Targeting Metastatic Triple Negative Breast Cancer Using Phage Display NanotechnologyCHRIS RAMHOLD & DR. VALERY PETRENKO11-MARCH-15DEPARTMENT OF PATHOBIOLOGY AUBURN UNIVERSITY CVM

General Outline Cancer

Significance Statistics Models

Phage Display Technology Introduction Selection Modification of pre-existing cancer nanomedicines Targeting

Summary Future Work

Highly invasive tumor embolus. UD- Bio-imaging Core Center

Why Cancer?

What Is Cancer? Product of malignant

progression Loss of proliferative

control Escape apoptotic signals Escape Hayflick limit

Loeb, Lawrence A. "Human cancers express mutator phenotypes: origin, consequences and targeting." Nature Reviews Cancer 11.6 (2011): 450-457.

Cancer (Incidence)

This year, the American Cancer Society estimates 232,670 new cases of breast cancer alone in the United States

Siegel, Rebecca, et al. "Cancer statistics, 2014." CA: a cancer journal for clinicians 64.1 (2014): 9-29

Cancer (Incidence) Incidence rates of breast cancer

are projected to stabilize Lung cancer projected to decline More management options are

needed

Siegel, Rebecca, et al. "Cancer statistics, 2014." CA: a cancer journal for clinicians 64.1 (2014): 9-29

Cancer (Deaths)

Deaths in the U.S. attributed to breast cancer are estimated at 40,000 annually Deaths worldwide are over 500,000

Breast Cancer (Metastatic) The 5-year relative survival

rates of invasive metastatic breast cancer is at 29%

High likelihood of recurrence

http://www.cancercenter.com/breast-cancer/statistics/tab/breast-cancer-survival-statistics/

Breast Cancer Survival Rates

http://www.cancer.org/acs/groups/content/@research/documents/document/acspc-042725.pdf

5-year relative survival rates are lower in younger women

Typically more aggressive Why are these types of cancer

so deadly?

“If we can put a man on the moon, why can’t we cure cancer?” Tumors are heterogeneous

Cancer genome unstable Cancer cells exhibit

plasticity

Wang, Anxin, et al. "Heterogeneity in cancer stem cells." Cancer letters 357.1 (2015): 63-68.

Two Models of Tumorigenesis

Girouard, S. D., & Murphy, G. F. (2011). Melanoma stem cells: not rare, but well done. Laboratory Investigation; a Journal of Technical Methods and Pathology, 91, 647–664. doi:10.1038/labinvest.2011.50

Cancer Stem Cell Hypothesis “Malignant tumors are initiated

and maintained by a population of tumor cells that share similar biologic properties to normal adult stem cells.” – Brenton Thomas Tan

Selective pressures induce evolution of cancer cells which may acquire mutations in the mechanism for EMT (epithelial-mesenchymal transition) allowing tumor formation.

Owens TW and Naylor MJ (2013) Breast cancer stem cells. Front. Physiol. 4:225. doi: 10.3389/fphys.2013.00225

CSCs by Any Other Name… Tumor populations are heterogeneous Not all cells are capable of forming new

tumors Some tumorigenic cells have been

characterized CSCs=Tumor cells with tumorigenic

potential

TobogganToboggan

CSCs and Tumor Initiators The metastases originate

with tumor initiating cells (TICs).

Metastases account for nearly all breast cancer related deaths

Goal: Target the TICs… Stop proliferation Stop metastases Stop recurrence

Cancer Stem Cells from http://www.currinbiotech.com/categories/20101004

Breast Cancer Molecular Subtypes

Prat, Aleix, and Charles M. Perou. "Mammary development meets cancer genomics." Nature medicine 15.8 (2009): 842-844.

Killing Cancer Chemotherapies

Prevent mitosis Induce apoptosis Effective against rapidly proliferating cells

Challenges Low weight- Cleared quickly Low accumulation in tumors Hydrophobicity- Large volume of distribution Tumors are heterogeneous Tumor vasculature is leaky Toxicity towards healthy cells

Art by JerryKongArt http://jerrykongart.deviantart.com/art/Killing-Cancer-Cells-185013819

How Do We Target Cancer?

