Prashant Raj Bhattarai, Dylan Vance, Ban-An Khaw
Graduate Category: Physical and Life Sciences Degree Seeking: PhD Abstract ID# 1816
Targeted Delivery of Polymer Pro-drug conjugates to overcome drug resistance in cancer Prashant Raj Bhattarai, Dylan Vance, Ban-An Khaw Results (contd.)
Opportunity Abstract:
Doxorubicin uptake Study
Purification and characterization of bispecific antibodies anti-HER2/neu X anti-DTPA Fab bispecific complex Multimeric complex Intact Anti-DTPA Bispecific Antibody complex Bispecific Antibody complex Anti-DTPA Fab
Affibody Multimeric complex
Figure 10: Combination therapy using 2 drugs (D-Dox-PGA and D-PtxPGA) after pre-targeting with 40µg/ml of biotinylated anti-DTPA Affibody
Figure 2: Elution profile of crude bispecific reaction product from size exclusion Zorbax Figure 3: SDS-PAGE for bispecific antibody complexes G-250 column eluted using 0.2M sodium phosphate buffer pH7.4
1
Determination of Biotinylation of Anti-DTPA
Figure 12: (A) MCF7-ADR cells pretreated with biotinylated anti-DTPA-FITC bsAbCx (1 h) followed by D-Dox-PGA
ELISA comparing Biotinylated-BSA vs Biotinylated-AntiDTPA
0.800
for 1 h (B) un-treated control cells - (a) bright field, (b) Hoechst nuclear stain, (c) FITC green, (d) Dox-fluorescence images of same cells, (e) superimposition of (b) and (d), (f) superimposition of (b) and (c), (g) superimposition of (c) and (d), and (h) superimposition of (b), (c) and (d) to demonstrate nuclear localization of the released Dox.
1.200
0.700
1.000
Doxorubicin Efflux Study
0.600
0.800
Absorbance 630
0.500 0.400 0.300 0.200
0.600
2
0.400
1
0.200 0.100
0.000
0.000 -0.100 1E-005
0.0001
0.001
0.01
0.1
1
-0.200 1E-007
10
1E-006
1E-005
0.0001
0.001
0.01
0.1
1
10
100
Concentration (µg/ml)
Ab Dilutions(µg/ml)
Figure 4:▲ =Biotinylated anti-DTPA, ■= unmodified anti-DTPA
Approach
Endocytosis inhibition Study
Biotinylated anti-DTPA bispecific complex
Absorbance 630
Rationale: Cancer cells over-express many tumor associated biomarkers. The potential of the pretargeting approach for targeting of single or multi-polymer pro-drug conjugates (PPDCs) to various cancer cells to overcome drug resistance is reported. Bispecific antibody complexes (bsAbCx) are used to pretarget drug resistant cancer cells and PPDCs are used for pro-drug therapy. Combination pro-drug polymer therapy conjugated with different drugs targeting different mechanisms of cell replication may result in highly efficient approach to overcome drug-resistance in cancer therapy. Methods: Bispecific antibody complexes consisting of either anti-DTPA Fab covalently-linked to anti-HER2 affibody via thioether bond or biotinylated intact anti-DTPA antibody were used to pretarget ovarian SKOV3 TR (paclitaxel resistant) and breast cancer MCF7 ADR (doxorubicin resistant) cell lines respectively. MCF7 ADR cells were used to generate spheroids by nonadhesive liquid overlay technique. Cell viability analyses utilizing pretargeting approach with single or combination PPDCs are carried out in both monolayer and 3D spheroid cancer cell models. Inhibition of endocytosis and drug delivery by epifluorescence microscopy are also undertaken to elucidate the mechanism of intracellular drug trafficking and release. Results: Targeted PPDCs (D-PTXL-PGA and D-DOX-PGA) after pretargeting with bsAbCx led to enhanced cytotoxicity in SKOV3 TR and MCF7 ADR cell lines respectively as compared to free drugs alone. Combination therapy utilizing the delivery of two PPDCs (D-DOX-PGA + D-PTXLPGA) led to significantly higher cell killing in both monolayer and spheroid cell culture models. Fluorescence microscopy showed improved intracellular and nuclear localization of DOX in MCF7 ADR cells using pretargeting approach. Conclusion: Utilization of the pretargeting approach for delivery of PPDCs enabled intracellular release of free drugs in the cytoplasm thereby avoiding the efflux pumps via the Pgp receptors that are cell membrane associated. The combination approach for the delivery of PPDCs leads to enhanced therapeutic effect as confirmed by greater tumor cell toxicity.
