Purpose: Non-muscle-invasive bladder cancer has few available treatments. The mainstay of treatment is surgery with a transurethral resection of the bladder tumor but the recurrence rate is as high as 75%. Mitomycin C is given peri-operatively to reduce the recurrence rate and when disease is high grade, Bacillus Calmette-Guerin (BCG) is used to prevent both recurrence and progression. However, these treatments often fail and when successful, they work through a poorly understood mechanism. Therefore, attention has been directed towards molecular alterations in bladder cancer to identify novel targets. Mutation analysis of 145 urothelial tumors found that FGFR3 and PIK3CA mutations were most commonly found in low grade non-muscle-invasive tumor (Sjodahl et al, 2011). In fact, the FGFR3 mutation is found in up to 80% in low-grade bladder tumors and up to 50% of invasive tumors. In addition, over-expression or amplification of EGFR is seen in the majority of urothelial tumors (Rotterud et al, 2005). However, targeted therapy with the monoclonal antibodies has been disappointing in bladder cancer. Multiple studies have been conducted with EGFR-targeted monoclonal antibodies and tyrosine kinase inhibitors without any significant clinical benefit despite encouraging pre-clinical results (Black et al, 2012). Recently, an FGFR3 monoclonal antibody was effective but primarily in invasive bladder cancer cell lines instead of the low grade bladder cancer where mutations in FGFR3 are more commonly found (Qing et al, 2009). Despite having these well-defined targets in non-muscle-invasive bladder cancer, there has been little success with targeted therapy alone. Recently, colleagues from the molecular imaging branch demonstrated successfully that humanized monoclonal antibodies could be used to target infrared light activated compounds selectively into colon cancer cells. Upon introduction of infrared light, these compounds become activated to induce cell death (Mitsunaga et al, 2011). These antibody-infrared light activated drug conjugates are able to capitalize on the targetable property of the antibodies but rely on the cytotoxicity of the drug conjugated to the antibody and not the antibody itself. Given the prevalence of EGFR and FGFR3 mutations in non-muscle-invasive bladder cancer and the disappointing results with standard targeted therapy, we postulate that antibody-infrared light activated drug conjugates will have significant activity in inducing cell death selectively in bladder cancer cells. This is novel as an infrared light could be attached to existing urologic equipment such as a urinary catheter or flexible cystoscope and be introduced into the bladder to activate such conjugates instilled into the bladder should pre-clinical work suggest a benefit of these agents in bladder cancer. We will further investigate molecular targeted photoimmunotherapy in bladder cancer by specifically targeting EGFR, FGFR3, and other related targets. Methods: Initially, bladder cancer cell lines rich in EGFR (UM-UC5, UM-UC9, and RT-4) and FGFR3 (UM-UC1, RT-4, RT-112, and UM-UC14) will be grown in cell culture with appropriate corresponding positive/negative control cell lines for these two receptors. For example, two colon cancer cell lines can be used for these controls in reference to EGFR: SW620 (EGFR negative) and HCT116 (EGFR positive) (Yang et al, 2007). Cell-surface immunofluorescence for all of these cell lines can be done through receptor specific antibodies by flow cytometry to verify and establish cell surface levels of EGFR and FGFR3 respectively. Once receptor expression has been confirmed for these cell lines, we will identify cell lines with the following relative characteristics to study further with our antibody-drug conjugates in conjunction with positive and negative controls: EGFR++/FGFR3++, EGFR--/FGFR3++, EGFR++/FGFR3--, and EGFR--/FGFR3--. In parallel to this cell culture and flow cytometry work, we will conjugate the infrared light-activated cytotoxic compound, IR-700 (purchased from Li-Cor Bioscience), to our monoclonal antibodies directed against EGFR and FGFR3. These monoclonal antibodies are panitumumab (purchased from Amgen), directed against human EGFR, and R3mab (to be donated by Genentech), directed against FGFR3. The purity of the conjugates will then be confirmed with size-exclusion HPLC and SDS-PAGE to ensure that no detectable monoclonal antibody aggregates are noted. Finally, the immunoreactivity of the mAb-IR700 conjugate will be confirmed through the use of a blocking assay where the monoclonal antibody is given first and then the mAb-IR700 is administered to see if any binding of the conjugate occurs. Using the various different cell lines and the pure antibody-drug conjugates, we will then proceed to assess target-specific cell death. We will look at dose-dependent relationships of conjugate and photoimmunotherapy and their impact on cell death (PI-FACS) and growth inhibition (thymidine uptake) in the various cell lines. Depending on this pre-clinical, in vitro work, we will expand these studies into a xenograft model by inducing EGFR and FGFR expressing tumors in vivo in nude mice. Successful data in this scenario, may introduce the possibility of a phase I clinical trial in the future. Update: We have successfully treated UMUC-5 and TCC-SUP cells with our Panitumumab-IR700 conjugate. This conjugate results in minimal cell death when activated by infrared light in normal urothelial cells (HUC-SV). Also, no cell death noted in the EGFR negative cell line, BalB3T3. We are now expanding cells for injection in mice for an in vivo model and we expect to see promising results. We have also successfully secured a MTA with genentech to study their FGFR3 mAb, R3mab.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011458-02
Application #
8763537
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2013
Total Cost
$281,478
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
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State
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