LOGO Dr. Nermin Hassan KSU Microbiology section

 Diagnostic Applications Biosensors & Microarrays  Therapeutic Applications Transplant rejection Muronomab-CD3 Cardiovascular disease Abciximab Cancer Rituximab Infectious Diseases Palivizumab Inflammatory disease Infliximab  Clinical Applications Purification of drugs, Imaging the target  Future Applications Fight against Bioterrorism

1.Blocking action of molecular targets  Can work antagonistically by binding a receptor to prevent activation  Can also bind the antigen and prevent activation 2.“Magic Bullet”  Compound with target specificity is coupled with various effector groups Toxins, radioactive, … 3.Signal molecules  Coupled to mediators of apoptosis, etc.

Diagnostic Uses:  Detection of:  Markers on lymphocytes and antigens on cells and micro-organisms a. Lymphocyte subsets determination (CD markers) b. Tissue typing c. Identification and typing of bacteria and viruses d. Detection of tumour related antigens

Diagnostic Uses: e.g. Detection of CEA (carcinoembrionic Ag), alpha foeto-protiens and other tumour markers e. Hormonal assay f. Radiolabelled monoclonal antibodies are used in vivo to detect or locate tumour antigens, e.g. for detection of breast cancer metastases.

To suppress the immune system 1- Anti-CD3 (OKT3) and two humanized anti-CD3 monoclonals. Bind to the CD3 molecule on the surface of T cells. Used to prevent acute rejection of organ, e.g., kidney, transplants. The humanized versions show promise in inhibiting the autoimmune destruction of beta cells in Type 1 diabetes mellitus.humanizedacute rejectionType 1 diabetes mellitus 2- Monoclonal antibodies, Binds to tumor necrosis factor- alpha (TNF-α). Shows promise against some inflammatory diseases such as rheumatoid arthritis (by blunting the activity of Th1 cells).tumor necrosis factor- alpharheumatoid arthritisTh1 cells Therapeutic Uses:

To suppress the immune system  3- Omalizumab (Xolair®). Binds to IgE thus preventing IgE from binding to mast cells. Shows promise against allergic asthma.mast cellsallergic asthma  4- Daclizumab (Zenapax®). Binds to part of the IL-2 receptors produced at the surface of activated T cells. Used to prevent acute rejection of transplanted kidneys. Has also showed promise against T-cell lymphoma.

 To kill or inhibit malignant cells  Rituximab (trade name = Rituxan®). Binds to the CD20 molecule found on most B-cells and is used to treat B-cell lymphomas.B-cells lymphomas  Zevalin®. This is a monoclonal antibody against the CD20 molecule on B cells (and lymphomas) conjugated to either  the radioactive isotope indium-111 (111In) or  the radioactive isotope yttrium-90 (90Y) MAGIC BULLET  Bexxar® (tositumomab ). This is a conjugate of a monoclonal antibody against CD20 and the radioactive isotope iodine-131 (131I). It, too, is designed as a treatment for lymphoma.  Although both Bexxar® and Zevalin® kill normal B cells, they don't harm the B-cell precursors because these do not express CD20. So, in time, the precursors can repopulate the body with healthy B cells. Therapeutic Uses:

T hree mechanisms that could be responsible for the cancer treatment. A.mAbs act directly when binding to a cancer specific antigens and induce immunological response to cancer cells. Such as inducing cancer cell apoptosis, inhibiting growth, or interfering with a key function. B.mAbs was modified for delivery of a toxin, radioisotope, cytokine or other active conjugates.toxinradioisotopecytokine C.it is also possible to design bispecific antibodies that can bind with their Fab regions both to target antigen and to a conjugate or effector cellbispecific

mAbs treatment for cancer cells ADEPT, antibody directed enzyme prodrug therapy; ADCC, antibody dependent cell-mediated cytotoxicity; CDC, complement dependent cytotoxicity; MAb, monoclonal antibody; scFv, single-chain Fv fragment. Carter P: Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer 2001;1:

 Herceptin® (trastuzumab ). Binds HER2, a receptor for epidermal growth factor (EGF) that is found on some tumor cells (some breast cancers, lymphomas). The only monoclonal so far that seems to be effective against solid tumors.HER2  Erbitux® (cetuximab ). Blocks HER1, another epidermal growth factor (EGF) receptor.HER1  Mylotarg®. A conjugate of a monoclonal antibody that binds CD33, a cell-surface molecule expressed by the cancerous cells in acute myelogenous leukemia (AML) but not found on the normal stem cells needed to repopulate the bone marrow.stem cells Therapeutic Uses:

 Mylotarg® is the first immunotoxin that shows promise in the fight against cancer.  LymphoCide. Binds to CD22, a molecule found on some B- cell leukemias.leukemias  Alemtuzumab (MabCampath®). Binds to CD52, a molecule found on white blood cells. Has produced complete remission of chronic lymphocytic leukemia (for 18 months and counting). Therapeutic Uses:

Why are there so few monoclonal therapy being used in human treatment more than quarter of a century after their discovery?

