Targeted Delivery of Polymer Prodrug Conjugates for Cancer Therapy

Investigator: Prashant Raj Bhattarai

Targeted Delivery of Polymer Prodrug conjugates for Cancer

therapy

Doctoral Thesis Dissertation Presented by

Prashant Raj Bhattarai

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

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:

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

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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

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

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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.

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.

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 Sensitive and resistant cells 64

Table 7: Hematological results in mice after different treatments (*p<0.05 when compared to saline group) 80

Table 8: Percentage of TUNEL positive cells (*p<0.05, **p<0.01 when compared to saline group) 82

Investigator: Prashant Raj Bhattarai

LIST OF FIGURES:

Figure 1: SDS-PAGE for Affibody characterization using 12.5% bis/acrylamide gel, Lane A) bacterial cell lysate before passing through column, Lane B) Flow through from the IMAC column, Lane C) Non-Reduced 34 purified Affibody, Lane D) Reduced purified Affibody

Figure 2: FACS analysis showing the binding of 5 µg/ml anti-HER2/neu affibody FITC to HER2/neu expressing SKOV3 and SKOV3 TR cell lines, and negative controls (MCF 7 and MDAMB-231 cell lines). 0 Cells were incubated with anti-HER2/neu affibody at final concentration of 5µg/ml for 30 min at 37 C. In 35 cells alone, cells were incubated with 1% BSA.

Figure 3: Epifluorescent microscopy for the determination of cell association of anti-HER2/neu affibody FITC with SKOV3 ovarian cancer cell line 40X magnification (A: Bright field, B: FITC, C: Hoechst Stain) 36 Figure 4: Figure 4: 4.1 Epifluorescent microscopy for the determination of cell association of anti-

HER2/neu affibody FITC with SKOV3 TR ovarian cancer cell line 4.2 Affibody FITC association study in HER2/ne negative MDAMB-231 breast cancer cells at 40X magnification (A: Bright field, B: FITC, C: 36 Hoechst Stain)

Figure 5: ELISA for the immunoreactivity of bromoacetylated anti-DTPA Fab (blue square) compared with the standard unmodified anti-DTPA Fab (red circle) 37

Figure 6: Purification and Characterization of Bispecific antibody complex; 5.1) SDS-PAGE for the identification of Lane A: Intact Anti-DTPA antibody, Lane B: Anti-DTPA Fab after Protein A purification, Lane C: crude bispecific extract after purification from IMAC Column, and Lane D: bispecific antibody complex after purification from size exclusion Zorbax G-250 column; 5.2) Elution profile of crude bispecific reaction product from size exclusion Zorbax G-250 column eluted using 0.2M sodium phosphate buffer pH 38 7.4.

Figure 7: In-Cell ELISA for demonstration of binding of anti-HER2/neu affibody X anti-DTPA Fab bispecific complex to HER2 positive SKOV3 and SKOV3 TR ovarian cancer cell lines. MCF7 breast cancer 39 cell line, which does not express HER2 receptors, is used as a negative control to demonstrate the specificity of bispecific antibody complex.

Figure 8: Determination for the presence of biotin on biotinylated anti-DTPA by ELISA (▲ =biotinylated 40 anti-DTPA, and ■= unmodified anti-DTPA) 1:4000 dilution of Streptavidin-HRP was used.

Figure 9: Quantitation of Biotin on biotinylated anti-DTPA by comparison to Biotinylated BSA standard (♦ = Biotinylated BSA and ▲ = biotinylated anti-DTPA) 1:8000 dilution of Streptavidin-HRP was used. 41

Figure 10: In-cell ELISA for demonstration of binding of biotinylated anti-DTPA to biotin receptors expressed in various cell lines 42 Figure 11: Elution profile of Blue Dextran and D-Dox-PGA superimposed together from Sephadex G25 column (1 X 35 cm) chromatography 43

Figure 12: Standard curve for the determination of concentration of Dox in D-Dox-PGA complex 44

Figure 13: Standard curve for the determination of melphalan concentration in D-Mph-PGA complex 45

Figure 14: Thin Layer Chromatography determination of conjugation of Ptxl to PGA 46

Figure 15: Standard Curve for the determination of concentration of Ptxl in D-Ptxl-PGA complex 47

Figure 16: Release profile of Paclitaxel from D-Ptxl-PGA in 0.1M PBS pH 7.4 (blue triangle); and 0.1M 47 sodium acetate buffer pH4 (red diamond)

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Investigator: Prashant Raj Bhattarai

Figure 17: Determination of presence of DTPA on Polymer drug conjugates by ELISA (Red circle: D-Dox- PGA, Blue diamond: D-Mph-PGA, and orange triangle: D-Ptxl-PGA) 48

Figure 18: Superimposition of standard positive control DTPA-BSA to the Polymer drug conjugates ELISA (Brown cross: DTPA-BSA, Red circle: D-Dox-PGA, Blue diamond: D-Mph-PGA, and orange triangle: D- Ptxl-PGA) 49

Figure 19: Cytotoxicity of non-targeted polymer drug conjugates in SKOV3 and SKOV3 TR cell lines at 48 hrs. Cells were incubated with D-Dox-PGA, D-Ptxl-PGA, D-Mph-PGA and PGA for 48 h at various concentrations. Data shown are representative of 2 independent experiments performed in triplicates, mean ± SD. 51

Figure 20: Western blot showing expression of Pgp protein in: A) SKOV3 sensitive, B) SKOV3 TR (resistant), C) MCF7 ADR (resistant) and D) MCF7 sensitive cell lines. β-actin bands showed that equal concentrations of cellular protein extracts were added to each lane. 52

Figure 21: Cytotoxicity of D-Ptxl-PGA targeted with 40 μg/ml of bispecific antibody complex compared to free Ptxl in SKOV3 cells. For targeted approach, cells were pretargeted with 40 µg/ml of bispecific complex for 1 hr. after which D-Ptxl-PGA at equivalent paclitaxel concentration was added. Data shown are representative of 2 independent experiments performed in triplicates, mean ± SD. 53 Figure 22: Cytotoxicity of targeted D-Ptxl-PGA after pretargeting with 40 μg/ml of bispecific antibody complex relative to free Ptxl and non-targeted D-Ptxl-PGA in SKOV3 TR cells. Data shown are representative of 2 independent experiments performed in triplicates, mean ± SD. 54 Figure 23: Cytotoxicity of D-Ptxl-PGA targeted with 20 μg/ml of bispecific complex compared to free paclitaxel and non-targeted D-Ptxl-PGA in SKOV3 TR cell line. For targeted approach, cells were treated with 10 µg/ml of chlorpromazine for 30mins before pretargeting step Data shown are representative of 2 independent experiments performed in triplicates, mean ± SD. 55 Figure 24: Cell viability of SKOV3 TR cells after 48 h incubation with targeted combination of 2 PPDCs (D-Ptxl-PGA+D-Dox-PGA) after pretargeted with two different bispecific antibody complex concentration A. 20 µg/ml of pretargeted bispecific antibody complex; B. 40 µg/ml of pretargeted bispecific antibody complex. Data shown are representative of 3 individual experiments carried out in triplicates, Mean ± SD *P<0.05. 58 Figure 25: Cell viability of SKOV3 TR cells after 48 h incubation with targeted combination of 3 PPDCs (D-Ptxl-PGA+D-Dox-PGA+D-Mph-PGA) versus targeted combination of 2 PPDCs (D-Ptxl-PGA and D- Dox-PGA) after preincubated with two different bispecific antibody complex concentration A. 20 µg/ml of pretargeted bispecific antibody complex; B. 40 µg/ml of pretargeted bispecific antibody complex. Data shown are representative of 3 individual experiments carried out in triplicates, Mean ± SD. 60 Figure 26: Cytotoxicity of targeted D-Dox-PGA after pretargeted with 20 μg/ml of biotinylated anti-DTPA bispecific antibody complex compared to free Dox in MCF7 cells. Data shown are representative of 3 individual experiments carried out in triplicates, Mean ± SD. 62 Figure 27: Figure 27: Cytotoxicity of targeted D-Dox-PGA after pretargeted with increasing concentration of biotinylated anti-DTPA bispecific antibody complex compared to free Dox in MCF7 ADR cells. Data shown are representative of 3 individual experiments carried out in triplicates, Mean ± SD. 63 Figure 28: Comparison of % cell viability of MCF7 ADR following the administration of free drugs, individually targeted D-Dox-PGA or D-Ptxl-PGA and combination therapy (D-Dox-PGA+D-Ptxl-PGA). Data shown are representative of 3 individual experiments carried out in triplicates, Mean ± SD. 64

Figure 29: Epifluorescent micrographs of MCF7 ADR cell after 1 h pretargeting with biotinylated anti- DTPA bispecific antibody followed by incubation with D-Dox-PGA for: A. 5 min, B. 10 min, C. 15 min, D. 30 min, E. 60 min, F. 120 min, G. 300 min, and H. 360 min (a: Bright field, b: Dox fluorescence, c: 66 Hoechst stain, d: superposition of b and c). Magnification = 40X.

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Investigator: Prashant Raj Bhattarai

Figure 30: Epifluorescent micrographs of MCF7 ADR after incubation with non-targeted D-Dox-PGA: A. 5 min, B. 10 min, C. 15 min, D. 30 min, E. 60 min, F. 120 min, G. 300 min, and H. 360 min (a: Bright 67 field, b: Dox fluorescence, c: Hoechst stain, d: superposition of images b and c). Magnification = 40X.

Figure 31: Epifluorescent micrographs of MCF7 ADR cells after incubation with Dox for: A. 5 min, B. 10 min, C. 15 min, D. 30 min, E. 60 min, F. 120 min, G. 300 min, and H. 360 min. (a: Bright field, b: Dox 68 fluorescence, c: Hoechst stain, d: superposition of b and c). Magnification = 40X.

Figure 32: A. Corrected total cell fluorescence (CTCF) values of MCF7 ADR cells after various treatments, B. CTCF values of the nuclei of MCF7 ADR cells after various treatments 70

Figure 33: Fluorescent Images of MCF7 ADR cells after: A. 1 h incubation with biotinylated anti-DTPA, followed by treatment with D-Dox-PGA for 1 h, followed by washing of the cells and incubation for 4 h in D-Dox-PGA free medium. B. Treatment with Dox for 1 h, followed by washing and incubation for 4 h in Dox free medium. C. Treatment with D-Dox-PGA alone for 1 h and the cells are treated as in A. (a: Bright 71 field, b: dox fluorescence, c: Hoechst stain, d: superposition of images b and c). Magnification = 40X.

Figure 34: A. CTCF values for MCF7 ADR cells after treatment with either Dox or pre-targeted D-DOX- PGA for 1 h followed by washing and replacing with Dox free medium compared to 1 h treatment without the washing step P<0.01, *** P<0.0001). B. CTCF values for the nuclei of MCF7 ADR cells after treatment with either Dox or pre-targeted D-DOX-PGA for 1 h followed by washing and replacing with Dox free 72 medium compared to 1 h treatment without the washing step (*, P<0.05, ** P<0.01).

Figure 35: A. Micrographs of MCF7-ADR cells pretreated with biotinylated anti-DTPA-FITC bispecific antibody complex for 1 h and then incubation with 20 μg/ml of D-Dox-PGA for 1 h relative to B. Non-treated control cells. a) Bright field, b) Hoechst stain, c) FITC green fluorescence, d) Dox fluorescence, e) superimposition of b) and c), and f) superimposition of b) and d) to demonstrate nuclear localization of the 73 released Dox. B) Same sequence of the micrographs as above in untreated control MDF7 ADR cells.

Figure 36: Fluorescent Images of MCF7 ADR cells after: A 30 min pre-treatment with 10 μg/mL of Chlorpromazine, followed by pre-targeting with Biotinylated anti-DTPA antibody for 1 h and 1 h incubation with D-Dox-PGA. B 30 min pre-treatment with 10 μg/mL of Chlorpromazine and then 1 h incubation with D-Dox-PGA. C 30 min pre-treatment with 10 μg/mL of Chlorpromazine followed by 1 h incubation with Dox. D Cells alone. (a: Bright field, b: Dox fluorescence, c: Hoechst stain, d: superposition of images b 74 and c). Magnification = 40X.

Figure 37: Spheroid formed using non-adhesive liquid overlay method shown in Day 3 and 5 in 100 µl of complete media. 75

Figure 38: Cytotoxicity of targeted D-Dox-PGA after pretargeting with various concentration of biotinylated anti-DTPA bispecific antibody complex compared to free Dox in MCF7 ADR spheroids. 76

Figure 39: Comparison in cytotoxicity of free Dox and Ptxl with individually targeted D-Dox-PGA or D- Ptxl-PGA and combination treatment (D-Dox-PGA+D-Ptxl-PGA) in MCF7 ADR spheroids. 76

Figure 40: Tumor growth inhibition studies in vivo in a mouse 4T1 xenograft model 79 Figure 41: Mouse bodyweight measurements during treatment 79 Figure 42: Representative images of excised tumors and tumor weights (n=5). 79 Figure 43: Fluorescence images of cardiac sections from mouse 4T1 tumor bearing Balb/c mice after systemic adminstration of different treatment groups. A) Dox treated groups, B) Targeted D-Dox-PGA treated group. Magnification 4X. 80

Figure 44: Immunohistochemical staining of 4T1 breast tumor tissue for induction of apoptosis using TUNEL assay 81

Investigator: Prashant Raj Bhattarai

LIST OF ACRONYMS

BSA: Bovine Serum Albumin. Dox: Doxorubicin. Ptxl: Paclitaxel. Mph: Melphalan. EDC: 1-ethyl -3-(3-dimethylaminopropyl) carbodiimide-HCL. PGA: Polyglutamic acid polymer. DTPA: Diethylene TetraPentaacetic Acid. D-Dox-PGA: Doxorubicin-loaded polyglutamic acid polymer. D-Ptxl-PGA: Paclitaxel-loaded polyglutamic acid polymer. D-Mph-PGA: Melphalan-loaded polyglutamic acid polymer. PPDCs: Polymer Pro-drug conjugates bsMAbcx: Bispecific antibody complex. EPR: Enhanced Permeability Retention. MAb: Monoclonal Antibody. BAAC: Bispecific Affibody-Antibody Complex. GPCR: G Protein Coupled Receptor. FITC: Fluorescein Isothiocyanate. HRP: HorseRadish Peroxidaze. EDTA: Ethylenediaminetetraacetic acid. PBS: Phosphate buffer saline. OD: Optical density. MDR: Multiple Drug Resistance. NaHCO3: Sodium bicarbonate. TUNEL Assay: Transferase (TdT)-mediated dUTP Nick End Labeling IPTG: Isopropyl β-D-1-thiogalactopyranoside

Investigator: Prashant Raj Bhattarai

1. INTRODUCTION:

1.1 Antibody targeted therapies:

Antibodies are glycoprotein endowed with exquisite specificity1. Discovery of the Hybridoma technology for efficient production of monoclonal antibodies by Kohler and Milstein in 1975, has led to a rise in use of antibodies in both diagnostic and therapeutic applications2. Monoclonal antibodies (MAbs) specific for over expressed surface biomarkers in cancer cells have been developed as alternate targeted tumor selective treatment approaches. Even though some MAbs have been successfully used in cancer therapy, most of the MAbs have not been found to be therapeutically effective on their own1. This deficiency has led to the development of Antibody-

Drug Conjugates (ADCs). Antibodies in ADCs are used as targeting agents to deliver potent cytotoxic compounds selectively to tumor cells, thereby improving the therapeutic index of the chemotherapeutic agent. Although ADCs have been reported to have some efficacy in cancer therapy, they are limited by the amount of the payload of the drugs that can be delivered by individual antibody molecules3.

1.2 Bispecific Antibodies and Pretargeting Approach:

Bispecific Antibodies (BispMAbs) are capable of binding two different antigens simultaneously4.

Dual targeting strategies of BispMAb can be applied in the following ways5:

1. BispMAb that bind directly to targets such as cell surface receptors can induce or inhibit

essential apoptotic signal cascade or cell growth signal transduction activation

respectively.

2. BispMAb can be used as pretargeting agents for targeted delivery of therapeutically active

compounds, such as effector molecules, or localization of effector cells on target cells.

Since the development of the first bispecific polyclonal antibodies by chemical coupling of two polyclonal antibodies by Nisonoff and Rivers in 1961, there has been a steady development and

Investigator: Prashant Raj Bhattarai increasing application of bispecific antibodies in biological research. Bispecific Antibodies have been developed by chemical conjugation of two monoclonal antibodies or fusion of two hybridomas cell lines to form quadromas6. Recent developments in genetic engineering have further the development of novel techniques such as “knobs into holes”, or the “Leucine zipper based dimerization of MAb fragments”7 for generation of bispecific antibodies. Bispecific antibodies have great potential in wide varieties of research and their current applications range from in vivo targeting, enzyme immunoassays, immunohistochemistry, radioimaging and radioimmunotherapy, and immunotherapy4.

Pretargeting strategies were first developed in the field of radioimaging and radiotherapy where high resolution between healthy and diseased tissue is essential for successful diagnosis or therapy8.

Due to their specificity MAbs can serve as excellent agents for delivery of radionuclides in vivo.

Their pharmacokinetic properties result in high antibody accumulation in the targets but due to slow blood clearance, use of MAb in diagnosis required long incubation time, leading to low tumor to blood activity ratios. In radioimmunoscintigraphic imaging, the low target to background activity can lead to equivocal diagnostic interpretations. In radaioimmunotherapy, high tumor activity can be achieved due to repeated circulation of the MAb into the tumors. However, long circulatory half-life of MAbs prolongs the exposure of healthy tissues and organs to excessive off- target radiation resulting in severe adverse effects, especially in radiosensitive tissues such as the bone marrow5,9. To overcome these problems, the pretargeting approach was developed where the delivery of the long circulating antibodies is separated from the delivery of therapeutic agents such as therapeutic radionuclides8. The pretargeting approach involves the use of bispecific antibody that can bind biomarkers expressed by pathological cells, and then the therapeutic or diagnostic payload attached to small carrier molecules is delivered. The payload is sequestered by the payload capturing arm of the bispecific antibody. Bispecific antibody after in vivo administration localizes at the diseased target sites. This step is referred to as the “pretargeting” step. Excess circulating bispecific antibody is cleared from the circulation either by the reticuloendothelial system (RES)

Investigator: Prashant Raj Bhattarai or renal excretion if the bispecific antibodies are modified to have molecular weight less than

50,000 daltons10. Radiolabeled monovalent or bivalent hapten or in our version of drug delivery, polymer loaded with diagnostic or therapeutic drugs are injected at a later time point, ideally when the level of the bispecific antibodies in the blood has reached negligible concentration. Due to the small molecular weight of the signaling or therapeutic agents conjugated to the carriers, they are cleared rapidly from the body but are selectively captured at sites of the disease due to the capture arm of the bispecific antibody that has specifically localized at the target sites. The pretargeting approach has been used successfully for imaging and radionuclide based therapy of cancer9.