Targeted delivery of the drug to the site of pathology by DDS

Pathology Pathology Pathology

<1% 10% 20-30%

Untargeted Drug Nanomedicine

Random distribution of the drug leading to side effects

Localization of nano-medicines in tumor due to vasculature defects

Targeted Nanomedicine

Development of Targeted Nanomedicines

Wang, Tao, et al. "On the mechanism of targeting of phage fusion protein-modified nanocarriers: only the binding peptide sequence matters." Molecular pharmaceutics 8.5 (2011): 1720-1728.

Phage Display Technology Genetically engineered to express random 9 amino acid insertion at the N-terminus of the pVIII protein. Billions of unique sequences constitute a phage display library

Phage Display using filamentous bacteriophage fd

Løset, Geir Åge, et al. "Expanding the versatility of phage display II: improved affinity selection of folded domains on protein VII and IX of the filamentous phage." PLoS One 6.2 (2011): e17433. * Modified

9-mer insert

Phage Display Library The library contains roughly one billion randomized clones. These clones are subjected to a process of affinity selection.

Negative selection- Depletion of non-specific binders such as plastic, serum, and a “normal” cell line.

Positive selection- Depletion of phage that doesn’t or weakly binds target cells and enrichment of phage that binds and penetrates target cells.

Following several rounds of selection, the phage enriched for binding target cells are characterized.

http://www.virology.wisc.edu/virusworld/ICTV8/1fd-enterobacteria-phage-fd.jpg

Phage Fusion Peptides Intrinsic membrane proteins Capable of self-integration N-terminus bearing targeting

peptide remains exterior to liposome

PEGylated liposomes

Jayanna, Prashanth K., et al. "Landscape phage fusion protein-mediated targeting of nanomedicines enhances their prostate tumor cell association and cytotoxic efficiency." Nanomedicine: Nanotechnology, Biology and Medicine6.4 (2010): 538-546.

Phage Fusion Protein Modified Nanomedicines

Leaky vasculature allows accumulation of liposomes at site of tumor Passive- Takes advantage of enhanced permeability and retention effect Active- Phage fusion protein targets cancer cells

Lammers, Twan, et al. "Drug targeting to tumors: principles, pitfalls and (pre-) clinical progress." Journal of controlled release 161.2 (2012): 175-187.

Phage DisplaySelection Overview

Selection- Round 1 (Depletion) Round 1 includes depletion against non-specific

binders (plastic, serum, “normal” cells) and incubates phage with cells at room temperature.

“Normal” cell line used was MCF-10A cell line (ATCC). Classified as non-tumorigenic. Derived from fibrocystic breast tissue (previously

termed “fibrocystic breast disease,” now replaced with fibrocystic breast condition as it is not a disease).

Morphology comparable to normal cells Immortalized due to a loss of p16 at both loci-

otherwise diploid and genetically stable

Botlagunta, Mahendran, Paul T. Winnard, and Venu Raman. "Neoplastic transformation of breast epithelial cells by genotoxic stress." BMC cancer 10.1 (2010): 343.

Selection- Round 1 (Target Cells)

MDA-MB-231 cell line was used as target cells. Tumorigenic- derived from metastatic site (primary

adenocarcinoma) Triple negative (no expression of estrogen and

progesterone receptors as well as HER2 oncogene). Falls into the claudin-low molecular subtype Enrichment for EMT markers Shares features with stem-like cells

Breast Cancer Molecular Subtypes Cell lines are heterogeneous CD44+/CD24- used as standard

detection of stem-like cells Not always tumorigenic CD44+/CD24-/ESA+ cells more

consistent Showed tumorigenicity across

33 cell lines Genomic profile associated

with EMT signaling, loss of proliferative control

Prat, Aleix, and Charles M. Perou. "Mammary development meets cancer genomics." Nature medicine 15.8 (2009): 842-844.

