Jason Pinkstaff

Synthesis of site-specific antibody-drug conjugates using unnatural amino acids

Antibody-drug conjugates (ADCs) allow selective targeting of cytotoxic drugs to cancer cells presenting tumor-associated surface markers, thereby minimizing systemic toxicity. Traditionally, the drug is conjugated nonselectively to cysteine or lysine residues in the antibody. However, these strategies often lead to heterogeneous products, which make optimization of the biological, physical, and pharmacological properties of an ADC challenging. Here we demonstrate the use of genetically encoded unnatural amino acids with orthogonal chemical reactivity to synthesize homogeneous ADCs with precise control of conjugation site and stoichiometry. p-Acetylphenylalanine was site-specifically incorporated into an anti-Her2 antibody Fab fragment and full-length IgG in Escherichia coli and mammalian cells, respectively. The mutant protein was selectively and efficiently conjugated to an auristatin derivative through a stable oxime linkage. The resulting conjugates demonstrated excellent pharmacokinetics, potent in vitro cytotoxic activity against Her2+ cancer cells, and complete tumor regression in rodent xenograft treatment models. The synthesis and characterization of homogeneous ADCs with medicinal chemistry-like control over macromolecular structure should facilitate the optimization of ADCs for a host of therapeutic uses.

Internal initiation of translation of five dendritically localized neuronal mRNAs

In neurons, translation of dendritically localized mRNAs is thought to play a role in affecting synaptic efficacy. Inasmuch as components of the translation machinery may be limiting in dendrites, we investigated the mechanisms by which translation of five dendritically localized mRNAs is initiated. The 5′ leader sequences of mRNAs encoding the activity-regulated cytoskeletal protein, the α subunit of calcium–calmodulin-dependent kinase II, dendrin, the microtubule-associated protein 2, and neurogranin (RC3) were evaluated for their ability to affect translation in the 5′ untranslated region of a monocistronic reporter mRNA. In both neural and nonneural cell lines, the activity-regulated cytoskeletal protein, microtubule-associated protein 2, and α-CaM Kinase II leader sequences enhanced translation, whereas the dendrin and RC3 5′ untranslated regions slightly inhibited translation as compared with controls. When cap-dependent translation of these constructs was suppressed by overexpression of a protein that binds the cap-binding protein eIF4E, it was revealed that translation of these mRNAs had both cap-dependent and cap-independent components. The cap-independent component was further analyzed by inserting the 5′ leader sequences into the intercistronic region of dicistronic mRNAs. All five leader sequences mediated internal initiation via internal ribosome entry sites (IRESes). The RC3 IRES was most active and was further characterized after transfection in primary neurons. Although translation mediated by this IRES occurred throughout the cell, it was relatively more efficient in dendrites. These data suggest that IRESes may increase translation efficiency at postsynaptic sites after synaptic activation.

A general approach to site-specific antibody drug conjugates

Significance Here we demonstrate the ability to genetically incorporate nonnative amino acids into proteins in mammalian cells using both transient and stable platform expression systems that provide yields and fidelities compatible with commercial applications. To illustrate the utility of this methodology we have generated chemically homogeneous antibody drug conjugates (NDCs) with precise control over the site and stoichiometry of drug conjugation. In rodent xenograft models these NDCs display improved properties, including half-life, efficacy and safety, relative to conventional heterogeneous ADCs. These advances allow the generation of therapeutic antibody drug conjugates with medicinal chemistry like control over structure, which should greatly facilitate the optimization of their pharmacological activities. Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oxime-bonded, site-specific NDCs were even more apparent when low–antigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicology study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These experiments support the notion that chemically defined antibody conjugates can be synthesized in commercially relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chemical modification.

GLP-1/Glucagon Coagonism Restores Leptin Responsiveness in Obese Mice Chronically Maintained on an Obesogenic Diet

We recently reported restoration of leptin responsiveness in diet-induced obese (DIO) mice using a pharmacologically optimized, polyethylene-glycolated (PEG)-leptin analog in combination with exendin-4 or FGF21. However, the return of leptin action required discontinuation of high-fat diet (HFD) exposure. Here we assess whether a single peptide possessing balanced coagonism at the glucagon-like peptide 1 (GLP-1) and glucagon receptors can restore leptin responsiveness in DIO mice maintained on a HFD. DIO mice were treated with PEG-GLP-1/glucagon (30 nmol/kg every fourth day) to induce an ∼15% body weight loss, upon which they were randomized to continue PEG-GLP-1/glucagon therapy or reassigned to receive supplemental daily PEG-leptin (185 nmol/kg/day). The addition of PEG-leptin to PEG-GLP-1/glucagon resulted in an ∼18% greater weight loss as compared with PEG-GLP-1/glucagon alone and was accompanied by further decreases in food intake and improved glucose and lipid metabolism. The beneficial effect of PEG-leptin supplementation occurred after an initial body weight loss similar to what we previously reported following reduced dietary fat along with PEG-leptin and exendin-4 or FGF21 cotreatment. In summary, we report that GLP-1/glucagon coagonism restores leptin responsiveness in mice maintained on a HFD, thus emphasizing the translational value of this polypharmacotherapy for the treatment of obesity and diabetes.

