UNIVERSITÀ DEGLI STUDI DI BRESCIA Monoclonal Antibodies as a novel class of cancer therapeutics Patrizia Alessi 07.01.2008 The concept of "magic bullets" for the treatment of disease site of disease (e.g., tumor) What are antibodies? Antibodies are immune system-related proteins called immunoglobulins produced by B lymphocyte/plasma cells in response to the presence in the body of antigens: (i.e., foreign proteins or polysaccharides derived form bacteria, toxins, yeast, viruses or other cells). Antibodies (or immunoglobulins ) generally assume one of two roles: 1) plasma membrane bound antigen receptors on the surface of a B-cell 2) free antibodies in cellular fluids functioning to intercept and eliminate antigenic determinants. Antibody structure Immunoglobulins are composed of four polypeptide chains: two "light" chains and two "heavy" chains. "Hypervariable" regions, or "Complementarity Determining Regions" (CDRs) are found within the variable regions of both the heavy and light chains. These regions serve to recognize and bind specifically to antigen. Antibody type The type of heavy chain determines the immunoglobulin isotype (IgA, IgD, IgG, IgE, IgM, respectively). Each chain has "constant" and "variable" regions as shown in the figure. Variable regions are contained within the amino (NH2) terminal end of the polypeptide chain (amino acids 1-110). Antibody: different parts with different functions Fab & Fc IgG Monoclonal antibody: classical procedure Hybridoma technique Hybridoma technique Pros - Reliable technique taking advantage of an “in vivo affinity maturation process” crucial for the generation of high affinity MAb. Cons -If the antigen is identical or very homologous between the rodent species and humans, such "anti-self" antibodies are difficult or impossible to obtain. Rodent immunoglobulins have sequences that are recognized as strong antigens by the human immune system, thus preventing repeated administration in vivo in humans (i.e., HAMA reactions: Human anti-Mouse antibody). Chimeric or Humanized MAb are thus necessary for clincal use. - Time consuming Monoclonal antibody: NEW TECHNIQUES (1) One promising way to increase antibody yield or develop new antibodies may be by using genetically altered animals. Abgenix, a company in Fremont, CA, has developed the transgenic “XenoMouse” in which the mouse antibody-producing genes have been inactivated and functionally replaced by approximately 90% of the human Ig gene loci in germline configuration, coding for the heavy and [kappa] light chains. Upon immunization with any specific human or nonhuman antigens, the “XenoMouse” generates MAbs, which are fully human Igs, with high affinities and antigen-binding specificities. “XenoMouse” strains producing specifically IgG1, IgG2, or IgG4 isotypes have also been created to generate panels of diverse and highly specific MAbs. Monoclonal antibody: NEW TECHNIQUES Kirin Brewery Company, Japan, has developed another transgenic mouse known as the “Trans-Chromo” mouse. The endogenous IgH and IgG loci of the “Trans-Chromo” mouse were inactivated, but it harbors 2 individual human chromosome fragments, derived from human chromosomes 2 and 14, that contain whole human Ig light- and heavy-chain loci, respectively. These mice are capable of producing every subtype of fully human Ig, including IgA and IgM. In these transgenic mouse models, human antibodies with high affinity to an immunized antigen are naturally selected by the murine immune system via an affinity maturation process, and thereby show increased diversity of the MAbs. Monoclonal antibody: NEW TECHNIQUES (1) At present at least 33 human Ab produced in transgenic mice are in clinical development, including 5 in Phase III clinical trials: 1) panitumumab Abgenix, Inc.and Amgen Inc., an epidermal-grow-factor receptor (EGFR) specific MAb for the treatment of colorectal cancer 2) ipilimumab Medarex, Inc. and Brystol-Myers Squibb Company, a cytotoxic T-lynphocyte antigen-4 (CTLA-4) for the treatment of melanoma 3) denosumab Amgen Inc. a receptor activator of nuclear factor-κB (RANK) ligand-specific MAb for post-menopausal osteoporosis and to avoid bone loss in hormone-ablation therapy during treatment of cancer 4) zanolimumab (HuMax-CD4) Genmab A/S and Serono S.A. a CD4 specific Ab for treatment of cutaneous T-cell lymphoma 