Targeted HPMA copolymer-gemcitabine conjugates for the treatment of ovarian carcinoma

Each year in the United States, over 14,000 women die from ovarian cancer. Anthracyclines-a class of chemotherapeutics-have long been the primary treatment for this and many other cancers, yet they often leave patients with cardiotoxicity, hepatotoxicity, and a number of other adverse effects. Polymer-drug conjugates using poly[N-(2-hydroxypropyl) methacrylamide] (pHPMA) are nanosized, water-soluble constructs that accumulate passively in solid tumors by the enhanced permeability and retention effect, as well as actively by targeting methods. As such, they have exhibited reduced toxicity in the body. The traditional approach used in conjugating antibody targeting moieties to the HPMA copolymer-drug backbone has proven ineffective in the case of gemcitabine, a chemotherapeutic and nucleoside analog that has demonstrated considerable effectivedfness in recent years treating ovarian cancer. The aims of this study were twofold: 1) to develop a novel pHPMA-OV-TL16 antibody fragment (Fab') conjugation apprach that is compatible with gemcitabine and 2) to optimize existing procedures for the synthesis of all monomeric precursors. The copolymer-gemcitabine conjugate was successfully developed following the synthesis of OVTL16 Fab', diblock chain-transfer agent (di-CTA), N-(2-(2-pyridyldithio)ethyl)methacrylamide (PDTEMA), and polymerizable gemcitabine derivative. Monomers were combined by reversible addition-fragmentation chain-transfer (RAFT) polymerization, and then the Fab' fragment was successfully bound to the copolymer backbone via a disulfide exhange reaction with PDTEMA. Future work will involve in virto and in vivo evaluation of the conjugate's therapeutic efficacy in nude mice bearing OVCAR3-exnografts.

TARGETED HPMA COPOLYMER-GEMCITABINE CONJUGATES FOR THE TREATMENT OF OVARIAN CARCINOMA By Shwan Javdan A Senior Honors Thesis Submitted to the Faculty of The University of Utah In Partial Fulfillment of the Requirements for the Honors Degree in Bachelor of Science In Biomedical Engineering Approved: ______________________ Thesis Faculty Supervisor ______________________ Patrick A. Tresco, Ph.D. Chair, Department of Bioengineering ______________________ Kelly Broadhead, Ph.D. Honors Faculty Advisor ______________________ Sylvia D. Torti, Ph.D. Dean, Honors College December 2015 Copyright © 2015 All Rights Reserved i ABSTRACT Each year in the United States, over 14,000 women die from ovarian cancer. Anthracyclines a class of chemotherapeutics have long been the primary treatment for this and many other cancers, yet they often leave patients with cardiotoxicity, hepatotoxicity, and a number of other adverse effects. Polymer-drug conjugates using poly[N-(2-hydroxypropyl) methacrylamide] (pHPMA) are nanosized, water-soluble constructs that accumulate passively in solid tumors by the enhanced permeability and retention effect, as well as actively by targeting methods. As such, they have exhibited reduced toxicity in the body. The traditional approach used in conjugating antibody targeting moieties to the HPMA copolymer-drug backbone has proven ineffective in the case of gemcitabine, a chemotherapeutic and nucleoside analog that has demonstrated considerable effectiveness in recent years treating ovarian cancer. The aims of this study were twofold: 1) to develop a novel pHPMA-OV-TL16 antibody fragment conjugation approach that is compatible with gemcitabine and 2) to optimize existing procedures for the synthesis of all monomeric precursors. The copolymer-gemcitabine conjugate was succ chain-transfer agent (di-CTA), N-(2-(2-pyridyldithio)ethyl)methacrylamide (PDTEMA), and polymerizable gemcitabine derivative. Monomers were combined by reversible addition-fragmentation chainwas successfully bound to the copolymer backbone via a disulfide exchange reaction with PDTEMA. Future work will involve in vitro and in vivo therapeutic efficacy in nude mice bearing OVCAR3-xenografts. ii TABLE OF CONTENTS ABSTRACT