Nick Knudsen

Optimized clinical performance of growth hormone with an expanded genetic code

The ribosomal incorporation of nonnative amino acids into polypeptides in living cells provides the opportunity to endow therapeutic proteins with unique pharmacological properties. We report here the first clinical study of a biosynthetic protein produced using an expanded genetic code. Incorporation of p-acetylphenylalanine (pAcF) at distinct locations in human growth hormone (hGH) allowed site-specific conjugation with polyethylene glycol (PEG) to produce homogeneous hGH variants. A mono-PEGylated mutant hGH modified at residue 35 demonstrated favorable pharmacodynamic properties in GH-deficient rats. Clinical studies in GH-deficient adults demonstrated efficacy and safety comparable to native human growth hormone therapy but with increased potency and reduced injection frequency. This example illustrates the utility of nonnative amino acids to optimize protein therapeutics in an analogous fashion to the use of medicinal chemistry to optimize conventional natural products, low molecular weight drugs, and peptides.

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.

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.

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.

Abstract 639: Site specific conjugation of ARX-788, an antibody drug conjugate (ADC) targeting HER2, generates a potent and stable targeted therapeutic for multiple cancers

Creation of ADCs by conjugating drug linkers and toxin payloads to available native lysines or cysteines generates ADCs with highly variable drug to antibody loading and linker stability profiles. Variable drug loading, amino acid position, conjugation chemistry and linker design affect the pharmacokinetic characteristics, potency and toxicity of the ADC. We describe here the synthesis and pharmacology of an ADC comprising a site-specifically conjugated dolastatin analog to an antibody targeting the EGF family receptor, HER2. To specify the site of conjugation and drug loading, a non-natural amino acid, para acetyl phenylalanine (pAcF), was incorporated at defined sites within the antibody primary sequence. This non-natural amino acid provides a linkable platform for covalent conjugation of a diverse array of payloads through a stable oxime bond. The dolastatin analog, MMAF, was coupled via a non-cleavable linker to the HER2 antibody at two specific sites to generate the ADC; ARX-788. In vitro cytotoxicity assays of ARX-788 generated picomolar IC509s across a spectrum of HER2 + cell lines from breast, ovarian, lung and gastric cancer. Multiple in vivo murine xenograft studies demonstrated a potent increase in anti-tumor activity when compared to T-DM1. A single injection of ARX-788 in established xenograft models of HER2 positive ovarian, gastric and breast cancer cell lines (SKOV3, BT474, N-87, and HCC1954) induced rapid regression in all models. ARX-788 induced regression in a Herceptin-resistant derived breast xenograft (JIMT-1) and was significantly more effective than T-DM1 at equivalent doses. Pharmacokinetic evaluation of ARX-788 in rodent and non-human primates (NHP) revealed long term stability of 12 and 8 days respectively. Importantly, the conjugated form of ARX-788 remained intact over the course of the 3 week study in NHP. Precise payload stability and effective half-life of ARX-788 due to site-specific conjugation provides an improved therapeutic window for this HER2-targeting ADC. ARX-788 is currently completing non-human safety and PK studies in anticipation of clinical trial evaluation in 2015. Citation Format: Robin C. Humphreys, Jessica Kirtely, Amha Hewit, Sandra Biroc, Nick Knudsen, Lillian Skidmore, Alan Wahl. Site specific conjugation of ARX-788, an antibody drug conjugate (ADC) targeting HER2, generates a potent and stable targeted therapeutic for multiple cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 639. doi:10.1158/1538-7445.AM2015-639

Abstract LB-222: Optimization and preclinical characterization of highly potent and stable anti-CD70 ADCs for the potential treatment of CD70 positive cancers

Antibody drug conjugates (ADCs) require coordinated optimization of antibody, linker, and payload to generate potent, specific, and stable cancer therapeutics. CD70 is an attractive ADC tumor target due to its limited expression in normal tissues and over-expression in many solid tumors and hematologic malignancies. Utilizing Ambrx9s site-specific EuCODE technology, we have generated and evaluated multiple ADCs with different combinations of linkers and payloads for treatment of CD70 positive cancers. In vitro activity identified specific CD70 ADCs with picomolar potency/activity against CD70 positive cancer cell lines. Conjugation of drug-linkers at different sites in the antibody and modification of linker design improved in vitro activity and in vivo stability of lead CD70 ADCs. Optimized anti-CD70 ADCs were evaluated for pharmacokinetic profile and demonstrated to be efficacious in reducing tumor burden and significantly prolonging survival in an in vivo orthotopic xenograft model of multiple myeloma. In summary, we have generated encouraging preclinical in vitro and in vivo data with stable, site-specific, DAR = 2 anti-CD70 ADCs for the potential treatment of CD70 positive cancers such as multiple myeloma. Citation Format: Lillian Skidmore, Kari Cox, Jessica Kirtley, Tiep Le, Juhi Firdos, Anthony Manibusan, Shiao-Yan Fang, Andy Beck, Shiva Bhowmik, Nick Knudsen, Jianing Wang, Ying Sun, Armando Ayala, Alyssa Powell, Alan Wahl, Feng Tian, Robin Humphreys. Optimization and preclinical characterization of highly potent and stable anti-CD70 ADCs for the potential treatment of CD70 positive cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-222.

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