Targeted Delivery of Polymer Prodrug Conjugates for Cancer Therapy
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Investigator: Prashant Raj Bhattarai Targeted Delivery of Polymer Prodrug conjugates for Cancer therapy Doctoral Thesis Dissertation Presented by Prashant Raj Bhattarai To The Bouvé Graduate School of Health Sciences in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Pharmaceutical Science NORTHEASTERN UNIVERSITY BOSTON, MASSACHUSETTS August 2018 i Investigator: Prashant Raj Bhattarai Northeastern University Bouvé College of Health Sciences Dissertation Approval Dissertation title: Targeted Delivery of Polymer Prodrug conjugates for Cancer therapy Author: Prashant Raj Bhattarai Program: PhD in Pharmaceutical Sciences Approval for dissertation requirements for the Doctor of Philosophy in: Pharmaceutical Science Dissertation Committee (Chairman): Dr. Ban-An Khaw Date: 8/07/2018 Other committee members: Dr. Vladimir Torchilin Date: 8/07/2018 Dr. Jonghan Kim Date: 8/07/2018 Dr. Eugene Bernstein Date: 8/07/2018 Dr. Joel Berniac Date: 8/07/2018 Dean of the Bouvé College Graduate School of Health Sciences: Date: ii Investigator: Prashant Raj Bhattarai TABLE OF CONTENTS ABSTRACT iii ACKOWLEDGEMENTS v LIST OF TABLES vi LIST OF FIGURES vii LIST OF ACRONYMNS x 1) INTRODUCTION 1.1 Antibody targeted therapies 1 1.2 Bispecific Antibodies and Pretargeting Approach 1 1.3 Rationale for using Antibody fragments 3 1.4 Rationale for using Affibody: 5 1.5 Rationale for using biotin as a second cancer-targeting agent 6 1.6 Polymer prodrug conjugates for Cancer Therapy 7 1.7 Multidrug Resistance in tumor 8 1.8 Combination therapy 9 1.9 Spheroid Cell Culture 10 2) SPECIFIC AIMS 12 3) MATERIALS AND METHODS 14 3.1 Purification and Characterization of anti-HER2/neu Affibodies 3.2 Preparation and Characterization of anti-HER2/neu X anti-DTPA Fab bispecific 18 complex 3.3 Preparation and characterization of biotinylated anti-DTPA bispecific antibody 21 complex 23 3.4. Synthesis and characterization of Polymer Prodrug conjugates iii Investigator: Prashant Raj Bhattarai 3.5 Tissue Cultures 27 3.6 Animal Studies 31 4) RESULTS 4.1 Purification and characterization of recombinant anti-HER2/neu Affibodies and demonstration of specificity of Fluorescein labeled Affibody for the HER2/neu 34 receptors on SKOV3 and SKOV3 TR ovarian cancer cell lines. 4.2 Preparation and characterization of anti-HER2/neu affibody X anti-DTPA Fab 35 bispecific antibody complex 4.3 Characterization of various polymer prodrug conjugates: a) D-Ptxl-PGA, b) D-Dox- 41 PGA and c) D-Mph-PGA 4.4 Evaluate in vitro cytotoxicity and drug resistance reversal in ovarian cancer SKOV3 and SKOV3 TR cell lines by delivery of PPDCs by pretargeting approach using anti- 49 HER2/neu affibody X anti-DTPA Fab bispecific antibody complex. 4.5 Evaluate in vitro cytotoxicity and drug resistance reversal in human breast cancer MCF7 and MCF7 ADR (doxorubicin resistant) cell lines by delivery of PPDCs by 60 pretargeting approach using biotinylated anti-DTPA bispecific complex. 72 4.6 3D Spheroid cell Culture 75 4.7 Tumor Growth inhibition in a 4T1 autologous graft model 82 5) DISCUSSION 89 6) CONCLUSION 90 7) REFERENCES ii Investigator: Prashant Raj Bhattarai ABSTRACT: Significant progress has been made in cancer therapy in last several years. However, improvements are needed to enhance therapeutic efficacy. Current standards of therapy include surgery, chemotherapy and radiation therapy. These treatments usually just result in temporary improvement in quality of life and not complete remission. Furthermore, development of multi drug resistance (MDR) of malignant cells is a common mechanism leading to failure of chemotherapy due to overexpression of drug efflux transporter pumps such as P-glycoprotein (Pgp) receptors. Effective cancer therapy should be the one with high specificity to malignant cells and show less normal cell toxicity. Since chemotherapeutic agents do not discriminate between normal and cancer cells, they are cytotoxic to both normal and malignant cells. Furthermore, development of MDR in cancer leads to the need for increased chemotherapeutic doses. Thus, targeted delivery of these cytotoxic agents to cancer cells becomes crucial to achieve higher rates of cancer cell killing and reduce non-targeted toxicities. Certain cancers overexpress normal membrane receptors. These overexpressed receptors could be used for targeted delivery of the chemotherapeutic agents. Polymers prodrug conjugates (PPDCs) have been developed to achieve reduction in non-target toxicities. Conjugation of cytotoxic agents to polymers changes the mechanism of uptake of these agents and can be pretargeted with bispecific antibodies for targeted delivery of these PPDCs. The objective of this thesis is to use the pretargeting approach