ii INTRODUCTION 1 METHODS 3 RESULTS 13 DISCUSSION 23 REFERENCES 26 iii 1 INTRODUCTION Each year in the United States, over 22,000 new cases of ovarian carcinoma are diagnosed and 14,000 women die [1]. Anthracyclines chemotherapeutics a class of common have long been the primary treatment for this and many other cancers, yet they often leave patients with cardiotoxicity, hepatotoxicity, and a number of other adverse effects [2]. Polymer-drug conjugates using poly[N-(2-hydroxypropyl) methacrylamide] (pHPMA) were among the first generation of nanomedicines developed as drug-delivery vehicles for the treatment of cancer with the ability to accumulate specifically in tumors and, as a result, reduce toxicity in the body [3]. When used in treatment, these conjugates can be advantageous compared to low molecular-weight drugs due to 1) passive accumulation at the tumor site by the enhanced permeability and retention (EPR) effect, and 2) active accumulation by targeting methods. The aforementioned EPR effect is the primary mechanism by which soluble vascular density, enhanced permeability of the blood vessels, and defective lymphatic drainage [4]. In addition to passive targeting by the EPR effect, some HPMA copolymerdrug conjugates can be designed to target tumor cell surface antigens for receptor-mediated endocytosis [5]. Once these nano-sized, water-soluble conjugates are internalized by cells, they are eventually localized to the lysosome where their drug attachments are enzymatically-degraded by cysteine proteases. In this way, HPMA copolymer-drug conjugates can specifically target a given drug to tumor cells, thereby mitigating many of the harmful effects of chemotherapy. 2 The traditional approach used in conjugating antibody targeting moieties to the HPMA copolymer-drug backbone [6] has proven ineffective in the case of gemcitabine, a chemotherapeutic and nucleoside analog that has demonstrated considerable effectiveness in recent years treating ovarian cancer [7], [8]. An issue arises during synthesis, as free amine groups classically used for the site of antibody fragment attachment will cleave gemcitabine from the copolymer backbone. For this reason, a novel approach must be used to synthesize a targeted drug delivery system using gemcitabine. Multiblock pHPMAgemcitabine conjugates have had success in recent years, particularly in combination therapies with paclitaxel. Studies by Larson et al. [9] and Zhang et al. [10] have found the efficacy of these combination treatments to be quite promising for treating ovarian cancer, and as such have demonstrated the importance in developing novel, targeted pHPMAgemcitabine conjugates. The aims of this study were twofold: 1) to develop a novel pHPMA-antibody conjugation approach that is compatible with gemcitabine and 2) to optimize existing procedures for the synthesis of all monomeric precursors. For the new approach, OV-TL16 antibody fragments targeted to ovarian carcinoma cell surface antigen OA3 [11], [12] were bound to the HPMA copolymer-gemcitabine backbone via disulfide exchange reaction with N-(2-(2-pyridyldithio)ethyl)methacrylamide (PDTEMA). Additionally, procedures were developed for the copolymerization of PDTEMA, HPMA, and a polymerizable gemcitabine derivative (MA-GFLG-GEM). These methods were verified by a number of analytical methods. These experiments create a foundation upon which new HPMA copolymer-drug conjugates may be developed, and from which subsequent in vitro and in vivo testing may lead to the improvement of ovarian carcinoma treatment. 3 METHODS Synthesis of Monomeric Precursors and Fab’ Fragment: A. Polymerizable Gemcitabine Derivative (MA-GFLG-GEM): As with most chemotherapeutics, gemcitabine is not polymerizable in its monomeric form. Thus, a polymerizable, backbone-degradable gemcitabine derivative known as methacryloyl-glycylphenylalanylleucylglycine-gemcitabine (MA-GFLG-GEM) was used (Fig. 1). This monomer had been previously synthesized and characterized in the laboratory, based on procedures detailed by Yang et al. [13]. Fig. 1. Structure of MA-GFLG-gemcitabine. A stock sample of MA-GFLG-GEM was used for this study. 