Anthony Manibusan

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.

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.

A CXCR4-Targeted Site-Specific Antibody-Drug Conjugate

A chemically defined anti-CXCR4-auristatin antibody-drug conjugate (ADC) was synthesized that selectively eliminates tumor cells overexpressing the CXCR4 receptor. The unnatural amino acid p-acetylphenylalanine (pAcF) was site-specifically incorporated into an anti-CXCR4 immunoglobulin G (IgG) and conjugated to an auristatin through a stable, non-cleavable oxime linkage to afford a chemically homogeneous ADC. The full-length anti-CXCR4 ADC was selectively cytotoxic to CXCR4(+) cancer cells in vitro (half maximal effective concentration (EC50 )≈80-100 pM). Moreover, the anti-CXCR4 ADC eliminated pulmonary lesions from human osteosarcoma cells in a lung-seeding tumor model in mice. No significant overt toxicity was observed but there was a modest decrease in the bone-marrow-derived CXCR4(+) cell population. Because CXCR4 is highly expressed in a majority of metastatic cancers, a CXCR4-auristatin ADC may be useful for the treatment of a variety of metastatic malignancies.

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.

Discovery of Pyrophosphate Diesters as Tunable, Soluble, and Bioorthogonal Linkers for Site-Specific Antibody–Drug Conjugates

As part of an effort to examine the utility of antibody-drug conjugates (ADCs) beyond oncology indications, a novel pyrophosphate ester linker was discovered to enable the targeted delivery of glucocorticoids. As small molecules, these highly soluble phosphate ester drug linkers were found to have ideal orthogonal properties: robust plasma stability coupled with rapid release of payload in a lysosomal environment. Building upon these findings, site-specific ADCs were made between this drug linker combination and an antibody against human CD70, a receptor specifically expressed in immune cells but also found aberrantly expressed in multiple human carcinomas. Full characterization of these ADCs enabled procession to in vitro proof of concept, wherein ADCs 1-22 and 1-37 were demonstrated to afford potent, targeted delivery of glucocorticoids to a representative cell line, as measured by changes in glucocorticoid receptor-mediated gene mRNA levels. These activities were found to be antibody-, linker-, and payload-dependent. Preliminary mechanistic studies support the notion that lysosomal trafficking and enzymatic linker cleavage are required for activity and that the utility for the pyrophosphate linker may be general for internalizing ADCs as well as other targeted delivery platforms.

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.

Novel Phosphate Modified Cathepsin B Linkers: Improving Aqueous Solubility and Enhancing Payload Scope of ADCs

In an effort to examine the utility of antibody-drug conjugates (ADCs) beyond oncology indications, a novel phosphate bridged Cathepsin B sensitive linker was developed to enable the targeted delivery of glucocorticoids. Phosphate bridging of the Cathepsin B sensitive linkers allows for payload attachment at an aliphatic alcohol. As small molecule drug-linkers, these aqueous soluble phosphate containing drug-linkers were found to have robust plasma stability coupled with rapid release of payload in a lysosomal environment. Site-specific ADCs were successfully made between these drug-linkers and an antibody against human CD70, a receptor specifically expressed in immune cells but also found aberrantly expressed in multiple human carcinomas. These ADCs demonstrated in vitro targeted delivery of glucocorticoids to a representative cell line as measured by changes in glucocorticoid receptor (GR) mediated gene mRNA levels. This novel linker expands the scope of potential ADC payloads by allowing an aliphatic alcohol to be a stable, yet cleavable attachment site. This phosphate linker may have broad utility for internalizing ADCs as well as other targeted delivery platforms.

Role of tRNA orthogonality in an expanded genetic code.