Negatively charged polymers loaded with either radionuclide or chemotherapeutic drugs are preferred as the targeting agents for pretargeting in our laboratory11. Negative charge of the polymers and negative charge of cell membranes lead to ionic repulsion thereby keeping non- specific interaction of the polymers to non-target tissues and organs to a minimum. This approach should maximize tumor targeting leading to higher target to background ratio in radioimaging and reduce bystander effect by minimizing non-targeted toxicity on healthy cells in radiotherapy or chemotherapeutic applications12. Since multiple drugs or payloads can be conjugated on the polymers, high specific activity polymer drug conjugates may be prepared. In addition, since the therapeutic drugs are covalently linked to the carriers such as biocompatible polymers, the covalently conjugated drugs are in the prodrug conformation, which will require release from the carriers prior to regaining the therapeutic potency, thereby further reducing off target toxicity during circulation in the blood.

1.3 Rationale for using Antibody fragments:

Intact MAbs of the IgG class are Y-shaped multidomain glycol-proteins with two antigen binding

Fab domains and an effector constant region Fc domain. The molecular weight of IgG MAb is approximately 150,000 Daltons. Intact antibodies have long circulating half-life due to the size of the antibody, which is greater than the size that can be eliminated by renal filtration13. This long

Investigator: Prashant Raj Bhattarai half-life leads to long wait times from injection to successful imaging. Nevertheless, the long half- life of antibodies is an advantage for therapeutic uses and a disadvantage for diagnostic uses as discussed previously.

Over 85% of the human cancers are solid tumors. Therefore, the ability of intact antibodies to penetrate solid tumors efficiently is an issue. Even though the tumor vascular is leaky, solid tumors are characterized by the presence of high interstitial fluid pressure13. Since the entry of MAbs into tumors is via diffusion the intra-tumor pressure gradient, becomes a hindrance because the rate of diffusion is inversely proportional to the cube root of the molecular weight. Therefore, large molecules such as MAbs would penetrate solid tumors very slowly14. Thus, out of the 20 MAbs approved for cancer therapy only 8 are specific for solid tumors, reflecting the current limitations in MAb therapy1.

Due to the modular nature of antibodies, separate domain can be generated via biochemical or genetic engineering means15. These approaches allow antibody drug designers to engineer various customized antibody fragments with pharmacologic properties optimized for specific applications.

Various antibody fragments such as antigen binding fragments Fab, F (ab’)2, Fab’ and Fv fragments can be generated by proteolytic cleavage of intact antibodies. Advancement in genetic engineering has led to generation of recombinant antigen binding fragments, including Fabs and ‘second generation’ single chain variable fragments (scFvs)16,15. Antibody fragments due to their small size can penetrate solid tumors more rapidly and deeper into the tumor tissues relative to intact MAbs.

These smaller fragments are also capable of binding to cryptic epitopes not accessible to intact

MAbs, such as immuno-evasive pathogen glycoproteins16. Of the 450 MAbs in clinical trials, 54 are antibody fragments. Of these 3 have been approved by the FDA in United States15.

Investigator: Prashant Raj Bhattarai

1.4 Rationale for using Affibody:

Antibody mimetics are antibody-like molecules, which possess high affinity for the cognate antigens similar to antibodies but are structurally unrelated to antibodies. They are usually comprised of artificial peptides or proteins with molecular weight between 3 to 20 kDa17. They provide a number of advantages over antibodies such as rapid tissue penetration, stability towards heat and enzymes, better solubility and comparatively low production costs. Several antibody mimetics such as affibodies, anticalins, DARPins, nanofitins, fynomers and avimers have been developed as therapeutic and diagnostic agents18.

Affibodies are small 6 kDa affinity proteins consisting of the three-helical-bundle Z domain, a stabilized variant of the B domain of staphylococcal protein A (SPA)17. Z domain scaffold is relatively short cysteine free peptide of 58 amino acids and has short folding time of 3μs. Various combinatorial phagemid libraries of affibody molecule has been generated by randomization of the

13 surface exposed amino acids located in helices 1 and 2 of Z domain from which affibody molecules with nanomolar and picomolar affinities have been produced using phage display technology specific for various targets such as human epidermal growth factor receptor 2

(HER2)7,8, epidermal growth factor receptor (EGFR)8, and tumor necrosis factor α (TNFα)9. Since affibody molecules lack cysteines, site-specific modification is possible for the introduction of cysteine residues. This modification enables site specific labeling with either fluorescent dyes or radionuclides as well as in the generation of bispecific complexes with antibody fragments. Thus, the low molecular weight of affibodies combined with its robust structure make them suitable for various applications such as diagnostic targeting agents and as inhibitors of receptor interactions18.

HER2/neu belongs to the epidermal growth factor receptor family. It is a 185-kDa glycoprotein having tyrosine kinase activity19. Certain normal tissues express low levels of HER2/neu, but it is overexpressed in many different types of cancer due to its important role in tumor cell proliferation, survival, maturation and mobility20. Overexpression of HER2/neu is observed in 23 to 30% of all

Investigator: Prashant Raj Bhattarai cases of patients with breast and ovarian cancers, and in significant fraction of lung, stomach, and bladder cancer patients. In breast cancer, HER2/neu overexpression is generally associated with poor prognosis, increased tumor aggressiveness, resistance to chemotherapies and higher mortality rates9. The discovery of HER2/neu in tumorogenesis made it a popular target for cancer therapy.

Trastuzumab, a humanized anti-HER2 monoclonal antibody was the first anti-HER2 therapy

21 developed that resulted in improved patient survival . Identification of affibody (His6-ZHER2/neu:4) which bound specifically to HER2 extracellular domain by Wikman et al. in 2004 led to wide use of anti-HER2 affibodies as efficient targeting agents for tumor imaging and therapy by conjugation with radionuclide or chemotherapeutic drug20,21.

1.5 Rationale for using biotin as a second cancer-targeting agent:

Every living cell require vitamin for survival and normal growth22. Since solid tumors consist of rapidly dividing cells they have very high requirement of certain types of vitamins, leading to overexpression of these vitamin receptors in many cancers. Thus, these receptors can be used for targeted drug delivery in receptor positive cancers. Folic acids, Vitamin B12, riboflavin and biotin are some of the vitamins that are essential for growth of normal healthy as well as cancer cells22,23.

Studies have shown that folate receptors can be used as an attractive candidate for targeting in imaging and for drug delivery to folate receptor overexpressing cancer cells. Tumor targeting via folate-modified liposomes has been used to target folate receptor overexpressing tumor cells24.

Doxorubicin and 5-fluorouracil loaded-liposomes targeting folate receptors have shown enhanced in vitro and in vivo cytotoxicity.

Biotin, belongs to Vitamin H family and is a growth promoter of cells. Even though cancerous cells overexpress biotin receptors, biotin as the ligand for drug targeting has not been studied extensively22,23. Studies have shown that biotin conjugated macromolecules were able to increase specific uptake of anticancer drug to the tumor cells24.

Investigator: Prashant Raj Bhattarai

1.6 Polymer prodrug conjugates for Cancer Therapy:

Cancer is a disease characterized by uncontrolled cell growth. These cancerous cells may invade nearby healthy cells and spread to various parts of the body via the blood and the lymphatic systems

(metastases) 24. Chemotherapy is the most common form of cancer therapy together with surgery and radiation therapy25. Anticancer drugs used for chemotherapy are antimetabolites, alkylating agents, DNA-complexing agents, mitosis inhibitors or hormones that exert their activity by either interfering with DNA replication, repair, translation or cell division26,27,28. These cytotoxic agents rely on the highly proliferative nature of cancerous cells for their cytotoxic effects. Thus, prolonged chemotherapy also results in lethal damage to proliferating healthy non-cancerous cells. Albert and

Harper first suggested the concept of the use of prodrug, which provides an alternative approach for the design of less cytotoxic form of the anticancer drugs. Prodrug remains in the inactive form during its delivery to the sites of action and is activated by specific conditions at the target sites29.

Conjugation of a drug to the polymer backbone forms the “Polymer prodrug conjugates”. Ringsdorf first saw the potential of the polymer prodrug and proposed the application of polymer prodrug conjugate in 197530. These biocompatible polymeric conjugates provided numerous advantages over those of the conventional precursors. These advantages include31: 1) increased bioavailability of poorly soluble or insoluble drugs by increasing their solubility, 2) improved pharmacokinetics of the drugs, 3) ability to provide passive or active targeting of the drugs to the sites of action, and

4) the able to carry the desired payload at the same time preserve the integrity of the drug during circulation and transport. Various polymers have been used as candidates for this purpose.

Polymers used for making polymer drug conjugates include synthetic polymers such as

Polyethylene glycol (PEG), N-(2-hydroxyporpyl) methacrylamide copolymers (HPMA); natural polymers such as dextran and chitosan, and pseudo synthetic polymers such as Polyglutamic acid

(PGA) and poly-L-lysine32. These polymer prodrug conjugates target via the enhanced permeability and retention (EPR) effect. Addition of cell-specific ligand would allow targeting to cancer cells.

Depending on the linkers used to conjugate drugs to the polymers, the release of the active drugs

Investigator: Prashant Raj Bhattarai from the prodrug form may occur on exposure to the acidic pH in the tumor microenvironment or by the action of lysozymes after internalization by endocytosis and fusion with lysosomes33,34.

1.7 Multidrug Resistance in tumor:

Multidrug resistance, a mechanism through which cancer develops resistance against chemotherapeutic drugs, is a major factor leading to failure of various conventional chemotherapies35. There are three mechanisms that lead to the development of drug resistance in cancer cells:

1) Decrease uptake of water-soluble drugs.

2) Increased energy dependent efflux of hydrophobic drugs that enter via diffusion to the cells.

3) Cellular changes that affect the ability of cytotoxic drugs to kill cells, including increased repair of DNA damage, reduced apoptosis and altered metabolism of drugs.

Of these three mechanisms, increased efflux of hydrophobic cytotoxic drugs mediated by energy dependent ATP-binding cassette (ABC) transporters is the most commonly encountered cause of drug resistance. P-glycoprotein (Pgp, also known as MDR1) belongs to the ABC transporter family10. Pgp transporter has broad substrate specificity for many cytotoxic drugs and overexpression of Pgp in cancer cells can induce MDR. Paclitaxel efflux from cancer cells generated by Pgp activity is a major factor affecting the clinical efficacy of paclitaxel36. Free drug enters cells via diffusion. Accumulation of drugs via diffusion occurs in the cytoplasm near the plasma membrane where Pgp pumps are located. This proximity lends itself to ready availability of the drugs for efflux by the Pgp pumps. However, when utilizing the pretargeted approach for drug delivery, internalization of the polymer drug conjugates is by endocytosis37. The endosomes then interact with lysosomes to form endolysosomes in the cytoplasm away from the proximity of the plasma membrane in cancer cells. Subsequently digestion of the biodegradable carrier polymers leads to release of the active drugs deep in the cells and results in greater availability to

Investigator: Prashant Raj Bhattarai induce the cytotoxic therapeutic effect whether they occur in the cytoplasm or in the nuclei38. This mechanism of active drug release is proposed to occur substantially away from the plasma membrane containing the efflux Pgp pumps thereby improving the therapeutic efficacy of the pretargeted polymer prodrug therapy in drug resistant cancers.

1.8 Combination therapy:

Cancer cells undergo a number of mutations in survival and pro-apoptotic pathways ensuring their continued growth and survival. It would therefore be advantageous to target a number of these pathways simultaneously using different therapeutic agents. A successful combination therapy should contain agents that38,39:

1) Have proven activity as a single agent

2) Have different target and mechanisms of action

3) Have non-overlapping dose limiting toxicities

4) Have non-overlapping resistance mechanisms

5) Can be delivered with an appropriate dosing schedule to achieve maximum efficacy.

There are numerous examples in the literature of effective usage of drug-drug combinations, siRNA-drug combinations and peptide-drug combinations40,41.

Investigator: Prashant Raj Bhattarai

1.9 Spheroid Cell Culture:

In vitro cell culture plays a very crucial role in screening of lead molecules for therapy, and for studying their mechanism of action at the molecular and cellular level. Monolayer cell culture, the current model of choice for cancer research, does not correlate well with results observed in vivo.

The monolayer cell culture model fails to represent behavior associated with tumor tissues such as cell-cell interaction, cell population at diverse cell cycle stages based on nutritional availability, low pH and hypoxic core 42,43. Since spheroids are model of solid tumors in cell culture that mimic the conditions stated above, interests in the use of spheroids for therapeutic screening in culture have increased significantly because the 3D spheroids contain extensive extracellular matrix

(ECM) and possess complex three-dimensional network of cell-to-matrix and cell-to-cell interactions44,45. These factors not only affect the distribution and function of cancer cells, but spheroids also affect drug penetration and action thereby making spheroids a better in vitro model than monolayers. Spheroids above 500µm diameter exhibit characteristics such as having a necrotic core surrounded by a layer of quiescent cells and an outermost layer of rapidly multiplying cells similar to the avascular early stage solid tumors. In 1977, Lucke-Huhle & Dertinger presented data demonstrating that V79 cells in spheroids were protected against hyperthermic damage when compared to exponentially growing cells in monolayer cultures. A genome wide gene-expression analysis of porcine hepatocytes revealed that more than 65 genes including those encoding hepatic specific transcription factors, express differently in spheroids thereby making the spheroids resemble more closely to liver tissue than monolayer hepatocytes46. Many more studies have established that spheroids mimic morphology and biochemical characteristics of solid tumors, which are not observed in a monolayer cultures. Thus, the 3D spheroid cultures may provide an in vitro approach more representative of the in vivo cancer model. Various methods to generate 3D spheroids are shown in Table 1.

Investigator: Prashant Raj Bhattarai

Table 1: Spheroid formation techniques along with their advantages and disadvantages

Methods Advantages Disadvantages Spinner Flasks47,48 ● Can produce large ● High shear force and easily accessible ● Specialized spheroids equipment necessary ● Mass production of ● Size and cell numbers spheroids possible in spheroids may vary Hanging drop49–52 ● Generate spheroids of ● Difficult for mass uniform shape and production size ● Time consuming and ● Fast and inexpensive no high throughput

Non Adhesive liquid ● Inexpensive ● Non uniform Overlay44,45 ● Easy to perform spheroid sizes ● No specialized equipment required 3D Scaffolds46,53 ● Provides extracellular ● Specialized support for generation equipment required of spheroids of cells for scaffold which don’t form fabrication spheroids easily

Investigator: Prashant Raj Bhattarai

2. SPECIFIC AIMS

The overall objective of this thesis is to evaluate the efficacy of the pretargeting approach to overcome drug resistance and/or improve therapeutic efficacy using single and combination drug therapy modalities. Conjugation of cytotoxic drugs to polymers changes the biodistribution, bioavailability as well as their mechanism of uptake and intracellular trafficking54. Bispecific antibody complexes are used as pretargeting agents for targeting of PPDCs to cancer cells. These pretargeted PPDCs are then taken up via receptor-mediated endocytosis different from delivery of free drugs. This route of prodrug polymer entry into the targeted cells should lead to endosomal compartmentalization, which then forms endolysosomal vesicles where the biocompatible carrier polymers are degraded and the active free drugs are released deeper within the cytoplasm. This altered mechanism of uptake and drug release should provide a mechanism to overcome Pgp mediated MDR by providing active drugs further away from the efflux pumps, thereby providing higher concentration of the active drug for more efficient induction of cytotoxicity in cancer masses35.

To achieve the above overall objectives, the following specific aims will be investigated.

Specific Aim 1: Evaluate expression of receptors on various cancer cell lines to be used in the pretargeting and targeting of PPDCs as a novel drug delivery approach:

a) Demonstrate overexpression of HER2/neu receptors in SKOV3 and SKOV3 TR (drug

resistant) ovarian cancer cell lines using fluorescein labeled anti-HER2/neu affibody

b) Demonstrate overexpression of Biotin Receptors on MCF7, MCF7 ADR, and other

cancer cells in vitro

Specific Aim 2: Prepare bispecific antibody complexes:

a) Anti-HER2/neu affibody X anti-DTPA Fab bispecific complex

b) Biotinylated anti-DTPA bispecific complex

Investigator: Prashant Raj Bhattarai

Specific Aim 3: Generate and characterize various polymer prodrug conjugates:

a) D-Ptxl-PGA,

b) D-Dox-PGA and

c) D-Mph-PGA

Specific Aim 4: Investigate in vitro cytotoxicity and drug resistance reversal after pretargeting with bispecific antibody complex by delivery of:

a) Single chemotherapeutic PPDCs

b) Combination chemotherapeutic PPDCs

Specific Aim 5: Demonstrate reversal of drug resistance reversal in vitro after pretargeted delivery of PPDCs in 3D spheroid model using drug resistant cancer cells.

Specific Aim 6: Evaluate anti-tumor activity following single and combination therapy in the pretargeting approach in an in vivo mouse model of breast cancer to investigate:

a) Therapeutic efficacy of single and combination targeted polymer prodrug therapy

b) Histological toxicology analysis on the heart and tumor tissues from the animals

subjected to various treatments

c) Hematological toxicity studies.

Investigator: Prashant Raj Bhattarai

3. MATERIALS AND METHODS

Monoclonal antibody anti-DTPA (2C31E11C7) is produced in house. Poly-L-glutamic acid

(Molecular weight 13,300 Da), bicyclic anhydride of Diethylene triamine pentaacetic acid, N- hydroxysuccinimide ester of Bromoacetic acid and were purchased from Sigma Aldrich (St. Louis,

MO). Dulbecco’s Modified Eagle Medium (DMEM), RPMI 1640 medium, Penicillin-

Streptomycin and Melphalan were purchased from Thermo Fisher Scientific. K-Blue is purchased from Neogen Corporation, KY. Doxorubicin hydrochloride and Paclitaxel were purchased from

LC labs (Woburn, MA). BALB/c mice were purchased from Charles Liver laboratory. Bispecific antibodies were generated in lab by chemical conjugation method via thioether linkages.

