Mark Tometsko Email & Phone Number

Greater Seattle Area

Greater Seattle Area

I was asked to return to Amgen to support a project that is near and dear to my heart. I came back as a contract employee to write study reports for the Investigational New Drug (IND) regulatory filing for Antibody Drug Conjugate (ADC) AMG 224. Reports summarized some really nice in vitro and in vivo data performed by team members from multiple departments including oncology, protein science, pathology and PKDM. Reports were completed in support of a first in human phase I multicenter open label study to evaluate the safety and tolerability of AMG 224 in subjects with relapsed or refractory multiple myeloma, targeted to begin in November 2015.https://clinicaltrials.gov/ct2/show/NCT02561962

Greater Seattle Area

Research included preclinical support for AMG 224: an ADC being evaluated as a potential treatment for multiple myeloma. Extensive data was compiled from early stage antibody campaign, to choosing a lead candidate based on in vitro data, to evaluation of the lead candidates in vivo. Concise results were necessary for the IND regulatory filing. In vitro experiments involved multi parameter flow cytometry to demonstrate expression of the target on cell lines, as well as primary patient tumor cells. ADC activity was demonstrated on cells in plate based assays including cell viability as well as induction of caspases and phospho-histone H3. Outstanding anti-tumor efficacy was observed in vivo with the lead candidate in bone tropic multiple myeloma models, which gives me great hope for the clinical success of this candidate.Additional research included the evaluation of ADC conjugation techniques. Conventional conjugation methods resulting in heterogeneous antibody conjugate products (with a mixture of different molar ratios of conjugated species to antibody linked at different sites), and site specific conjugation methods resulting in homogeneously loaded ADCs better suited to clinical development, were compared. ADC panels were evaluated in vitro in multiple plate based assays including cell viability, induction of caspases and metaphase arrest. Comparison was also performed in vivo, anti-tumor efficacy models as well as PK studies. Other exciting work included a collaboration with PKDM to achieve novel discovery of ADC catabolism. We were able to demonstrate the kinetics of ADC catabolism in tumor cells, the detection of catabolites in the lysosome, and the discovery of a novel lysosomal transporter critically involved in this process.

Greater Seattle Area

My oncology research was focused on contributing in vitro and pharmacological data in support of the regulatory filing for AMG 162, a fully human antibody to RANKL. Extensive expression analysis was performed on multiple tumor cell lines including breast, prostate and lung. Protein expression was performed by multi-parameter flow cytometry of tumor cell lines and primary cells for RANK, RANKL and OPG. We also demonstrated the direct effects RANKL has on tumor cells in vitro and in vivo. Retroviral transduction was used to overexpress RANK on tumor cells. Orthotopic bone tropic models were used to demonstrate RANK involvement in bone metastasis and osteolytic lesion formation. Bioluminescent imaging techniques were used to show tumor progression to the hind limbs of tumor cells that were transduced with luciferase expressing lentivirus. X-ray analysis was used to reveal the formation of osteolytic lesions. Blocking RANKL in these models led to reduction in tumor burden and osteolytic lesion formation. Preclinical data contributed to the regulatory filing and approval of Denosumab (Xgeva) in 2010 as a treatment for the prevention of bone metastasis.

Greater Seattle Area

Research at Immunex and Amgen led to the discovery of novel members of the TNFR/TNF superfamily. RANK and RANKL were discovered based on homology to this superfamily of proteins. Early work established the ability of RANKL to activate NFkB and this was demonstrated by Western Blot analysis. Reporter assays were also performed to show activation of RANK. Building DNA constructs, transfecting cells, and labeling with 125I was commonplace. We also studied the role of the TRAF proteins and mapped the TRAF binding site on RANK, using in vitro transcription/translation and peptide inhibition of GST fusion protein binding. The real excitement occurred when it was realized that RANKL was the critical mediator of osteoclast differentiation, activity and survival. RANKL could be used in vitro to differentiate a macrophage cell line into a bone resorbing osteoclast. I performed many osteoclast differentiation assays, and still am amazed at the process. After exposing RAW 264.7 cells (or later, primary cells from mouse bone marrow) to RANKL we were able to cover a tissue culture plate with huge, multi-nucleated, TRAP positive, bone resorbing osteoclasts. Seeing what these cells could do in vitro to calcium phosphate, or whale dentin demonstrated how destructive osteoclasts could be to bone when they are up-regulated in the tumor microenvironment. It was equally amazing how osteoclast differentiation could be totally inhibited if the RANKL pathway was blocked.

Greater Seattle Area

Immunex was a place where I realized I could enjoy working on interesting problems with great people. It seems kind of old school now, but this is where I learned about oligonucleotide synthesis, DNA sequencing, plasmid preps, restriction mapping and cloning. We routinely used 32P to kinase oligos to use as probes on Southern Blots hybridizing overnight in big coffin-like water baths. Sometimes I really miss the beeping of the geiger counter.... I also learned the art of isolating RNA (before it became RNeasy), running Northern Blots, and making cDNA libraries. This was a great time to hone my molecular biology skills…I actually saved money so I could get my own copy of the classic Sambrook&Manniatis textbook: Molecular Cloning. I really enjoyed working in the working on discovery research in the molecular biology department…looking at the TNFR/TNF superfamily, and hoping for the approval of Enbrel as a treatment for rheumatoid arthritis.