Ingrid Joseph

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.

Antitumor activity of orally bioavailable farnesyltransferase inhibitor, ABT-100, is mediated by antiproliferative, proapoptotic, and antiangiogenic effects in xenograft models

Purpose: To evaluate the preclinical pharmacokinetics, antitumor efficacy, and mechanism of action of a novel orally active farnesyltransferase inhibitor, ABT-100. Experimental Design:In vitro sensitivity of a panel of human cell lines was determined using proliferation and clonogenic assays. In vivo efficacy of ABT-100 was evaluated in xenograft models (flank or orthotopic) by assessing angiogenesis, proliferation, and apoptosis in correlation with pharmacokinetics. Efficacy of the racemate of ABT-100 (A-367074) was also compared with R115777 (tipifarnib). Results: ABT-100 inhibited proliferation of cells in vitro carrying oncogenic H-Ras (EJ-1 bladder; IC50 2.2 nmol/L), Ki-Ras (DLD-1 colon, MDA-MB-231 breast, HCT-116 colon, and MiaPaCa-2 pancreatic; IC50 range, 3.8-9.2 nmol/L), and wild-type Ras (PC-3 and DU-145; IC50, 70 and 818 nmol/L, respectively) as well as clonogenic potential. ABT-100 shows 70% to 80% oral bioavailability in mice. ABT-100 regressed EJ-1 tumors (2-12.5 mg/kg/d s.c., every day for 21 days) and showed significant efficacy in DLD-1, LX-1, MiaPaCa-2, or PC-3 tumor-bearing mice (6.25-50 mg/kg/d s.c. once daily or twice daily orally). A-367074 showed equivalent efficacy to R115777 given at approximately one-fourth the total dose of R115777 for a shorter duration (EJ-1 and LX-1). Antitumor activity was associated with decreased cell proliferation (Ki-67), increased apoptosis (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling), and decreased angiogenesis. A reduction in tumor angiogenic cytokine levels (vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8) correlated with a reduction in tumor vascularity (CD31). Conclusions: Overall, ABT-100 has an acceptable pharmacokinetic profile, is well tolerated, and possesses broad-spectrum antitumor activity against a series of xenograft models similar to farnesyltransferase inhibitors in clinical development; therefore, it is an attractive candidate for clinical evaluation.

Angiogenesis and Prostate Cancer

Prostate cancer is the most commonly diagnosed nonskin malignancy in American men, with an estimated 184,000 new cases projected for 2000 (8). It will be responsible for more than 34,000 deaths this year, ranking it second only to lung cancer in cancerrelated mortality (8). Two features make prostate cancer relatively unique among neoplasms—its androgen sensitivity and its relatively low growth fraction. It has been known for almost 60 years that androgen deprivation through surgical or chemical castration results in significant tumor regression (29,162,169). Recently, the mechanism for this regression has been demonstrated to be caused by induction of programmed cell death in the androgen responsive prostate cancer cells (38). Unfortunately, this effect is transient, and inevitably selects for cell populations that can grow independent of androgens. It is believed that prostate cancer begins as a single androgen sensitive cell and expands clonally (38). Over time, this clonal population develops genetic alterations, some of which lead to androgen independence. Thus, by the time of clinical detection, these cancers are composed of heterogeneous populations ranging from androgen dependent to androgen sensitive and independent cells. At this point, although androgen ablation can result in significant cell death of the androgen dependent cells, it eventually selects for androgen sensitive and independent cancer cells. In reference to the low-growth fraction, at any given time the vast majority of the prostate cancer cells are in a proliferatively quiescent (i.e., nondividing) state termed G0, which makes them relatively resistant to many of the traditional chemotherapeutic agents that are targeted at dividing cells. Normal prostate glandular cells have an extremely low proliferation rate (approx 0.2%/d), which is balanced by the rate of death, and thus no net growth of the gland occurs (17). In metastatic prostate cancer patients, the median proliferation rate of cancer cells within lymph nodes is 3.1%/d, and the median death rate is about 1.1%/d (17). In the bone, the median proliferation rate of prostate cancer cells is low—about 2.2%/d, and the death rate is about 2.1% d (17). These figures demonstrate that the proliferation rate for metastatic prostate cancer cells is remarkably low (<3%/d), and this explains why antiproliferative chemotherapy has had limited value against metastatic disease.