4 B. Peptide Di-Arm Chain Transfer Agent (di-CTA): In the process of reversible addition-fragmentation chain transfer (RAFT) polymerization, a chain transfer agent is needed to extend the polymer chain. In clinical application, the cleavable amino acid sequence, GFLG, needs to be incorporated into the copolymer backbone for backbone degradation by cysteine proteases in the lysosome. An enzyme-sensitive di-arm chain transfer agent (di-CTA) was thus synthesized by solidphase peptide synthesis (Fig. 2) according to the procedures detailed by Pan et al. [14]. The purity of the di-CTA batch was then verified using analytical high-performance liquid chromatography (HPLC), and the identity was confirmed by mass spectrometry (M.W. = 1417.55). Fig. 2. Synthesis steps for peptide di-CTA. Fmoc protecting groups secured the terminal amine, then methanol was used to saturate the unreacted bead-chlorine conjugates. Piperidine then acted as a deprotecting group to remove the Fmoc. Amino acids were then continually added in this manner until the desired GFLG peptide was synthesized. Finally, -diisopropylcarbodiimide (DIC) and hydroxybenzotriazole (HOBt) were used for uncoupling, followed by tetrafluoroethylene (TFE) and dichloromethane (DCM) for cleavage. 5 C. N-(2-(2-pyridyldithio)ethyl)methacrylamide (PDTEMA): N-(2-(2-pyridyldithio)ethyl)methacrylamide (PDTEMA) was the polymerizable molecule used to conjugate the antibody fragment targeting moiety to the copolymer backbone via a disulfide exchange reaction. There existed very little literature on the synthesis of PDTEMA, and none that was well-established. To this end, methods were initially adapted from Wang et al. [15], but were modified in a number of ways to increase both product yield and purity. Through many trials using small and large batches, modifying the solvent ratios used during key steps, an optimized procedure was developed for the synthesis and purification of PDTEMA (detailed below). A general outline for this procedure is shown in Fig. 3. Fig. 3. dipyridyldisulfide (DPDS) to yield 2-aminoethyl 2-pyridyl disulfide (AEPD) and 2thiopyrodone. The nucleophilic acyl substitution of methacryloyl chloride yielded N-[2-(2pyridyldithio)]ethyl methacrylamide (PDTEMA). Prior to PDTEMA synthesis, anhydrous pyridine was prepared by reflux and .W. = 220.31, 2.0 g, 9 mmol) was dissolved in ethanol (95%, 24 mL). A solution of cysteamine 6 (MW = 77.15, 490 mg, 6.3 mmol) was then prepared in deionized water (pH = 8.5, 24 mL), followed by dropwise addition of the DPDS solution (stirring at room temperature) to turn the mixture yellow. This new solution was left at room temperature (1.5 h.) before eventually removing the solvent by rotary evaporation (35°C, 30 min.). The product was obtained as a syrup, which was dried by co-evaporation (3x, 15 min. each) with anhydrous pyridine (2.5 mL). Pyridine was then evaporated using an oil (vacuum) pump. The dried crude product was dissolved in a mixture of anhydrous DMF (10 mL) and anhydrous pyridine (10 mL), then bubbled with N2 (30 min., while stirring) to remove the oxygen in the bottle. The container was then sealed tightly with a rubber stopper. Methacryloyl chloride (1.22 mL, 12.5 mmol) was added slowly (5 min., over ice bath) using a syringe, then left to stir at room temperature (2 h., color change to red). The reaction was then quenched by slowly adding saturated NaHCO3 (160 mL) while cooling (color change to yellow-green). The product was extracted twice in a separatory funnel with ethyl acetate (2x, 160 mL, top fraction = EtOAc + PDTEMA, bottom fraction = NaHCO3). The combined organic phases were washed with deionized water (2x 40 mL) and then dried using anhydrous Na2SO4 (Mg2SO4 also works) before being placed in a cold room (24 h.). The PDTEMA product was filtered into a RB flask to remove the drying agent, then evaporated to dryness by rotary evaporation (35°C) and oil pump (for