Novel Anti-HER2 Fragments for Better Detection and Therapy

Institution: University of California, Los Angeles
Investigator(s): Shannon Sirk, BA -
Award Cycle: 2008 (Cycle 14) Grant #: 14GB-0157 Award: $38,000
Award Type: Dissertation Award
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure



Initial Award Abstract (2008)

HER2-positive tumors account for 25-30% of all breast cancers diagnosed and are often aggressive and highly metastatic. Currently, HER2 status is determined by immunohistochemistry of a tumor biopsy, which is both invasive and time-consuming. Whole body or breast imaging of HER2-positive tumors would aid in earlier and more accurate detection and diagnosis. Trastuzumab (Herceptin), an intact “humanized” monoclonal antibody, binds HER2 and has been used to treat HER2-positive breast cancer. Due to its large molecular size and the presence of an Fc antibody region, trastuzumab has a long half-life that is ideal for therapy. However, trastuzumab has far too much “background” (non-tumor localization at any time point) for applications such as imaging.

Antibody engineering makes it possible to tailor the size and composition of an antibody for specific purposes. We propose to create an anti-HER2 “cys-diabody”, which is one third the size of the parental trastuzumab and lacks the antibody’s Fc portion, resulting in superior pharmacokinetics (drug absorption, distribution, metabolism and excretion) for imaging applications. This molecule will contain an amino acid (cysteine) modification, enabling site-specific coupling of functional groups for imaging and therapy using simple thiol chemistry. Because a large percentage of HER2-positive tumors become refractory to trastuzumab therapy, targeted delivery of a drug via the cys-diabody presents an attractive alternative to traditional chemotherapy with higher dose delivery to the tumor, while sparing normal tissue. Thus, the aims of this study are 1) to create and characterize the anti-HER2 cys-diabody, a small antibody fragment capable of HER2 targeting and site-specific conjugation to imaging and therapeutic moieties; 2) to specifically modify anti-HER2 cys-diabodies with cargo for fluorescent imaging, and analyze their ability to demonstrate bi-functionality – HER2-binding and fluorescence; and 3) to conjugate anti-HER2 cys-diabodies to therapeutic nanoparticles and assess their effect on breast cancer cells and whole animal tumor models .

The potential impact on breast cancer of the anti-HER2 cys-diabody would be to improve non-invasive detection, diagnosis, and treatment of HER2-positive breast cancer. The use of a targeted probe for imaging HER2-positive tumors, as opposed to traditional imaging methods, decreases the level of uncertainty that a physician or patient must deal with when interpreting results. More importantly, in cases of trastuzumab-refractory disease, targeted therapy using drugs or therapeutic nanoparticles conjugated to anti-HER2 cys-diabody presents an appealing alternative to systemically administered chemotherapy.




Final Report (2009)

Note: the PI resigned the grant after 1-yr because she graduated with a Ph.D. degree from UCLA.

Tumor targeting antibodies specifically recognize cancer cells, but not normal tissue. Attaching toxic payloads to therapeutic antibodies increases the tumor-killing effect. Full-size antibodies remain in the circulation for weeks, leading to tumor accumulation but high background in imaging applications. Engineering antibodies into smaller fragments produces tumor targeting proteins with rapid clearance profiles. High contrast images can be obtained at earlier time points. More efficient screening could lead to better diagnoses and treatments, especially in the case of HER2-positive breast cancer. The goal of this work was to create small engineered HER2-targeting antibody fragments to deliver a variety of cargo to HER2-positive breast tumors, specifically for diagnostic imaging and targeted therapeutics. The cys-diabody is a bivalent antibody fragment of 50 kDa containing a C-terminal cysteine modification for site-specific modification via thiol chemistry.

The first aim was to create a HER2-targeting cys-diabody and characterize binding and in vivo delivery potential. These goals were accomplished. The second aim was to site-specifically modify the cys-diabody with fluorescent tags, and examine in vitro and in vivo behavior. The in vitro studies were completed but the in vivo studies were not carried out due to the more promising PET applications that required large amounts of protein. The third aim was to site-specifically modify the anti-HER2 cys-diabody with a therapeutic nanoparticle and study it in vitro and in vivo. This aim was not completed, as the nanoparticle could not be provided by our collaborators. Instead, two studies using copper-labeled cys-diabody were performed, one using a random and one using a site-specific labeling approach. The results demonstrated the ability of the cys-diabody to target HER2-positive tumors in vivo. The similarity between the results showed that the site-specific labeling technique is a viable method for radiolabeling a cys-diabody. Additionally, a novel method for fluorine-18 labeling of the cys-diabody was developed with potential for rapid production of biomolecule PET tracers. The most important barrier that was overcome involved re-focusing the scientific questions once the therapeutic nanoparticle arm of the studies did not work out.

The major accomplishments of this research are the creation of a novel HER2-targeting biomolecule that can carry cargo to HER2-positive tumors in vivo, and the development of a streamlined method for radiolabeling biomolecules for same-day, high-contrast imaging applications. The project will be continued with further investigation into fluorine-18 labeling methods, focusing on site-specific conjugation to the C-terminal cysteines. There is no other breast cancer funded research in the lab.



Cys-diabody Quantum Dot Conjugates (ImmunoQdots) for Cancer Marker Detection.
Periodical:Bioconjugate Chemistry
Index Medicus: Bioconjug Chem
Authors: Barat B, Sirk SJ, et al, and Wu AM
Yr: 2009 Vol: 20 Nbr: 8 Abs: Pg:1474-81

Site-specific, thiol-mediated conjugation of fluorescent probes to cysteine-modified diabodies targeting CD20 or HER2.
Periodical:Bioconjugate Chemistry
Index Medicus: Bioconjug Chem
Authors: Sirk SJ, Olafsen T, Barat B, Bauer KB, Wu AM
Yr: 2008 Vol: 19 Nbr: 12 Abs: Pg:2527-34