Why MDA-MB-231? Nearly 100% of MDA-MB-231

cells are CD44+/CD24- MDA-MB-231 cell line has one of

the highest % for CD44+/CD24-/ESA+

Fillmore, Christine M., and Charlotte Kuperwasser. "Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy." Breast cancer res 10.2 (2008): R25.

Hypothesis Targeting breast cancer

stem-like cells will decrease the population of tumorigenic cells, increasing cytotoxic effects and reducing recurrence and metastasis.

Reya, Tannishtha, et al. "Stem cells, cancer, and cancer stem cells." nature414.6859 (2001): 105-111.

Selection- Round 1 (Phage Titers)

After incubation of phage with target cells (1 hr), unbound or weakly bound phage are removed by a series of sequential washes

Input- Starting phage concentration

Unbound and washes- non-specific and weakly binding phage

Dilute eluate through lysate- phage specific for target cells, both surface binding and membrane penetrating

Input

Unbou

nd

1st wash

2nd w

ash

3rd wash

4th wash

5th wash

6th wash

7th wash

8th wash

9th wash

10th

wash

Dilute E

luate

1st PE

wash

2nd P

E wash

PE su

pernata

ntLys

ate1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

Phage Titers Throughout Round 1 of Selection

Phage Fractions

CFU

(Tite

r x V

olum

e)

Selection- Quantification of Phage Phage recovered from both

the eluate and lysate fractions were amplified for use as inputs for subsequent rounds of selection.

Concentration measured by absorbance @ 269nm

Selection- Round 1 (Phage Recovery % Yield)

Low % recovery is typical as the large pool of ~ 1 billion clones is not enriched for the target cells

Phage recovered from the eluate vs lysate fractions is more pronounced in Round 1 as incubation at room temperature creates unfavorable conditions for phage to penetrate into target cell membranes

Successive rounds of selection should… Indicate enrichment of target-specific clones Have an increase in % recovery for both the

eluate and lysate fractions

Eluate yield (%) Lysate yield (%)0

0.001

0.002

0.003

0.004

0.005

0.006

0.007

Phage % Recovery from Round 1 of Se-lection against MDA-MB-231 cells

Phage Fraction

% P

hage

Rec

over

y (O

utpu

t/In

put)

x100

Selection- All Rounds(Eluate Phage Titers)

Round 2 & 3 incubation steps are carried out at 37oC providing an environment conducive to membrane penetrating phage

Results comparable to Round 1

Enrichment of targeted phage

Input

Unbou

nd

1st wash

2nd w

ash

3rd wash

4th wash

5th wash

6th wash

7th wash

8th wash

9th wash

10th

wash

Dilute E

luate

1st PE

wash

2nd PE

wash

PE su

pernata

ntLys

ate1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

Phage Titers Throughout Rounds 1-3 of Selection

Round 1 Round 2 Round 3

Selection- Comparison of Phage % Recovery

Was there enrichment of target specific clones? Yes, an increase in % recovery is seen

each round Caveat* some of the phage may simply

be “fast growers” Did the % of phage recovered in the

lysate fraction increase with an increase in temperature? Higher temperatures allowed more

phage to penetrate target cells as shown by an increase in the amount of phage recovered through cell lysis

Eluate Lysate0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Phage % Recovery Comparison

Round 1Round 2Round 3

Phage Fraction

% P

hage

Rec

over

y (O

utpu

t/In

put)

x100

Isolation of Phage Clones Well isolated, individual colonies of infected K91 Blue

Kan E. coli cells from the Eluate, Lysate, and Post elution wash titer plates were chosen at random and inoculated on a 100 grid plate. (100 colonies from eluate, lysate, and 50 each from the post elution wash plates).

