated carbohydrate antigens. Such constructs could, in principle, serve as superior mimics of cell surface antigens and, hence, as potent cancer vaccines. Here we report preclinical ELISA-based evaluation of a TF–Ley–Tn bearing construct (compound 3) with native mucin glycopeptide architecture and a Globo-H–Ley–Tn glycopeptide (compound 4) with a nonnative structure. Mice were immunized with one or the other of these constructs as free glycopeptides or as keyhole lymphet hemocyanin conjugates. Either QS-21 or the related GPI-0100 were coadministered as adjuvants. Both keyhole lymphet hemocyanin conjugates induced IgM and IgG antibodies against each carbohydrate antigen, however, the mucin-based TF–Ley–Tn construct was shown to be less antigenic than the unnatural Globo-H–Ley–Tn construct. The adjuvants, although related, proved significantly different, in that GPI-0100 consistently induced higher titers of antibodies than QS-21. The presence of multiple glycans in these constructs did not appear to suppress the response against any of the constituent antigens. Compound 4, the more antigenic of the two constructs, was also examined by fluorescence activated cell sorter analysis. Significantly, from these studies it was shown that antibodies stimulated in response to compound 4 reacted with tumor cells known to selectively express the individual antigens. The results demonstrate that single vaccine constructs bearing several different carbohydrate antigens have the potential to stimulate a multifaceted immune response.

Vaccination of patients with small-cell lung cancer with synthetic fucosyl GM-1 conjugated to keyhole limpet hemocyanin.

Purpose: Immunotherapy directed toward cell surface antigens may provide a novel approach to the eradication of chemoresistant micrometastatic disease in patients with small-cell lung cancer (SCLC). Studies in SCLC cell lines and human tissues suggest that the ganglioside fucosyl GM1 is an abundant yet specific target. A prior clinical study demonstrated the potent immunogenicity of fucosyl GM-1 derived from bovine thyroid gland, conjugated to keyhole limpet hemocyanin (KLH) and administered with QS-21 adjuvant. Experimental Design: We tested the immunogenicity of three different doses of a synthetic version of fucosyl-GM1 in patients with SCLC after a major response to initial therapy. The primary end point was to establish the lowest effective dose capable of inducing antibody production. Results: Five of six patients at the 30-μg dose and three of five patients at the 10-μg dose mounted IgM responses of 1:80 or greater. These antibodies were confirmed by flow cytometry in seven of eight cases. None of the patients at the 3-μg dose had titers above 1:80. One patient at the 30-μg dose had an IgG response with a titer of 1:80. The sera from six of the eight responders induced potent complement-mediated cytotoxicity of tumor cells. Conclusions: Vaccination with the synthetic fucosyl GM1-KLH conjugate induces an IgM antibody response against fucosyl GM1 and tumor cells expressing fucosyl GM1, comparable with the response induced by the bovine derivative. We plan to combine synthetic fucosyl GM1 vaccine at a dose of 30 μg with vaccines against three other antigens—GM2, Globo H, and polysialic acid—to test in patients with SCLC after initial chemotherapy.

Vom Labor zur Klinik: vollsynthetische Antitumor‐Impfstoffe auf Kohlenhydratbasis

