Antibody

Antibodies, also known as immunoglobulins, are large protein molecules produced by the body’s immune system in response to antigenic stimuli that are either infectious agents, such as a bacteria, fungi, viruses, or parasites, or other molecular components recognized by the body’s immune system as foreign. Antibodies consist of two identical pairs of polypeptide chains, comprised of a heavy chain and a light chain. The type of heavy chain determines the immunoglobulin isotype (IgA, IgD, IgG, IgE, or IgM). These polypeptide chains are arranged in a large Y-shaped protein. Both heavy and light chains have constant and variable regions. Variable regions are contained within the amino (NH₂) terminus of the polypeptide chain where complementarity determining regions (CDRs) are found. CDRs serve to recognize and bind specifically to antigen. Monoclonal antibodies are antibody molecules with identical amino acid sequences expressed by a single clone of cells.

Molecular Target of Trastuzumab

HER2 (also known as neu and ErbB2) is encoded by ERBB2/neu gene and is a member of the HER family of receptor tyrosine kinases. HER family receptors are composed of four type I receptors: EGFR/HER1/ErbB1, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4. All receptors share a similar structure composed of an extracellular ligand-binding region, a single transmembrane lipophilic segment, and a cytoplasmic tyrosine kinase-containing domain. The extracellular ligand-binding region of HER family receptors is composed of four domains (I–IV). Domains I and III are important for ligand binding. Domain II mediates receptor dimerization. Domain IV forms intramolecular interactions with domain II and thus blocks dimerization. Ligand binding to the extracellular domain of HER family members disrupts the autoinhibition conformation. This results in receptor homo- or heterodimerization and transphosphorylation followed by the activation of the downstream signaling pathways. A ligand for HER2 has not been identified yet. However, the HER2 extracellular domain adopts a fixed conformation that resembles a ligand-activated state that permits it to form a dimer in the absence of a ligand. This conformation of HER2 probably explains why HER2 is the preferred dimerization partner for the other HER family members. Moreover, although none of the ligands for the HER family receptors directly binds to HER2, activation of EGFR, HER3, or HER4 by their ligands can facilitate transactivation of HER2 through ligand-induced heterodimerization.

Overexpression of HER2 as a result of amplification of the HER2 gene (ERBB2/neu) appears to mediate the initiation, progression, and metastasis of many types of human cancer, including breast and gastric cancers. HER2 is overexpressed in approximately 20–25% of breast cancers and is associated with poor disease-free survival and poor response to chemotherapy. Gene amplification is the most common mechanism resulting in HER2 overexpression in breast cancer. HER2-positive expression was also observed in 22.1% metastatic gastric or gastroesophageal junction (GEJ) patients.

Murine Anti-HER2 Monoclonal Antibody (mAb) 4D5

The murine anti-HER2 monoclonal antibody, 4D5, was shown to recognize human HER2 and to suppress the growth of HER2-overexpressing tumor cells, as well as to enhance the sensitivity of tumor cell killing by the host immune system. Further studies demonstrated that radiolabeled 4D5 localized to HER2-overexpressing tumors in patients. In order to make a therapeutic monoclonal antibody that would maintain the half-life and effector functions of human antibodies and not be recognized as foreign by a patient’s immune system, 4D5 was humanized by engineering the 4D5 CDR heavy and light chain sequences into the framework of a consensus human monoclonal antibody IgG1 isotype. The humanized version of 4D5 (also known as rhuMabHER2; later named trastuzumab) alone or in combination with other chemotherapy agents showed significant inhibitory effects in HER2-overexpressing breast cancer cells and in mouse xenograft models of HER2-overexpressing breast cancer.