We found that Methanocaldococcus jannaschii DSM2661 tyrosyl-tRNA synthetase (Mj E9RS), specifically evolved to charge its cognate tRNA with the unnatural amino acid p-acetylphenylalanine (pAcF) in E. coli, misaminoacylates the endogenous E. coli prolyl-tRNAs with pAcF at a low level (0.5% per proline frequency) in both the absence or presence of its co-evolved amber suppressor tRNA (M. jannaschii tyrosyl-tRNA, tRNACUAMjTyr). In contrast to other E. coli tRNAs, the identity elements for recognition of the proly tRNAs by the E. coli prolyl-tRNA synthetase (C1, G72, and A73) are similar to those in tRNACUAMjTyr. Although the unique acceptor stem identity elements of the prolyl-tRNAs likely lower their recognition by the other endogenous aaRSs in E. coli, resulting in enhanced fidelity in the wild type strain, they lead to misaminoacylation by the archae-derived E9RS. Misincorporation of pAcF for proline was resolved to below detectable levels by overexpression of the endogenous E. coli prolyl-tRNA synthetase (proS) gene in combination with additional genomic manipulations to further increase the intracellular ratio of the ProS over its cognate proline tRNAs. These experiments suggest another mechanism by which the cell maintains the high fidelity of protein biosynthesis.

Abstract 5691: Novel site-specific antibody drug conjugates based on novel amino acid incorporation technology have improved pharmaceutical properties over conventional antibody drug conjugates

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL We demonstrate here for the first time generation of ADCs by using EuCODETM technology that site-specifically incorporates novel amino acids into proteins in mammalian cells. Conventional conjugation strategies for making ADCs rely on conjugating the payload to the antibody via the side chains of lysines or cysteines: this procedure produces a heterogeneous mixture of ADCs exhibiting a range of biological activities. We believe that making ADCs using the novel amino acid incorporation technology (NDCs) will enable production of homogenous site specific conjugates that may have improved pharmaceutical properties, and simplified analytics compared to ADCs made using conventional conjugation strategies. 5T4 is an oncofetal antigen, that is selectively overexpressed on various cancers and is rapidly internalized upon antibody binding. Using anti-5T4 antibody (A1) as a model system, NDCs were made that contain the novel amino acid, para-acetylphenylalanine (pAF) at designated sites of the antibody molecule. Toxin payload, monomethyl auristatin D (MMAD), a potent tubulin inhibitor, was then conjugated via oxime linkages to the two engineered pAF residues in the anti-5T4 antibody to give homogenous NDCs with antibody:drug ratio of 1:2. Compared to the native form of the A1 antibody, pAF site-specific mutants and NDC conjugates made thereof do not alter the antibody binding and internalization kinetics on 5T4 positive tumor cells. Further, NDCs preserve the cytotoxicity observed with conventional ADCs. In vivo, NDCs demonstrated substantially improved pharmacokinetics in mice relative to conventional ADCs. For example, the mean systemic exposure for the NDC with a non-cleavable linker (nc) (A1-HS115pAF-ncMMAD) was significantly greater (2-4 fold) than that of A1-mcMMAD ADC (made using conventional cysteine-conjugation strategy). Most importantly, the NDCs had similar or better PK compared to the unconjugated Ab, suggesting that by using NDC technology, the payload is not lost from the Ab in circulation. In efficacy experiments, we compared NDCs and conventional ADCs head to head, using the same payload-linker. In two xenograft models, NDCs demonstrated equivalent or slightly better efficacy compared to conventional ADCs. Further, in a single dose rat toxicology studies, NDCs were well tolerated up to 90 mg/kg, the highest dose tested. To demonstrate the scalability of NDC production, a stable CHO clone for the corresponding mutated Ab of NDC HA114pAF was created and shown to support antibody production at > 1 g/L using a fed-batch process. In summary, NDCs demonstrated improved in vivo efficacy and pharmacokinetic stability relative to ADCs prepared using conventional conjugation approaches. Based on these preclinical data, NDC technology could serve as another avenue for making site specific homogenous ADCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5691. doi:1538-7445.AM2012-5691

Development of Anti-CD74 Antibody–Drug Conjugates to Target Glucocorticoids to Immune Cells

Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody-drug conjugates (ADCs) using antibodies containing site-specific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen. However, mechanism of action studies pointed to accumulation of free payload in the tissue culture supernatant as the dominant driver of activity and indeed administration of the ADC to human CD74 transgenic mice failed to activate GR target genes in splenic B cells. Suspecting dissipation of released payload, we designed an ADC bearing a novel GR agonist payload with reduced permeability which afforded cell-intrinsic activity in human B cells. Our work shows that antibody-targeting offers significant potential for rescuing existing and new dose-limited drugs outside the field of oncology.