Development of ASG-15ME, a Novel Antibody–Drug Conjugate Targeting SLITRK6, a New Urothelial Cancer Biomarker

SLITRK6 is a member of the SLITRK family of neuronal transmembrane proteins that was discovered as a bladder tumor antigen using suppressive subtractive hybridization. Extensive immunohistochemistry showed SLITRK6 to be expressed in multiple epithelial tumors, including bladder, lung, and breast cancer as well as in glioblastoma. To explore the possibility of using SLITRK6 as a target for an antibody–drug conjugate (ADC), we generated a panel of fully human mAbs specific for SLITRK6. ADCs showed potent in vitro and in vivo cytotoxic activity after conjugation to Monomethyl Auristatin E or Monomethyl Auristatin F. The most potent ADC, ASG-15ME, was selected as the development candidate and given the product name AGS15E. ASG-15ME is currently in phase I clinical trials for the treatment of metastatic urothelial cancer. This is the first report that SLITRK6 is a novel antigen in bladder cancer and also the first report of the development of ASG-15ME for the treatment of metastatic bladder cancer. Mol Cancer Ther; 15(6); 1301–10. ©2016 AACR.

AGS16F is a Novel Antibody Drug Conjugate Directed Against ENPP3 for the Treatment of Renal Cell Carcinoma

Purpose: New cancer-specific antigens are required for the design of novel antibody–drug conjugates (ADC) that deliver tumor-specific and highly potent cytotoxic therapy. Experimental Design: Suppression subtractive hybridization identified ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3 or CD203c) as a potential human cancer-specific antigen. Antibodies targeting the extracellular domain of human ENPP3 were produced and selected for specific binding to ENPP3. Expression of ENPP3 in normal and cancer tissue specimens was evaluated by immunohistochemistry (IHC). ADCs comprising anti-ENPP3 Ab conjugated with maleimidocaproyl monomethyl auristatin F via a noncleavable linker (mcMMAF) were selected for therapeutic potential using binding and internalization assays, cytotoxicity assays, and tumor growth inhibition in mouse xenograft models. Pharmacodynamic markers were evaluated by IHC in tissues and ELISA in blood. Results: ENPP3 was highly expressed in clear cell renal cell carcinoma: 92.3% of samples were positive and 83.9% showed high expression. By contrast, expression was negligible in normal tissues examined, with the exception of the kidney. High expression was less frequent in papillary renal cell carcinoma and hepatocellular carcinoma samples. AGS16F, an anti-ENPP3 antibody–mcMMAF conjugate, inhibited tumor growth in three different renal cell carcinoma (RCC) xenograft models. AGS16F localized to tumors, formed the active metabolite Cys-mcMMAF, induced cell-cycle arrest and apoptosis, and increased blood levels of caspase-cleaved cytokeratin-18, a marker of epithelial cell death. Conclusions: AGS16F is a promising new therapeutic option for patients with RCC and is currently being evaluated in a phase I clinical trial. Clin Cancer Res; 22(8); 1989–99. ©2015 AACR.

Apoptosis in Hormone-Responsive Malignancies

Publisher Summary The breast and prostate cancer belong to the spectrum of hormone-related malignancies. So, this chapter seeks to review current knowledge about the role of programmed cell death in the normal development and function of the mammary and prostate glands, the carcinogenic process, and the prevention and therapy of breast and prostate cancers. Its suitability as a target for new therapies for both cancers is also explored. Although the focus of the chapter is on breast and prostate cancers, it seems likely that lessons learned from these two neoplasms is also of relevant to a variety of other epithelial malignancies. Breast and prostate represent two excellent model tissues in which to study the balance between cell proliferation and cell death in the normal setting. Research suggests that steroid hormones are key factors that promote proliferation and block death in both tissues. In addition, the pivotal role of programmed cell death in the normal lactational cycle of the breast has been identified. Furthermore, hormone-responsive breast and prostate cancers retain a similar programmed cell death pathway(s) that can be triggered by hormone withdrawal. Similar or identical pathways also exist in endocrine-unresponsive prostate and breast cancer cells even though hormonal manipulation is no longer capable of triggering these pathways. As the components of these pathways, their activators, and their inhibitors are delineated, these programmed cell death pathways provide opportunities for the development of new therapeutic approaches.