3.1. Purification and Characterization of anti-HER2/neu Affibodies:

3.1.1 Affibody Production:

Affibodies were expressed as 6-His tag fusion proteins from pET28b vector encoding for affibody gene between NcoI and HINDIII restriction sites in E.coli strain BL2112. An aliquot of 15μl of bacteria was inoculated in 100 ml of Lucia-broth (LB) media containing 30μg/ml of kanamycin in sterile 500 ml Erlenmeyer flask and incubated overnight at 370C shaker. 100μl of the E. coli were taken from the overnight culture and inoculated in fresh LB media (300 ml) containing 30μg/ml kanamycin and grown at 370C. When absorbance at 600 nm reaches 0.8, gene expression was induced by the addition of 3 ml of isopropyl b-D-thiogalactoside (IPTG) to a final concentration of

1 mM. After allowing bacteria to grow overnight at 370C in a shaker, E. coli were harvested by ultracentrifugation (30,000 rpm, 30mins, 40C). The bacteria were then resuspended in 30 ml of binding buffer (50 mM Sodium phosphate, 0.3M NaCl, 5mM Imidazole, 0.1% Triton X 100 1mM

PMSF, pH 8) and lysed by 5 cycles of freeze thaw procedure using liquid nitrogen. After lysis, ultracentrifugation (30,000 rpm, 30mins, 40C) was carried out to separate the bacterial lysate from the bacterial debris.

Investigator: Prashant Raj Bhattarai

3.1.2 Affibody Purification:

The 6-His-Her2/neu fusion proteins were recovered using ProfinityTM Immobilized metal affinity chromatography (IMAC) Ni2+- charged resin (Bio-Rad). An aliquot of 1ml of IMAC resin slurry was taken and the storage solution was removed using the magnetic rack. IMAC resin was then washed with 3 column volumes of distilled water and then enough distilled water was added to make a 50% slurry. An aliquot of 30 ml of the cell lysate containing 6-His-6-Her2/neu proteins was then added to the prepared resin slurry and the mixture swirled gently. Resin-lysate mixture was then incubated at 40C for 30 minutes and then the mixture was loaded to the column (10 cm). After the resin had settled down in the column, the flow through volume of the lysate in the column was collected and the column was washed with 5 volumes of binding/washing buffer (50 mM Sodium

Phosphate, 0.3M NaCl, 5mM Imidazole, pH8). After thorough washing when the eluate showed no absorbance at 280 nm, 6-His-Her2/neu fusion proteins were eluted using 5 ml of the elution buffer (50 mM Sodium phosphate, 0.3M NaCl, 0.5M Imidazole, pH8). The protein concentration was determined using Pierce Bicinchoninic acid assay (Pierce biotechnology) kit with bovine serum albumin (BSA) as the protein standard for quantitation.

3.1.3 SDS PAGE identification of anti-HER2/neu Affibody:

Bio-Rad mini-PROTEAN Tetra cell kit was used for the characterization of purified Affibody using

SDS PAGE55. The affibody molecules contain a C-terminal cysteine residue. The free sulfhydryl residue, tend to oxidize and form dimers. Therefore, both reduced (treated with 20 % β mercaptoethanol) and non-reduced samples were analyzed in by SDS-PAGE. Acrylamide/Bis- acrylamide with 12.5% resolving gel and 4% stacking gel (around 2cm) was prepared. Aliquots of

6μg of protein samples in the loading buffer containing 10% SDS and bromophenol blue tracking dye were prepared and heated at 950C for 10 minutes before loading samples to the polymerized acrylamide gel. SDS-PAGE running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) was used.

Investigator: Prashant Raj Bhattarai

Electrophoresis was performed at 90V for about 95 minutes. After completion of electrophoresis, gel was removed from the gel cassette, rinsed with deionized water for 10 minutes and then stained with 0.1% Coomassie brilliant blue for 30 minutes. The gel was then destained with three changes of the de-staining solution (50% deionized water, 40% methanol, 10% glacial acetic acid). The gel was then rinsed with deionized water and transferred to a wet chromatographic filter paper followed by overlaying with plastic sheet. The assembly was then transferred to Bio-Rad gel dryer (Model

No. 583) and dried for 2 h under vacuum.

3.1.4 Anti-HER2/neu affibody labeling with FITC:

The cysteine residue at the C-terminal of the affibody was used for site specific labeling of the affibody with thiol-reactive Fluorescein-maleimide fluorophore. An aliquot of 0.5 mg/ml of affibody was incubated with 20 mmol/L of dithiothreitol (DTT) at pH 7.4 for 2 h at room temperature. After the reduction of oxidized cysteine, affibody solution was dialyzed extensively against 0.1M PBS buffer containing 10 mM EDTA for 24 h at 370C. Fluorescein-maleimide dye

(5mg) was dissolved in DMSO (500μl) and the aliquot was added to the reduced affibody while vigorously stirring. The reaction of the mixture was allowed to proceed overnight at 40C. Unreacted fluorophores were then removed by Sephadex G-10 desalting column chromatography using the centrifugation separation protocol56.

3.1.5 Epi-fluorescent microscopy for characterization of HER2/neu receptors in SKOV3 and

SKOV3 TR cell lines:

SKOV3 and SKOV3 TR (Paclitaxel resistant) cell lines were obtained from Dr. Torchilin’s lab and were cultured in RPMI 1640 medium with 10% Fetal clone (Thermo Fisher, USA), penicillin (1000

0 units/ml) and streptomycin (1000 units/ml) at 37 C with 5% CO2. An aliquot of 500μl of culture media containing approximately 80,000 SKOV3 and SKOV3 TR cells were added to the 12 well culture plates containing coverslip in the wells and incubated overnight. Cells that grew on the

Investigator: Prashant Raj Bhattarai coverslips were then washed with 0.1M PBS, then fixed and permeabilized by adding 500μl of

Acetone to the coverslips for 10 minutes at room temperature. The coverslips were blocked with

3% BSA for 2 h and washed again. The aliquots of 100μl of 5 μg/ml of Affibody-FITC were added to each coverslip and incubated in dark for 1 hour in a humidity chamber. Coverslips were washed

5X times with PBS-T followed by PBS and were counterstained with Hoechst (1 µg/ml), and the coverslips were mounted on microscope slides with Fluoromount-G (Southern Biotech). Slides were then sealed using clear nail polish and were stored in slide box at -200C in the dark for subsequent epifluorescence microscopic examination (Nikon Eclipse from Dr. Torchillin’s lab).

3.1.6 Flow Cytometry studies of HER2/neu receptors in SKOV3 and SKOV3 TR cell lines:

SKOV3 and SKOV3 TR cells were cultured in 6 well plates starting at 40,000 cell/well. After each well has reached ~ 80% confluency, cells were trypsinized and neutralized with RPMI 1640 cell culture medium. Then, the cell pellets were suspended in 100μl of 0.1M PBS. The cells were then treated with either 100μl of 5μg/ml Affibody-FITC or 1% BSA alone and incubated at 40C for 30 minutes. The cells were then washed 3X with ice cold 0.1M PBS. Samples were then assessed by flow cytometry (FACS Calibur instrument, BD Biosciences, San Jose, CA) equipped with an argon-ion laser and an optional second red diode laser (source energy, 15 mW; detection time, 500 counts per second). Data were live gated for 10,000 cells each by Forward light scatter (FSC) and

Side light scatter by FL1 (blue laser, 488 nm). Cell Quest pro software was used for data acquisition and analysis (BD Biosciences, San Jose, CA).

Investigator: Prashant Raj Bhattarai

3.2. Preparation and Characterization of anti-HER2/neu X anti-DTPA Fab bispecific complex:

3.2.1 Preparation of anti-DTPA Fab:

Intact monoclonal anti-DTPA (2C31E11C7) antibody was subjected to enzymatic digestion with immobilized papain beads (Pierce) to prepare Fab fragments according to the manufacturer’s specifications. An Aliquot of 3 mg/ml of the intact anti-DTPA was dialyzed overnight against the sample buffer (20mM sodium phosphate, 10mM EDTA, and pH 7). Immobilized papain beads were then equilibrated in digestion buffer containing 20 mM Sodium phosphate, 10mM EDTA,

20mM cysteine hydrochloride pH 7 and then added to the dialyzed sample followed by incubation for 20 h at 370C in a shaking water bath. After incubation, the crude digest containing Fab and Fc fragments were separated from the immobilized papain beads, and mixed with 1 ml of 1.5 M Tris-

HCl pH 7.5. Crude digest was then dialyzed overnight against the binding buffer (20mM Sodium phosphate, 0.15M NaCl, pH 8) for the Protein A affinity purification of Fab fragments from Fc and undigested intact anti-DTPA antibody. After dialysis, the crude digest was applied to the Protein-

A column and the pure anti-DTPA Fab fragment was collected in the fall through fractions whereas

Fc and undigested intact anti-DTPA were retained by the affinity column. Anti-DTPA Fab fragments were then characterized using SDS-PAGE and ELISA.

3.2.2 Immunoreactivity ELISA for anti-DTPA Fab:

The immunoreactivity of anti-DTPA Fab fragments was assessed by ELISA using 100μl aliquots of 1μg/ml DTPA-BSA as the antigen to coat 96 well microtiter plates (BD Falcon). The plates were incubated at 370C for 1 hour followed by washing 5X with 0.1M PBS-T (Tween) and then blocked by adding aliquots of 200 μl of 3% BSA for 1 hour at 370C. After, blocking the plate was washed with 0.1M PBS-T (5X) and then 100μl of serial dilutions of anti-DTPA Fab fragments starting with 0.001 to 1 μg/ml were loaded to the plate. Intact purified anti-DTPA antibody was used as the positive control and anti-myosin antibody was used as the negative control. Plates were

Investigator: Prashant Raj Bhattarai then incubated for 1 hour at 370C, followed by washing with 0.1M PBS-T (5X). Aliquots of 50

μl/well of Secondary antibody Goat anti-Mouse antibody conjugated to HRP (1:500 dilutions) were added to the plate and incubated for 1 h at 370C and washed again with 0.1M PBS-T (5X). Then aliquots of 50μl of K-Blue substrate were added to each well and incubated in dark at room temperature for 15 minutes. Plate was then read at OD 630 nm (BioTek, Winooski, VT) and results were analyzed using GEN5.0 software.

3.2.3 SDS PAGE analysis of anti-DTPA:

Generation and purity of anti-DTPA Fab was characterized by using SDS-PAGE as described previously in 3.1.3.

3.2.4 Preparation of anti-HER2/neu - anti-DTPA Fab bispecific complex:

Anti-HER2/affibody purified in step 3.1.2 and anti-DTPA Fab prepared in step 3.2.1 was used for the generation of the bispecific complex. Anti-DTPA Fab fragment (1 mg/ml) in 0.1 M PBS pH

7.4 was modified with 100x molar excess of N-hydroxy succinimide ester of Bromoacetic acid and the reaction was allowed to proceed for 6 h at 40C. Modified anti-DTPA was then purified using

Sephadex G-25 prepacked column (GE Healthsciences) using centrifugation protocol. 0.1M PBS pH 7.4 was used as the elution buffer. The extent of modification of anti-DTPA was assessed by the 2, 4, 6- Trinitrobenzene sulfonic acid assay12 and anti-DTPA ELISA was performed to assess the immunoreactivity of the modified anti-DTPA as described in step 2.2. Briefly, dimeric anti-

HER2/neu affibody was reduced with 20mM DTT for 2 h at room temperature followed by dialysis overnight against 4 liters of 0.1M PBS, 10mM EDTA pH 7.4. Equi-molar concentration of bromoacetylated anti-DTPA and reduced affibody (with free thiol groups) were mixed together and incubated overnight at 40C to form the bispecific antibody conjugates was via the thioether linkage.

Investigator: Prashant Raj Bhattarai

3.2.5 Purification of bispecific antibody complex:

Crude reaction mixture was passed through the ProfinityTM IMAC column. Unreacted anti-DTPA

Fab fragment did not bind to the column and was obtained in the fall through fractions. Bound multimeric and bispecific complexes along with the free unconjugated affibody were eluted from the column using 1ml of the elution buffer (50 mM Sodium phosphate, 0.3M NaCl, 0.5M

Imidazole, pH8). The eluate was then extensively dialyzed against 4L of 0.2 M phosphate buffer pH7.4 overnight using a 20,000-kDa molecular weight cutoff membrane (SpectraPore Dialysis membrane, Spectrum Laboratories, Rancho Dominguez CA). HPLC size exclusion chromatography was then performed for the separation of the bispecific complex from the multimeric complexes. A Zorbax GF-250 column (9.4 X 250mm) (Agilent Technologies, size exclusion limits = 400,000 Daltons to 10,000 Daltons) equilibrated with 0.2M phosphate buffer was used. An aliquot of 400 μl of the sample was applied to the column and 250μl aliquot fractions were collected. Absorbance at 280 nm was read to determine the elution profile.

3.2.6 SDS PAGE analysis:

Crude reaction mixture and purified bispecific antibody complex were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions as described earlier and compared with the molecular weight standards.

3.2.7 In-Cell ELISA for binding of anti-HER2/neu X anti DTPA Fab bispecific antibody complex to SKOV3 and SKOV3 TR ovarian cancer cell lines:

Aliquots of 5000 cells/well of SKOV3, SKOV3 TR and MCF7 were seeded in 96 well cell culture plates. The cells were allowed to attach overnight. The cells were fixed by adding 100μl of 8% paraformaldehyde solution to the plate containing the 100 μl of cell culture media for 15 minutes at room temperature. Paraformaldehyde solution was carefully removed from the plate and washed

Investigator: Prashant Raj Bhattarai

3X gently with 0.1M PBS pH 7.4. Aliquots of 200μl of 3% BSA blocking solution were added to plate and incubated for 1 h at 370C. Plates were then washed and with 100 μl of serial dilution of

1, 0.1, 0.01 μg/ml of anti-HER2/neu affibody X anti-DTPA Fab bispecific complex, anti-DTPA

Fab and affibody alone as negative controls were added to the wells. The wells were then incubated overnight at 40C. Next day, the plates were washed and 100 μl aliquots of 1:1000 dilution of secondary antibody GAM-HRP (MP Biochemicals, Santa Anna CA) were added to all the wells.

After incubating the plate for 1 h at 370C, the wells were washed 5X and then aliquots of 50μl/well of K-Blue substrate were added to the wells and incubated in the dark at room temperature for 15 minutes. Reaction was the stopped by adding of aliquots of 50μl of 0.5M HCl and absorbance recorded at OD 450 nm.

3.3. Preparation and characterization of biotinylated anti-DTPA bispecific antibody complex:

3.3.1 Biotinylation of Anti-DTPA antibody:

An aliquot of 0.4 mg/ml of anti-DTPA was dialyzed in 0.1M PBS pH 7.4 overnight. A stock solution of 20 mM NHS-PEG4-Biotin was prepared and then 20 molar excess of NHS-PEG4-Biotin was added to the antibody solution. The reaction mixture was incubated for 2 h on ice, and non- reacted NHS-PEG4-Biotin was removed by dialysis using a 3,500 Dalton molecular weight cut off dialysis membrane (SpectraPore Dialysis membrane, Spectrum Laboratories, Rancho Dominguez

CA), overnight in 0.1M PBS pH 7.4.

3.3.2 Characterization of biotinylated Anti-DTPA antibody by ELISA:

A 96 well microtiter plate (BD Falcon) was coated with 100μl aliquots of serial dilutions (1, 1, 0.1,

0.01, 0.001, 0.0001 μg/ml) of biotinylated bovine serum albumin (BSA) in triplicates. Additional wells of the microtiter plate are coated with 100μl aliquots of serial dilutions (1, 1, 0.1, 0.01, 0.001

0.0001 μg/ml) of Biotinylated anti-DTPA in triplicates. The plate is incubated for 2 h in a 37°C

Investigator: Prashant Raj Bhattarai water bath, then it is washed 5X with 200μl of PBST, followed by blocking with aliquots of 3%

BSA as previously described. Then, 100μl aliquots of Streptavidin-HRP (1:4000 or 1:8000 dilutions, MP Biochemicals, Santa Anna CA) were added to each well and the plate was incubated for 1 h at 37°C. After additional washing, 50μl aliquots of substrate K-Blue were added to each well. The plate was incubated at room temperature in the dark for 15 minutes and then absorbance recorded at OD 630 nm was obtained and the results were analyzed using GEN5.0 software.

3.3.3 Characterization of biotin receptors in cancer cell lines using in-cell ELISA:

Aliquots of 5000 cells/well of SKOV3, SKOV3 TR, MCF7, MCF7 ADR, 4T1 and H9C2 were seeded in 96 well cell culture plates and were allowed to attach overnight at 370 C. The next day, the cells were fixed to the wells by addition of equal volume of 8% paraformaldehyde solution to the plate containing cell culture media for 15 minutes at room temperature. Paraformaldehyde solution was carefully removed from the plate and washed 3X gently with 0.1M PBS pH 7.4. Then aliquots of 200μl of 3% BSA blocking solution were then added to plate and incubated for 1 h at

370C. The plates were then washed. After washing as previously described, 100 μl aliquots of

1μg/ml of biotinylated anti-DTPA or unmodified anti-DTPA as negative controls were added. The plates were then incubated overnight at 40C. Next day the plates were washed and 100μl aliquots of 1:1000 dilution of secondary antibody GAM-HRP were added to each well. After incubating the wells for 1 h at 370C, unreacted secondary antibody was washed 5X and then 50 μl/well aliquots of K-Blue substrate were added to the wells and incubated in dark at room temperature for 15 minutes. The reaction was the stopped by the addition of aliquots of 50μl of 0.5M HCl, followed by determination of the absorbance at OD 450 nm.

Investigator: Prashant Raj Bhattarai

3.4. Synthesis and characterization of Polymer Prodrug conjugates:

3.4.1 Conjugation of DTPA to PGA:

A solution of 50 mg/ml of PGA in 0.1 M sodium bicarbonate pH 8.6 is prepared. An aliquot of 3X excess of DTPA dissolved in a minimum volume of DMSO was added drop wise to the PGA solution while vortexing vigorously. The mixture was incubated at room temperature for 4 h and then dialyzed overnight at 40C in 4 liters of 0.1M phosphate buffered saline. Conjugation of DTPA to PGA is analyzed using 2, 4, 6- Trinitrobenzene sulfonic acid assay (TNBS). TNBS reacts with free amine groups to form a chromogenic derivative, which is quantitatively assessed by measuring the absorbance at OD 420 nm. Unmodified PGA was used as the standard for comparison.

