Transcript
Page 1: Aimee Skrei Melendy Poster

The University of Minnesota is an equal opportunity educator and employer. This publication/material is available in alternative formats upon request. Printed on recycled and recyclable paper with at least 10 percent postconsumer material.

Introduction•Even with increased awareness, diagnostic advances, and

molecularly-targeted therapies, morbidity and mortality associated

with breast cancer remain high.

•Patients presenting with a metastatic disease have a 5-year

survival rate of just 25%.1,2 This dramatic reduction in survival rates

due to metastasis suggests a need to focus on the development of

technologies that can detect and eliminate metastatic cancer.

•In order to aid in the treatment of metastasized breast cancer, the

mentor’s group has discovered that the proteoglycan HSPG2 can be

used as a unique cell surface biomarker for triple negative breast

cancer (TNBC) cells that have undergone EMT. They have

developed an antibody that will recognize HSPG2 expressed on

EMT-phenotypic cells in a mouse model of human TNBC.

•The objective of this research project is to create an antibody-drug

conjugate utilizing a known chemotherapeutic agent, paclitaxel, to

the anti-HSPG2 antibody.

•The antibody-drug conjugate’s anticancer effectiveness will be

examined through comparison with commercially available

Abraxane® (albumin paclitaxel, a main-stay treatment for metastatic

breast cancer).

•We hypothesize that HSPG2 is a key target for delivering

chemotherapeutic agents specifically to metastases in TNBC

patients.

Methods

Design and develop a synthetic route for preparing

an antibody-drug conjugate

•The antibody-drug conjugate will be synthesized by first modifying

the lysine residues on the antibody using 2-iminothiolane in order to

add a thiol group. The number of thiol groups added to the antibody

will be determined using the AAT Bioquest Amplite Flurometric Total

Thiol Quantitation Assay Kit- Green Fluorescence.4

•Simultaneously, the paclitaxel molecule or other drug molecule will

be modified to incorporate a reactive maleimide group.4 Completion

of the reaction will be confirmed by NMR. Synthetic techniques for

the conversion of paclitaxel to paclitaxel-maleimide is previously

published.5

•Other drug molecules of interest include doxorubicin and

salinomycin.

•Maleimide functionalized paclitaxel will then be conjugated to the

imino-thiolated antibody to generate the antibody-drug conjugate.

The conjugate will be characterized by mass spectroscopy.

•The antibody-drug conjugate will then be tested using in vitro cell

culture studies to ensure that the chemical modifications have not

affected its binding ability to the antigen of interest.

•In addition, the cytotoxic efficacy of the conjugate will be

determined in vitro against a panel of TNBC cell lines to obtain

preliminary proof for the anticancer effectiveness of the conjugate.

SignificanceThe antibody-drug conjugate is expected to provide a safe and

effective approach to treat metastatic breast cancer. Studies

suggest that conventional chemotherapies are not effective in

eradicating metastases. Antibody-drug conjugates have the ability to

deliver drugs in a targeted manner. This precise delivery of drug to

cancer cells could lead to improved effectiveness and reduced

toxicity. This research has a substantial societal impact by providing

a powerful approach to address the dismal survival rate for

metastatic breast cancer.

Results and Discussion•To accomplish our goal of completing an antibody-drug conjugate,

many hurdles regarding purifying and testing conjugate components

had to be overcome.

• Using Traut’s Reagent to thiolate specific lysine residues present

on the antibody, a linear relationship of “thiols per antibody” was

found

•Unexpected complications were encountered upon trying to

activate the drug molecule, Paclitaxel, in order to allow for

conjugation

•Several different drug molecules were obtained from different

laboratories , but the conjugation could not be completed

•Further methods to be explored to confirm conjugation include

absorption spectroscopy, NMR, size-exclusion chromatography,

mass spectrometry, particle sizing.

ConclusionWith the completion of this project, a detailed procedure was

devised for the thiolation of a specific antibody. An antibody-drug

conjugate was not able to be completed due to unforeseen

complications with activating the drug molecule. Future laboratory

studies can be completed using different drug molecules which will

allow for simplified activation, and thus conjugation with the

antibody.

References1. Morrow, Monica and Goldstein, Lori. Surgery of the Primary Tumor in

Metastatic Breast Cancer. Journal of Clinical Oncology. 2006; 24: 2694-2696.

2. Heerboth, Sarah, et al. EMT and tumor metastasis. Clinical and Translational

Medicine. 2015; 4:6.

3. Yu, F., et al. MicroRNA 34c gene down-regulation via DNA methylation

promotes self-renewal and epithelial-mesenchymal transition in breast tumor-

initiating cells. J Biol Chem. 2012; 287: 465-473.

4. Hermanson, Greg. Bioconjugate Techniques, 3rd ed.; Academic Press: San

Diego, 2013.

5. Greenwald, R., Choe, Y., McGuire, J., Conover, C. Effective drug delivery by

PEGylated drug conjugates. Advanced Drug Delivery Review. 2003; 55:

217-250.

6. Pierce Traut’s Reagent (2-iminothiolane). Life Technologies. Thermo Fisher

Scientific Inc. 2015.

Antibody-drug conjugate for the treatment of metastatic breast cancer

Aimee Skrei, Stephen Kalscheuer, Jayanth Panyam, PhD

University of Minnesota, Minneapolis, MN, 55455

Figure 1. Reaction Scheme of Traut’s Reagent. The above

reaction depicts the thiolation of antibody that was achieved with

the first portion of the reaction.

Figure 4. Reproducibility and linearity of thiolation results.

The 20X reaction was completed separately from the 1X, 5X, 10X,

and 40X reactions. The actual sample yielded an incorporation of

3.1 thiols per antibody. Using the plot of “thiol per antibody versus

2-IT molar excess—excluding 20x”, the predicted incorporation

was 3.8 thiols per antibody.

Molar Excess 2-IT Thiols per Antibody

1X 0.47

5X 0.97

10X 1.87

20X 3.08

40X 7.40

Figure 3. Results of

thiolation of breast

cancer specific antibody.

The resulting number of

thiols per antibody

molecule at varying 2-

iminothiolane reaction

input levels.

Figure 2. Reaction Scheme of Maleimide Reaction. The

maleimide functional group present on the drug molecule can react

with the sulfhydrol group present on the antibody molecules to

create a drug-antibody conjugate.

Figure 5. Influence of antibody thiolation on specific binding to metastatic breast cancer cell line. Isotype control human IgG, and a

relevant therapeutic human IgG targeting metastatic breast cancer cell line MDA-MB-231-LM2 were thiolated under 40x molar excess 2-IT.

Following removal of excess 2-IT, the influence of thiolation on antibody specific binding capacity was evaluated by flow cytometry. 100nM of non-

thiolated (grey histogram) and thiolated (red histogram) antibodies were separately incubated with target cells. Following wash steps, cell binding

was evaluated with a secondary anti-human Dylight650 conjugated antibody. (A) Binding of isotype human IgG to target cells is negligible, and

does not change as a result of thiolation. (B) Therapeutic IgG binding capacity is maintained with the addition of free thiols. A Minor increase in

binding of thiolated antibody that was observed is considered negligible.

A B