Figure 5:♦ = Biotinylated BSA, ▲ = biotinylated anti-DTPA
3 2
In-vitro cytotoxicity studies of polymer-drug conjugates in SKOV3 Monolayer Cell culture
Method: Utilization of 2-step Pretargeting approach: Step 1: Targeting with bispecific antibody Step 2: Delivery of Polymer drug conjugates/radiolabeled haptens
4 3
Figure 6: Without pre-targeting with bispecific antibody no cell cytotoxicity was observed with polymer drug conjugates
Figure 11: Fluorescence microscopic images demonstrating the effect of 30mins of pre-incubation of 10μg/ml of Chlorpromazine in blocking delivery of pre-targeted delivery of D-DOX-PGA on DOX retention in MCF7 ADR cell lines. MCF7 ADR cells were treated with 10 μg/ml of 1)biotinylated anti-DTPA targeted D-DOX-PGA , 2) D-DOX-PGA, 3) Doxorubicin alone and 4) Cells alone. a) brightfield, b) dox fluorescence, c) Hoeschst stain and d) Merged images. Figure 13: Fluorescence microscopic images demonstrating the effect of pre-targeted delivery of D-DOX-PGA on DOX retention in MCF7 ADR cell lines. MCF7 ADR cells were treated with 10 μg/ml of 1) Doxorubicin alone, 2) biotinylated anti-DTPA targeted D-DOX-PGA and 3) D-DOX-PGA without targeting for 1 hr and then reincubated in fresh medium for 4 hrs. a) brightfield, b) dox fluorescence, c) Hoeschst stain and d) Merged images
In-vitro cytotoxicity studies in MCF7 ADR Spheroid Cell culture
Spheroid Generation
1
Figure 7: In SKOV3 TR cell lines, increase in concentration of pre-targeting bispecific complex led to increase in the cell cytotoxicity with D-PTXL-PGA as compared to free paclitaxel. Ÿ● Free Paclitaxel,■ 20μg/ml of bsMAbCx + Targeted D-Ptxl-PGA,▲ 40μg/ml of bsMAbCx + Targeted D-Dox-PGA and □ Non-targeted D-Dox-PGA DOI: 10.1158/1078-0432.CCR-1004-0009
In-vitro cytotoxicity studies in MCF7 Monolayer Cell culture
2
Figure 14: Spheroids were generated using Liquid Overlay technique. Figures represent the development of spheroids with 1) showing spheroid at Day 3 after seeding and 2) representing same spheroid at Day 5. Day 5 spheroids have well developed black necrotic core and was used for cytotoxicity studies.
Results
Figure 15: Pre-targeted delivery of D-DOX-PGA using biotinylated antiDTPA leads to increased tumor toxicity in MCF7 ADR spheroid cell culture compared to Dox alone.
Western Blot for Pgp expression
Impact Figure 8: Cell cytotoxicity studies after pre-targeting with 10µg/ml of Biotinylated Anti-DTPA
Figure 1: Western Blot assay was performed to determine and compare the expression level of P-gp receptors in two different sensitive and drug resistant cell lines. A- SKOV3 sensitive, B- SKOV3 TR (PTXL resistant), C- MCF7, and D- MCF7 ADR (Dox resistant)
Figure 9: Cell cytotoxicity studies after pre-targeting with various concentration of biotinylated anti-DTPA bispecific complex
Value Proposition The unique feature about my research is: the use of the pre-targeting approach for the delivery of polymer (multi-)pro-drug conjugates. This addresses the problem of: Multidrug resistance generally arise in cancer therapy. Our research shows that utilizing bispecific antibody pre-targeting approach and polymer drug conjugate drug delivery result in overcoming drugresistance by delivery of higher concentrations of active drugs in cancer cells even in the presence of P-gp efflux pumps involved in drug resistance. This is further confirmed by significantly higher tumor cell toxicity in drug resistant cancer cells with our pretargeting approach compared to conventional free drugs therapy.
Figure 16: In MCF7 ADR spheroid culture, increase in concentration of pretargeting bispecific complex led to increase in the tumor cytotoxicity with DDOX-PGA
REFERENCES • Li, C. et al. Complete regression of well-established tumors using a novel water-soluble poly (L-glutamic acid)-paclitaxel conjugate. Cancer Res. 58, 2404–2409 (1998). • Wong, H. L. A Mechanistic Study of Enhanced Doxorubicin Uptake and Retention in Multidrug Resistant Breast Cancer Cells Using a Polymer-Lipid Hybrid Nanoparticle System. J. Pharmacol. Exp. Ther. 317, 1372–1381 (2006). • Kim, S. Y., Cho, S. H., Lee, Y. M. & Chu, L.-Y. Biotin-conjugated block copolymeric nanoparticles as tumor-targeted drug delivery systems. Macromol. Res. 15, 646–655 (2007).