Dieser Aufsatz gibt einen Uberblick uber die Untersuchungen unserer Arbeitsgruppe zur Synthese von Oligosacchariden und Glycokonjugaten mit dem Ziel der Herstellung und Erprobung von Impfstoffen auf der Basis kohlenhydrathaltiger Tumorantigene. Unser Ausgangspunkt war die bekannte Tendenz transformierter Zellen, auf der Zelloberflache vergleichsweise selektiv Kohlenhydratmotive in Form von Glycoproteinen oder Glycolipiden zu exprimieren. Angeregt wurden diese Untersuchungen dadurch, dass derartige Antitumor-Impfstoffe effektive Targets fur Immunerkennung und -angriff sein konnten; es ist jedoch schwierig, ausreichende Substanzmengen aus naturlichen Quellen zu gewinnen. Nachdem die Totalsynthese tumorassoziierter Kohlenhydratantigene gelungen war, war im klinischen Umfeld zu klaren, ob das menschliche Immunsystem auf solche vollsynthetischen Antigene zielgerichtet und sinnvoll antworten kann. Dafur wurden die Ressourcen von Chemie und Immunologie zu einem gemeinsamen Angriff zusammengefuhrt. Die Synthese von tumorassoziierten Kohlenhydratantigenen sowie die Immunkonjugation und die Ergebnisse der Impfung von Mausen mit solchen Konstrukten werden beschrieben. Hierfur war die Konjugation an einen immunogenen Trager notwendig, wofur sich KLH-Konjugate als geeignet erwiesen. In praklinischen und klinischen Studien mit synthetischen Kohlenhydratkonjugaten als Impfstoffen lies sich die Induktion einer IgM- und IgG-Antikorper-Reaktion zeigen. Eine andere Strategie bestand in der Verwendung von Glycopeptidclustern als Targets fur den Immunangriff. Anfanglich richtete sich unsere Aufmerksamkeit auf mucinahnliche, O-glycosidisch gebundene Glycopeptide. Synthetische trimere Cluster von Glycoepitopen, die sich von den Tn-, TF- und Ley-Antigenen ableiteten, erwiesen sich nach geeigneter Biokonjugation als immunogen. Die Hoffnung ist, dass Patienten, die mit den synthetischen Kohlenhydrat-Impfstoffen in Kombination mit einem Adjuvans immunisiert werden, Antikorper gegen Tumorzellen produzieren und dass die Produktion solcher Antikorper der Ausbreitung des Tumors entgegenwirkt, wodurch die Uberlebenschancen und die Prognose fur die „Lebensqualitat” verbessert werden.

A Second Generation Synthesis of the MBr1 (Globo‐H) Breast Tumor Antigen: New Application of the n‐Pentenyl Glycoside Method for Achieving Complex Carbohydrate Protein Linkages

A new synthesis of the hexasaccharide MBr1 antigen (globo-H) is reported. A revised construction with improved efficiency was necessary because an anti-cancer vaccine containing this antigen is entering phase II and phase III clinical trials for prostate cancer. The key feature of this second generation synthesis is the preparation of globo-H as its n-pentenyl glycoside. This group serves as an anomeric protecting group and as a linker for bioconjugation to carrier protein. The resultant synthesis allows for the production of suitable quantities of globo-H for clinical trials.

Rapid identification of a novel small molecule phosphodiesterase 10A (PDE10A) tracer.

A radiolabeled tracer for imaging therapeutic targets in the brain is a valuable tool for lead optimization in CNS drug discovery and for dose selection in clinical development. We report the rapid identification of a novel phosphodiesterase 10A (PDE10A) tracer candidate using a LC-MS/MS technology. This structurally distinct PDE10A tracer, AMG-7980 (5), has been shown to have good uptake in the striatum (1.2% ID/g tissue), high specificity (striatum/thalamus ratio of 10), and saturable binding in vivo. The PDE10A affinity (K(D)) and PDE10A target density (B(max)) were determined to be 0.94 nM and 2.3 pmol/mg protein, respectively, using [(3)H]5 on rat striatum homogenate. Autoradiography on rat brain sections indicated that the tracer signal was consistent with known PDE10A expression pattern. The specific binding of [(3)H]5 to rat brain was blocked by another structurally distinct, published PDE10A inhibitor, MP-10. Lastly, our tracer was used to measure in vivo PDE10A target occupancy of a PDE10A inhibitor in rats using LC-MS/MS technology.

Discovery of clinical candidate 1-(4-(3-(4-(1H-benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)ethanone (AMG 579), a potent, selective, and efficacious inhibitor of phosphodiesterase 10A (PDE10A).