5) golimumab Centocor, Inc a tumor necrosis factor (TNF) specific MAb for treatment of rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis. Transgenic mouse Pros: Fully human antibodies with high affinity to an immunized antigen. (i.e., are naturally selected by the immune system via an affinity maturation process, and thereby show increased diversity of the MAbs). Cons: - If the antigen is a self protein for humans, such "anti-self" antibodies are difficult to obtain. - More time consuming (immune response is less robust in transgenic animal) - Difficult to obtain species/crossreactive Ig - Technology nor available to the majority of Lab worldwide. - Expensive technology Monoclonal antibody: NEW TECHNIQUES (2) Antibody Phage Display One human antibody isolated by phage-display has been approved widely for therapeutic use: adalimumab (Humira) Abbott Lab a TNF-specific MAb used for treatment of rheumatoid and psoriatic arthritis Monoclonal Antibodies as Novel anti-Cancer Therapies Therapeutic antibody Monoclonal antibodies for cancer. ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment. MAbs for cancer immunotherapy Since the mid-1990, antibodies have emerged as an important new drug class. Indeed, 18 antibodies are now approved (17 have been marketed and 1 withdrawn) for therapeutic use in the United States across diverse clinical setting, including oncology, chronic inflammatory diseases, transplantation, infectious diseases and cardiovascular medicine. These approved antibody therapeutics include 14 unmodified IgG, 1antibody-drug conjugate and 1 Fab. At least 150 antibodies are in clinical development. Some of these are chimeric or humanized antibodies developed in the early days of antibody engineering, whereas others are derived from novel technologies. Bioparmaceutical in the pipeline 2006 Potent MAb therapeutics by design Abs belong to a well-established drug class that has a high success rate from the first use in humans to regulatory approval: 29% for chimeric Abs, 25% for humanized Abs (only 11% small-molecule drug). Abs are generally well tolerated by humans, although infusion reaction (first dose) are common but manageable (e.g.: rituximab). Their clinical potential can readily be increased by improving their existing activity or by endowing them with new activities. Restriction of the targets of those on the surface or exterior of host cells or Invading pathogens. For the antigen, factors that determine the efficacy of the targeting of the antibody include specific tissue expression, abundance, availability, number of antigens per cell, and antigen (receptor) shedding. Antibody drugs are expensive, usage limitation. Adverse events (natalizumab, Tysabri) Iterative design of antibody therapeutics Goals for antibody therapeutics Improve efficacy = improve antitumor activity and patient survival (combination therapy) Improve safety = increasing the potency and the half life in plasma (dose or frequency of administrator can be reduced) Reduce cost = use of alternative host for production FDA approved MAb for cancer immunotherapy Muromonab-CD3 Johnson & Johnson New Brunswick, New Jersey Orthoclone OKT3 Murine, IgG2a, anti-CD3 Rituximab Genentech Rituxan Chimeric, IgG1 , anti-CD20 Daclizumab Hoffmann-La Roche Basel Zenapax Humanized, IgG1 , anti-CD25 Basiliximab Novartis Basel Simulect Chimeric, IgG1 , anti-CD25 Palivizumab MedImmune Gaithersburg, Maryland Synagis Humanized, IgG1 , anti-respiratory syncytial virus Infliximab Centocor Remicade Chimeric, IgG1 , anti-tumor necrosis factor (TNF ) Trastuzumab Genentech Herceptin Humanized, IgG1 , anti-HER2 Gemtuzumab ozogamicin Wyeth Madison, New Jersey Mylotarg Humanized, IgG4 , anti-CD33; immunotoxin Alemtuzumab Genzyme Cambridge, Massachusetts Campath-1H Humanized, IgG1 , anti-CD52 Ibritumomab tiuxetan Biogen Idec Zevalin Murine, IgG1 , anti-CD20; radiolabeled (Yttrium 90) Adalimumab Abbott Deerfield Park, Illinois Humira Human, IgG1 , anti-TNF Tositumomab-I131 Corixa Seattle Bexxar Murine, IgG2a , anti-CD20; radiolabeled (Iodine 131) Cetuximab Imclone Systems New York Erbitux Chimeric, IgG1 , anti-Epidermal growth factor receptor Bevacizumab Genentech Avastin Humanized, IgG1, anti-vascular endothelial growth factor Tunable properties of antibodies Immunigenicity is influenced by biochemical (sequence, origin, stability, modification), clinical parameter (dose, route and frequency of administration) and patient-specific factors (disease and immune status, MHC haplo-type and concomitant medication. Immunogenicity Hybridoma technique 3% success Poor effector functions Poor half life in plasma MAbs therapeutics generation Tunable properties of antibodies Immunigenicity is influenced by biochemical (sequence, origin, stability, modification), clinical parameter (dose, route and frequency of administration) and patient-specific factors (disease and immune status, MHC haplo-type and concomitant medication. Antigen binding specificity: cross-species reactivity for evaluation of efficacy and toxicity. Antigen binding-affinity: increasing Kd (display technologies) Biological activities associated with variable domains : increasing efficacy and biological potency by functional screening after affinity maturation, correct selection of the Ab format. Antibody formats How to improve performances Effector functions: the right isotype Intact antibodies targeted to bacteria, viruses or cells can mediate target elimination through the interaction of macrophages, neutrophils and natural killer cells, which induce phagocytosis and antibody-dependent cellular cytotoxicity (ADCC), as well as through activation of the complement system (CDC). Because the interaction between antibodies and effector molecules, such as Fc receptors or complement, is mediated by the Fc part of the antibody molecule, antibody fragments without Fc cannot attract such effectors. A prerequisite for effective target elimination comprises crucial elements both within the antigen and the antibody Antibody effector functions Tunable properties of antibodies Pharmacokinetics: the terminal half life of antibodies in plasma can be tuned over a wide range to fit clinical goals. A way is to tailor the interaction Fc and FcRn. Another is the PEGylation… Molecular architecture: the modular domain architecture or No Fc effector functions PEGylation More efficient penetration of tumor Faster clearance Greater tumor uptake Binding without crosslinking (monovalent) Small size High affinity, solubility and robust expression Camels, Llamas and sharks ? Mode of actions for Mabs 1980-2005 Tunable properties of antibodies Internalization is desirable for certain applications, such as for the delivery of cytotoxic drug and immunoliposomes Biophysical properties such as stability, solubility and folding kinetics (role of the framework) Cancer Immunotherapy with MAb Examples of Monoclonal antibody endowed with therapeutic efficacy against human tumors: ERBITUX® (Cetuximab) = Anti-EGF-r (EU 2004) Herceptin (Trastuzumab) = Anti-HER2/neu (EU 2000) Avastin (bevacizumab) = Anti-VEGF (EU 2005) HER2/neu and Cancer • The human HER-2/neu (c-erbB2) gene product, like the epidermal growth factor receptor, is a transmembrane receptor protein that includes an intracellular tyrosine kinase domain. • In normal cells, two copies of the gene per cell (one on each chromosome 17) are present. However, in the presence of HER-2/neu amplification there may be as many as 50 or 100 HER-2/neu genes per tumor cell. This gene amplification results in overexpression of p185 HER-2/neu at both the mRNA and protein levels. As a result, instead of ~ 20,000 to 50,000 HER-2/neu molecules per cell, there can be as many as 2,000,000 molecules per cell in neoplastic tissues. • HER2/neu overexpression has been previously suggested to represent a major prognostic factor in several human cancers including endometrial, breast and ovarian carcinomas and to be correlated with resistance to chemotherapy HER2/neu and Breast Cancer • An estimated 211,240 women will be diagnosed with breast cancer in the United States in 2005. Of these, about 30 percent have lymph node-positive breast cancer, and about 20 percent of these tumors overexpress the HER2 protein, the target for trastuzumab. • Breast cancer is the most commonly diagnosed cancer in women and the second leading cause of cancer-related death in women in this country. An estimated 40,110 deaths from female breast cancer will occur in 2005 in the United States, accounting for about 15 percent of all cancer-related deaths in women in the nation. What is Herceptin ? Herceptin® (Trastuzumab) is the only FDA-approved therapeutic for HER2 