Targeted Delivery of Polymer Pro-drug conjugates to overcome drug resistance in cancer Prashant Raj Bhattarai, Dylan Vance, Ban-An Khaw Results (contd.)
Opportunity Abstract:
Doxorubicin uptake Study
Purification and characterization of bispecific antibodies anti-HER2/neu X anti-DTPA Fab bispecific complex Multimeric complex Intact Anti-DTPA Bispecific Antibody complex Bispecific Antibody complex Anti-DTPA Fab
Affibody Multimeric complex
Figure 10: Combination therapy using 2 drugs (D-Dox-PGA and D-PtxPGA) after pre-targeting with 40µg/ml of biotinylated anti-DTPA Affibody
Figure 2: Elution profile of crude bispecific reaction product from size exclusion Zorbax Figure 3: SDS-PAGE for bispecific antibody complexes G-250 column eluted using 0.2M sodium phosphate buffer pH7.4
1
Determination of Biotinylation of Anti-DTPA
Figure 12: (A) MCF7-ADR cells pretreated with biotinylated anti-DTPA-FITC bsAbCx (1 h) followed by D-Dox-PGA
ELISA comparing Biotinylated-BSA vs Biotinylated-AntiDTPA
0.800
for 1 h (B) un-treated control cells - (a) bright field, (b) Hoechst nuclear stain, (c) FITC green, (d) Dox-fluorescence images of same cells, (e) superimposition of (b) and (d), (f) superimposition of (b) and (c), (g) superimposition of (c) and (d), and (h) superimposition of (b), (c) and (d) to demonstrate nuclear localization of the released Dox.
1.200
0.700
1.000
Doxorubicin Efflux Study
0.600
0.800
Absorbance 630
0.500 0.400 0.300 0.200
0.600
2
0.400
1
0.200 0.100
0.000
0.000 -0.100 1E-005
0.0001
0.001
0.01
0.1
1
-0.200 1E-007
10
1E-006
1E-005
0.0001
0.001
0.01
0.1
1
10
100
Concentration (µg/ml)
Ab Dilutions(µg/ml)
Figure 4:▲ =Biotinylated anti-DTPA, ■= unmodified anti-DTPA
Approach
Endocytosis inhibition Study
Biotinylated anti-DTPA bispecific complex
Absorbance 630
Rationale: Cancer cells over-express many tumor associated biomarkers. The potential of the pretargeting approach for targeting of single or multi-polymer pro-drug conjugates (PPDCs) to various cancer cells to overcome drug resistance is reported. Bispecific antibody complexes (bsAbCx) are used to pretarget drug resistant cancer cells and PPDCs are used for pro-drug therapy. Combination pro-drug polymer therapy conjugated with different drugs targeting different mechanisms of cell replication may result in highly efficient approach to overcome drug-resistance in cancer therapy. Methods: Bispecific antibody complexes consisting of either anti-DTPA Fab covalently-linked to anti-HER2 affibody via thioether bond or biotinylated intact anti-DTPA antibody were used to pretarget ovarian SKOV3 TR (paclitaxel resistant) and breast cancer MCF7 ADR (doxorubicin resistant) cell lines respectively. MCF7 ADR cells were used to generate spheroids by nonadhesive liquid overlay technique. Cell viability analyses utilizing pretargeting approach with single or combination PPDCs are carried out in both monolayer and 3D spheroid cancer cell models. Inhibition of endocytosis and drug delivery by epifluorescence microscopy are also undertaken to elucidate the mechanism of intracellular drug trafficking and release. Results: Targeted PPDCs (D-PTXL-PGA and D-DOX-PGA) after pretargeting with bsAbCx led to enhanced cytotoxicity in SKOV3 TR and MCF7 ADR cell lines respectively as compared to free drugs alone. Combination therapy utilizing the delivery of two PPDCs (D-DOX-PGA + D-PTXLPGA) led to significantly higher cell killing in both monolayer and spheroid cell culture models. Fluorescence microscopy showed improved intracellular and nuclear localization of DOX in MCF7 ADR cells using pretargeting approach. Conclusion: Utilization of the pretargeting approach for delivery of PPDCs enabled intracellular release of free drugs in the cytoplasm thereby avoiding the efflux pumps via the Pgp receptors that are cell membrane associated. The combination approach for the delivery of PPDCs leads to enhanced therapeutic effect as confirmed by greater tumor cell toxicity.