DMF, pyridine removal). To purify the product, the crude PDTEMA was dissolved in methanol (2-3 mL, in parts) and absorbed to a silica gel column (50 x 3.5 cm, packed with 70:30 hexanes:ethyl acetate, volumetric ratio). The column was eluted first with 70:30 hexanes:ethyl acetate to collect the first fraction, then with 1:1 hexanes:ethyl acetate to draw out the second fraction 7 containing the purified PDTEMA product (continuous monitoring by TLC plating). The PDTEMA large batch was confirmed by HPLC, 1H-NMR, and mass spectrometry. D. OVIn order to actively target the copolymer drug delivery system to ovarian carcinoma cells, the OVantigen OA3 targeted to ovarian carcinoma cell surface was utilized. In order to maximize the number of targeting moieties on the immunoglobulin G (IgG) fragments were regularly used as models for OV-TL16 where needed due to their similar size, shape, and reaction chemistry. Fig. 4. Overall scheme for digestion and reduction of OV-TL16 antibody. The OV-TL16 IgG antibody was first digested with pepsin, then reduced by tris(2carboxyethyl)phosphine (TCEP). 8 OV-TL16 IgG (15 mg.) was dissolved in citric buffer (pH = 4.0, 3.75 mL). A small sample (100 µL) was taken into phosphate buffer solution (PBS with azide, 1 mL) for analysis by fast protein liquid chromatography (FPLC, S200a column) and ultraviolet (UV) spectroscopy. Pepsin (10%, 1.5 mg) a digesting agent was added dropwise to the OV- TL16 IgG solution, which was then mixed thoroughly by vortexing and placed in a shaking water bath (37°C, 1.5 h.) to allow for digestion of the Fc fragment r 2 product was filtered by Amicon ultrafiltration (3x, 30 kDa MWCO) to remove the Fc fragment and residual pepsin, as well as to change the buffer solution from citric buffer to PBS (pH = 7.0). The solution volume was increased (15 mL) to p 2 aggregation. A sample was again taken for FPLC and UV analysis to assess the sample purity. 2 -carboxyethyl)phosphine (TCEP, MW = 250.19, 10 mM) was added to a concentrated solution of th 2 (4.5 mL by ultrafiltration volume reduction). The reduction (37°C, Stir 6, 1 h.) was allowed to then immediately used for conjugation to the polymer backbone. RAFT Polymerization and Fab’ Fragment Attachment: A. Di-CTA Incorporation by RAFT Polymerization Peptide di-CTA was incorporated into the N-(2-hydroxypropyl)methacrylamide (HPMA) polymer backbone by reversible addition-fragmentation chain transfer (RAFT) 9 polymerization, as detailed by Moad et al. [16]. V-501 initiator was used alongside HPMA and di-CTA for the polymerization, which was done in an oil bath (18 h. 70°C). The copolymer was precipitated with a mixture of cold acetone and diethyl ether (2:1), vortexed briefly, and then centrifuged (8 min., 5300 RCF, 4° C). Incorporation of di-CTA into the polymer backbone was confirmed by enzymatic cleavage by Cathepsin B or papain, a cysteine protease similar to that in the lysosome of the cell. A standard procedure for enzymatic cleavage was used, as detailed by Zhong et al. [17]. Fast-protein liquid chromatography (FPLC, S6a column) was used to monitor the cleavage process by quantifying the molecular weights of the polymers before and after protease treatment. B. Copolymerization of HPMA, PDTEMA, and MA-GFLG-GEM Although RAFT polymerization has been previously demonstrated with HPMA and gemcitabine, the copolymerization has never been done with PDTEMA. A novel 30 kDa HPMA copolymer-gemcitabine conjugate with PDTEMA incorporated into the polymer backbone was therefore synthesized. HPMA (129 mg), PDTEMA (11 mg, 4%), and MA-GFLG-GEM (46 mg, 6%) were combined by RAFT polymerization protocols to create the novel P-SS-GEM (Fig. 5). 