In Vitro and In Vivo Evaluation of Cysteine and Site Specific Conjugated Herceptin Antibody-Drug Conjugates

Antibody drug conjugates (ADCs) are monoclonal antibodies designed to deliver a cytotoxic drug selectively to antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity and development of the ADC. The cytotoxic drugs or payloads used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for a IgG1 antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non target related toxicities. In these studies we incorporated a non-natural amino acid, para acetyl phenylalanine, at two unique sites within an antibody against Her2/neu. We covalently attached a cytotoxic drug to these sites to form an ADC which contains two drugs per antibody. We report the results from the first direct preclinical comparison of a site specific non-natural amino acid anti-Her2 ADC and a cysteine conjugated anti-Her2 ADC. We report that the site specific non-natural amino acid anti-Her2 ADCs have superior in vitro serum stability and preclinical toxicology profile in rats as compared to the cysteine conjugated anti-Her2 ADCs. We also demonstrate that the site specific non-natural amino acid anti-Her2 ADCs maintain their in vitro potency and in vivo efficacy against Her2 expressing human tumor cell lines. Our data suggests that site specific non-natural amino acid ADCs may have a superior therapeutic window than cysteine conjugated ADCs.

Recruiting Cytotoxic T Cells to Folate-Receptor-Positive Cancer Cells**

Herein, we describe the synthesis of a chemically defined anti-CD3 Fab-folate conjugate that targets cytotoxic T cells to folate receptor positive (FR+) tumors. The unnatural amino acid pacetylphenylalanine (pAcPhe) was site-specifically incorporated into an anti-CD3 Fab and conjugated to folate via the formation of a stable oxime linkage. The anti-CD3 Fab-folate conjugate was able to promote T cell mediated killing of FR+ cancer cells in culture. Moreover, the anti-CD3 Fab-folate conjugate potently eliminates tumor xenografts in mice. This approach can likely be generalized to other ligands that bind cancer and other pathogenic cells.

Comparison of bioanalytical methods for the quantitation of PEGylated human insulin

PURPOSE The quality of bioanalytical data is dependent upon selective, sensitive, and reproducible analytical methods. With evolving technologies available, bioanalytical scientists must assess which is most appropriate for their molecule through proper method validation. For an early stage PEGylated insulin program, the characteristics of four platforms, ELISA, ECL, Gyrolab, and LC-MS/MS, were evaluated using fit-for-purpose method development and validation, while also evaluating costs. METHOD Methods selected for validation required acceptable performance based on satisfaction of a priori criteria prior to proceeding to subsequent stages of validation. LBA pre-validation included reagent selection, evaluation of matrix interference, and range determination. LC-MS/MS pre-validation included selection of a signature peptide; optimization of sample preparation, HPLC, and LC-MS/MS conditions; and calibration range determination. Pre-study validation tested accuracy and precision (mean bias criteria±30%; precision≤30%). Pharmacokinetic (PK) parameters were estimated for an in vivo study with WinNonlin noncompartmental analysis. Statistics were performed with JMP using ANOVA and Tukey-Kramer post hoc analysis. A cost analysis was performed for a 200-sample PK study using the methods from this study. RESULTS All platforms, except Gyrolab, were taken through validation. However, a typical Gyrolab method was included for the cost analysis. Ranges for the ELISA, ECLA, and LC-MS/MS were 8.52-75, 2.09-125, and 100-1000 ng/mL, respectively, and accuracy and precision fell within a priori criteria. PK samples were analyzed in the 3 validated methods. PK profiles and parameters are similar for all methods, except LC-MS/MS, which differed at t=24h and with AUC0-24. Further investigation into this difference is warranted. The cost analysis identified the Gyrolab platform as the most expensive and ELISA as the least expensive, with method specific consumables attributing significantly to costs. CONCLUSIONS ECLA had a larger dynamic range and sensitivity, allowing accurate assessment of PK parameters. Although this method was more expensive than the ELISA, it was the most appropriate for the early stage PEGylated insulin program. While this case study is specific to PEGylated human insulin, it highlights the importance of evaluating and selecting the most appropriate platform for bioanalysis during drug development.