for active targeting of PPDCs to cancer cell to overcome multidrug resistance, and demonstrate that targeted delivery of combination PPDCs consisting of different chemotherapeutic agents affecting different mechanisms of cell replication may result in more efficient in cancer therapy. To achieve this objective, we developed two bispecific antibody complexes, i) anti HER2/neu affibody X anti-DTPA Fab, and ii) biotinylated anti-DTPA targeting the overexpressed HER2 and biotin receptors in cancer cells respectively. In-Cell ELISA developed in the lab showed that iii Investigator: Prashant Raj Bhattarai modified affibody and biotin retained the binding capacity to their respective cognate receptors. In Vitro studies demonstrated that by utilizing the pretargeted approach we were able to overcome drug resistance in both paclitaxel (Ptxl) and doxorubicin (Dox) cells seen by increased cell death as compared to free drugs treatments. On evaluation of the uptake mechanism of the PPDCs, pretreatment of cells with Chlorpromazine inhibited the cytotoxicity associated with the PPDCs. This confirmed the endocytic uptake of PPDCs and these results were further corroborated using the fluorescent microscopic studies. Combination therapy, involving the delivery of D-Dox-PGA and D-Ptxl-PGA resulted in significantly higher cancer cell toxicities in vitro. In vivo, evaluation of targeted PPDCs in 4T1 murine breast cancer model showed inhibition of tumor growth by 80% (D-Dox-PGA) and 55% (D-Ptxl-PGA). However, combination therapy with targeted D-Dox-PGA and D-Ptxl-PGA treatment was most effective in tumor growth inhibition relative to untreated controls by 92%. Towards the end of the treatments, significant differences were seen between the tumor sizes of combination treatment group relative to free drugs and untreated groups. No change in the bodyweight of mice treated with D-Dox-PGA, D-Ptxl-PGA and combination therapy was observed indicating lack of non-targeted toxicity. Mice treated with Dox lost approximately 25% of bodyweight and all succumbed by day 24. Examination of the heart section for fluorescence of Dox in Dox or D-Dox-PGA treated mice confirmed the Dox fluorescence only in free Dox treated mice. All other mice treated with D-Dox-PGA alone or in combination showed no red fluorescence in the heart sections. This confirmed that cardiotoxicity is observed only in mice treated with free Dox. TUNEL staining of tumor sections showed significantly greater apoptosis in tumor treated with individual or combination targeted PPDCs treatments relative to saline and free drug treatment groups. Overall, this study highlighted the ability of pretargeted approach for the targeted delivery of PPDCs to optimally kill wide variety of cancer types and its potential to overcome multidrug resistance. iv Investigator: Prashant Raj Bhattarai ACKNOWLEDGEMENTS Firstly, I would like to express my sincere gratitude to my research advisor and mentor, Dr. Ban- An Khaw. During my time in his he has been a constant source of inspiration and responsible for my development as a scientist, as well as a person. His approach towards his work and discipline has always inspired me. I have always valued every moment of my time spent with him and will continue to do so forever. I would like to extend my special thanks to members of my committee, Dr. Jonghan Kim, Dr. Vladimir Torchilin, Dr. Eugene Bernstein and Dr. Joel Berniac for taking out their valuable time for being in my thesis committee. I am extremely thankful to Dr. Can Sarisozen for all his help and allowing me the access and training to use various instruments in Dr. Torchilin’s lab. I would also like to thank Dylan Vance whose energy and hard work helped immensely in my studies. I would also like to thank all the other lab members who worked in my lab, especially Ankita Pandey and Na Yoon Kim who were always there when I needed any help from them. I would like to acknowledge the Department of Pharmaceutical Sciences at Bouve College of Health Sciences for healthy and challenging environment. The help provided by Sarom Lay and Rosalee Robinson will always be kindly remembered. Finally I would like to dedicate this thesis to my parents and entire family. Without their continuous support and encouragement I would never have been able to pursuit my higher education. v Investigator: Prashant Raj Bhattarai LIST OF TABLES Table 1: Spheroid formation techniques along with their advantages and disadvantages 11 Table 2: TNBS Assay to determine the percent modification of anti-DTPA Fab 38 Table 3: Determination of concentration of Dox in D-Dox-PGA complex 44 Table 4: Determination of concentration of paclitaxel in D-Ptxl-PGA complex 46 Table 5: Zeta potential values of Polymer compared with various Polymer drug conjugates 49 Table 6: IC50 Values for MCF7