10 Fig. 5. Overall scheme for synthesis of novel P-SS-GEM conjugate. HPMA, PDTEMA, and MA-GFLG-GEM were combined by RAFT polymerization. The quantified PDTEMA and gemcitabine content in the 30 kDa polymer were assessed by two separate experiments involving the release of either 2-thiopyridone or gemcitabine in samples taken from the large batch (Fig. 6). To assess PDTEMA content, cysteamine was added (in excess) to release 2-thiopyridone for every PDTEMA in the copolymer backbone. Based on a calibration curve of 2-thiopyridone using UV max = 360 nm), the PDTEMA content per polymer chain was determined using the Beer= 268 nm) using an excess of 2-aminopropanol. max 11 Fig. 6. Method for assessing PDTEMA and gemcitabine content in P-SS-GEM. Gemcitabine was released from the polymer backbone using an excess of papain or 2max = 268 nm). The same was done for assessing PDTEMA content, using cysteamine to release 2max = 360 nm). C. Conjugation of OVThe OV- via a disulfide exchange reaction with PDTEMA (Fig. 7). A 60 kDa pHPMA/PDTEMA sample was prepared by RAFT polymerization [13], and polymer solution (2.2 mg in 200 -SH:PDTEMA attachment sites) and incubated at room temperature (2 h.) before placing in the cold room (24 h.). 12 Fig. 7. Conjugation of OV- via a disulfide exchange reaction with the molecule, PDTEMA. assessed by fast-protein liquid chromatography (FPLC, S200a column) and UV spectroscopy, then compared to profiles for the un- 2 Key Terms: di-CTA di-arm chain transfer agent MA-GFLG-GEM polymerizable gemcitabine derivative OVovarian carcinoma Immunoglobulin G fragment PDTEMA N-(2-(2-pyridyldithio)ethyl)methacrylamide pHPMA poly[N-(2-hydroxypropyl) methacrylamide] 13 RESULTS Peptide Di-Arm Chain Transfer Agent (di-CTA): Peptide di-CTA was synthesized by solid-phase peptide synthesis, a multistep process that resulted in a percent yield of 24.02%. In addition to confirmation by mass spectrometry (M.W. = 1417.55), the product was confirmed by high performance liquid chromatography (HPLC). After the crude product was synthesized, a preparative HPLC column (C18) allowed for the isolation of the prominent di-CTA peak at 26 min. elution time. The purified di-CTA was then confirmed by an analytical HPLC column (Fig. 8). Fig. 8. Analytical HPLC profile for purified di-CTA sample. This HPLC profile of absorbance (mAU) vs. elution time (min.) shows the detection of a signal peak at 26.796 min. which is proportional to the amount of di-CTA in the product. The lack of other signal peaks would indicate product purity, as only the di-CTA absorbance peak was detected. 14 N-(2-(2-pyridyldithio)ethyl)methacrylamide (PDTEMA): The synthesis of PDTEMA, after numerous small and large batches attempting to optimize the procedure, resulted in a product yield as high as 70.6% with a large batch. Analytical HPLC confirmed the purity of the sample (Fig. 9), and mass spectrometry confirmed its identity (Fig. 10). Finally, 1H-NMR provided a third form of product confirmation to rule out any undesired stereoisomers that may have also had a molecular weight of 254 g/mol (Fig. 11). Fig. 9. Analytical HPLC profile for purified PDTEMA sample. A single peak was detected at 13 min. elution time, with no other major peaks in the profile. 15 Fig. 10. Mass spectrometry profile for purified PDTEMA sample. The predominant peak, with 100% signal intensity (y-axis) had a mass/charge [m/z] value of 255.13 (x-axis), representing the protonated PDTEMA product (MW = 254.05). 16 Fig. 11. 1H-NMR profile for purified PDTEMA sample. The profile is signal intensity (yaxis) vs. ppm on the delta scale (x-axis). Each peak is representative of a hydrogen atom on the PDTEMA molecule, with their downfield (leftward) shifting based on their proximity to electron-withdrawing groups. As can be seen, the peaks can be matched up to each hydrogen (demonstrated with matched letters a-j) on PDTEMA, with no additional peaks representative of potential byproducts or stereoisomers. OV-TL16 Antibody Fragment (Fab’ ): OV-TL16 Immunoglobulin G (IgG), targeted to ovarian carcinoma cell surface copolymer backbone. This digestion and reduction process was monitored by fast-protein liquid chromatography (FPLC) profiles (Fig. 12). The combination of elution volume (similar to elution time from HPLC) and integration under the peaks allowed for calculation 17 of -TL16 IgG (elution 2 (elution volume = 13.9 mL, 50kDa). Fig. 