3.4.2 Anti-DTPA ELISA for the detection of DTPA-PGA:

A 96 well microtiter plate (BD Falcon) was coated with 100μl aliquots of DTPA-BSA (1μg/ml) in each of the 12 wells in row A and B of the plate. Row C and D are coated with 100μl of DTPA-

PGA (1μg/ml) and incubated at 370C water bath for 2 h. Plates are then washed 5X with 200μl of

0.1M PBS containing 0.1% Tween 20 (PBST) pH7.4 and then aliquots of 200μl of 3% bovine serum albumin were added for blocking. After incubating the plate at 370C for 1 h, plate was again washed as before and serial dilution of primary antibody 2C31E11C7 (10, 1, 0.1, 0.01, 0.001 μg/ml) was added in aliquots of 100μl in quadruplicates (n=4). Plate was then incubated at 370C and washed with 0.1M PBST (pH 7.4). A 50μl aliquot of 1:500 dilution of secondary antibody Goat anti-mouse horseradish peroxidase (GAM-HRP) was then added to each well and incubated at

370C. The wells were washed with 0.1M PBST (pH7.4). Then 50μl aliquots of substrate K-Blue were added to each well. Plates are incubated at dark for 15 minutes at room temperature and the wells were read using BioTek microplate reader at OD 630 nm. The results were analyzed using

GEN 5 software as described.

Investigator: Prashant Raj Bhattarai

3.4.3 Preparation and characterization of D-Dox-PGA complex:

3.4.3.1 Conjugation of Doxorubicin to DTPA-PGA:

An aliquot of 1 ml of 10 mg/ml of DTPA-PGA in 0.1M PBS pH7.4 was mixed with 9.6mg of doxorubicin (24 molar excess) dissolved in minimum amount of DMSO (300μl). Then 17.2 mg of

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) dissolved in a minimal volume (300 µl) of DMSO was added drop wise to the mixture of DTPA-PGA and doxorubicin while vortexing vigorously. EDC activates the carboxylic group on PGA, which then reacts, with the free amine group of doxorubicin to form an amide bond. The reaction mixture is then incubated at 40C for 2 h, followed by overnight incubation at room temperature at dark.

3.4.3.2 Separation of free Dox from D-Dox-PGA:

Free unconjugated doxorubicin was separated from the DTPA-doxorubicin-PGA conjugate by gel filtration chromatography using Sephadex G-25 columns (1X35 cm column). The fractionation range of Sephadex G-25 is 1000-5000 Da. Blue dextran was used to determine the void volume of the column. Separation of Dox from D-Dox-PGA was then achieved by this column chromatography collecting 1 ml (20 drops) fractions. The elution profile was determined by obtaining the absorbance of each fraction at 490 nm. The concentration of doxorubicin in D-Dox-

PGA conjugate was then determined using the doxorubicin standard curve at OD 490 nm55,57.

3.4.3.3 Conjugation of Melphalan to DTPA-PGA:

An Aliquot of 1 ml of 10 mg/ml of DTPA-PGA in 0.1M PBS pH7.4 was mixed with 4.2 mg of melphalan dissolved in minimum volume of DMSO (300μl). An aliquot of 17.2 mg of 1-ethyl-3-

(3-dimethylaminopropyl) carbodiimide (EDC) dissolved in minimal volume (300µl) of DMSO was added drop wise to the mixture of DTPA-PGA and melphalan while vortexing vigorously. The reaction mixture was incubated at 40C for 2 h, followed by overnight incubation at room temperature in the dark. Free unconjugated melphalan is then separated from the DTPA-melphalan-

Investigator: Prashant Raj Bhattarai

PGA conjugate by extensively dialyzing it against 4 liters 0.1M PBS pH7.4 overnight at 40C. The concentration of melphalan in D-Mph-PGA conjugate was then determined using the melphalan standard curve at OD 260 nm12.

3.4.3.4 Conjugation of Paclitaxel and DTPA to PGA:

An aliquot of 32 mg of PGA was dissolved in 1.5 ml of dry N, N - dimethylformamide. To this solution, 11 mg of Paclitaxel, 15 mg of Dicyclohexylcarbodiimide, and a trace amount (3 mg) of dimethylaminopyridine were added58. The reaction was allowed to proceed overnight at room temperature and then thin layer chromatography was performed to determine conjugation of paclitaxel to the polymer. Addition of the reaction mixture into chloroform stopped the reaction and polymer drug conjugate was then extracted using Rotavapor. The resulting precipitate was dissolved in 0.5 M sodium bicarbonate buffer (pH 9.6) and then dialyzed extensively overnight against 4 liters of 0.1 M sodium bicarbonate buffer (pH 9.6). An aliquot of 20 X excess of DTPA dissolved in minimum volume of DMSO (200μl) was added drop wise to the dialyzed Paclitaxel-

PGA solution. The reaction mixture was incubated for 2 h at room temperature and then dialyzed extensively against 0.1 M PBS pH 7.4 overnight at 40C. Anti-DTPA ELISA was carried out to determine the conjugation of DTPA to the polymer and standard curve of Paclitaxel (227 nm) was plotted to determine the concentration of Paclitaxel in the D-Ptxl-PGA conjugate.

3.4.3.5 Stability Studies of D-Ptxl-PGA:

Stability of D-Ptxl-PGA was carried out in the various buffer systems at pH 4 and 7.4. An aliquot of 1 ml of D-Ptxl-PGA solution was placed in the dialysis membrane bag with molecular cutoff of

3000 Da and placed in either into 50 ml of 0.1 M PBS (pH 7.4) or 50 ml of 0.1 M sodium acetate buffer (pH4). The flasks contained the dialysis bags in 0.1M PBS or acetate buffer solutions were placed at 370C with continuous mixing using magnetic stirring bar and a stirring base. At various predetermined time intervals, 1 ml of samples were drawn from the release media and analyzed

Investigator: Prashant Raj Bhattarai spectrophotometrically at 227 nm. Absorbance was determined 3 times for each sample and then the samples were returned to respective flasks. Release of paclitaxel from the polymers was determined using the standard curve for Paclitaxel59.

3.4.3.6 Measurement of Zeta Potential of Polymer prodrug conjugates:

Zeta potential of the polymer prodrug conjugates were assessed using Zeta Plus (zeta potential analyzer) Brookhaven Instruments Corporation (Holtsville, NY) equipped with a palladium electrode with acrylic support was used. BIC zetapw32 software was used and all the measurements were taken at 250C using High Precision Mode.

3.4.3.6 Demonstration of presence of DTPA in Polymer pro-drug conjugates:

A 96 well micro-titer plate was coated with 100µl of 10 μg/ml DTPA-bovine serum albumin

(BSA)/well as a positive control or D-Dox-PGA at concentration of 1 µg/ml equivalent. The plate was incubated for 1 h at 37ºC. The micro-titer plate was washed with 0.1M PBS-T (3 X) followed by addition of 5% blocking FCS and incubated for 1 h at 37ºC. The micro-titer plate was then washed with 0.1 M PBS-T (3X) and the wells were loaded with 100µl aliquots of serial dilutions of anti-DTPA antibody from 1 to 0.0001 µg/ml of the antibody. The micro-titer plate was incubated for 1 h at 37ºC, washed 3 X with 0.1 M PBS-T and 50µl aliquots of GAM-HRP (1/1000 dilution) were added to each well. Incubation and washing was again carried out as previously described. K- blue substrate was added (50µl each well) as a chromogen. Absorbance at OD 630 nm was obtained. The assay was carried out in quadruplicates and analyzed using GEN5 software.

Investigator: Prashant Raj Bhattarai

3.5. Tissue Cultures:

3.5.1 Western Blot analysis for Pgp overexpression in cancer cell lines:

The Pgp overexpressing SKOV3 TR and MCF7 ADR and parental cell lines SKOV3 and MCF7 were obtained from Dr. Torchilin’s lab. Western Blot analysis was performed to allow the comparison of the Pgp levels of cell lines used. SKOV3, SKOV3 TR, MCF7 and MCF7 ADR cell lines are cultured in 25 mm Petri dishes for preparation of protein extracts. Cells were washed in ice cold PBS, and lysed in RIPA buffer supplemented with complete miniprotease inhibitor cocktail tablets (complete Mini, Roche). Protein concentrations are determined using bicinchoninic acid

(BCA) protein assay (Pierce Biotechnology) and samples containing equal amount of protein (50

μg) are used for immunoblotting, after addition of 5× concentrated sample buffer. Samples are then heated for 5 min at 95°C and subjected to 10% SDS-PAGE. The protein bands are transferred electrophoretically to polyvinylidene fluoride membrane (Amersham Hybond™-P, GE

Healthecare). Prestained molecular weight protein markers (Precision Plus Protein Dual Color

Standards, BioRad) were included in the SDS–PAGE gels. The membrane is blocked for 1 h at room temperature in PBS containing 0.1% Tween-20 (PBS-T) and 5% BSA. The membrane is incubated with the primary antibody overnight at 4°C (Anti-P-Glycoprotein Mouse monoclonal antibody, Calbiochem). After washing for 1 h in PBS-T with 1% BSA, the membrane is incubated for 1 h at room temperature with horseradish peroxidase-linked anti-mouse IgG+IgM secondary antibody (Sigma, 1:10,000 in PBS-T with 1% BSA). Immunoblot is developed using the Enhanced

Chemi-Fluorescence system (ECF; GE Healthcare) and a Storm device (Molecular Dynamics, GE

Healthcare). The membrane is then re-probed and tested for β-actin using anti-β-Actin Mouse monoclonal antibody, (Sigma, 1:5000) to confirm that equivalent concentrations of protein extracts were compared.

Investigator: Prashant Raj Bhattarai

3.5.2 Cell Viability Assays:

CellTiter Blue® (Promega, Madison, WI) is used for the assessment of cell viability according to the manufacturer’s instructions. Aliquots of 5000 cells/well were seeded in the 96 well cell culture plates and incubated at 37°C for 24 h in a 5% CO2 incubator. Cells are then treated with the various formulations (free drug, polymer drug conjugate alone and targeted polymer drug conjugates) in complete DMEM medium for 48 h. For targeted treatment, SKOV3 cells were treated with anti-

HER2/neu affibody X anti-DTPA Fab bispecific complex (10, 20 or 40 μg/ml) for 1h. 4T1 and

MCF7 cells were treated with biotinylated anti-DTPA bispecific antibody complex (10, 20, or 40

μg/ml) for 1 h. After pre-treatment, media were removed and the plate was washed 2X with 200μl of complete medium. Then, Ptxl or Dox, D-Ptxl-PGA or D-Dox-PGA at 0.001 to 15 μg/ml was added to the medium. The drug treated cells are incubated at 37°C for 48 h. The cells are then washed and incubated with 50μl aliquots of 1:5 dilution of CellTiter Blue® reagent for 2 h. Cell viability is evaluated by measuring the fluorescence (excitation 530 nm, emission 590 nm) using a

Synergy HT multi-21 detection microplate reader (Biotek, Winooski, VT). Cells treated with complete medium alone were used as a control to calculate 100% cell viability.

3.5.3 Inhibition of endocytosis:

Chlorpromazine, an inhibitor of endocytosis is used to demonstrate that endocytosis is the process of internalization of D-Ptxl-PGA associated with enhanced cytotoxicity in SKOV3 TR cells60.

SKOV3-TR cells (5,000) are pre-incubated with 10 μg/mL of chlorpromazine for 30 min before incubation with bispecific antibody followed by incubation with D-Ptxl-PGA or free Ptxl. Cell viability studies were carried out as described in 3.5.2.

Investigator: Prashant Raj Bhattarai

3.5.4 Fluorescence microscopy for drug and bispecific antibody uptake into breast cancer cells:

Aliquots of 40,000 MCF7 ADR cells were seeded into coverslip and allowed to attach overnight.

For the pretargeted approach, media was replaced with fresh media containing of 10 μg/ml of biotinylated anti-DTPA bispecific complex and incubated for 1 h at 37° C. After 1 h, the medium was removed and replaced with medium containing 20 μg/ml of DTPA-Dox-PGA, and incubated for various time-points (5, 10, 15, 30 min, 1, 2, 5, and 6 h). Controls consisted of wells with coverslips with MCF7 ADR cells incubated with 20 μg/ml of D-Dox-PGA and Dox alone for the same time-points described above.

For doxorubicin efflux studies coverslips coated with coverslips were incubated with the biotinylated anti-DTPA bispecific antibody complex and treated with 20 μg/ml of D-Dox-PGA for

1 h. Supernatant was removed at the end of 1 h treatment, and cells were washed with ice-cold

PBS. The wells were filled with fresh drug-free medium, and cells were incubated for 4 h at 37°C to facilitate cellular drug efflux. This is repeated for the Dox and D-Dox-PGA alone.

At various times of treatment with D-Dox-PGA, the cells were washed with chilled PBS. Cells were then fixed with 4% paraformaldehyde for 15 min at room temperature. The coverslips are washed 2 times each with PBS-T and PBS for 5 min each and counterstained with 1 μg/ml of

Hoechst to stain the nuclei. Coverslips are then mounted on a clean microscope slides with

Fluoromount-G mounting medium and were sealed and stored in −20 °C freezer until fluorescent microscopic examination (Nikon Eclipse E400) was performed61. The excitation wavelength of

528–553nm, DM 565nm and emission wavelength of (BA) 600–660nm were set for fluorescent microscopy. Ocular CFI 10X22M and the objective Plan 40X 0.40 PH1 DL were used.

Investigator: Prashant Raj Bhattarai

3.5.5 Preparation of Spheroids:

Non-adhesive liquid overlay technique was used for the generation of spheroids44,45,62. Each well in 96 well plate was coated with 50µl of sterile 1.5% agar in serum free DMEM to provide a non- adherent coating. Addition of 50µl of agar is important to cover complete surface of a well and formation of concave surface. After the coating, the plates were allowed to cool for 40 minutes before addition of cell suspension. To prepare the cell suspension, MCF7 ADR cells were grown in monolayer till they were 70% confluent. Cells were then trypsinized and centrifuged at 1000 rpm for 2 minutes to get a cell pellet. The pellet was re-suspended followed by cell counting. The cell suspension was adjusted to 150,000 cells/ml. For formation of a spheroid in each well, 100µl of this suspension was added to pre-coated wells. Plates were then centrifuged at 1500 g for 15 minutes at 4o C. Centrifugation resulted in the formation of a single layer of cell pellet. 96 well plate containing such single layered cell pellets was incubated at 37o C for the formation of spheroids.

3.5.6 Cell viability studies in Spheroids:

After generation of spheroids described 3.5.5 fresh aliquot of 100μl of medium was added after 2 days. Medium was replaced every 2 days by removing 100μl of old medium and replacing it with the same volume of fresh serum containing media without disturbing the spheroids. After spheroids reached their optimum compactness (5 days after initial seeding) around 15 spheroids were treated with each treatment type (5 spheroids per group). Treatment included free Dox, D-Dox-PGA without pretargeting and biotinylated Anti-DTPA bispecific complex pretargeted D-Dox-PGA.

Plates were then incubated for 48 h. After the incubation period, spheroids from each of the group were collected in separate Eppendorf tubes. Each spheroid was washed with 1X PBS pH 7.4 and

200 µl of Accumax® Cell Dissociation solution was added to each Eppendorf tube and incubated at 37°C for 5 minutes. After the spheroids were dissociated to individual cells, an equal volume of

FBS was added to stop the reaction. The tubes were then centrifuged to form cell pellets.

Investigator: Prashant Raj Bhattarai

Supernatant was discarded and the pellet was resuspended with 200µl of the following: CellTiter-

Glo® ratio of 1:12. Cells were then lysed by vortexing for 10 minutes. From this suspension, 100µl was added to a 96-well black plate with clear bottom and luminescence was measured after 10 minutes. Cell viability was calculated relative to the luminescence of the untreated spheroids

(controls) and expressed as % cell viability63.

3.6. Animal Studies:

All animal studies were performed in 6 weeks old Balb/C mice obtained from Charles River

Laboratories, Wilmington, MA. Experimental studies were carried out using protocol # - 17-0101R that was approved by the Institutional Animal Care and Use Committee at Northeastern University in accordance to NIH guidelines. All procedures for the study were followed in accordance with

NIH guidelines. The animals were given food and water ad libitum.

3.6.1 In vivo breast cancer therapy model:

Balb/C mice were injected subcutaneously in the right shoulder region with 2 X 106 4T1 murine breast cancer cells in serum-free DMEM media. Tumors were allowed to develop until they were approximately 100 mm3 (Day 13). The animals were then randomized into 7 groups of 6 animals each and weekly therapeutic intervention was carried out. Mice were pretargeted with 20 µg of biotinylated anti-DTPA bispecific complex. Following the clearance of unbound bispecific complex after 24 h, mice in this group were treated with D-Dox-PGA (5mg/kg doxorubicin equivalent dose), D-Ptxl-PGA (10mg/kg paclitaxel equivalent dose) or combination (D-Dox-PGA

+ D-Ptxl-PGA). Treatment was repeated every 7days. Control comparator groups consisted of mice treated with Dox, Ptxl, or saline. Mice were dosed for a total of 4 times weekly. To check for evidence of toxicity, mice were weighed daily. Tumor dimensions were measured using Vernier calipers and the volume was calculated using the formula 40,

Volume = (Width2 x Length)/2.

Investigator: Prashant Raj Bhattarai

Once the tumors in the control saline treated group reached an average size of 1000 mm3, all the animals were sacrificed except the dual polymer-drug conjugated treated group, which was allowed to go for 1 more week. After dissection and removal of the tumor tissues, the tumors were washed in sterile PBS, weighed and stored frozen in OCT embedding medium at −80°C.

For TUNEL staining to assess the extent of apoptosis in the tumors, some tumors were fixed in formalin.

3.6.2 Epifluorescence microscopy to study doxorubicin localization in mice hearts:

The hearts were collected from all animals and were stored in OCT embedding medium at -80 °C until processed. The frozen hearts were processed for the preparation of tissue slices using Microm

HM550 (provided by Dr. Barbara Waszczak’s lab). The temperature of the chamber was set at -20

°C. The chucks were then put in the freezing station. A base was build up on the chuck with NEG-

50 freezing medium. The base was then allowed to freeze. The hearts were placed longitudinally on the frozen chuck. A base of NEG-50 was build up on top of the heart covering the entire tissue and mount angle was set at position 6. The glass anti-roll device was adjusted so that the frozen chuck containing the heart was mounted properly. The mount was then directed up and down to cut 10 µm thick sections. The sections were taken up on a glass slide using a clean brush. The sections were then fixed on microscope slides by acetone drying and were examined using epifluorescence microscopy for the presence of Dox.

3.6.3 TUNEL staining for the detection of apoptosis in tumors:

Formalin-fixed tumor tissues were embedded in OCT medium and 10 µm thick sections were cut.

DeadEnd™ Colorimetric Apoptosis Detection System (Promega, Madison, WI) was used to detect apoptosis in the tumor sections placed on slides according to the manufacturer's protocol.