Clinical Indications

For the past 20 years, the development of monoclonal antibodies targeting HER family receptor tyrosine kinases has been intensely pursued as an important cancer therapeutic strategy. Based on results from a phase III investigational clinical trial of trastuzumab, which showed that trastuzumab in combination with chemotherapy increased time to disease progression and response rates compared to chemotherapy alone, trastuzumab received FDA approval in September 1998 for use in women with HER2-overexpressing metastatic breast cancer. Trastuzumab is indicated for treatment of patients both as first-line therapy in combination with paclitaxel chemotherapy and as a single agent for those who have received one or more chemotherapy regimens. Trastuzumab was the first HER2 targeted treatment for metastatic breast cancer. Dako’s HercepTest™, which is a semi-quantitative immunohistochemical (IHC) assay for determination of HER2 protein overexpression in breast cancer tissues, was approved simultaneously to aid in the identification of patients eligible for trastuzumab treatment. On November 16, 2006, the FDA granted approval to trastuzumab as part of a treatment regimen containing doxorubicin, cyclophosphamide, and paclitaxel for the adjuvant treatment of women with early-stage HER2-positive and node-positive breast cancer. This approval was based on evidence of a significant prolongation in disease-free survival in women receiving trastuzumab and chemotherapy compared to those receiving chemotherapy alone. On October 20, 2010, the FDA granted approval for trastuzumab in combination with cisplatin and a fluoropyrimidine (either capecitabine or 5-fluorouracil) for the treatment of patients with HER2-overexpressing metastatic gastric or GEJ adenocarcinoma who have not received prior treatment for metastatic disease. This approval is based on results of a single international multicenter open-label randomized clinical trial BO18255 (ToGA trial), which enrolled 594 patients with locally advanced or metastatic HER2-overexpressing adenocarcinoma of the stomach or GEJ.

HER2 Status

Trastuzumab is used for the treatment of breast or gastric/GEJ cancers where HER2 is overexpressed. Two testing methodologies can be used clinically to determine the HER2 status of tumor samples: immunocytochemistry (IHC) and fluorescence in situ hybridization (FISH). IHC detects the level of HER2 protein in cancer samples, whereas FISH detects the level of HER2 gene amplification. The American Society of Clinical Oncology/College of American Pathologists guideline recommends the use of both IHC and FISH testing to determine the HER2 status of human breast cancer. According to this guideline, a positive HER2 result is the IHC staining of 3+ and a FISH result of more than six HER2 gene copies per nucleus or a FISH ratio [HER2/chromosome 17 (CEP17)] of more than 2.2. In the ToGA trial, patients were eligible for trastuzumab treatment if their tumor samples were scored as 3+ on ICH or if they were FISH positive (HER2:CEP17 ratio ≥2). HER2 heterogeneity in gastric/GEJ tissue is greater than in breast cancer tissue, which may cause discordance between FISH and IHC.

Mechanisms of Action of Trastuzumab

Trastuzumab directly binds to the extracellular domain IV of HER2 to mediate an inhibitory effect on cancer cells. While the mechanisms by which trastuzumab induces regression of HER2-positive breast cancers are still being investigated, it is currently believed that the binding of trastuzumab to HER2 contributes to its therapeutic effect either by direct modulation of proliferative and pro-survival signaling downstream of HER2 or by its effect on angiogenesis and immune cell recruitment. Binding of trastuzumab to the extracellular domain of HER2 directly inhibits HER2 signaling by (a) prevention of the cleavage of HER2 extracellular domain by metalloproteinase ADAM10, (b) inhibition of either HER2 homodimerization or heterodimerization. and (c) induction of HER2 endocytosis followed by receptor degradation. Taken together, binding of trastuzumab to HER2 leads to the inhibition of pro-survival and proliferative pathways, such as the phosphatidylinositol 3-kinase (PI3K) pathway, mitogen-activated protein kinase (MAPK) pathway, and cell cycle progression (Fig. 1). Trastuzumab has also been shown to inhibit tumor angiogenesis, resulting in the decreased microvessel density of tumor in vivo and reduced endothelial cell migration in vitro. Trastuzumab has been demonstrated to kill tumor cells not only by its direct action on tumor cell signaling but also through antibody-dependent cell-mediated cytotoxicity (ADCC). More specifically, trastuzumab is an IgG₁ isotype and its constant region is capable of binding to Fc receptors presented on certain immune cells, which activates antibody effector functions. These immune cells release enzymes and factors that kill the tumor cells. Studies have demonstrated that when trastuzumab is present, immune cells preferentially target HER2-overexpressing cancer cells compared to cancer cells that do not overexpress HER2.