We report the identification of a PDE10A clinical candidate by optimizing potency and in vivo efficacy of promising keto-benzimidazole leads 1 and 2. Significant increase in biochemical potency was observed when the saturated rings on morpholine 1 and N-acetyl piperazine 2 were changed by a single atom to tetrahydropyran 3 and N-acetyl piperidine 5. A second single atom modification from pyrazines 3 and 5 to pyridines 4 and 6 improved the inhibitory activity of 4 but not 6. In the in vivo LC-MS/MS target occupancy (TO) study at 10 mg/kg, 3, 5, and 6 achieved 86-91% occupancy of PDE10A in the brain. Furthermore, both CNS TO and efficacy in PCP-LMA behavioral model were observed in a dose dependent manner. With superior in vivo TO, in vivo efficacy and in vivo PK profiles in multiple preclinical species, compound 5 (AMG 579) was advanced as our PDE10A clinical candidate.

Design, Optimization, and Biological Evaluation of Novel Keto-Benzimidazoles as Potent and Selective Inhibitors of Phosphodiesterase 10A (PDE10A).

Our development of PDE10A inhibitors began with an HTS screening hit (1) that exhibited both high p-glycoprotein (P-gp) efflux ratios in rat and human and poor metabolic stability. On the basis of cocrystal structure of 1 in human PDE10A enzyme, we designed a novel keto-benzimidazole 26 with comparable PDE10A potency devoid of efflux liabilities. On target in vivo coverage of PDE10A in rat brain was assessed using our previously reported LC-MS/MS receptor occupancy (RO) technology. Compound 26 achieved 55% RO of PDE10A at 30 mg/kg po and covered PDE10A receptors in rat brain in a dose-dependent manner. Cocrystal structure of 26 in PDE10A confirmed the binding mode of the novel scaffold. Further optimization resulted in the identification of keto-benzimidazole 34, which showed an increased in vivo efficacy of 57% RO in rats at 10 mg/kg po and an improved in vivo rat clearance and oral bioavailability.

Discovery of potent, orally bioavailable phthalazinone bradykinin B1 receptor antagonists.

The bradykinin B1 receptor is rapidly induced upon tissue injury and inflammation, stimulating the production of inflammatory mediators resulting in plasma extravasation, leukocyte trafficking, edema, and pain. We have previously reported on sulfonamide and sulfone-based B1 antagonists containing a privileged bicyclic amine moiety leading to potent series of 2-oxopiperazines. The suboptimal pharmacokinetics and physicochemical properties of the oxopiperazine sulfonamides led us to seek B1 antagonists with improved druglike properties. Using a pharmacophore model containing a bicyclic amine as anchor, we designed a series of amide antagonists with targeted physicochemical properties. This approach led to a novel series of potent phthalazinone B1 antagonists, where we successfully replaced a sulfonamide acceptor with a cyclic carbonyl unit. SAR studies revealed compounds with subnanomolar B1 binding affinity. These compounds demonstrate excellent cross-species PK properties with high oral bioavailability and potent activity in a rabbit biochemical challenge pharmacodynamic study.

Unfolded Protein Response in Cancer: IRE1α Inhibition by Selective Kinase Ligands Does Not Impair Tumor Cell Viability.

The kinase/endonuclease inositol requiring enzyme 1 (IRE1α), one of the sensors of unfolded protein accumulation in the endoplasmic reticulum that triggers the unfolded protein response (UPR), has been investigated as an anticancer target. We identified potent allosteric inhibitors of IRE1α endonuclease activity that bound to the kinase site on the enzyme. Structure-activity relationship (SAR) studies led to 16 and 18, which were selective in kinase screens and were potent against recombinant IRE1α endonuclease as well as cellular IRE1α. The first X-ray crystal structure of a kinase inhibitor (16) bound to hIRE1α was obtained. Screening of native tumor cell lines (>300) against selective IRE1α inhibitors failed to demonstrate any effect on cellular viability. These results suggest that IRE1α activity is not essential for viability in most tumor cell lines, in vitro, and that interfering with the survival functions of the UPR may not be an effective strategy to block tumorigenesis.