Equilibration buffer was added to the slides and incubated for 10 minutes followed by 10-minute incubation in 20 µg/ml proteinase K solution. The sections were washed in PBS and incubated with

Investigator: Prashant Raj Bhattarai

TdT enzyme at 37°C for 1 hour in a humidified chamber for incorporation of biotinylated nucleotides at the 3′- OH ends of DNA. Slides were then incubated in horseradish peroxidase- labeled streptavidin to bind the biotinylated nucleotides followed by detection with stable chromogen diaminobenzidine (DAB). Three slides per group were stained and the brown staining identified the apoptotic cells.

3.7. Data Analysis:

Data were generated in triplicates and reported as mean ± SD. Student’s t-test was used for the comparison of two groups and one-way ANOVA for more than two groups. P value < 0.05 was considered statistically significant.

Investigator: Prashant Raj Bhattarai

4. RESULTS:

4.1 Purification and characterization of recombinant anti-HER2/neu affibodies and

demonstration of specificity of Fluorescein labeled affibody (anti-HER2/neu affibody

FITC) for the HER2/neu receptors on SKOV3 and SKOV3 TR ovarian cancer cell

lines.

4.1.1 Characterization of Affibodies:

Expression of recombinant anti-Her2/neu affibody was induced with IPTG at a final concentration

of 1mM. Following purification of the affibodies using an affinity column chromatography, BCA

assay was used for determination of the recombinant protein concentration using BSA as the

standard. High concentration of anti HER2/neu affibodies at 1.5mg/ml was obtained. SDS PAGE

was then carried out for the characterization and determination of purity of affibodies.

A B C D D

Affibody multimeric complex

Affibody Dimer

Affibody Monomer

Figure 1: SDS-PAGE for Affibody characterization using 12.5% bis/acrylamide gel, Lane A) bacterial cell lysate beforeAs expected passing throughdue to the IMAC presence column, of Laneterminal B) F cysteinelow through residue, Lane affibody C) Non- Reducedexisted in purified dimeric Affibody and , Lane D) Reduced purified Affibody. As expected due to the presence of terminal cysteine residue affibody existed as dimeric and

multimeric forms as seen in Figure 1. After reducing the sample with the 10mM DTT, Figure 1,

lane D showed presence of two bands, one band at 9 kDa indicating monomeric affibody and a

second band at about 18kDa showing that the sample was not fully reduced. Molecular weight

Investigator: Prashant Raj Bhattarai estimation of Affibody monomer was confirmed by calculating the Rf values of the monomer and dimer form against the standard molecular weight markers.

4.1.2 Cell Association studies of anti-HER2/neu Affibody:

Ani-Her2/neu affibodies were labeled with fluorescein and the cell binding studies of these affibodies to HER2/neu positive SKOV3 and SKOV3 TR was carried out using flow cytometry

(Figure 2). There was no significant cell association of the anti-HER2/neu with HER2/neu negative

MDAMB-231 cells and MCF7 ADR cell lines. The fluorescence intensity was equivalent to the background level obtained with cells without the fluorescein labeled affibody. However higher cell association of anti-HER2/neu affibodies with SKOV3 and SKOV3 TR was observed. The data suggest that both SKOV3 and SKOV3 TR ovarian cancer cell lines possess HER2/neu receptors and that anti-HER2/neu affibody could be used for targeting HER2 receptors in these cancer cell lines.

FACS analysis showing binding of anti-HER2/neu affibody FITC to HER2/neu positive and negative cell lines. 70

Geometric Geometric Mean 20

0 Cells Alone SKOV3 SKOV3 TR MDAMB-231 MCF 7 ADR Figure 2: FACS analysis showing the binding of 5 µg/ml anti-HER2/neu affibody FITC to HER2/neu expressing SKOV3 and SKOV3 TR cell lines, and negative controls (MCF 7 and MDAMB-231 cell lines). Cells were incubated with anti-HER2/neu affibody at final concentration of 5µg/ml for 30 min at 370C. In cells alone, cells were incubated with 1% BSA.

Investigator: Prashant Raj Bhattarai

A B C

A B C 4.1

4.2

Figure 4: 4.1 Epifluorescent microscopy for the determination of cell association of anti-HER2/neu affibody FITC with SKOV3 TR ovarian cancer cell line 4.2 Affibody FITC association study in HER2/ne negative MDAMB-231 breast cancer cells at 40X magnification (A: Bright field, B: FITC, C: Hoechst Stain).

Figure 3 and 4 show confirmatory data that both SKOV3 and SKOV3 TR ovarian cancer cells had

HER2/neu receptors by epifluorescence microscopy. The intensity of fluorescence determined by computer planimetry showed that the concentration of HER2/neu receptors on SKOV3 TR cells

(Figure 4) is higher than that in SKOV3 Paclitaxel sensitive cells (Figure 3). No fluorescence associated with affibody-FITC was seen in HER2/neu negative MDAMB-231 cells confirming the specificity of affibody for HER2/neu receptors.

Investigator: Prashant Raj Bhattarai

4.2 Preparation and characterization of anti-HER2/neu affibody X anti-DTPA Fab bispecific complex

4.2.1 Immunoreactivity ELISA for anti-DTPA Fab:

Anti-DTPA Fab was generated by the papain digestion of the intact anti-DTPA antibody. Following the purification of the anti-DTPA Fab fragments from the protein A affinity column, ELISA was carried out to determine the immunoreactivity of these fragments. Figure 5 below shows that the anti-DTPA Fab fragments retained their immunoreactivites to bind DTPA even after the enzymatic digestion as compared to the intact antibodies.

Figure 5: ELISA for the immunoreactivity of bromoacetylated anti-DTPA Fab (blue square) compared with the standard unmodified anti-DTPA Fab (red circle).

4.2.2 TNBS Assay to determine the modification of anti-DTPA Fab:

Anti-DTPA Fab was modified with N-hydroxy succinimide ester of bromoacetic acid. The % bromoacetylation of the anti-DTPA Fab was assessed by TNBS assay and was determined to be

73.1% relative to the unmodified standard anti-DTPA Fab.

Investigator: Prashant Raj Bhattarai

Table 2: TNBS Assay to determine the percent modification of anti-DTPA Fab

4.2.3 Purification and Characterization of Bispecific Antibody complex: 6.1 A B C D 6.2

Multimeric complex Bispecific antibody complex Intact Anti-DTPA

Bispecific Antibody complex

Anti-DTPA FAb

Affibody

Multimeric complex

Affibody

Figure 6: Purification and Characterization of Bispecific antibody complex; 6.1) SDS-PAGE for the identification of Lane A: Intact Anti-DTPA antibody, Lane B: Anti-DTPA Fab after Protein A purification, Lane C: crude bispecific extract after purification from IMAC Column, and Lane D: bispecific antibody complex after purification from size exclusion Zorbax G-250 column; 6.2) Elution profile of crude bispecific reaction product from size exclusion Zorbax G-250 column eluted using 0.2M sodium phosphate buffer pH 7.4.

As seen in Figure 6.2, the elution profile of the crude extract obtained after passing through the

Zorbax GF-250 column resulted in the three peaks: 1) multimeric complex, 2) bispecific antibody complex, and 3) free unconjugated affibody. The bispecific antibody complex eluted around 8.67 minutes whereas the free affibody eluted at around 11.7 minutes. Further the SDS-PAGE analysis shown in Figure 6.1 confirmed the presence of all molecular species eluted from Zorbax GF-250 columns.

Investigator: Prashant Raj Bhattarai

4.2.4 In-Cell ELISA for demonstration of binding of anti-HER2/neu X anti DTPA

Fab bispecific antibody complex to SKOV3 and SKOV3 TR ovarian cancer cell lines:

0.5 Anti-DTPA Fab Anti-HER2/neu Affibody X Anti-DTPA Fab bispecific Affibody alone 0.45 0.4 0.35 0.3

nm 0.25 450 0.2 0.15 0.1

Absorbance 0.05 0 1 0.1 0.01 1 0.1 0.01 1 0.1 0.01 -0.05 SKOV3 SKOV3 TR MCF7 Serial dilution of anti-DTPA Fab, bispecific antibody and affibody alone (µg/ml)

Figure 7: In-Cell ELISA for demonstration of binding of anti-HER2/neu affibody X anti-DTPA Fab bispecific complex to HER2/neu positive SKOV3 and SKOV3 TR ovarian cancer cell lines. MCF7 breast cancer cell line, which does not express HER2/neu, is used as a negative control to demonstrate the specificity of bispecific antibody complex.

Bispecificity of anti-HER2/neu affibody X anti-DTPA Fab bispecific complex is demonstrated by the in-cell ELISA (Figure 7). Binding of anti-HER2/neu affibody arm of bispecific complex to

Her2 receptors of either SKOV3 or SKOV3 TR cell lines allowed the binding of GAM-HRP with the anti-DTPA Fab arm of the bispecific complex. Moreover, high binding was achieved using the bispecific antibody complex as compared to the non-specific background activity of anti-DTPA

Fab or affibody alone. Also, HER2/neu negative MCF7 was used as the negative control wherein there was no difference in binding between anti-DTPA antibody and affibody alone. Thus, this confirmed the specificity of bispecific antibody complex to HER2/neu positive cell lines.

Investigator: Prashant Raj Bhattarai

4.2.5 Characterization of biotinylated anti-DTPA bispecific complex

Figure 8: Determination for the presence of biotin on biotinylated anti-DTPA by ELISA (▲ =biotinylated anti-DTPA, and ■= unmodified anti-DTPA) 1:4000 dilution of Streptavidin-HRP was used. ELISA showed the presence of biotin in anti-DTPA as shown in the Figure 8. Unmodified anti-DTPA showed no binding to streptavidin-HRP as compared to biotinylated anti-

DTPA. Figure 8 confirms the presence of biotin on biotinylated-anti-DTPA bispecific antibody complex. Biotinylated BSA prepared in a similar way is used as a standard to determine the degree of biotinylation of antibody. Utilizing the TNBS assay, biotinylated

BSA is assessed to have 18 moles of biotin per mole of BSA. Figure 9 shows the comparison of binding of biotinylated anti-DTPA to Streptavidin relative to biotinylated

BSA. The binding curves were used to estimate the number of moles of biotin per mole of antibody, which was calculated to be 2 moles of biotin per mole of antibody. The difference in the maximum absorbance between Figure 8 and 9 is due to difference dilution of 1:4000 and 1:8000 of the secondary antibody, goat anti-murine streptavidin-HRP used. The 1:8000

Investigator: Prashant Raj Bhattarai dilution of the secondary antibody is used because absorbance at 630 nm for biotinylated-

BSA is above 1 OD unit read at 630 nm.

Figure 9: Quantitation of biotin on biotinylated anti-DTPA by comparison to biotinylated BSA standard (♦ = biotinylated BSA, and ▲ = biotinylated anti-DTPA) 1:8000 dilution of Streptavidin-HRP was used.

4.2.6 Specificity of biotinylated anti-DTPA complex for tumor cells by In-Cell ELISA

In vitro cell binding assay showed that SKOV3, 4T1, MCF7, BT20 and MCF7 ADR cells contained different concentrations of biotin receptors expressed on the cell surfaces.

SKOV3 human ovarian cancer and 4T1 mouse mammary cancer cells incubated with 10

µg/ml of biotinylated anti-DTPA bispecific complex showed significantly greater binding to cancer cells than to control H9C2 rat embryonic cardiomyocytes (Figure 10).

Biotinylated anti-DTPA bispecific complex also bound to MCF7 and MCF-ADR human breast cancer cells but at a lower concentration to SKOV3 and 4T1 cells.

Investigator: Prashant Raj Bhattarai

In-Cell ELISA for demonstration of binding of biotinylated anti- DTPA(10ug/ml) to biotin receptors expressed in various cell lines

0.8 nm 0.7 450 0.6 0.5 0.4 0.3

Absorbance at 0.2 0.1 0 SKOV3 4T1 MCF7 MCF7 ADR BT 20 H9C2 Cell lines

Figure 10: In-cell ELISA for demonstration of binding of biotinylated anti-DTPA to biotin receptors expressed in various cell lines

Investigator: Prashant Raj Bhattarai

4.3 Characterization of various polymer prodrug conjugates (PPDCs): a) D-Ptxl-

PGA, b) D-Dox-PGA and c) D-Mph-PGA

Purification and Characterization of D-Dox-PGA complex:

Sephadex G25 size exclusion chromatography was used for the separation of the D-Dox-PGA complex from free Dox. Blue dextran was first passed through the column to determine the void volume. The length and width of the column is approximately 16 x 1 cm. Figure 11 shows the elution profile of the blue dextran (blue square) and D-Dox-PGA (red triangle). D-Dox-PGA is eluted in the void volume due to its high molecular weight compared to free Dox. Fraction number

17-21 were pooled and were extensively dialyzed against 4L 0.1M PBS (pH7.4) buffer overnight.

Standard curve of Dox was generated using the serial dilution of known concentration of free Dox

(Table 3). The concentration of Dox in the D-Dox-PGA complex was then extrapolated using the standard curve (Figure 12). The concentration of doxorubicin in D-Dox-PGA was calculated to be approximately 438.98 µg/ml.

2.5

1.5 Blue Dextran (OD 280) D-DOX-PGA (OD 490)

1 Absorbance 490nm 0.5

0 0 20 40 60 80 100 Fraction Number Figure 11: Elution profile of Blue Dextran (blue) and D-Dox-PGA superimposed together from Sephadex G25 column (1 X 35 cm) chromatography

Investigator: Prashant Raj Bhattarai

Table 3: Determination of concentration of Dox in D-Dox-PGA complex

Doxorubicin concentration Mean Absorbance (µg/ml) 490nm 125 0.565666667 62.5 0.307666667 31.25 0.166333333 15.625 0.091666667 7.8125 0.049 3.90625 0.027666667 Standard 1.953125 0.017 0.9765625 0.008666667 0.48828125 0.005666667 0.244140625 0.006 0.122070313 0.003333333 0.061035156 0.003 0.030517578 0.001333333 0.015258789 0.001333333 0 0 Unknown D-DOX-PGA sample 1:10 dilution 0.209333333

Figure 12: Standard curve for the determination of concentration of Dox in D-Dox-PGA complex

Investigator: Prashant Raj Bhattarai

4.3.1 Purification and Characterization of D-Mph-PGA complex:

D-Mph-PGA was separated from the free melphalan by extensively dialyzing the conjugate against

0.1 M PBS pH 7.4. The absorbance of the conjugate was taken before and after dialysis at 260 nm.

Standard curve for the melphalan (Figure 13) was then used to calculate the concentration of the melphalan in the polymer conjugate complex. The concentration of melphalan after dialysis was calculated to be 350 µg/ml relative to 355 µg/ml before dialysis, indicating almost 100% incorporation of melphalan on the polymers.

Melphalan Standard Curve y = 0.0584x + 0.0497 3.5 R² = 0.9971 3

nm 2.5

260 2

1.5

1 Absorbance 0.5

0 0 10 20 30 40 50 60 Concentration (µg/ml)

Figure 13: Standard curve for the determination of melphalan concentration in D-Mph-PGA complex

4.3.2 Purification and Characterization of D-Ptxl-PGA Conjugate:

Thin Layer chromatography was carried out for the overnight reaction mixture of PGA and paclitaxel to analyze the efficiency of the conjugation reaction. Figure 14 shows the TLC results on silica TLC plates developed with CHCl3: methanol (10:1). Samples were spotted on the TLC plate and run at room temperature following which the Rf values were calculated. The Rf value of

Ptxl was 0.65 compared to the Ptxl-PGA (Rf = 0.07). The TLC assessment shows that nearly 100% of paclitaxel was conjugated to PGA. Ptxl-PGA complex was then precipitated and DTPA was conjugated to the complex to obtain D-Ptxl-PGA complex. Standard curve of Ptxl (Table 4 and

Investigator: Prashant Raj Bhattarai

Figure 15) was used to determine the concentration of paclitaxel in the D-Ptxl-PGA complex. The concentration of Ptxl in the conjugate was calculated to be 475 µg/ml. Moles Ptxl per mole of the polymer in the conjugate was determine to be 20:1.

Free Ptxl

Ptxl-PGA PGA

Figure 14: Thin Layer Chromatography determination of conjugation of Ptxl to PGA

Table 4: Determination of concentration of paclitaxel in D-Ptxl-PGA complex

Absorbance Concentration (µg/ml) 227nm 20 0.511 Standards 15 0.388 10 0.262 5 0.133 2.5 0.049 Unknown D-Ptxl-PGA Sample 1:1000 dilution 0.475

Investigator: Prashant Raj Bhattarai

Paclitaxel Standard Curve y = 0.0261x - 0.0052 R² = 0.9982 0.6

0.5 nm

0.4 227 0.3

0.2

Absorbance Absorbance 0.1

0 0 5 10 15 20 25 Concentration (µg/ml)

Figure 15: Standard Curve for the determination of concentration of Ptxl in D-Ptx-PGA complex

Stability study of D-Ptxl-PGA at different buffer systems:

Paclitaxel stability study at different buffer system 100

% % released drug 20

0 0 10 20 30 40 50 60 70 80

-20 Time (hrs)

Figure 16: Release profile of Paclitaxel from D-Ptxl-PGA in 0.1M PBS pH 7.4 (blue triangle); and 0.1M sodium acetate buffer pH4 (red diamond)

Investigator: Prashant Raj Bhattarai

Figure 16 shows the release profile of paclitaxel in pH 7.4 and 4.0 buffers. D-Ptxl-PGA is stable in

0.1M PBS pH 7.4 buffer but 80% of Ptxl on D-Ptxl-PGA dissociated in the acidic buffer (0.1M sodium acetate pH 4) in less than 10 h. Only approximately 6.2% of the Ptxl was released from D-

Ptxl-PGA after 72 h in pH 7.4 0.1 M PBS buffer, whereas, more than 50% of Ptxl is released within

1 h. in 0.1 M sodium acetate buffer. The release profiles of D-Ptxl-PGA showed that in physiological pH there is minimal dissociation of Ptxl from the polymer conjugates but at pH 5 (pH in the endolysosomes), Ptxl release is dramatically accelerated. Similar release profile was observed for D-Dox-PGA conjugate12.

4.3.3 ELISA for the incorporation of DTPA in different Polymer prodrug conjugates:

Indirect anti-DTPA ELISAs used to determine the presence of DTPA on PGA is shown in Figures

17 and 18. D-BSA generated in the lab was used as positive control (Figure 18). Aliquots of D-

Dox-PGA, D-Mph-PGA and D-Ptxl-PGA showed equivalent binding to anti-DTPA antibody concentration at 10 μg/ml (Figure 17). Since the PGA molecule has only one amino group, there can only be one mole of DTPA for one mole of PGA. The presence of many amino groups allowed more DTPAs to be conjugated to BSA (Figure 18). This is confirmed by the result in Figure 18 where the DTPA-polymer conjugates was about 1% of maximal binding of D-BSA with anti-DTPA antibody.