Abstract 2650: Ags67e, an anti-cd37 monomethyl auristatin e antibody (mmae) drug conjugate as a potential therapeutic for non-hodgkin's lymphoma, chronic lymphocytic leukemia and acute myeloid leukemia

We have developed AGS67E, an antibody drug conjugate that targets CD37, a tetraspanin highly expressed on malignant B cells, for the potential treatment of non-Hodgkin9s lymphoma (NHL), chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). AGS67E is a fully human anti-CD37 monoclonal IgG2 antibody conjugated to the potent microtubule-disrupting agent, MMAE, via reduced cysteines and the protease cleavable linker, maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl. AGS67E exhibits potent in vitro binding, internalization and cytotoxicity on a variety of NHL, CLL and AML models and patient-derived samples, including CD34+CD38- leukemic stem cells. AGS67E also demonstrates potent anti-tumor responses, including complete tumor regressions in a variety of NHL, CLL and AML xenografts, including Rituxan refractory models and patient-derived samples. In general, CD37 was highly expressed across all models and a strong correlation was observed between the in vitro and in vivo efficacy of AGS67E. To confirm binding of AGS67E in a variety of normal and patient-derived NHL, CLL and AML samples, we developed flow cytometry and immunohistochemistry (IHC) assays which have confirmed reported CD37 expression data in NHL & CLL. In normal hematopoietic cells, AGS67E bound strongly to B cells and to a much lesser extent to monocytes, T cells, neutrophils and NK cells. AGS67E also bound with high and similar affinity to cynomolgus monkey B cells and was equally cytotoxic to these and human B cells. In other normal tissues, AGS67E binding was only evident where lymphoid structures were apparent such as in the spleen and lymph node. With respect to CD37 expression in NHL, CLL and AML, AGS67E was found to bind to >80% of NHL and 100% of CLL and AML samples. Taken together, our findings suggest that AGS67E may serve as a potential therapeutic for NHL, CLL and AML. To our knowledge, this body of work is also the first demonstration that CD37 is well expressed and potentially drug-able in AML. Citation Format: Daniel S. Pereira, Claudia Guevara, Alla Verlinsky, Cyrus Virata, J Hsu Ssucheng, Zili An, Chungying Zhang, Nick Dinh, Hector Avina, Lisa Do, Sher Karki, Joseph Abad, Peng Yang, Jimmy Ou, Karen Morrison, Sing-Ju Moon, Faisal Malik, Liqing Jin, Michael Choi, Christina Wu, Banmeet Anand, Scott Cooper, Ingrid Joseph, Xiao-Chi Jia, Kendall Morrison, Pia Challita-Eid, Fernando Donate, Thomas Kipps, John Dick, David Stover. Ags67e, an anti-cd37 monomethyl auristatin e antibody (mmae) drug conjugate as a potential therapeutic for non-hodgkin9s lymphoma, chronic lymphocytic leukemia and acute myeloid leukemia. [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 2650. doi:10.1158/1538-7445.AM2014-2650

Abstract 1976: MDR expression/ activity may serve as potential biomarker in developing therapeutic drugs for AML therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Multidrug resistance may play a critical role in developing cancer therapeutics for acute leukemia. The expression of the ATP binding cassette (ABC) transporter Pgp/MDR1 (also known as ABCB1) has been reported in AML patients and correlates with poor clinical outcome. Additional transporters like MRP1 and BCRP also appeared to contribute towards this resistance mechanism. We have profiled a panel of AML tumor cell lines and tumor xenografts for both expression and activity of Pgp/MDR1 via FACS and MSD;. Our profiling also includes analysis of the known SNPs associated with these transporters in key models. We have evaluated the sensitivity of the AML models to a series of standard of care therapies, including auristatins, in both in vitro and in vivo studies. These payloads alone or when conjugated to antibodies as antibody drug conjugates (ADCs) are known substrates of the ABC transporters, Using Pgp inhibitors, we could restore the sensitivity of the MDR expressing cells to the cytotoxic drugs and ADCs, which confirmed that the drugs were MDR substrates. These inhibitors could also block the activity of the Pgp pump. We have also observed limited activity in vivo with these ADCs when used in a MDR positive model. Our initial observation indicates that a threshold level of activity of MDR1 may be critical to confer resistance to these standard of care molecules and antibody drug conjugates using similar payloads. Currently, we are investigating the contribution of other efflux pumps in this mechanism of drug resistance MRP1 and BCRP, in similar manner. This can also aid in identifying cytotoxic drugs that are able to bypass the resistance mediated by these efflux pumps. Additional work is underway to characterize patient samples and identify MDR threshold level of activity as a potential predictive biomarker in developing AML therapeutics. Citation Format: Nandini Rudra-Ganguly, Christine Lowe, Mukta Shiwalkar, Claudia I. Guevara, Christopher Kemball, Min Michelle Wu, Cyrus Virata, Alla Verlinsky, Ssucheng J Hsu, Michael Mattie, William Yeh, Peng Yang, Sung-Ju Moon, Ingrid Joseph, David R. Stover, Daniel S. Pereira, Dowdy Jacksosn. MDR expression/ activity may serve as potential biomarker in developing therapeutic drugs for AML therapy. [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 1976. doi:10.1158/1538-7445.AM2014-1976