The document describes research into creating an antibody-drug conjugate (ADC) to treat metastatic triple negative breast cancer. Key points: - Researchers developed an antibody that recognizes HSPG2, a biomarker expressed on metastatic breast cancer cells, and aims to link this antibody to the chemotherapy drug paclitaxel. - The ADC would be synthesized by modifying the antibody with thiol groups and paclitaxel with maleimide groups, allowing them to conjugate. - While the antibody thiolation was successful, complications arose in activating paclitaxel for conjugation. Other drug molecules will be explored to complete the ADC synthesis.

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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.
Significance
The 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.
Conclusion
With 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.
References
1. 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