1. Therapeutic agents_Monoclonal Ab Romana Siddique

2. 2 Therapeutic Agents Recombinant Proteins Monoclonal Antibodies Clinical Applications Transplantation – muronomab (OKT3) 1986, basiliximab 1998 Cardiovascular disease – abciximab 1994 Cancer – rituximab 1997, trastuzumab 1998 Viral infection – palivizumab 1998 Inflammatory diseases – infliximab 1998, etanercept 1999 Side effects: Transfusion reactions ( any adverse event which occurs because of a blood transfusion) Infections, immunosuppression Cardiac, respiratory arrest ( discontinuation of breathing) Pharmacological toxicity

3. 3 Therapeutic Agents Recombinant Proteins Monoclonal Antibodies Production Monoclonal antibodies results from a clone of a B lymphocyte producing a single antibody which will bind to a specific epitope of an antigen. Monoclonal antibodies are produced: – Fusion of a myeloma (B cell which has become cancerous) with a spleen cell that is immunized with a specific antigen. The resulting hybridomas are tested for the production of a monoclonal antibodies. Diluted to one cell cultures Hypoxanthine Guanine Phosphoribosyl Transferase (HGPT) negative myeloma cells Grown in Hypoxanthine Aminopterin Thymidine (HAT) medium Only successful fusion cells grow (rare)

4. 4 Therapeutic Agents Recombinant Proteins Monoclonal Antibodies Production: Major Problems with non-human ABs: Immunological Responses  ”Humanization” of ABs

5. 5 Therapeutic Agents Recombinant Proteins Monoclonal Antibodies Chimeric Ab 65 – 90% human and consist of the mouse variable regions and substituting the mouse Fc region of the antibody with that from human Fc DNA that encodes the binding portion of monoclonal mouse antibodies and merges it with human antibody-producing DNA. Use mammalian cell cultures to express this DNA and produce these half-mouse and half-human antibodies. (Bacteria cannot be used for this purpose, since they cannot produce this kind of glycoprotein.) Depending on how big a part of the mouse antibody is used, one talks about chimeric antibodies or humanized antibodies.mammaliancell cultures glycoprotein 95% human, and are made by grafting the hypervariable region (or CDR) of the chimeric antibody –which determines Ab specificity Humanized Ab

6. Production of Humanized monoclonal Antibody

7. Human Monoclonal Antibody Xenomouse: In which 1.The mouse atibody production machinery is inactivated and 2.All of the human immunoglobulin loci (both L and H chains) are integrated into mouse chromosome.  The human H chain genes and L chain genes were cloned into a YAC vector.  YAC vector with the cloned genes were then introduced into mouse embryonic stem cells by fusing YAC-containing yeast spheroplasts with the embryonic stem cells.  This produces a large number of embryonic stem cells in which all of the introduced human immunoglobulin genes have become stably integrated into the chromosomal DNA.  These tranfected cells were used to generate mice containing human immunoglobulin gene loci.

8. Crossbreeding of two mice lines,one carrying both both mouse and human immunoglobulin genes and the other carrying only the deleted mouse immunoglobulin genes,produces a mouse strain (Xeno Mouse) that express expresses only human human immunoglobulins. After immunization XenoMouse will produce a fully humanized immunoglobulin.

9. Antibody fragments Single Chain Ab(ScFv) contains only VL and VH domain. ScFv have molecular weight of of approx. 27 Kdas compared with approx. 150 Kdas for IgG molecules. Because of their small size ScFv can penetrate and distribute in large tumors more readily than intact antibodies. A protein coding sequence can be linked to a ScFv sequence to create a dual function molecule that can both bind to specific target and deliver a toxin or some other specific activity to a cell.

10. 10 Therapeutic Agents Recombinant Proteins Antibodies for human therapy derived without using mice: -> uses various "display" methods (primarily phage display) as well as methods that exploit the elevated B- cell levels that occur during a human immune response.phage display Create new variations of antibodies – New combinations of heavy and light chains mRNA from immunized individual PCR H and L chains Clone into vector in new H/L combinations -> create library -> screening by display

11. 11 Therapeutic Agents Recombinant Proteins Antibodies for human therapy derived without using mice: Screening by Phage display

12. 12 Therapeutic Agents Recombinant Proteins Antibodies for human therapy derived without using mice: only one heavy-chain variable domain and one light-chain variable domain  covalently linked by peptide Single-chain Fixed variable Single-Chain Combinatorial Antibody Library

13. 13 Therapeutic Agents Recombinant Proteins Monoclonal Antibodies Application of single-chain fixed variable-> Immunotoxins Protein toxin connected to Fv region Single-chain or S-S linked Fv region Toxin localized to antigen-expressing cells

14. 14 Therapeutic Agents Recombinant Proteins Monoclonal Antibodies Examples: 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

15. 15 Therapeutic Agents Recombinant Proteins Monoclonal Antibodies Examples: Target Angiogenesis -> to prevent tumor growth Dr. Judah Folkman (1971) Tumor secretes factors -> promote blood vessel growth to the tumor

16. 16 Therapeutic Agents Recombinant Proteins Monoclonal Antibodies Examples: Target Angiogenesis -> to prevent tumor growth Recombinant humanised monoclonal antibody target ing the angiogenic factor VEGF (93% human, 7% mouse) Bevacizumab (Avastin ® )

17. Monoclonal Ab for drug delivery Anticancer antibody Chemitherapeutic: Irinotecan Cellular toxin: auristatin 17

18. Figure 10.40 Making monoclonal antibodies even more effective therapeutic agents: two ways

19. Antianthrax antibody Caused by Bacillus anthracis: a spore forming, gram positive, rod shaped bacteium. produces an exotoxin ( anthrax toxin) made up of three proteins: 1.Protective antigen( PA) 2.Edema factor ( EF) 3.Lethal factor ( LF)  Once in the cell cytosol anthrax toxin disrupt cell signalling pathways, thereby evade immune system, proliferate and kill host animal.  PA binds the cell receptor and forms a pore through which it delivers LF and EF into the cytosol.  LF is a Zinc dependent protease –resulting in lysis of macrophages.  Ca-calmodulin dependent adenylate cyclase causing edema.  PA together with LF results in formation of lethal toxin, while PA with EF forms edema toxin.  PA is the target component in current anthrax vaccine & target antigen for most monoclonal ad against anthrax.

20. As there were concerns about the long term efficacy of this approach. So it was decided to develop anti EF-monoclonal antibody using them either alone or in concert with anti-PA mab. Flow chart:

21. Antiobesity antibody Ghrelin stimulates food intake. In humans, ghrelin levels increase during dieting which actually facilitates weight regain and impedes sustainable weight loss. Catalytic ab can be designed against ghrelin.

22. Enzymes Dnase I Alginate lyase Α1-Antitrypsin