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Mechanisms of Trastuzumab Resistance

Treatment with trastuzumab has significantly improved the outcome in women with HER2-positive breast cancer. However, tumor resistance to trastuzumab poses a significant hurdle in breast cancer therapy. Clinical data has shown that approximately two thirds of HER2-positive metastatic breast cancer patients demonstrated primary resistance to single-agent trastuzumab and that the majority of patients with HER2-positive breast cancer who achieve an initial response to trastuzumab acquire resistance within 1 year. While the mechanisms of trastuzumab resistance are still being investigated, the following are proposed mechanisms based on clinical and preclinical studies: (1) overexpression of membrane-associated glycoprotein mucin-4 (MUC4), which may mask the epitope of HER2 recognized by trastuzumab; (2) formation of homodimers and heterodimers among EGFR, HER2, and HER3 due to overexpression of HER family ligands, which interferes with trastuzumab-mediated growth inhibition; (3) upregulation of Rac1, a member of Rho family small GTPases, which may impair trastuzumab-induced HER2 endocytic downregulation; (4) increased heterodimerization between HER2 and IGF-1R, which may interfere with trastuzumab-mediated induction of the cell cycle progression inhibitor, cyclin-dependent kinase p27 (kip1); (5) activation of PI3K either by constitutive activation, such as PIK3CA mutant, or by loss of PTEN which results in activation of PI3K signaling; (6) cyclin E amplification/overexpression; (7) increased cleavage of HER2 extracellular domain, which results in the formation p95HER2, a hyperactive membrane anchored fragment that drives breast cancer progression in vivo; and (8) loss of HER2 expression in HER2-overexpressing breast cancer cells.

Trastuzumab-Induced Cardiotoxicity

HER2 signaling in the heart is essential for cardiac development and function, as well as for the prevention of dilated cardiomyopathy. Deletion of HER2 gene in a mouse model has been shown to result in early death of the animal. In the adult heart, HER2 may continue to have an important function in modifying the cardiac response to stress. It is possible that trastuzumab treatment results in a loss of HER2-mediated signaling in cardiomyocytes and that this interferes with the heart’s ability to respond to stress. Molecular mechanisms by which trastuzumab induces cardiac dysfunction still remain elusive.

Trastuzumab cardiotoxicity in the metastatic clinical trial: Cardiotoxicity was initially reported in a phase III trial, which tested the efficacy of combining chemotherapy with trastuzumab versus chemotherapy alone in metastatic breast cancer disease. This study found that the combination of anthracyclines and cyclophosphamide (AC) alone was associated with a rate of overall cardiac dysfunction (CD) of 8% and a New York Heart Association (NYHA) class III/IV rate of 4%. However, when trastuzumab was added to AC, the overall CD rate was 27% with a rate of NYHA class III/IVCHF of 16%. Paclitaxel alone was associated with a CD rate of 1% and a NYHA class III/IV rate of 1%. The addition of trastuzumab to paclitaxel resulted in a CD rate of 13% with a NYHA class III/IV rate of 2%. Trastuzumab associated CD is manifested as severe congestive heart failure (CHF) and a significant decrease in left ventricular ejection fraction (LVEF).

Trastuzumab cardiotoxicity in the adjuvant trials: Adjuvant trastuzumab prolonged survival, including both overall survival (OS) and disease-free survival (DFS), among women with HER2-positive early breast cancer, but the benefits were accompanied by the risks of cardiac toxicity. Based on the assessment of several major adjuvant trastuzumab clinical trials, including the National Surgical Adjuvant Breast and Bowel Project (NSABP) B-31, North Central Cancer Treatment Group N9831, Herceptin Adjuvant, Breast Cancer International Research Group 006, and Finland Herceptin trials, up to 4% of patients experienced severe CHF during treatment. However, a large number of patients on these trials experienced some form of cardiotoxicity that ultimately required discontinuation of trastuzumab. Approximately 14% of patients in the NSABP B-31 trial were reported to discontinue trastuzumab treatment due to asymptomatic decrease in LVEF. A systematic review of eight clinical trials, which involved 11,991 women with HER2-positive operable breast cancer who were treated with trastuzumab or the standard therapy (with no trastuzumab), found that breast cancer mortality was reduced by one third, but the risk of cardiac toxicity (i.e., CHF and LVEF) was five times more likely for women receiving trastuzumab than women receiving standard therapy alone. The incidence of cardiac toxicities may be associated with duration of trastuzumab administration, such that the longer treatment (1 year) may involve a greater risk of severe heart toxicities than shorter treatment (6 months or less).

Trastuzumab-associated cardiotoxicity is believed to be reversible upon stopping the treatment. However, the concept that trastuzumab-related cardiotoxicity is reversible has been challenged due to the lack of sufficient long-term follow-up cardiac data. Additionally, in NSABP B-31trial among those diagnosed with a cardiac event, two thirds of patients continued to receive cardiac medications and 71% had a decrease in LVEF relative to baseline on follow-up, suggesting persistent cardiac dysfunction.