The main difficulty is that mouse antibodies are "seen" by the human immune system as foreign, and the human patient mounts an immune response against them, producing HAMA ("human anti- mouse antibodies"). These not only cause the therapeutic antibodies to be quickly eliminated from the host, but also form immune complexes that cause damage to the kidneys. Problems with monoclonal therapy

 Using genetic engineering it is possible to make mouse-human hybrid antibodies in an attempt to reduce the problem of HAMA. Chimeric antibodies.  The antibody combines the antigen-binding parts (variable regions) of the mouse antibody with the effector parts (constant regions) of a human antibody.variable regionsconstant regions  The antibody combines only the amino acids responsible for making the antigen binding site (the hypervariable regions) of a mouse antibody with the rest of a human antibody molecule thus replacing its own hypervariable regions.hypervariable regions

 mAb are genetically engineered using a molecular approach  Chimeric Ab are obtained by genetically fusing the mouse variable domains to human constant domains  The antibody combines the antigen-binding parts (variable regions) of the mouse antibody with the effector parts (constant regions) of a human antibody.  Variable regions are Isolated using polymerase chain reaction (PCR). 17 Chimeric mAb

 Mouse monoclonal antibodies have been genetically engineered to replace all of the antibody molecule with human counterparts except the hypervariable regions directly involved with antigen binding.  the complementary regions (CDRs), which are the responsible for antigen binding within the variable regions, have been transferred to human frameworks creating ‘‘CDR- grafted’’ or ‘‘humanized’’ antibodies. This is a human Ab with small segments containing mouse ab genes. 18 Humanized Monoclonal Antibodies

Chimeric mAb 19

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mAbs development A. Recombinant monoclonal antibodies.  Recombinant antibody engineering involves the use of viruses or yeast to create antibodies, rather than mice.  Phage display library: construction of VH and VL gene libraries and expression of them on a filamentous bacteriophage.  The phage expressing an antigen-bonding domain specific for a particular antigen. 21

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B. Transgenic animals:  transgenic (genetically engineered) mouse to produce more human-like antibodies.  Such mice have human antibody gene loci inserted into their bodies (using the embryonic stem cell method) 23 mAbs development

Production of Human Antibodies

Evolution of Therapeutic Antibodies 1. TRANSGENIC DNA SPLICING / GENE KNOCK OUT 2. LIBRARIES a.BACTERIOPHAGE b. mRNA c. Cell Surface

Evolution of Therapeutic Antibodies

FDA APPROVED MONOCLONAL ANTIBODIES H2003PsoriasisRaptiva ™ Genentech/Xoma C1997Non-Hodgkin’s LymphomaRituxan®BiogenIdec/Genentech/Roche M2003Non-Hodgkin’s LymphomaBexxar®Corixa/GlaxoSmithKline C2004Colorectal CancerErbitux ™ BMS/ImClone Systems H2003AsthmaXolair®Novartis/Genentech/Tanox PD2002Rheumatoid ArthritisHumira ™ Abbott/CAT H2001Chronic Lymphocytic LeukemiaCampath®Schering /ILEX Oncology H2000Acute Myleoid LeukemiaMylotarg ™ Wyeth C1998Acute Transplant RejectionSimulect®Novartis C1998Crohn’s, Rheumatoid ArthritisRemicade®J & J HHHH Breast Cancer Colorectal Cancer Herceptin® Avastin ® Genentech/Roche H1998Viral Respiratory DiseaseSynagis®MedImmune/Abbott H1997Acute Transplant RejectionZenapax®PDLI M2002Non-Hodgkin’s LymphomaZevalin ™ BiogenIdec C1994Acute Cardiac ConditionsReoPro®J&J/Eli Lilly M1986Organ Transplant RejectionOrthoclone-OKT®Ortho Biotech Antibody Type (2) Date of FDA ApprovalIndicationsName of Product (1) Company Name

 Raymond Hamers  Discovered in camels that antibodies Completely lack the light chain!  Same antigen affinity as their four-chain counterparts  Structure makes them more resistant to heat and pH  May lead to development of oral nanobody pills

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