Pharmaceutical Biotechnology Lecture Notes PDF

11/3/2024 Pharmaceutical Biotechnology Prof. Dr. Azza S. Zakaria Professor of Microbiology and Immunology Faculty of Pharmacy, Alexandria University Email: [email protected] 1 2 2...

11/3/2024 Pharmaceutical Biotechnology Prof. Dr. Azza S. Zakaria Professor of Microbiology and Immunology Faculty of Pharmacy, Alexandria University Email: [email protected] 1 2 2 1  11/3/2024 1. in diagnostics specific proteins, antigens, or hormones. HCG indicative of pregnancy. 2. research experiments ELISA 3. target certain cells tumor cells monoclonal antibodies against surface markers on the cells. a toxic anti-cancer agent decreasing the harmful side effects of the anti-cancer agent increased specificity. 3 3 4. radiolabelled diagnostic imaging immunoscintigraphy γ-emitter parenteral administration radiolabelled Mab tumor can be visualized γ-ray detection equipment. 5. Therapeutic use neutralizing the effect of certain inflammatory mediators in humans Infliximab rheumatoid arthritis and Crohn’s disease. 4 4 2  11/3/2024 5 5 6 6 3  11/3/2024 in mice foreign antigens immune response   Therefore, the therapeutic efficacy of murine monoclonals is limited to the first and, at most, the second dose administered. 7 7 a relatively short half-life Poor recognition N.B.: 8 8 4  11/3/2024 Polyclonal antibodies Monoclonal Antibodies Produced by: Many B cell clones A single B cell clone Bind to: Multiple epitopes of all A single epitope of a single antigens used in the antigen immunization Antibody class: A mixture of different All of a single Ab class Ab classes (isotypes) Ag-binding sites: A mixture of Abs with All Abs have the same antigen different antigen-binding binding site sites 9 9 Different cancer types: therapy and diagnostics. Some Mabs have become first-line treatment in certain lung cancers Autoimmune diseases: Rheumatoid arthritis, inflammatory bowel diseases (e.g, Crohn’s disease, ulcerative colitis), psoriasis Migraine (prevention) Familial hypercholesterolemia Prophylaxis of acute organ rejection in transplants Multiple sclerosis (MS) Hemophilia A Systemic lupus erythematosus (SLE) Postmenopausal women with osteoporosis Atopic dermatitis Severe refractory asthma 10 5  11/3/2024 Very few available Mabs against infectious diseases 1 Respiratory syncytial virus (prophylactic) 2 Anthrax (Anti-Toxin) 3 Clostridium difficile (Anti-Toxin B) 4 HIV (anti-CD4, approved March 2018, entry inhibitor) 11  A solution to the above limitations of murine Mabs may be through the creation of “Chimeric antibodies”[the variable region is of mouse origin while the constant part is of a human antibody]  Chimeric Mabs: chimers combine the human constant regions with the intact rodent variable regions. Affinity and specificity are unchanged. However, it can cause human anti-chimeric antibody response (since about 30% of it is of murine source) 12 12 6  11/3/2024 13 13  Mouse monoclonal antibodies have been genetically engineered to replace all of the antibody molecules with human counterparts except the hypervariable regions directly involved with antigen binding.  the complementary regions (CDRs), which are 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.  Creation of chimeric or humanized antibodies are done using recombinant DNA technology. 14 14 7  11/3/2024 Chimeric antibody Humanized antibody White areas are of mouse origin while black areas are of human origin. 15 15 16 16 8  11/3/2024  Chimeric antibodies repeated administration of chimerics eventually raises an immune response in most recipients  Humanized antibodies  Fully Human Monoclonal antibodies 17 17 Percentage of human sequence in the different antibody types - Colored blocks: Mouse origin - Blank blocks: Human origin 18 18 9  11/3/2024    19 19 the use of transgenic mice. transgenic mice human immunoglobulin genes 20 20 10  11/3/2024  immunization of a transgenic mouse.  The mouse has been genetically engineered for the expression of human immunoglobulins.  The B cells harvested after immunization can be immortalized by fusion with a myeloma cell line  The hybridomas can then be screened for specific antibodies. 21 Nature Reviews Drug Discovery 2, 52-62 (January 2003) 21 Other approaches for the production of fully human Mabs include:  human hybridoma cells  In-vitro human antibody library through screening using the Phage Display technology. 22 11  11/3/2024  The phage display technique relies on generating a library of millions of bacteriophages that have been genetically engineered to display different peptides or proteins (Fab region) on their surface.  This is achieved by inserting a gene encoding a protein of interest into the phage's protein shell (a direct physical link between DNA sequences and their encoding proteins).  the modification aims to generate a molecule that can mimic a natural human immunoglobulin.  Several types of phages are used for the purpose of phage display. Filamentous bacteriophages are the most popular.  23 24 12  11/3/2024  The genetically modified phages are assembled into a library for use as a platform to screen different antigens.  Screening is performed by the addition of the phage-display library to the wells of a microtiter plate that contains immobilized target proteins or DNA sequences.  The plate is then incubated for some time to allow the phages to bind with the target of interest and then washed to flush away any non-binding phages.  Any phages that remain attached to the wells are then removed and inserted into other bacteria for replication.  