Investigator: Prashant Raj Bhattarai

Figure 17: Determination of presence of DTPA on Polymer drug conjugates by ELISA (red circle: D-Dox- PGA, blue diamond: D-Mph-PGA, and orange triangle: D-Ptxl-PGA)

Figure 18: Superimposition of standard positive control D-BSA to the Polymer drug conjugates ELISA (brown cross: DTPA-BSA, red circle: D-Dox-PGA, blue diamond: D-Mph-PGA, and orange triangle: D- Ptxl-PGA)

Investigator: Prashant Raj Bhattarai

4.3.4 Zeta Potential values of various Polymer prodrug Conjugates:

Table 5 shows the zeta potentials of various polymer prodrug conjugates. All polymers showed negative zeta potentials.

Table 5: Zeta potential values of Polymer compared with various Polymer drug conjugates

Polymer Drug Conjugate Zeta Potential

PGA -21.425 ± 2.34

D-Dox-PGA -17.25 ± 1.25

D-Mph-PGA -16.37 ± 0.98

D-Ptxl-PGA -15.754 ± 2.13

Investigator: Prashant Raj Bhattarai

4.4 Evaluate in vitro cytotoxicity and drug resistance reversal in SKOV3 and SKOV3

TR ovarian cancer cell lines by delivery of PPDCs by pretargeting approach using anti-HER2/neu affibody X anti-DTPA Fab bispecific antibody complex.

SKOV3 and SKOV3 TR cells were treated with different formulations at various doses of polymer alone and polymer drug conjugates without pretargeting with bispecific antibody complex. PGA alone, D-Dox-PGA, D-Mph-PGA and D-Ptxl-PGA treatments showed very little inherent toxicity for both the cell lines as shown in Figure 19 A and B below. This confirms that the Polymer-drug conjugates are in the prodrug form with no inherent toxicity.

Investigator: Prashant Raj Bhattarai

A Cytotoxicity at 48 hrs SKOV3

120 100 80 60 40

% % Cell Viability 20 0

Drug concentration (µg/ml)

D-Mph-PGA D-Ptx-PGA D-Dox-PGA PGA

B Cytotoxicity at 48 hrs in SKOV3 TR 120 100 80 60 40

% % Cell Viability 20 0 7.5 3.75 1.875 0.9375 0.46875 0.234375 0.1171875 Drug Concentration (µg/ml)

D-Mph-PGA D-Ptx-PGA D-Dox-PGA PGA

Investigator: Prashant Raj Bhattarai

4.4.1 Western Blot analysis for Pgp expression:

Figure 20 shows the Western Blot analysis of Ptxl and Dox resistant SKOV3 TR and MCF7 ADR cells respectively. Each cell line expresses significantly greater concentration of Pgp (~170kDa)

(Figure 20 Lanes B and C). Sensitive variants SKOV3 and MCF7 showed minimal Pgp expression

(Figure 20 A and D). The internal standard β-actin shows that equivalent concentrations of protein extract are loaded in each electrophoretic lane.

Investigator: Prashant Raj Bhattarai

4.4.2 Cell Viability studies using single polymer prodrug conjugates:

To determine the extent of reversal of multidrug resistance SKOV3 and SKOV3 TR cells were treated with Ptxl alone, non-targeted D-Ptxl-PGA and targeted D-Ptxl-PGA after pre-incubation of

1h with 40 µg/ml of bispecific complex at different concentration of paclitaxel for 48 hours. After incubation cytotoxicity analysis was carried out to determine the IC50 values for various preparations of paclitaxel.

Cytotoxicity at 48h SKOV3 sensitive

120

100

% % Cell viability 40

0 0 1 2 3 4 5 6 7 8 Ptxl concentration(μg/ml)

Non-targeted D-Ptxl-PGA Ptxl 40 µg/ml of bispecific complex + D-Ptxl-PGA

Figure 21 shows that free Ptxl is highly effective in inducing cell death in SKOV3 sensitive cells and no advantage was gained in cytotoxicity using pretargeted delivery of D-Ptxl-PGA. Even at high bispecific antibody concentration of 40 µg/ml there was no significant difference in tumor toxicity between free Ptxl, and targeted D-Ptxl-PGA.

Investigator: Prashant Raj Bhattarai

Cytotoxicity at 48h SKOV3 TR

120

100

% % Cell viability 40

0 0 1 2 3 4 5 6 7 8 Ptxl concentration(μg/ml)

Non-targeted D-Ptxl-PGA Ptxl 40 µg/ml of bispecific complex + D-Ptxl-PGA

Figure 22: Cytotoxicity of targeted D-Ptxl-PGA after pretargeting with 40 μg/ml of bispecific antibody complex relative to free Ptxl and non-targeted D-Ptxl-PGA in SKOV3 TR cells. Data shown are representative of 2 independent experiments performed in triplicates, mean ± SD.

Figure 22 shows that resistant variant SKOV3 TR is more susceptible to targeted D-Ptxl-PGA treatments as seen by significantly higher cytotoxicity (lower viability) as compared to free Ptxl treatments (p<0.01). The pretargeting approach increases the delivery of D-Ptxl-PGA resulting in an increase in cell death.

IC50 values of Ptxl in SKOV3 TR cells was 0.936 µg/ml which was approximately 10.5 times higher relative to 0.089 µg/ml in SKOV3 sensitive cells. However, the IC50 of SKOV3 and SKOV3 TR cells pre-incubated with 40 µg/ml of the bispecific antibody complex and targeted with D-Ptxl-

PGA after was 0.069 and 0.172 µg/ml for SKOV3 and SKOV3 TR cell lines respectively. Thus,

IC50 of Ptxl in the pretargeted treatment of SKOV3 TR cells (0.172 µg/ml) was significantly less than that with free Ptxl alone (0.936 µg/ml, p< 0.05) by 5.6 times.

Investigator: Prashant Raj Bhattarai

4.4.3 Endocytosis Inhibition study:

Treatment of SKOV3 TR cell lines with 10 μg/ml of chlorpromazine for 30 minutes before pre- incubation with bispecific antibody complex led to the reversal of cytotoxicity of targeted D-Ptxl-

PGA as observed in Figure 23. Chlorpromazine is a known inhibitor of endocytosis. Therefore, the mechanism of intracellular entry of the polymer prodrug conjugates pretargeted with bispecific antibody complexes is via endocytosis after the bispecific antibody complex binds the HER2/neu receptors and the PPDCs. Free drugs enter cells via diffusion so no effect on tumor cytotoxicity of free Ptxl is seen after pre-treatment with chlorpromazine (blue squares Figure 23). Intracellular internalization of the PPDCs in our pretargeting approach via endocytosis may be why reduction in cytotoxicity by Pgp efflux pumps in drug resistant tumors cells can be overcome.

Cytotoxicity at 48h SKOV3 TR 120

100

40 % % Cell viability

0 0 1 2 3 4 5 6 7 8 Ptxl concentration(μg/ml) Non-targeted D-Ptxl-PGA Ptxl 10 μg/ml Chlorpromazine+20 μg/ml of bispecific antibody complex+D-Ptxl-PGA Figure 23: Cytotoxicity of D-Ptxl-PGA targeted with 20 μg/ml of bispecific complex compared to free paclitaxel and non-targeted D-Ptxl-PGA in SKOV3 TR cell line. For targeted approach, cells were treated with 10 µg/ml of chlorpromazine for 30mins before pretargeting step Data shown are representative of 2 independent experiments performed in triplicates, mean ± SD.

Investigator: Prashant Raj Bhattarai

4.4.4 Targeted Combination drug treatment for cancer therapy:

Various clinical trials have used combination drug treatment involving paclitaxel and doxorubicin for refractive drug resistant cancer therapy. Doxorubicin inhibits DNA repair and promotes formation of free radicals whereas paclitaxel stabilizes the mitotic spindle thus arresting the cell cycle26. However, this treatment is limited by the cardiotoxic effect of Dox and other drug- associated side effects. Khaw et al12 showed previously that PGA doxorubicin conjugates were able to overcome cardiotoxic effects even at high equivalent doxorubicin concentration. Melphalan belongs to the nitrogen mustard class of chemotherapeutic agent that exerts its action by alkylation of DNA. Since the mechanism of action of Paclitaxel, Doxorubicin and melphalan is different, it is proposed that synergistic cancer killing by combination targeted delivery of 2 PPDCs (D-Ptxl-PGA and D-Dox-PGA) and 3 PPDCs (D-Ptxl-PGA, D-Dox-PGA and D-Mph-PGA) after pretargeting with bispecific antibody complex and subsequent complete reversal of multidrug resistance, could be achieved.

Investigator: Prashant Raj Bhattarai

A Cytotoxicity of two combined PPDCs vs single PPDC at 48 h in SKOV3 TR pretargeted with 20 µg/ml of bispecific antibody complex

100

90 *

80 * 70 * 60 *

50 * * %Cell %Cell Viability 40 * 30 20 10 0 7.5 3.75 1.875 0.9375 0.46875 0.234375 0.1171875 Drug Concentration (ug/ml) 20μg/ml of bispecific antibody complex+D-Dox-PGA+D-Ptxl-PGA 20μg/ml of bispecific antibody complex+D-Dox-PGA 20μg/ml of bispecific antibody complex+D-Ptxl-PGA Dox Dox+Ptxl

Investigator: Prashant Raj Bhattarai

B Cytotoxicity of two combined PPDCs vs single PPDC at 48 h in SKOV3 TR pretargeted with 40 µg/ml of bispecific antibody complex

100 90

80 70

% % Cell Viability 40

20 10

0 7.5 3.75 1.875 0.9375 0.46875 0.234375 0.1171875 Drug concentration (ug/ml)

40μg/ml of bispecific antibody complex+D-Dox-PGA+D-Ptxl-PGA 40μg/ml of bispecific antibody complex+D-Dox-PGA 20μg/ml of bispecific antibody complex+D-Ptxl-PGA Figure 24: Cell viability of SKOV3 TR cells after 48 h incubation with targeted combination of 2 PPDCs (D-Ptxl-PGA+D-Dox-PGA) after pretargeted with two different bispecific antibody complex concentration A. 20 µg/ml of pretargeted bispecific antibody complex; B. 40 µg/ml of pretargeted bispecific antibody complex. Data shown are representative of 3 individual experiments carried out in triplicates, Mean ± SD *P<0.05.

Figure 24 A and 24 B show the in vitro cytotoxicity of pretargeted combination therapy using D-

Ptxl-PGA and D-Dox-PGA compared to individually targeted polymer drug conjugates in SKOV3

TR cells. Cells were pre-incubated with two different concentration of bispecific antibody complex at 20 (Figure 24A) and 40 µg/ml (Figure 24B). Both concentrations of pretargeting bispecific antibody complexes resulted in significant cell killing observed in the 2 drug combination therapy relative to combination of free drugs (Dox + Ptxl) and individual D-Dox-PGA or D-Ptxl-PGA treatments (p<0.05) up to the highest drug concentration used in the study. However, tumor cell cytotoxicity was significant greater in SKOV3 TR cells treated with higher concentration of the pretargeting bispecific antibody complex (Figure 24 B). There were significant differences at all

Investigator: Prashant Raj Bhattarai concentrations of the preparations of polymer drug conjugates except at 7.5 and 0.234375 µg/ml relative to cells targeted with D-Dox-PGA and pretargeted with 20 g/ml of the bispecific antibody complex. With increase in concentration of bispecific antibody used, the total cytotoxicity in

SKOV3 TR was significantly greater than individual PPDC treatment at all drug concentrations.

The results show that more efficient tumor cell killing in drug-resistant ovarian cancer cells can be achieved by targeted delivery of 2 different drugs in the prodrug forms and optimal cytotoxicity may be achieved by modulation of both the pretargeting bispecific antibody complex concentration and the drug concentration.

A Cytotoxicity of 3 combined PPDCs vs 2 PPDCs at 48h in SKOV3 TR cell line after pretargeting with 20 µg/ml of bispecific antibody complex 100 90 80 70 60 50 40

%Cell %Cell Viability 30 20 10 0 7.5 3.75 1.875 0.9375 0.46875 0.234375 0.1171875 Drug Concentration ug/ml 20μg/ml of bispecific antibody complex+D-Dox-PGA+D-Ptxl-PGA+D-Mph-PGA 20μg/ml of bispecific antibody complex+D-Dox-PGA+D-Ptxl-PGA

Investigator: Prashant Raj Bhattarai

B Cytotoxicity of 3 combined PPDCs vs 2 PPDCs at 48h in SKOV3 TR cell line after pretargeting with 40 µg/ml of bispecific antibody complex 100 90 80 70 60 50 40

% Cell Viability Cell % 30 20 10 0 7.5 3.75 1.875 0.9375 0.46875 0.234375 0.1171875 Drug concentration (ug/ml) 40μg/ml of bispecific antibody complex+D-Dox-PGA+D-Ptxl-PGA+D-Mph-PGA 40μg/ml of bispecific antibody complex+D-Dox-PGA+D-Ptxl-PGA Figure 25: Cell viability of SKOV3 TR cells after 48 h incubation with targeted combination of 3 PPDCs (D-Ptxl-PGA+D-Dox-PGA+D-Mph-PGA) versus targeted combination of 2 PPDCs (D-Ptxl-PGA and D- Dox-PGA) after preincubated with two different bispecific antibody complex concentration A. 20 µg/ml of pretargeted bispecific antibody complex; B. 40 µg/ml of pretargeted bispecific antibody complex. Data shown are representative of 3 individual experiments carried out in triplicates, Mean ± SD.

Figure 25 A shows significantly greater cell killing at low drug concentration of 0.2343 and 0.11789

µg/ml in targeted three drug combination therapy as compared to the two drugs combination at the concentration of pretargeted bispecific antibody complex of 20 µg/ml. But at higher drug concentration of 3.75, 1.875, and 0.9375 µg/ml, targeted 2 drug combination therapy was much better as compared to the 3 drug combination therapy. This may be attributed to high concentration of PPDCs, which may lead to competition for the binding site of the capture antibody or to steric hindrance. This may lead to decrease in the delivery of Ptxl, Dox or Mph conjugated polymer drug conjugates due to competition between the 3 different polymer drug conjugates for a limited concentration of the bispecific antibody complexes on cancer cells leading to a decreased internalization of the 2 most cytotoxic drug conjugates.

Investigator: Prashant Raj Bhattarai

Furthermore, when the concentration of the pretargeting bispecific antibody complex was increased to 40 µg/ml there was not significant change in the cell killing between targeted combination of two and three drug therapy at low drug concentrations but at the highest conjugated drug concentrations, competition for binding to the pretargeted bispecific antibody complexes showed slight reduction when 3 drugs polymer-drug conjugates were used (0.9375 to 7.5 g/ml drug concentrations, Fig. 25 B).

Investigator: Prashant Raj Bhattarai

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 polymer prodrug conjugates by pretargeting approach using biotinylated anti-DTPA bispecific complex.

MCF7 sensitive cell lines pretargeted with biotinylated anti-DTPA bispecific complex for the delivery of D-Dox-PGA showed no significant increase in cytotoxicity as compared to free Dox treatment. Both had very similar IC50 values of 0.0261 and 0.00875 µg/ml respectively (Figure 26) and Dox was very effective in inducing cell death in MCF sensitive cells.

Cytotoxicity at 48 hr MCF7 sensitive

120

100

% % cell viability 40

0 0 2 4 6 8 Dox concentration (µg/ml) Free Dox Non-targeted D-Dox-PGA 20ug/ml of biotinylated anti-DTPA + D-Dox-PGA

Figure 26: Cytotoxicity of targeted D-Dox-PGA after pretargeted with 20 μg/ml of biotinylated anti-DTPA bispecific antibody complex compared to free Dox in MCF7 cells. Data shown are representative of 3 individual experiments carried out in triplicates, Mean ± SD.

Investigator: Prashant Raj Bhattarai

Cytotoxicity at 48 hr MCF7 ADR 120

100

% % Cell viability 20

0 0 5 10 15 20 25 Dox Concentration (µg/ml) Free DOX Non-Targeted D-DOX-PGA 20µg/ml of biotinylated anti-DTPA + D-Dox-PGA 40µg/ml of biotinylated anti-DTPA + D-Dox-PGA 60µg/ml of biotinylated anti-DTPA+D-Dox-PGA

Figure 27: Cytotoxicity of targeted D-Dox-PGA after pretargeted with increasing concentration of biotinylated anti-DTPA bispecific antibody complex compared to free Dox in MCF7 ADR cells. Data shown are representative of 3 individual experiments carried out in triplicates, Mean ± SD.

However, in MCF7 ADR cells, the pretargeted approach induced significantly greater cell death than treatment with free Dox (p<0.001) (Figure 27). By increasing the pretargeting bispecific concentration from 20 μg/ml to 40 μg/ml tumor toxicity increased. Furthermore, by increasing the concentration of the bispecific antibody concentration to 60 μg/ml did not enhance the cytotoxicity.

This may be due to saturation of biotin receptors at about 40 μg/ml bispecific antibody concentration.

Investigator: Prashant Raj Bhattarai

Table 6: IC50 Values for MCF7 Sensitive and resistant cells Cell line Treatment IC50 (µg/ml) Dox Alone 0.00875±0.00157 MCF7 Sensitive 20 µg/ml Biotinylated anti-DTPA + D- Dox-PGA 0.0261±0.0094 Dox Alone 11.9±0.05743 10 µg/ml Biotinylated anti-DTPA + D- Dox-PGA 3.499±0.454 MCF7 ADR (Resistant) 20 µg/ml Biotinylated anti-DTPA + D- Dox-PGA 1.392±0.652 40 µg/ml Biotinylated anti-DTPA + D- Dox-PGA 0.442±0.0892 Table 6 shows that in MCF7 ADR resistant cells IC50 of D-Dox-PGA (0.442 µg/ml) pretargeted with 40 µg/ml of bispecific antibody complex is 27 times less than that of free Dox treatment (IC50

= 11.9 µg/ml).