12. Analytical FPLC profiles for digestion and reduction of OV-TL16 IgG. Profiles are of absorbance (mAU) vs. elution volume (mL). OV-TL16 IgG (elution volume = 12.9 2 - 18 Di-CTA Incorporation by RAFT Polymerization: Peptide di-CTA was used in the synthesis of a 100 kDa pHPMA/di-CTA copolymer by RAFT polymerization to confirm its intended mechanism of action to cleave high molecular weight HPMA copolymers into smaller ones by cysteine proteases in the cell. The pHPMA/di-CTA copolymer was synthesized with 65.67% yield. Fast-protein liquid chromatography (FPLC) profiles before and after cleavage by papain demonstrated degradation of the copolymer from ~94 kDa to ~55 kDa (Fig. 13), indicating successful incorporation of di-CTA into the copolymer backbone. Fig. 13. Analytical FPLC profiles for enzymatic cleavage of pHPMA/di-CTA copolymer. (A) FPLC profile of pHPMA with di-CTA before degradation, with an elution volume of (B) FPLC profile of pHPMA with di-CTA after degradation, by papain. 19 Copolymerization of HPMA, PDTEMA, and MA-GFLG-GEM: The copolymerization of HPMA, PDTEMA, and MA-GFLG-GEM process demonstrating PDTEMA spacer compatibility with gemcitabine a novel was confirmed with the successful synthesis of a 30 kDa PDTEMA/HPMA-gemcitabine copolymer (PSS-GEM). P-SS-GEM was verified by separately releasing PDTEMA and gemcitabine from the copolymer backbone, then measuring the UV absorbance of the released monomers to calculate content using the Beer-Lambert Law. Calibration curves were made for 2- max max = 268 nm) (Fig. 14), the products being measured by UV absorbance, and the equation for the best-fit line was used to find PDTEMA and gemcitabine percent by weight. From this, the number of monomers per polymer chain could be calculated. The PDTEMA content was found to be 3.05% by weight, or 7.7 PDTEMA molecules per polymer chain. The gemcitabine content was found to be 9.6% by weight, or 6.4 gemcitabine molecules per polymer chain. Thus, both monomers were simultaneously incorporated into the polymer backbone, a novel process. 20 A B Fig. 14. Calibration curves for 2-thiopyridone and free gemcitabine using UV spectroscopy. Various concentrations of the free monomers were measured using UV spectroscopy to create calibration curves that would allow for the quantification of PDTEMA and gemcitabine content in the 30 kDa P-SS-GEM conjugate. (A) The UV absorbance of 2-thiopyridone released from the P-SS-GEM conjugate was 0.264 AU, leading to a concentration of 5.1 µg/mL (7.7 PDTEMA/chain) using the calibration equation. (B) The UV absorbance of free gemcitabine was 2809.856 AU, leading to a concentration of 153.1 µg/mL (6.4 GEM/chain). 21 Conjugation of OV-TL16 Fab’ Fragment to HPMA Polymer Backbone: The OV- via disulfide exchange reaction with PDTEMA in a 60 kDa pHPMA/PDTEMA copolymer. The copolymer- -protein liquid chromatography (FPLC, S200a column), and compared with a control sample of PLC profile revealed a number of byproducts in the copolymer2 (likely produced by the re-association of unstable ine-tetraacetic acid (EDTA) from the buffer solution. A low-absorption, very wide peak was found in the region for the copolymer- -13 mL. Given that the control sample had a sharp peak at 11.8 mL elution volume, the wide peak on the copolymer- to each polymer chain (i.e. some chain may have 2-3, or even none at all). After isolating the conjugate region using a preparative FPLC column (S200p), the purified copolymerconfir 2 in solution (Fig. 16). 22 Fig. 15. Analytical FPLC profile for 60 kDa copolymer2, as well as EDTA from the buffer. The wide elution volume range for the copolymerdemonstrates that copolymer- Fig. 16. Analytical FPLC profile of purified copolymer2 was removed, and the extent of polydispersity was reduced. 