protein overexpression metastatic breast cancer. Herceptin is a therapy for women with metastatic breast cancer whose tumors have too much HER2 protein. For patients with this disease, Herceptin is approved for first-line use in combination with paclitaxel, and as a single agent for those who have received one or more chemotherapy regimens. Lowering immunogenicity Technologies for antibody optimization Construction of humanized gene. humAb4D5 variants In vitro potency Inhibition of SK-BR-3 (breast carcinoma cells) proliferation In vivo efficacy In vitro cytotoxicity in combination with paclitaxel In vivo therapeutic properties Dose finding Combination therapy Data of complete tumor regression Synergistic interaction at clinically relevant [drug] Combination therapy How does Herceptin work Frequency of efficacy end point events in the Herceptin® Adjuvant (HERA) trial Is Herceptin effective against Human cancer? Results from two large randomized clinical trials for patients with HER-2 positive invasive breast cancer show that those patients with early-stage breast cancer who received Herceptin® (trastuzumab) in combination with chemotherapy had a significant decrease in risk for breast cancer recurrence compared with patients who received the same chemotherapy without trastuzumab. Patients in the clinical trials who received trastuzumab in combination with standard combination chemotherapy had a 52 percent decrease in disease recurrence compared to patients treated with chemotherapy alone. This difference is highly statistically significant. Among the specific findings from the study were: At a median follow-up of two years, there were 261 events (such as return of the cancer, second primary cancer, or death before recurrence) in the control group and 133 events in the trastuzumab group. The percentage of patients alive and disease-free at three years was 87.1 percent in the trastuzumab group and 75.4 percent in the control group. After four years, the respective percentages were 85.3 percent for trastuzumab vs. 67.1 percent for those on standard chemotherapy. Women taking trastuzumab with a particular chemotherapy regimen had a 33 percent reduction in risk of death (October 20, 2005 New England Journal of Medicine). From the trastuzumab era to new target therapies: beyond revolution In the October 2005 edition of The New England Journal of Medicine, Gabriel Hortobagy claimed that ‘‘the results of trastuzumab adjuvant trials are not evolutionary but revolutionary’’ It is obvious that there are certain limitations in the use of this extraordinary drug. The first is that trastuzumab can only be used in a small number of patients; the majority (HER-2 negative) cannot benefit from its use. The second problem is the potential cardiotoxicity of this treatment. The third dilemma is whether the duration of therapy should be short or prolonged. The fourth problem concerns the high cost of trastuzumab treatment to the National Health Service, which is not to be disregarded. Acquired and de novo resistance to trastuzumab is a significant clinical challenge. Biopharmaceutical benchmarks 2006 What is fueling the biotech engine? Trends in US sales of MAb Top companies Background Nearly a century ago, Paul Ehrlich first espoused the concept of "magic bullets" for the treatment of disease. By exploiting inherent differences between healthy and diseased cells, therapeutic agents could be designed to specifically target diseased cells while leaving healthy cells unperturbed. While traditional therapeutic modalities, such as chemotoxic drugs or ionizing radiation, are very effective at killing tumor cells, they often fail to adequately discriminate between normal and malignant tissues. Thus, these treatments exact a heavy toll on the patient and can severely limit the dose or duration of treatment to suboptimal levels, resulting in poor outcomes for the management of disease, quality of life, and overall survival. Clearly, elucidating novel therapeutic strategies to target cytotoxicity exclusively to malignant cells would greatly reduce deleterious side effects to normal tissues, enhance the tolerance of the patient to therapy, and result in more effective treatment. Christiansen JJ et al., Molecular Cancer Therapeutics, 3(11):1493-501, 2004.
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