Figure 5:♦ = Biotinylated BSA, ▲ = biotinylated anti-DTPA
3 2
In-vitro cytotoxicity studies of polymer-drug conjugates in SKOV3 Monolayer Cell culture
Method: Utilization of 2-step Pretargeting approach: Step 1: Targeting with bispecific antibody Step 2: Delivery of Polymer drug conjugates/radiolabeled haptens
4 3
Figure 6: Without pre-targeting with bispecific antibody no cell cytotoxicity was observed with polymer drug conjugates
Figure 11: Fluorescence microscopic images demonstrating the effect of 30mins of pre-incubation of 10μg/ml of Chlorpromazine in blocking delivery of pre-targeted delivery of D-DOX-PGA on DOX retention in MCF7 ADR cell lines. MCF7 ADR cells were treated with 10 μg/ml of 1)biotinylated anti-DTPA targeted D-DOX-PGA , 2) D-DOX-PGA, 3) Doxorubicin alone and 4) Cells alone. a) brightfield, b) dox fluorescence, c) Hoeschst stain and d) Merged images. Figure 13: Fluorescence microscopic images demonstrating the effect of pre-targeted delivery of D-DOX-PGA on DOX retention in MCF7 ADR cell lines. MCF7 ADR cells were treated with 10 μg/ml of 1) Doxorubicin alone, 2) biotinylated anti-DTPA targeted D-DOX-PGA and 3) D-DOX-PGA without targeting for 1 hr and then reincubated in fresh medium for 4 hrs. a) brightfield, b) dox fluorescence, c) Hoeschst stain and d) Merged images
In-vitro cytotoxicity studies in MCF7 ADR Spheroid Cell culture
Spheroid Generation
1
Figure 7: In SKOV3 TR cell lines, increase in concentration of pre-targeting bispecific complex led to increase in the cell cytotoxicity with D-PTXL-PGA as compared to free paclitaxel. Ÿ● Free Paclitaxel,■ 20μg/ml of bsMAbCx + Targeted D-Ptxl-PGA,▲ 40μg/ml of bsMAbCx + Targeted D-Dox-PGA and □ Non-targeted D-Dox-PGA DOI: 10.1158/1078-0432.CCR-1004-0009
In-vitro cytotoxicity studies in MCF7 Monolayer Cell culture
2
Figure 14: Spheroids were generated using Liquid Overlay technique. Figures represent the development of spheroids with 1) showing spheroid at Day 3 after seeding and 2) representing same spheroid at Day 5. Day 5 spheroids have well developed black necrotic core and was used for cytotoxicity studies.
Results
Figure 15: Pre-targeted delivery of D-DOX-PGA using biotinylated antiDTPA leads to increased tumor toxicity in MCF7 ADR spheroid cell culture compared to Dox alone.
Western Blot for Pgp expression
Impact Figure 8: Cell cytotoxicity studies after pre-targeting with 10µg/ml of Biotinylated Anti-DTPA
Figure 1: Western Blot assay was performed to determine and compare the expression level of P-gp receptors in two different sensitive and drug resistant cell lines. A- SKOV3 sensitive, B- SKOV3 TR (PTXL resistant), C- MCF7, and D- MCF7 ADR (Dox resistant)
Figure 9: Cell cytotoxicity studies after pre-targeting with various concentration of biotinylated anti-DTPA bispecific complex
Value Proposition The unique feature about my research is: the use of the pre-targeting approach for the delivery of polymer (multi-)pro-drug conjugates. This addresses the problem of: Multidrug resistance generally arise in cancer therapy. Our research shows that utilizing bispecific antibody pre-targeting approach and polymer drug conjugate drug delivery result in overcoming drugresistance by delivery of higher concentrations of active drugs in cancer cells even in the presence of P-gp efflux pumps involved in drug resistance. This is further confirmed by significantly higher tumor cell toxicity in drug resistant cancer cells with our pretargeting approach compared to conventional free drugs therapy.
Figure 16: In MCF7 ADR spheroid culture, increase in concentration of pretargeting bispecific complex led to increase in the tumor cytotoxicity with DDOX-PGA
REFERENCES • Li, C. et al. Complete regression of well-established tumors using a novel water-soluble poly (L-glutamic acid)-paclitaxel conjugate. Cancer Res. 58, 2404–2409 (1998). • Wong, H. L. A Mechanistic Study of Enhanced Doxorubicin Uptake and Retention in Multidrug Resistant Breast Cancer Cells Using a Polymer-Lipid Hybrid Nanoparticle System. J. Pharmacol. Exp. Ther. 317, 1372–1381 (2006). • Kim, S. Y., Cho, S. H., Lee, Y. M. & Chu, L.-Y. Biotin-conjugated block copolymeric nanoparticles as tumor-targeted drug delivery systems. Macromol. Res. 15, 646–655 (2007).