23 DISCUSSION The aims of this study were to develop a novel pHPMA-OV-TL16 antibody conjugation approach that is compatible with gemcitabine, and to optimize existing procedures for the synthesis of all monomeric precursors. All monomers were synthesized and verified using a number of analytical techniques. These monomers were then combined to confirm the successful novel conjugation approach using N-(2-(2- pyridyldithio)ethyl)methacrylamide (PDTEMA). Synthesis of peptide di-CTA was successful, with yield (24%) and purity that are sufficient for future experiments (Fig. 8). This chain transfer agent allowed for synthesis of copolymer-drug conjugates of high molecular weight, thus taking full advantage of the enhanced permeability and retention (EPR) effect. The protocol for synthesizing the PDTEMA monomer was also improved, with product yield increased from ~50% (the yield obtained from the literature protocol) to ~70% with the optimized procedure. This came at no cost to purity, which was evident by analytical HPLC, mass spectrometry, and the 1HNMR profile (Figs. 9-11). The OVand its purity was confirmed by fast-protein liquid chromatography (FPLC) (Fig. 12). Incorporation of di-CTA into the HPMA copolymer backbone was confirmed via a cleavage experiment with papain, a cysteine protease. Cleavage of the 100 kDa copolymer to 50 kDa (Fig. 13) demonstrated successful incorporation of the cleavable amino acid sequence GFLG into the copolymer backbone. In addition to optimizing existing procedures for synthesis of a number of monomeric precursors, one of the primary aims of this research was to develop a novel 24 pHPMA-antibody conjugation approach that was compatible with gemcitabine. The dual presence of the PDTEMA spacer and gemcitabine in the 30 kDa P-SS-GEM conjugate was confirmed by UV spectroscopy (Fig. 14). This result is rather noteworthy, as it supports the compatibility of PDTEMA with gemcitabine during synthesis, unlike in the classical approach in which gemcitabine is cleaved from the backbone. While the classical use of a maleimide group for antibody conjugation [6] is still quite useful for a number of applications, the use of PDTEMA as an alternative spacer molecule is a valid choice in cases where free amine groups may cleave other molecules chemotherapeutic such as the chosen from the copolymer backbone. In attempting to conjugate the OV-TL16 antibody fragment to the copolymer backbone via disulfide exchange with PDTEMA, the results appeared to confirm - copolymer backbone (hence the wide FPLC peak). This polydispersity was mitigated after purification (Fig. 16), but improvements can still be made to create a monodisperse fragment may be a limitation that requires further assessment. Overall, the novel pHPMA-antibody conjugation approach with gemcitabine one that is compatible paves the way for the synthesis of future HPMA copolymer-drug conjugates. In other cases of maleimide spacer incompatibility, it may be possible to use PDTEMA as a valid replacement spacer molecule. In vitro experiments using the novel conjugate, including half-maximal inhibitory concentration (IC50) tests and cytotoxicity assays, are currently underway. Future work will involve in vivo evaluation of the targeted 25 pHPMAcarcinoma xenografts. Ultimately, by building upon current knowledge of targeted HPMA copolymer-drug conjugates, an improved treatment for ovarian carcinoma may one day be achieved. ACKNOWLEDGEMENTS I would like to thank Stephan Rudolph for his assistance with the optimization of the PDTEMA synthesis protocol, as well as in the synthesis of peptide di-CTA. Additionally, I would like to thank Dr. Jiyuan Yang mentorship throughout the research process. for their guidance and 26 REFERENCES [1] http://www.cancer.org/cancer/ovariancancer/detailedguide/ovarian-cancer-keystatistics, 2013. [2] molecular advances and pharmacologic developments in antitumor activity and cardioto -229, 2004. [3] -149, 2010. [4] Maeda, H., Wu J., Sawa T., Matsu Release, vol. 65, pp. 271-284, 2000. [5] chemotherapy and photodynamic therapy with Fab' fragment targeted HPMA vol. 5, no. 5, pp. 696-709, 2008. [6] t [7] 30, pp. 8507-8515, 2007. -1104, 1999. 27 [8] Oncology/Hematology, vol. 48, no. 1, pp. 81-88, 2003. 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