Following an overnight culture, 95 colonies were then used in PCR to provide… Confirmation that the colony contains phage with major

coat protein Product to be sequenced for peptide identification

Electrophoresis of PCR Products

Band indicating presence of major coat protein Primer Dimers Clear Negative control

Eluate Clones 1-24 LtR TtB Eluate Clones 25-48 LtR TtB Eluate Clones 49-72 LtR TtB Eluate Clones 73-95 LtR TtB

Solid Contaminant

Sequencing Results (Peptide Sequences)

Unique Clones from Eluate (Repeats) Unique Clones from Lysate (Repeats)

AGLNYNVDQ DYDSLHINS (15) GMVSGQADD (4) VDYSEVGSLDARQGVMME EFGGPDYDT GPSYSENPD (2) VEPGGWTGDDGSGSLDGD EPYSGSISN GSLDEALNQ (12) VGENGGSAD (2)DGYRSSEDS (4) ERFQEGGTD (4) GSQTVMTDD (5) VGSDYGTGD (16)DLDLPGVND EVDAHVNLD GSSMSFQDT VLRDFLDTDDLQAQWAGD (15) EWNRSELGD (2) GSYDEVSSA (2) VNHETLTAD (3)DMQWSSGDT (3) EYSQGQEGS VADSAVGHD VPPDFLDTD (6)DSGWERNVD (2) GDYTEAVGA VAVSEPGMD (3) VSEPTSNES (2)DSSMSWSGD (63) GETGGADND (3) VDADRFSGD VSYMESETD (2)DTGMLEGGN GLNGGNWDD (8) VDTAEISSL (4) VTASGMSDD (2)

AGSNNEGMT (2) ETSRYSDID (2)AGSYGDMDT (11) GAEYVGDTT (2)DFAVGPGSD (2) GFNTEFGDT (2)DFNLHDAMD (2) GSEQSWTGD (2)DGIWEHGDS (2) GSLLSSQED (2)DHGGGGHDS (2) GYDPVNDYN (2)DMTNGSVPE (2) VDIAEQSTA (2)DSVYDEENS (2) VGGHGDDFD (2)DVPRETGLD (2) VGSMSDGYN (4)ESALWGGDS (2) VSTSSDFDP (2)

Sequencing Results (Families)(A)GS(Y) (E/N)GG(N) GGH NVD (W)TGD YSEAGSNNEGMT DTGMLEGGN DHGGGGHDS AGLNYNVDQ GSEQSWTGD GPSYSENPD

AGSYGDMDT ERFQEGGTD VGGHGDDFD DSGWERNVD VEPGGWTGD VDYSEVGSL

GSYDEVSSA VGENGGSAD GLN QEG VGSDYGTGD V**DFLDTD

AVG GLNGGNWDD AGLNYNVDQ ERFQEGGTD VDA VLRDFLDTD

DFAVGPGSD EPG GLNGGNWDD EYSQGQEGS EVDAHVNLD VPPDFLDTD

GDYTEAVGA VAVSEPGMD GSD SGD VDADRFSGD Orphans

VADSAVGHD VEPGGWTGD DFAVGPGSD DMQWSSGDT VGS DARQGVMME

DFD / DYD EQS VGSDYGTGD DSSMSWSGD VDYSEVGSL DFNLHDAMD

VGGHGDDFD GSEQSWTGD GSL(D) VDADRFSGD VGSDYGTGD DLQAQWAGD

VSTSSDFDP VDIAEQSTA GSLLSSQED SGS VGSMSDGYN DMTNGSVPE

DYDSLHINS ETG VDYSEVGSL DGSGSLDGD VND ETSRYSDID

EFGGPDYDT DVPRETGLD DGSGSLDGD EPYSGSISN DLDLPGVND EWNRSELGDDGY GETGGADND GSLDEALNQ (S)SMS GYDPVNDYN GMVSGQADD

DGYRSSEDS GD(S/T) HGD DSSMSWSGD VSEP GSQTVMTDD

VGSMSDGYN DGIWEHGDS DGIWEHGDS GSSMSFQDT VAVSEPGMD VDTAEISSL

EFG DMQWSSGDT VGGHGDDFD VGSMSDGYN VSEPTSNES VNHETLTAD

EFGGPDYDT ESALWGGDS MSD YDE VSYMESETD

GFNTEFGDT GAEYVGDTT VGSMSDGYN DSVYDEENS

GFNTEFGDT VTASGMSDD GSYDEVSSA

Sequencing Results (Potential Targets)