4.5.1 Combination therapy:

MCF 7 MDR cytotoxicity studies 48hrs 100 90 80 70 60 50 40 30

% Cell Viability Cell % 20 10 0 23.2 11.6 5.8 2.9 1.45 0.725 0.3625 Equvalent drug concentration (µ g/ml) Free Dox Free Paclitaxel 40µg/ml Biotinylated Anti-DTPA+ D-Dox-PGA 40µg/ml Biotinylated Anti-DTPA+ D-Ptxl-PGA 40µg/ml Biotinylated Anti-DTPA+(D-Dox-PGA+D-Ptxl-PGA)

Figure 28: Comparison of % cell viability of MCF7 ADR following the administration of free drugs, individually targeted D-Dox-PGA or D-Ptxl-PGA and combination therapy (D-Dox-PGA+D-Ptxl-PGA). Data shown are representative of 3 individual experiments carried out in triplicates, Mean ± SD.

Investigator: Prashant Raj Bhattarai

Combination therapy involving targeted delivery of D-Dox-PGA and D-Ptxl-PGA showed significantly higher cytotoxic effects compared to the free Dox or single targeted polymer drug conjugates (Figure 28). Combination therapy resulted in significantly higher cell killing as compared to free Dox (p<0.01) at all drug concentrations. Most interesting is the significantly high cytotoxicity seen with combination therapy at low total drug concentration of 0.725 and 0.3625

μg/ml than in higher drug concentrations compared with treatment groups targeted with either D-

Dox-PGA or D-Ptxl-PGA alone. Comparing these individual treatments of D-Dox-PGA and D-

Ptxl-PGA to the combination treatment shows more than additive effects. This provides evidence for the potential utility of co-delivery of these agents.

4.5.2 Fluorescence Microscopy Studies:

Figure 29 shows the increase in internalization of D-Dox-PGA in Dox resistant MCF7 ADR cells after pretargeting with biotinylated anti-DTPA antibody bispecific complex (20 μg/ml) from 5 min to 6 h of incubation with media containing D-Dox-PGA. Increase in intracellular fluorescence with increasing incubation time is observed. However, non-targeted D-Dox-PGA accumulation over the same period of incubation shows significantly less fluorescence indicative of minimal non-specific sequestration of D-Dox-PGA in Dox resistant MCF7 ADR cells (Figure 30). Dox treatment alone in MCF7 ADR resistant cells (Figure 31), leads to higher intracellular localization of Dox than in cells treated with non-targeted D-Dox-PGA. However, intracellular accumulation of Dox in these cells is less than in MCF7 ADR cells pretargeted and targeted with D-Dox-PGA (Figure 29). Figure

32 A&B represents the quantitation of fluorescent intensity reflective of Dox concentration in

MCF7 ADR cells and the nuclei of these cells. A major difference in Dox concentration was observed in the total cell assessment (Figure 32A and 34A) and in the nuclei (Figure 32B and 34B).

With free Dox treatment, most of the fluorescence signal was confined in cytoplasm. However, by utilizing pretargeted approach MCF7 ADR cells treated with D-Dox-PGA, the nuclear regions apparently showed much higher fluorescent intensity (Figure 34B).

Investigator: Prashant Raj Bhattarai

Figure 29: Epifluorescent micrographs of MCF7 ADR cell after 1 h pretargeting with biotinylated anti- DTPA bispecific antibody followed by incubation with D-Dox-PGA for: A. 5 min, B. 10 min, C. 15 min, D. 30 min, E. 60 min, F. 120 min, G. 300 min, and H. 360 min (a: Bright field, b: Dox fluorescence, c: Hoechst stain, d: superposition of b and c). Magnification = 40X.

Investigator: Prashant Raj Bhattarai

Figure 30: Epifluorescent micrographs of MCF7 ADR after incubation with non-targeted D-Dox-PGA: A. 5 min, B. 10 min, C. 15 min, D. 30 min, E. 60 min, F. 120 min, G. 300 min, and H. 360 min (a: Bright field, b: Dox fluorescence, c: Hoechst stain, d: superposition of images b and c). Magnification = 40X.

Investigator: Prashant Raj Bhattarai

Figure 31: Epifluorescent micrographs of MCF7 ADR cells after incubation with Dox for: A. 5 min, B. 10 min, C. 15 min, D. 30 min, E. 60 min, F. 120 min, G. 300 min, and H. 360 min. (a: Bright field, b: Dox fluorescence, c: Hoechst stain, d: superposition of b and c). Magnification = 40X.

Investigator: Prashant Raj Bhattarai

Fluorescence Intensity in the MCF7 ADR Cells 1600000

1400000

1200000 y = 213492ln(x) - 178747 R² = 0.9318 1000000

800000 y = 65660ln(x) + 226552 CTCF R² = 0.6441 600000

400000 y = 42987ln(x) - 58086 R² = 0.6661 200000

0 1 51 101 151 201 251 301 351 401 Time (minutes) 20 µg/ml Biotinylated anti-DTPA + D-Dox-PGA Dox D-Dox-PGA Alone

Investigator: Prashant Raj Bhattarai

Fluorescence Intensity in the MCF7 ADR Nuclei

600000

y = 84364ln(x) - 54087 500000 R² = 0.876

400000 y = 26516ln(x) + 181318 R² = 0.3792

300000 CTCF

200000 y = 25917ln(x) - 22318 R² = 0.6428

100000

0 1 51 101 151 201 251 301 351 401 Time (Minutes) 20 µg/ml Biotinylated anti-DTPA + D-Dox-PGA Dox D-Dox-PGA Alone Figure 32: A. Corrected total cell fluorescence (CTCF) values of MCF7 ADR cells after various treatments, B. CTCF values of the nuclei of MCF7 ADR cells after various treatments.

4.5.3 Dox Efflux studies:

MCF7 ADR cells were pretargeted with 40 µg/ml of bispecific antibody complex and targeted with

20 µg/ml of D-Dox-PGA, followed by washing and incubation for 4 h in drug free media. Figure

33 shows the fluorescence micrographs showing the role of the efflux pumps Pgp in regulating the intracellular concentration of Dox. The Dox fluorescence intensities in various treatment groups are compared. Strong fluorescence intensity was detected in MCF7 ADR cells pretargeted with bispecific antibody complex. The intensity of Dox fluorescence did not decrease substantially even after 4 h of the potential action of the efflux pumps. In comparison, MCF7 ADR cells treated with free Dox showed significant fluorescent intensity after 1 h incubation but the intensity faded dramatically and became minimally detectable after the 4 h drug efflux period. Fluorescence

Investigator: Prashant Raj Bhattarai appeared more granular in pretargeted treatment whereas fluorescence in free Dox treated cells was more evenly distributed. Furthermore similar localization of Dox and Hoechst stain seen in cells with pretargeted treatment confirms the nuclear delivery and localization of Dox by this approach.

Figure 33: Fluorescent Images of MCF7 ADR cells after: A. 1 h incubation with biotinylated anti-DTPA, followed by treatment with D-Dox-PGA for 1 h, followed by washing of the cells and incubation for 4 h in D-Dox-PGA free medium. B. Treatment with Dox for 1 h, followed by washing and incubation for 4 h in Dox free medium. C. Treatment with D-Dox-PGA alone for 1 h and the cells are treated as in A. (a: Bright field, b: Dox fluorescence, c: Hoechst stain, d: superposition of images b and c). Magnification = 40X.

Investigator: Prashant Raj Bhattarai

Comparison of 1 hr Treatment of MCF7 ADR Nuclei after Washing at 1 hr 500000 ** 450000 400000 350000 *** 300000 250000

CTCF 200000 150000 100000 50000 0 Biotinylated anti- Dox at 1 hr Biotinylated anti- Dox with Wash at DTPA DTPA 1 hr pretargeted + pretargeted + Targeted D-Dox- Targeteted D- PGA at 1 h Dox-PGA with wash at 1 hr

Comparison of 1 hr Treatment of MCF7 ADR Cells after Washing at 1 hr 1200000

1000000 *

800000 **

600000 CTCF 400000

200000

0 Biotinylated anti- Dox at 1 hr Biotinylated anti- Dox with Wash at DTPA DTPA 1 hr pretargeted + pretargeted + Targeted D-Dox- Targeteted D- PGA at 1 h Dox-PGA with wash at 1 h

Figure 34: A. CTCF values for MCF7 ADR cells after treatment with either Dox or pretargeted D-Dox-PGA for 1 h followed by washing and replacing with Dox free medium compared to 1 h treatment without the washing step P<0.01, *** P<0.0001). B. CTCF values for the nuclei of MCF7 ADR cells after treatment with either Dox or pretargeted D-Dox-PGA for 1 h followed by washing and replacing with Dox free medium compared to 1 h treatment without the washing step (*, P<0.05, ** P<0.01).

Investigator: Prashant Raj Bhattarai

Figure 35: A. Micrographs of MCF7-ADR cells pretreated with biotinylated anti-DTPA-FITC bispecific antibody complex for 1 h and then incubation with 20 μg/ml of D-Dox-PGA for 1 h relative to B. Non-treated control cells. a) Bright field, b) Hoechst stain, c) FITC green fluorescence, d) Dox fluorescence, e) superimposition of b) and c), and f) superimposition of b) and d) to demonstrate nuclear localization of the released Dox. B) Same sequence of the micrographs as above in untreated control MDF7 ADR cells.

To show that the biotinylated anti-DTPA bispecific antibody complex and the D-Dox-PGA are both internalized intracellularly, but only Dox released from the digestion of D-Dox-PGA is sequestered in the nuclei, cells pretreated with biotinylated anti-DTPA-FITC and treated with D-

Dox-PGA are examined for the green and red fluorescence by epifluorescent microscopy and compared to the corresponding nuclear stain. Figure 35 shows that the green fluorescence remained in the cytoplasm (Figure 35A, b, c and f), whereas red fluorescence of Dox was seen to sequester primarily in the nuclei (Figure 10A, b, d and e). Figure 35 f. is also shown enlarged in the right panel. Figure 35A, g represents superimposition of Figure 35 A c and d and figure 35A, h represents superimposition of Figure 35A, b, c and d. No specific fluorescence is seen in the untreated control cells (Figure 35B).

Investigator: Prashant Raj Bhattarai

Figure 36: Fluorescent Images of MCF7 ADR cells after: A. 30 min pre-treatment with 10 μg/mL of Chlorpromazine, followed by pretargeting with biotinylated anti-DTPA antibody for 1 h and 1 h incubation with 20 μg/ml of D-Dox-PGA. B. 30 min pre-treatment with 10 μg/mL of Chlorpromazine and then 1 h incubation with D-Dox-PGA. C. 30 min pre-treatment with 10 μg/mL of Chlorpromazine followed by 1 h incubation with Dox. D. Cells alone. (a: Bright field, b: Dox fluorescence, c: Hoechst stain, d: superposition of images b and c). Magnification = 40X.

When the MCF7 ADR cells are pre-incubated with Chlorpromazine, an inhibitor of endocytosis, no D-Dox-PGA is internalized in the cells pretargeted with Biotinylated anti-DTPA bispecific antibody complex (Figure 36A), similar to that of MCF ADR cells treated with D-Dox-PGA without pretargeting antibody (Figure 36B) or that of the untreated control (Figure 36D). However, internalization of free Dox was not affected by pre-incubation with Chlorpromazine (Figure 36C).

Investigator: Prashant Raj Bhattarai

4.6 3D Spheroid cell Culture:

Figure 37 shows MCF7 ADR spheroids at day 3 and 5, grown using non-adhesive liquid overlay method with 10,000 cells/well and centrifugation at 1500 g for 15 minutes. The size of spheroids at day 5 was determined to be 542 µm utilizing the Spot advanced software. Non-liquid overlay technique produced consistent spheroids with respect to shape and size. Day 5 spheroids were chosen for the cell viability studies.

Figure 37: MCF7 ADR spheroids formed using non-adhesive liquid overlay method shown in Day 3 and 5 in 100µl of complete media.

4.6.1 Cell viability studies in MCF7 ADR spheroids:

Cell viability in MCF7 ADR spheroids following the treatments with either free Dox or targeted

D-Dox-PGA was assessed using CellTiter-Glo® (Figure 38). As expected, targeted D-Dox-PGA showed significantly higher cytotoxic effects compared to that of free Dox due to Dox resistant nature of this cell line. Increasing the concentration of the pretargeting bispecific complex resulted in higher cytoxicity associated with D-Dox-PGA but plateaued out at 60 µg/ml consistent with the earlier data.

Investigator: Prashant Raj Bhattarai

MCF7 ADR Spheroids Cytotoxicity Studies 48 h

140

120

100

Average% Cell Viability 20

0 0 20 40 60 80 100 Dox Concentration (μg/ml)

Free Dox alone 20μg/ml of biotinylated anti-DTPA+D-Dox-PGA

40μg/ml of biotinylated anti-DTPA+D-Dox-PGA 60μg/ml of biotinylated anti-DTPA+D-Dox-PGA Figure 38: Cytotoxicity of targeted D-Dox-PGA after pretargeting with various concentration of biotinylated anti-DTPA bispecific antibody complex compared to free Dox (blue diamond) in MCF7 ADR spheroids.

Drug combination studies in MCF7 ADR spheroids 48 h 160 40μg/ml of biotinylated 140 anti-DTPA+D-Dox- PGA+D-Ptxl-PGA 120 40μg/ml of biotinylated anti-DTPA+D-Dox-PGA 100 40μg/ml of biotinylated 80 anti-DTPA+D-Ptxl-PGA

60 Free Dox

% % Cell viability 40 20 Free Ptxl 0 92 46 23 11.5 5.75 2.875 1.4375 Free Dox+Ptxl Equivalent Drug concentration (μg/ml)

Figure 39: Comparison in cytotoxicity of free Dox and Ptxl with individually targeted D-Dox-PGA or D- Ptxl-PGA and combination treatment (D-Dox-PGA+D-Ptxl-PGA) in MCF7 ADR spheroids.

Investigator: Prashant Raj Bhattarai

Next, combination therapy was carried out for the comparison to results seen in monolayer MCF7 cells (Figure 39). Combination treatment resulted in significantly higher cytotoxicity (p<0.01) compared to free paclitaxel and Dox. But no significant difference was observed in cytotoxicity between combination treatments compared to individual targeted treatment groups.

4.7 Tumor Growth inhibitions in a 4T1 Autologous graft model:

Due to its well-characterized biotin receptors expression level and ease of growing tumor in mice, murine 4T1 was chosen as the autologous tumor model for subsequent in vivo studies in Balb/c mice. Mice subcutaneously injected with 1x105 4T1 cells in 50% (v/v) matrigel in their right shoulder for tumor formation were used for this study. When tumors reached an average volume of about 100 mm3 mice were randomized into treatment groups. At day 0, mice were injected intravenously with 20µg of pretargeting biotinylated anti-DTPA bispecific antibody complex.

After 8 h of bispecific antibody complex injection, D-Dox-PGA and D-Ptxl-PGA at equivalent doses of 5 mg/kg and 10 mg/kg (100µl injection volume) respectively were injected once a week, with a total of 4 injections. Daily tumor volume measured for all mice in each treatment group is shown in Figure 40. Control saline treated group showed maximal tumor growth. Free Dox and

Ptxl treated groups showed significant tumor growth inhibition of 25% and 21% respectively

(p<0.05). However, between free Dox and paclitaxel treated groups no significant difference in tumor growth inhibition was observed. Individually targeted D-Dox-PGA and D-Ptxl-PGA showed significantly higher tumor growth inhibition of 43 % and 55 % respectively (p<0.01). In comparison, combination treatment was shown to be superior in achieving tumor growth inhibition relative to free or individually targeted Dox and Ptxl in subcutaneous mouse 4T1tumor model

(70%).

Investigator: Prashant Raj Bhattarai

Animal bodyweight change commonly used as an indicator of in vivo toxicity shows that there was no reduction in the total body weights in all groups except in the free Dox treatment group (Figure

41). All mice receiving Dox succumbed by day 24 of the study. They received total Dox dose of

15mg/kg by day 21. The death in this group is most probably associated with cardiotoxicity.

Furthermore, this group of mice treated with Dox showed approximately 25% decrease in total bodyweight by time of its death. No such toxicity was observed in other treatment groups including targeted D-Dox-PGA group confirming change in mechanism of uptake of doxorubicin. The data indicate that D-Dox-PGA is non-toxic in vivo even at cumulative Dox dose of 20 mg/kg.

Hematological toxicity determined by White Blood cell counts in blood samples obtained at time of death or termination of the experiment in various treatment groups is shown in Table 7. Free

Ptxl treatment significantly decreased the WBC count in the 4T1 tumor bearing mice compared with saline treated control group (p<0.01). WBC count in Dox treated groups (8.91+0.505 [109 /L blood]) was lower than the saline group (12.08 + 0.381 [109 cells/L blood]) and significantly different (p<0.05). All mice treated with PPDCs showed no statistically significant reduction in the white blood cell counts.

The tumors were harvested at sacrifice of each group and photographs taken of all tumors and each tumor was weighed on 28 days at time of sacrifice, except for the combination polymer drug therapy group, which was allowed to survive for one more week due to the very small size of the tumors. Representative images of the excised tumors are shown in Figure 42. Targeted D-Dox-

PGA and D-Ptxl-PGA showed significant reduction in tumor weights as compared to saline treated groups (p<0.01), free Dox (p<0.05) or free paclitaxel treated groups (p<0.05). Similar to the in vivo measurement of the tumor volumes (Figure 40) the actual measurement of the tumors after excision confirmed the greatest efficacy in mice treated with the combination therapy (Figure 42 right).

Investigator: Prashant Raj Bhattarai

saline 1200 4T1 in vivo tumor studies dox

1000 combined

Ptxl 800

) Targeted D-Ptxl- 3 PGA 600 Targeted D-Dox- PGA ↑ IV injection 400

Tumor Tumor volume (mm 200

0 0 5 ↑ 10 ↑15 20↑ 25 ↑ 30 35 40 ↑ Days post initiation of therapy

Figure 40: Tumor growth inhibition studies in vivo in a mouse 4T1 xenograft model

Bodyweight Comparisons 30

20 dox Saline 15 ptxl

Weight gms in Targeted D-Dox-PGA 10 Targeted D-Ptxl-PGA combined 5

0 0 5 10 15 20 25 30 Days

Figure 41: Mouse bodyweight measurements during treatment

Investigator: Prashant Raj Bhattarai

Table 7: Hematological results in mice after different treatments (*p<0.05, **p<0.01 when compared to saline group) Groups WBC (109/L) Saline 12.08 ± 0.381 Free Dox 8.91 ±0.505* Free Ptxl 5.54 ± .310** Targeted D-Ptxl-PGA 9.5 ±0.82 Targeted D-Dox-PGA 10.17 ± 0.44 Combined (D-Dox-PGA + D-Ptxl-PGA) 9.89 ± 0.072

Figure 42: Representative images of excised tumors and tumor weights (n=5).