The cycle is repeated until only phage-displaying proteins highly specific for the target remain.  Once the whole process is completed, the gene coding for the specific protein is isolated and purified from the phage so that it can be used for different applications. 25 26 26 13  11/3/2024 Adalimumab (Humira ®). 27 - 6th Annual Conference of the Department of Microbiology and Immunology, 29th Sep. 2018 Suffix of Mabs: -omab: Murine -ximab: Chimeric -zumab: Humanized -umab: Human 28 14  11/3/2024  Detection of markers on lymphocytes and antigens on cells and microorganisms  Identification and typing of bacteria and viruses  Hormonal assay  Detection of tumor markers  Radiolabeled monoclonal antibodies are used in vivo to detect or locate tumor antigens, e.g. for detection of breast cancer metastases. 29 29 A. To suppress the immune system  Transplant rejection (Muronomab-CD3) a murine anti-CD3 monoclonals bind to the CD3 molecule on the surface of T cells. Used to prevent acute rejection of organs, e.g., kidney transplants.  The humanized type shows promise in inhibiting the autoimmune destruction of beta cells in type 1 diabetes mellitus.  Monoclonal antibodies bind to tumor necrosis factor-alpha (TNF-α) and show promise against some inflammatory diseases such as rheumatoid arthritis.  30 30 15  11/3/2024  Xolair®, omalizumab). Binds to IgE thus preventing IgE from binding to mast cells. It shows promise against allergic asthma.  (Zenapax®, daclizumab). Binds to part of the IL-2 receptor produced at the surface of activated T cells. Used to prevent acute rejection of transplanted kidneys. Has also shown promise against T-cell lymphoma. 31 31 antibody-dependent cellular cytotoxicity (ADCC) complement-dependent cytolysis (CDC). Antibody-Directed Enzyme Single chain Prodrug Therapy (ADEPT) variable fragment 32 32 16  11/3/2024 B. To kill or inhibit malignant cells  Rituximab is used to treat certain autoimmune diseases and types of cancer by targeting CD20 on B cells It is used to treat B-cell lymphomas  This is a conjugate of a monoclonal antibody against CD20 and the radioactive isotope iodine-131. It is designed as a treatment for non-Hodgkin’s lymphoma.  binds HER2, a receptor for epidermal growth factor (EGF) that is found on some tumor cells (some breast cancer lymphomas). It is the only monoclonal so far that seems to be effective against solid tumors. 33 33  Erbitux®, cetuximab Blocks HER1, another epidermal growth factor (EGF) receptor antagonist. It is used to treat head and neck cancer and metastatic colorectal cancer.  Gemtuzumab (Mylotarg®), is an antibody-drug conjugate (a drug-linked monoclonal antibody) that is used to treat acute myeloid leukemia (AML). C. Other therapeutic benefits  Cardiovascular disease (Abciximab)  Infectious Diseases (Palivizumab)  Inflammatory disease (Infliximab ) 34 34 17  11/3/2024 35  1993 camel serum mouse serum  camel antibodies only heavy chains Heavy chain antibodies 36 36 18  11/3/2024  Camelids Camelidae Dromedary camels, Bactrian camels and llamas.  heavy chain antibodies Dromedary camel= Bactrian camel Llama Arabian camel 37 37  Camelid antibodies lack light chains the first heavy chain constant region (CH1).  (VHH)  38 19  11/3/2024  Intensive research is ongoing to exploit camelid antibodies for diagnostic and therapeutic use.  This research involves the use of the VHH fragment of the camelid antibody using recombinant DNA technology  Ablynx company is developing VHH fragments and called it Nanobody®  Nanobodies VHH fragments= VHHs are under intensive research and have been cloned in several expression systems. 39 39 40 40 20  11/3/2024 1. They can survive elevated temperatures thermostable 2. They are highly soluble 3. They can penetrate more quickly than normal antibodies through cell layers in tissue to reach their targets. 4. The genetic engineering procedures to select and clone VHHs are much easier than those for conventional antibodies 5. Unlike conventional antibodies which require mammalian cell lines for their proper expression and glycosylation, Nanobodies (VHHs) are usually conveniently produced in E. coli and yeast. This is an important advantage due to the ease and low cost of large-scale production. 41 41 6- VHHs can be easily genetically manipulated to produce multivalent nanobodies with mono- or multi-specificities. By binding several VHH domains together using a linker peptide, we can obtain multivalent Nanobody, trivalent, bi-specific nanobody. e.g. Ozoralizumab (under development by Ablynx company to treat rheumatoid arthritis by neutralizing TNF-alpha. Anti-human serum albumin (anti-HSA) VHH domain will increase the half-life time of Ozoralizumab (due to increased plasma protein Ozoralizumab binding) 42 21  11/3/2024 Although there is a great similarity between VHH of camelids and VH of human antibodies (more than 80%), an immune response against nanobodies can occur upon administration to humans Consequently, developed Nanobodies are routinely humanized to reach a homology above 90% with the human VH domain. 2- Nanobodies are rapidly excreted in urine due to their small molecular size resulting in short-half life This can be overcome by the conjugation anti- human serum albumin (anti-HSA) to VHH domain to increase plasma protein binding Another approach is to pegylate (chemical binding to polyethylene glycol PEG) nanobodies to increase half-life time. 43 43 antibody-dependent cellular cytotoxicity (ADCC) complement-dependent cytolysis (CDC). genetically engineering VHHs to be linked to Fc domains of human antibodies 44 44 22  11/3/2024 45 23