Utilizing BLAST MimoDB, peptides that are similar or identical to the sequenced phage peptides can be matched, giving clues to potential targets.

Our phage from selection against MDA-MB-231 cells may bind to… Hepatocellular carcinoma line Mahlavu Vβ1, Vβ3, and Vβ6 integrins Phosphorylated/Unphosphorylated Erβ EGFR PC3 prostate carcinoma cell line Human lewis lung carcinoma cells TNF-α SK-OV-3 (Human ovarian tumor cell

line)

Breast cancer tumor (Human- in vivo) OS-732 (osteosarcoma cell line) LNCaP (Prostate carcinoma cells) NCI-H1299 non-small cell lung cancer

cell line 9L Glioma cell line HT29 colon cancer cell line

Binding Assays 2 mL propagation of phage clones. 7b1 phage used as negative control. MDA-MB-231 used as target cell

line. Media containing serum (DMEM/F12

+ 10% FBS + 1% Ab/Am) was used for baseline comparison.

  1 2 3 4 5 6 7 8 9 10 11 12

A                        

B                        

C                        

D                        

E                        

F                        

G                        

H                        

Binding Assay (Comprehensive)

7b1

DLQAQWAGD

VDADRFSGD

DHGGGGHDS

DYDSLH

INS

DGSGSLD

GD

VNHETLTA

D

VDYSEV

GSL

VDTAEIS

SL

DFAVGPG

SD

DGYRSS

EDS

DSSMSW

SGD

DLDLPG

VND

GLNGGNWDD

ERFQ

EGGTD

VAVSEPG

MD

AGSNNEG

MT

GSLLSS

QED

GSQTV

MTDD

GAEYVGDTT

DFNLH

DAMD

DGIWEH

GDS

EFGGPD

YDT

VGENGGSA

D

DVPRET

GLD

ESALW

GGDS

DSVYD

EENS

0.00E+00

2.00E-02

4.00E-02

6.00E-02

8.00E-02

1.00E-01

1.20E-01

1.40E-01

1.60E-01

Binding Assay Complete

% Yield Target % Yield Serum

Phage Clones

% Y

ield

Binding Assay (Top 15 Yields)

GAEYVGDTT

VSTSS

DFDP

DFNLH

DAMD

GETGGADND

DGIWEH

GDS

DMTNGSV

PE

EFGGPD

YDT

AGLNYN

VDQ

VGENGGSA

D

GDYTEA

VGA

DVPRET

GLD

VLRDFLD

TD

ESALW

GGDS

GYDPV

NDYN

DSVYD

EENS

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

Binding Assay (Top 15 % Yields)

% Yield Target % Yield Serum

Phage Clones

% Y

ield

Binding Assay (Top Target Specificity *16)

GSQTV

MTDD

DVPRET

GLD

GSYDEV

SSA

GYDPV

NDYN

AGLNYN

VDQ

DYDSLH

INS

VGSDYG

TGD

VPPDFLD

TD

GSSMSFQ

DT

VLRDFLD

TD

GDYTEA

VGA

EFGGPD

YDT

DGIWEH

GDS

VGENGGSA

D

ESALW

GGDS

DSVYD

EENS

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

Binding Assay Clones With Target Specificity > 10

% Yield Target % Yield Serum

Phage Clones in order of increasing target specificity (Target % yield / Serum % yield)x100

% Y

ield

Potential Champion Clones (10)