4.7.1 Epifluorescence microscopy to study doxorubicin localization in mice hearts:

Due to the potential of cardiotoxicity distribution of Dox in all the hearts was evaluated by fluorescence microscopy. Cryotome 10 µ thick sections of the hearts from free Dox and targeted

D-Dox-PGA treated groups were examined by fluorescence microscopy of (Figure 43). Green fluorescence represents auto fluorescence associated with the heart sections. In the tissue sections obtained from Dox treated groups, red fluorescence associated with the Dox merged with green to give rise to orange color (seen in Figure 43 A). There was no visible red epifluorescence in the tissues sections obtained from mice treated with targeted D-Dox-PGA as evidenced by the absence of orange color in Figure 43 B. Magnification of an area of the heart sections for free Dox treated

Investigator: Prashant Raj Bhattarai mice show that almost all nuclei of the cardiocytes were fluorescent red indicative of Dox accumulation in the hearts. However, mice treated with pretargeted D-Dox-PGA showed no nuclear staining in the heart sections. These cryotome sections demonstrated that there was no Dox accumulation in the heart of mice treated with pretargeted D-Dox-PGA. This is consistent with our observation that there is no cardiotoxicity when D-Dox-PGA is the vehicle for delivery of Dox prodrug polymers. This lack of cardiotoxicity with D-Dox-PGA has been reported by Panwar et al from our lab55.

A B

C D

Figure 43: Fluorescence images of cardiac sections from mouse 4T1 tumor bearing Balb/c mice after systemic adminstration of different treatment groups. A) Dox treated groups, B) Targeted D-Dox-PGA treated group, C) and D) are the magnified images of portion of A) and B) respectively to show red Dox fluorescence Magnification 4X.

Investigator: Prashant Raj Bhattarai

4.7.2 TUNEL Staining:

To explore the mechanisms of tumor inhibition in vivo, tumors were collected after mice were sacrificed. The presence of the apoptotic cells was identified using TUNEL assay (Figure 44). The apoptotic tumor cells are stained brown. Tumors from the control saline treated groups and free drug treatment groups showed less apoptotic cells (TUNEL positive) compared to those tumors from D-Dox-PGA and D-Ptxl-PGA treatment groups. The highest number of apoptotic cells was found in tumors from mice in the combination treatment group. Quantitative analysis of stained slides was performed by counting 100 cells from 6 random microscopic fields and the results are shown in Table 8. Single targeted delivery of either D-Dox-PGA or D-Ptxl-PGA induced DNA fragmentation (brown staining), which was further significantly increased (p<0.01) by the combination treatment. The combination treatment led to the apoptosis in 87.32 ± 2.86 percent of tumor cells, whereas free Dox and Ptxl induced apoptosis in 29.65 ± 3.22 and 23.34 ± 1.7 percent of tumor cells respectively.

Table 8: Percentage of TUNEL positive cells (*p<0.05, **p<0.01 when compared to saline group) Treatment Groups % TUNEL positive cells Saline 11.43 ± 1.94 Free Dox 29.65 ±3.22 Free Ptxl 23.34 ± 1.7 Targeted D-Ptxl-PGA 47.52 ±02.3* Targeted D-Dox-PGA 62.65 ± 1.75* Combined (D-Dox-PGA + D-Ptxl-PGA) 87.32 ± 2.86**

Investigator: Prashant Raj Bhattarai

Figure 44: Immunohistochemical staining of 4T1 breast tumor tissue for induction of apoptosis using TUNEL assay

Investigator: Prashant Raj Bhattarai

5. DISCUSSION

Pretargeting strategies were initially developed for radio-immuno-imaging and radio-immuno- therapy8,56. Bispecific antibody complexes are ideal for the pretargeting approach. Bispecific antibodies can be made either by chemical cross-linking8,12,15, quadroma technology (fusion of hybridoma cells with two different specificities) or via recombinant technology6,64. The use of appropriate technology for the production of bispecific antibodies depends on one’s needs.

Bispecific antibodies have two different antigen specific binding sites; one for the target antigen

(tumor specific or associated antigen) and other for the effector compounds or also referred to as the capture unit. Tumor is targeted via the tumor specific arm of bispecific antibodies. Following clearance of the circulating bispecific antibodies, effector compound, which binds to the effector capture arm of the bispecific antibodies is injected leading to tumor localization. These effector compounds can be radiolabeled mono- or di-valent haptens56 or polymers in our laboratory for diagnostic application12,55 or PPDCs for tumor therapy. Pretargeting with bispecific antibodies and targeting with mono-and di-valent haptens enabled achievement of very high target to background activity but is limited by very low non-target activity sequestration11,12,17. Bombesin bispecific antibody and anti-HER2-affibody-anti-DTPA Fab (BAAC) bispecific antibody complexes were used to demonstrate targeted delivery of radiolabeled polymers for preclinical in vivo cancer diagnostic imaging12. Subsequent studies from our laboratory extended delivery of PPDCs to treatment of HER2 positive human mammary cancer in vivo as well as in vitro demonstration of the enhanced potential for cancer therapy12, 65. This pretargeting approach has now been extended to overcome multidrug resistance in cancer as demonstrated in this thesis and publication65.

Anti-HER2 neu affibody X anti-DTPA Fab bispecific complex is prepared using a chemical conjugation method via thioether bonds. Immunoreactivity of the monoclonal antibody must be maintained after modification with heterobifunctional cross linker N-hydroxy succinimide ester of bromoacetic acid (Figure 5) to enable optimal in vivo targeting. Size exclusion chromatography

Investigator: Prashant Raj Bhattarai was used to purify the monomeric bispecific antibody complex from multimeric bispecific complexes. Depending on the targeting and capture units employed for preparation of the bispecific antibody complexes, very high yield of the bispecific complexes, e.g. almost 100% crosslinking of affibody to Fab can be achieved. No free, unconjugated anti-DTPA Fab was evidenced in the SDS

PAGE (Figure 6).

This thesis also reports the potential of the pretargeting approach and targeting with PPDCs to treat drug resistant cancers. One of the mechanisms of multidrug resistance development in cancer is over expression of the membrane transporter P-glycoprotein (Pgp). Pgp are ATP dependent ABC cassette proteins that are responsible for continuous efflux of drugs that have entered the cancer cells54. Maintaining high intracellular level of the therapeutic drugs in Pgp over expressing cancer cells is a major challenge. To enable maintenance of effective intracellular concentration of drugs in drug resistant cancer cells, various approaches have been developed. These approaches to enhance delivery of various chemotherapeutics agents into drug-resistant cancers include use of: 1)

MDR transporter inhibitors41,66 2) microRNA and RNA interference for inactivation of MDR associated genes67 and 3) nanoparticles such as dendrimers66, polymer-drug conjugates34,65, liposomes68,69 and micelles41,70,71. Nanoparticles have also been used for combination therapy by encapsulation of anticancer drugs together with MDR inhibitors and RNAi molecules to overcome drug resistance72. Western blot analysis in Figure 20 confirms overexpression of Pgp receptors in

SKOV3 TR and MCF7 ADR cell lines.

We have concentrated on using Doxorubicin and Paclitaxel because they are the two frontline chemotherapeutic agents for cancer therapy. Nevertheless, Dox, is limited by its cardiotoxicity at its optimal therapeutic dosage. Myelosupression, nephrotoxicity, and hepatotoxicity are some non- target toxicities associated with Doxorubicin therapy55,57,61. On the other hand, Ptxl is highly lipophilic and has very poor aqueous solubility. Most common commercial formulation of paclitaxel requires cremophor as a solvent73. Cremophor causes severe side effects such as peripheral neuropathy, immunological reactions, and nausea24,58.

Investigator: Prashant Raj Bhattarai

One of our goals was to generate water soluble D-Ptxl-PGA conjugate and specifically target the

PPDCs (D-Dox-PGA and D-Ptxl-PGA) to cancer cells via the pretargeting approach using bispecific antibody complex and attempt to overcome multidrug resistance in cancer. PGA is chosen as the polymeric drug carrier because of its biocompatibility and presence of large number of carboxylic residues that can be modified for conjugation with either Doxorubicin or

Paclitaxel12,58. Conjugation of these drugs to polymers increases the solubility of hydrophobic drugs as well as provides a high specific activity pro-drug delivery mechanism with increased bioavailability. PGA also possess a net negative charge which is repelled by the negatively-charged cell surfaces leading to reduction of off-target interaction and hence off-target toxicity11. Dox and

Ptxl were conjugated covalently on PGA via the amide bond using EDC chemistry. Each PGA has an N-terminal modified DTPA permitting site-specific capture by the bispecific antibody complex.

PGA with weight of 11.3 a molecular kDa was chosen due to the molecular size that allows sufficiently rapid blood clearance via renal excretion at the same time permit sufficient blood circulation to allow high tumor localization. As stated above these 2 PPDCs of PGA i.e. D-Dox-

PGA and D-Ptxl-PGA, have negative zeta potentials and therefore also reduce off-target sequestration. Khaw et al (2006) have shown that use of negatively charged PPDCs reduced non- specific ionic interaction between the negatively charged cell surfaces and the PPDCs11.

Stability studies also showed that D-Ptxl-PGA was stable in 0.1M PBS. Release profile of Ptxl from D-Ptxl-PGA occurred in acidic (pH 4) but not at neutral pH 7.4. Ptxl was observed at pH 7.4 after 72 h incubation at 370C to release only about 7%. However, more than 50% Ptxl is released within 1 h at pH 4 in the sodium acetate buffer. Similar release profile has been shown for the Dox in D-Dox-PGA in previous studies by Khaw et al12. Thus, for in vivo delivery, we propose that targeted PPDCs must be internalized by endocytosis and free drugs are released only following fusion of endosomes with lysosomes. Lysozyme activity and the acidic lysosomal microenvironment of endolysomes containing the PPDCs allow facilitated release of the active drugs intratumorally leading to enhanced targeted tumor killing. The role of endocytosis is

Investigator: Prashant Raj Bhattarai demonstrated in our study shown in Figure 23. When endocytosis is inhibited with chlorpromazine pretreatment, tumor cytotoxicity was not observed.

Furthermore, non-targeted PPDCs had no cytotoxic effects on sensitive or resistant SKOV 3 ovarian cancer, MCF7 human or 4T1 murine mammary cancer cell lines. This is probably due to the prodrug state of Ptxl and Dox after conjugation to PGA. In tumor cells, toxicity was enhanced only after pretargeting with bispecific antibody complexes. In both the SKOV3 and MCF7 drug sensitive cell lines, the pretargeting approach followed by targeting with either D-Ptxl-PGA or D-

Dox-PGA showed no significant improvement in tumor cytotoxicity relative to treatment with free drugs. In drug resistant cell lines however, higher tumor toxicity was observed with pretargeted therapy relative to free drugs alone. Tumor toxicity of targeted PPDCs depended on the concentration of the pretargeting bispecific antibody complex used. Increase in the concentration of the pretargeting bispecific antibody complex led to higher tumor cytotoxicity (Figure 27).

However, at 40 μg/ml of biotinylated anti-DTPA complex treatment, near receptor binding saturation was reached in the cancer cells tested. Increasing the concentration of the pretargeting bispecific antibody to 60 μg/ml showed no significant change in cytotoxicity. Therefore, our data is consistent with the concept that enhanced cytotoxicity in drug resistant SKOV3 TR and MCF7

ADR cells after pretargeting with bispecific antibody complexes and targeting with PPDCs is due to receptor targeting. Although receptor binding saturation appears to occur in in vitro cell studies, it may not be a major factor in in vivo therapeutic applications since tumor burden will essentially be greater and clearance of the bispecific antibody complexes will potentially not allow the bispecific pretargeting units to reach saturation on cancer cells.

The pretargeted approach (Figure 29) shows a prolonged ability to deliver the PPDCs over 6 h. At

5 min there was initiation of binding of D-Dox-PGA to the extracellular surface of the cells followed by increase in the intensity of intracellular Dox fluorescence with time. Quantitatively, the whole cell intensity representing localization of D-Dox-PGA increases as time of incubation increases and reached corrected total cell fluorescence (CTCF) values of approximately 1 million

Investigator: Prashant Raj Bhattarai by 6 h incubation relative to approximately 500,000 CTCF of cells incubated for the same period of time with free Dox (Figure 32 A).

The CTCF values following Dox treatment for 1 h was also less (500,700 ± 105,700 ) than that in the pretargeted D-Dox-PGA MCF7 ADR cells (822,300 ± 246,600 CTCF, P < 0.001) indicating that more Dox in the form of D-DOX-PGA or newly released Dox, is first sequestered and retained by MFC7 ADR cells. Therefore, delivery of PPDCs after pretargeting resulted in approximately 2 times the drug concentration obtained with Dox alone (Figure 34 A, P <0.001). In the drug retention studies with MCF7 ADR cells, where the medium is replaced with Dox free medium after 1h incubation with either Dox or pretargeted D-Dox-PGA and further incubated for 4 h, nearly 80% of Dox is retained in the pretargeted MCF7 ADR cells relative to approximately only 35% for Dox

(Figure 33 and 34). These results combined with Dox uptake studies in Figures 29, 31 and 34 are consistent with our hypothesis that pretargeted delivery of D-Dox-PGA leads to higher uptake and retention of Dox in MCF ADR resistant cell lines. Even though the pretargeted approach does not directly inhibit the activity of the Pgp, it helps the PPDCs to bypass the efflux action of Pgp receptors.

Nuclei of the cells are targets of Dox. Our study showed that very high Dox fluorescence intensity was associated with the nuclei in the pretargeted cells relative to treatment of the same cancer cells with free Dox (Figure 32 B, 34 B). Figures 29 and 31 substantiate the superiority of the pretargeted approach to achieve higher nuclear localization of Dox in MCF7 ADR resistant cells. In addition, the use of FITC labeled biotinylated anti-DTPA permitted tracking of the pretargeting bispecific antibody complex relative to localization of the PPDCs. The green fluorescence of the FITC labeled bispecific antibody complex is internalized into the cytoplasm but is not observed to sequester to the nuclei of the treated cells (Figure 35 Ac and e), whereas the red fluorescence of Dox is initially internalized along with the FITC- bispecific antibody complex and subsequently, is localized in the nuclei, consistent with the concept that following internalization of D-Dox-PGA, Dox is released which then is sequestered to the nuclei (Figure 35 Ad).

Investigator: Prashant Raj Bhattarai

Furthermore, the mechanism of internalization of the pretargeted PPDCs is consistent with endocytosis. Inhibition of endocytosis with pretreatment of cancer cells with chlorpromazine

(Figure 36) resulted in the inhibition of uptake of D-Dox-PGA after pretargeting with bispecific antibody complex as seen by the absence of the red Dox fluorescence. This observation further support the proposal that uptake of PPDCs after pretargeting the biomarker receptors is via endocytosis and that the cell cytotoxicity is abrogated after pre-incubation of cancer cells with chlorpromazine (Figure 36 A). No inhibition of cytotoxicity is observed in the MCF7 ADR cells pretreated with chlorpromazine and treated with Dox, suggesting that the mechanisms of internalization of Dox and pretargeted D-Dox-PGA are not the same. Similar results were observed in the SKOV3 TR cells (Figure 23) thus confirming the role of endocytosis in PPDCs uptake in the pretargeting approach. Internalization of antibodies by the targeted cells has been reported to be via clathrin-mediated endocytosis. Our results are similar to those of Minko et al 1998, who reported that HPMA-copolymer Adriamycin conjugates entered cancer cells via endocytosis circumventing the Pgp efflux pumps relative to free drug entry by diffusion and efflux of the free drug54.

The final goal of the thesis to test the efficacy of PPDCs targeted via pretargeted approach in vivo.

4T1 mouse mammary breast cancer autologous graft model showed that the tumor growth inhibition was initiated at an early stage of tumor growth in mice treated with the pretargeted- targeted PPDC therapeutic approach. A 5 mg/Kg Dox dose and 10 mg/Kg Ptxl dose was chosen as per the literature reports40,57,74. After inoculation of animals with tumor cells, therapeutic intervention protocols were initiated when the tumors reach an average size of 100 mm3. Both the individually pretargeted-targeted D-Dox-PGA and D-Ptxl-PGA showed significant tumor growth inhibition by 43% and 55% respectively as compared to the saline and free drugs treated groups throughout the study (Figure 40). However, combination therapy with targeted D-Dox-PGA and

D-Ptxl-PGA treatment was most effective in tumor growth inhibition as determined by tumor weights by 92% (Figure 42). Towards the end of the treatments, significant differences were seen

Investigator: Prashant Raj Bhattarai between the tumor sizes of combination treatment group and free drugs and saline treated groups

(Figure 42). Mice injected with 5mg/Kg free Dox showed a sharp decrease in bodyweight by about

10% in 10 days probably due to toxicity of Dox. Approximately 25% loss of the initial bodyweight occurred in Dox treated mice and all mice died by 24 days. Targeted D-Dox-PGA treated mice that received cumulative dose of 20mg/Kg of Dox showed no signs of toxicity. Although, D-Dox-PGA treated mice showed no loss of weight they also did not gain weight as in untreated saline controls.

Furthermore, epifluorescence photomicrographs of the 10 µ frozen sections of the harvested murine hearts demonstrated that Dox treated mice had substantially more red fluorescence in the nuclei of the cardiocytes. On the other hand, no fluorescence was observed in the tissue sections from mice that were treated with pretargeted or non-targeted D-Dox-PGA.

Induction of apoptosis is significantly increased in tumors of mice treated with the combination treatment compared to the saline and free drug treated groups (Figure 44). Since apoptosis is an important pathway associated with anticancer activity of any protocols in cancer therapy, our pretargeting approach for targeting with PPDCs especially combination PPDCs may result in significantly enhanced cancer therapy. Furthermore, higher drug concentrations can be used in therapy in the targeted prodrug format with minimal or no off target toxicities, this approach may result in a potentially highly desirable therapeutic approach that may eradicate nearly all cancer cells in solid tumors.

Investigator: Prashant Raj Bhattarai

6. CONCLUSIONS:

Overall, single targeted and combination PPDCs therapies showed significant increases in cell cytotoxicity compared to the untargeted PPDCs and free drugs in drug resistant tumor cell lines in vitro. There was no significant difference in tumor toxicity between free drugs and targeted PPDCs in vitro in drug sensitive cell lines. In in vivo studies, significantly higher toxicity was observed via targeted PPDCs in in vivo 4T1 model. This study highlights the potential of the pretargeting approach to overcome the multidrug resistance in cancer. It is also evident that although in vitro experimental designs allow for easier evaluations of formulations as well as higher throughput, it is imperative that these formulations be investigated in vivo to fully understand and assess their therapeutic potentials.

Investigator: Prashant Raj Bhattarai

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