Abstract 668: Folate conjugated site-specifically to anti-human-CD3-Fab is efficacious in mouse models of ovarian cancer

Ovarian cancer affects ∼22,000 women in the US each year. Folate receptor (FOLR1) is overexpressed in a high proportion of ovarian tumors and is generally associated with advanced stage disease. We report a first-in-class bi-specific conjugate for ovarian cancer by using RECODE™ technology that site-specifically incorporates novel amino acids into proteins in bacterial cells. The Ambrx bi-specific conjugate (Fol-aCD3) is developed for epithelial ovarian cancer and consists of (i) Folate that can bind to FOLR1 receptor on ovarian cancer cells and (ii) Anti-human CD3ϵ Fab that can bind to the CD3ϵ receptor on cytotoxic T cells. The potential engagement of T cells by Fol-aCD3 provides for T cell recruitment especially in an immune evasive, suppressive and tolerogenic tumor microenvironment. Para-acetyl phenylalanine (pAcF) containing anti-CD3ϵ Fab is conjugated to hydroxyl-amine-folate drug-linker. The resulting compound was analyzed by LC-MS with >95% conjugation of a single folate per Fab and >95% main peak by size-exclusion chromatography. By an in vitro LDH cytotoxicity assay using activated human T-cells, Fol-aCD3 killed SKOV-3 and Ov-90 cells with an EC50 of 7.4 and 4.4 ng/mL, respectively. The pharmacokinetic half-life in CD1 mice is 20 and 60 minutes for IV and IP administration, respectively. Fol-aCD3 delivered IV at 5, 0.5 or 0.05 mg/kg daily for 5 days inhibited tumor growth in a dose-dependent manner for Ov-90 co-implanted subcutaneously with activated T-cells in NOD-SCID mice on a folate deficient diet. There was no body weight loss (BWL). Fol-aCD3 was tested in a peritoneal dissemination mouse model of ovarian cancer where the cancer cells, Fol-aCD3, and activated T-cells were injected IP. At a dose of 1 mg/kg of Fol-aCD3 delivered every 3rd day simultaneously with activated T-cells, the NOD-SCID mice exhibited transient BWL but recovered by the 4th day. The treated mice experienced 30% and 50% increase in life span (ILS) for SKOV-3 and OV-90, respectively. The increased survival was seen for mice treated immediately upon tumor cell inoculation (prevention mode) as well as for mice treated 17 days after inoculation (treatment mode). Thus, Fol-aCD3 is being considered for development as a therapeutic for ovarian cancer.. Citation Format: Sandra L. Biroc, Marco Gymnopoulos, Shailaja Srinagesh, Brad Hayes, Nick Knudsen, Anthony Manibusan, Jason Pinkstaff, Tim Buss, Kari Cox, Robin Marsden, Lillian Skidmore, Jinming Xia, Ying Sun, Ning Zou, Tsotne Javahishvili. Folate conjugated site-specifically to anti-human-CD3-Fab is efficacious in mouse models of ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 668. doi:10.1158/1538-7445.AM2014-668

Abstract LB-20: PEGylated TRAIL/Apo2L shows greatly improved circulating half life and improved anti-tumor efficacy in xenograft models for colon and pancreatic cancer

PURPOSE TRAIL/Apo2L is a death receptor (DR) agonist, inducing apoptosis through the extrinsic pathway in tumor cells expressing DR4 or DR5. Agonistic antibodies or the naked ligand have been tested in the clinic to treat solid tumors. Owing to its extremely short serum half life, TRAIL must be administered by daily IV infusions. Attaching polyethylene glycol (PEG) to the ligand could afford a longer half life and systemic exposure, thus requiring fewer hospital visits for the patient. However, as TRAIL is a functional homotrimer with complex ligand-receptor interactions, non-specific PEGylation will likely result in diminished potency. Using Ambrx9s proprietary site specific PEGylation, we evaluated the potency and circulating half life of several PEGylated TRAIL variants. METHODS Using Ambrx proprietary technology, PEG can be attached at a precise location so as not to interfere with receptor binding by engineering insertion of a non-natural amino acid, pAF, which provides a site-specific reactive moiety for attaching other molecules. Several trimeric-PEG-TRAIL analogs were generated and tested for anti-proliferation in vitro and for pharmacokinetic (PK) parameters in the rat. Finally, a PEG-TRAIL analog was tested for anti-tumor activity in pancreatic and colon cancer xenograft model. RESULTS PEGs of varying length (3k, 5k, 10k, and 20k) were attached to site-specifically incorporated pAf to yield one, two or three PEGs per trimeric molecule. One selected PEG analog showed only minimal loss of anti-proliferative activity when tested against a panel of cell lines including representatives from colon, pancreatic, lung and lymphoma cancers. The rat serum half life and AUC of PEG-TRAIL was approximately 6 and 21 fold better as compared to wild type (wt) TRAIL. Administering PEG-TRAIL qdx1 resulted in identical efficacy in pancreatic and colon xenografts as wt TRAIL administered qdx5. Finally, PEG-TRAIL was dramatically more efficacious than Mapatumumab at 1/5 the dose or wt TRAIL at equivalent dose, but had similar efficacy to Lexatumumab. Thus, site specific PEGylation of TRAIL homotrimer produces a drug that maintains in vivo potency and allows intermittent rather than daily dosing. CONCLUSIONS Modifying TRAIL by attaching a single PEG moiety to the homo-trimeric molecule increases rat serum half life compared to wild type TRAIL without decreasing agonistic potency or anti-tumor efficacy in xenograft animal models. There is potential for administering PEGylated TRAIL to patients with pancreatic or colon cancer with a once weekly instead of a daily dosing regimen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-20. doi:10.1158/1538-7445.AM2011-LB-20