EFGGPDYDT AGLNYNVDQ VGENGGSAD GDYTEAVGA DVPRETGLD VLRDFLDTD ESALWGGDS GYDPVNDYN DSVYDEENS0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

Potential Champion Clones

% Yield Target % Yield Serum

Phage Clones

% Y

ield

Specificity Assay

DFPPSS

AE

VGENGGSA

D

VLRDFLD

TD

GSSMSFQ

DT

VPPDFLD

TD

VGSDYG

TGD

DYDSLH

INS

AGLNYN

VDQ

GYDPV

NDYN

GSYDEV

SSA

DVPRET

GLD

GSQTV

MTDD

GDYTEA

VGA

EFGGPD

YDT

DGIWEH

GDS

VGENGGSA

D

ESALW

GGDS

DSVYD

EENS

0.00E+00

5.00E-02

1.00E-01

1.50E-01

2.00E-01

2.50E-01

Specificity Assay

% Yield 231 % Yield MCF7 % Yield MCF10A % Yield Serum

Phage Clones

% Y

ield

Future Work Designate champion clones from best binding clones Isolate protein of champion clones and modify Lipodox®

Characterize modified nanomedicine (size, zeta potential, cytotoxicity, uptake)

Isolate breast cancer stem-like cells (potentially select CD44+high/CD24-low /ESA+ cells)

Select phage specific for breast cancer stem-like cells Modify nanomedicine to target tumor initiating cells Cytotoxicity assay on CSC population in cell lines representing

all molecular subtypes Potential for in vivo applications

Acknowledgements

Special thanks to lab members Dr. Valery Petrenko Dr. Anatoliy Puzyrev James Gillespie Amanda Gross Logan Stallings

Funding from AURIC Graduate Research

Fellowship NIH Grant

ReferencesFillmore, Christine M., and Charlotte Kuperwasser. "Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy." Breast cancer res 10.2 (2008): R25.Girouard, S. D., & Murphy, G. F. (2011). Melanoma stem cells: not rare, but well done. Laboratory Investigation; a Journal of Technical Methods and Pathology, 91, 647–664. doi:10.1038/labinvest.2011.50http://www.virology.wisc.edu/virusworld/ICTV8/1fd-enterobacteria-phage-fd.jpghttp://www.cancer.org/acs/groups/content/@research/documents/document/acspc-042725.pdfhttp://www.cancercenter.com/breast-cancer/statistics/tab/breast-cancer-survival-statistics/Jayanna, Prashanth K., et al. "Landscape phage fusion protein-mediated targeting of nanomedicines enhances their prostate tumor cell association and cytotoxic efficiency." Nanomedicine: Nanotechnology, Biology and Medicine6.4 (2010): 538-546.Lammers, Twan, et al. "Drug targeting to tumors: principles, pitfalls and (pre-) clinical progress." Journal of controlled release 161.2 (2012): 175-187.Loeb, Lawrence A. "Human cancers express mutator phenotypes: origin, consequences and targeting." Nature Reviews Cancer 11.6 (2011): 450-457.Løset, Geir Åge, et al. "Expanding the versatility of phage display II: improved affinity selection of folded domains on protein VII and IX of the filamentous phage." PLoS One 6.2 (2011): e17433. * ModifiedOwens TW and Naylor MJ (2013) Breast cancer stem cells. Front. Physiol. 4:225. doi: 10.3389/fphys.2013.00225Prat, Aleix, and Charles M. Perou. "Mammary development meets cancer genomics." Nature medicine 15.8 (2009): 842-844.Siegel, Rebecca, et al. "Cancer statistics, 2014." CA: a cancer journal for clinicians 64.1 (2014): 9-29Wang, Anxin, et al. "Heterogeneity in cancer stem cells." Cancer letters 357.1 (2015): 63-68.Wang, Tao, et al. "On the mechanism of targeting of phage fusion protein-modified nanocarriers: only the binding peptide sequence matters." Molecular pharmaceutics 8.5 (2011): 1720-1728.