Date of Award

January 2011

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Mark Mamula

Subject Area(s)

Medicine

Abstract

EFFECTS OF ERBB2 VACCINATION ON THE MURINE HEART. Sumayya Ahmad, Renelle Gee, Kerry Russell, and Mark J. Mamula. Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510.

ErbB2, a member of the epidermal growth factor receptor family, is found in many tissues of the body. It is known to potentiate growth via vascularization and anti-apoptotic signals, amongst other things. Its presence on breast cancer cells results in a particularly poor prognosis. A novel monoclonal antibody targeting this receptor, Herceptin, allows precise targeting of these cells and results in longer life spans for these patients. However, early trials of the biologic agent demonstrated that up to one third of patients taking Herceptin get depressed cardiac function when taking the drug in conjunction with Doxorubicin, a common first-line chemotherapeutic agent. Since its creation, the mechanism of action of Herceptin on the heart, and in particular its affinity for the cardiac ErbB2 receptor, has been profoundly explored. However, potential autoimmune mechanisms of cardiac dysfunction with anti-ErbB2 agents have not been investigated previously.

We examined the effects of a peptide vaccine (p601) targeting a 16 amino-acid sequence on the extracellular region of ErbB2 on immune responses and cardiac function in mice. Unpublished data from our lab showed that genetically engineered mice with ErbB2+ tumors demonstrated tumor decline when immunized with this vaccine. To accomplish this, we performed echocardiography on immunized mice to assess cardiac function and serologic and immunohistochemistry studies to examine the immune reactivity to ErbB2 and to heart muscle.

We found that immunization of Balb/c mice with p601 did not result in echocardiographic abnormalities. However, p601-immunized FVB mice in which ErbB2 was up-regulated demonstrated arrhythmias not found in any other mice. Furthermore, ErbB2-immunized mice sera as well as Herceptin bound to the extracellular domain of ErbB2 as well as heart lysate not found in PBS controls. After analyzing the western blot via spectrometry, we discovered that the bound region contained alpha cardiac myosin heavy chain. A BLAST search revealed a homologous 8 amino acid peptide region between myosin and ErbB2, suggesting that anti-ErbB2 agents may be binding a component of myosin. Immunofluorescence staining revealed increased non-specific anti-IgG binding to immunized mice hearts compared to PBS controls. Furthermore, H&E stain revealed increased leukocyte adhesion to the surface of arterioles in a Balb/c immunized control. FVB immunized mice that demonstrated arrhythmias also had sarcomere destruction in addition to leukocyte adhesion to the surface of arterioles.

Because mice immunized with two synthetic peptides spanning the homologous region of ErbB2 and myosin did not develop ErbB2 reactivity or echocardiographic abnormalities, we looked for evidence of complement deposition in mouse hearts immunized with the p601 and the anti-ErbB2 and myosin peptides. Examination of tissues by immunohistochemistry revealed increased C3 staining in small pockets of ventricular tissue in p601-immunized controls as compared to both PBS and anti-ErbB2 and myosin peptides. Since such staining is not typically found in autoimmune heart disease, it may indicate another process of immunological destruction, such as immune complex deposition. Our studies suggest an autoimmune mechanism for the action of anti-ErbB2 agents on the heart, in contrast to previous studies that have focused on the effects of receptor binding and signal transduction on the cardiac ErbB2 receptor.

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