Applications of Ab Molecules. Chapter 4 Monoclonal Ab (p.99) Chapter 5 Ab genes and Ab Engineering (p.128)

Transcription

1 Applications of Ab Molecules Chapter 4 Monoclonal Ab (p.99) Chapter 5 Ab genes and Ab Engineering (p.128)

2 Monoclonal Antibodies

3 Clonal Selection of B Lymphocytes

4 Hybridoma Köhler and Milsten (1975) - continuous culture of specific antibody-forming cells Hybrid = lymphoblast x myeloma cells -oma = tumor

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6 Formation and Selection of Hybridoma Cells

7 Myeloma Cell Lines Commonly Used to Make Hybridomas cell line Ig produced P3-X63Ag8 (Ag8) γ1, κ NS1/1-Ag4.1 (NS1) κ (not secreted) Sp2/0-Ag14 (Sp2/0) none X63-Ag8.653 (Ag8.653) none Y3-Ag1.2.3 (Y3) - rat κ

8 Principle of Selection HGPRT - hypoxanthine guanine phosphoribosyl transferase HAT - hypoxanthine, aminopterin, thymidine Lymphocyte - HGPRT (+), can grow in HAT medium not immortalized myeloma cell HGPRT ( ), cannot grow in HAT medium immortalized hybridoma - HGPRT (+), can grow in HAT medium immortalized

9 5-Amino Imidazole- 4-Carboxy Ribonucleotide * PRPP PP 5-Formido-Imidazole- 4-Carboxamine Ribonucleotide Hypoxanthine Inosine Monophosphate Hypoxanthine Guanine Phosphoribosyl Transferase (HGPRT) Guanine Guanosine Monophosphate (GMP) PRPP PP Thymidine GDP dgdp Thymidine kinase RNA GTP dgtp dtmp dtdp d TTP DNA * Thymidylate Synthetase UDP dutp dump dctp datp

10 Metabolic pathways relevant to hybrid selection in medium containing hypoxanthine, aminopterin and thymidine (HAT medium). When the main synthetic pathways are blocked with the folic acid analogue aminopterin (*), the cell must depend on the salvage enzymes HGPRT and TK (thymidine kinase). HGPRT ( ) cells cannot grow in HAT medium unless they are fused with HGPRT (+) cells.

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12 Production of mab

13 Procedures 1. Immunization of BALB/c mice 2. Fusion of spleen cells and myeloma cells with polyethylene glycol (PEG) 3. Selection of hybrid cells in HAT medium 4. Screening of antibody-producing cells 5. Cloning 6. Large-scale production of antibodies

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15 Characterization 1. Determination of Ab class 2. Determination of Ab specificity 3.Analysis of antigens recognized by Ab

16 Applications 1. Study of antigens, e.g., microbial antigens, histocompatibility antigens, tumor antigens, differentiation antigens, etc. 2. Immunoglobulin structure and function 3. Immunodiagnosis 4. Immunotherapy 5. Affinity purification

17 Advantages of mab 1. A monoclonal antibody reacts with a single antigenic determinant (epitope). 2. Cross reactions are consistent. 3. Monoclonal antibodies are available in unlimited supply. 4. We can produce antibodies to single molecules in complex mixtures. 5. Monoclonal antibodies may detect components in a mixture that are present in small quantities not detectable by conventional antisera. 6. Antibodies can be biologically modified.

18 Disadvantages of mab 1. Monoclonal antibodies cross-react due to structural relatedness among antigens. 2. Biological function may be limited by heavy chain class. 3. Most monoclonal antibodies will not precipitate in immunodiffusion due to failure of cross-linking. 4. Single affinity and specificity may be more influenced by ph, temperature, etc. 5. Sometimes, a monoclonal antibody may be too specific.

19 Clinical Uses for mab Diagnosis, imaging, and therapeutic reagents Immunotoxins: mab conjugated to toxins, such as ricin, Shigella toxin, and diphtheria toxin

20 : inhibitory toxin chain : binding component of the toxin

21 toxin receptor

22 Catalytic mab (Abzymes) - A mab that has catalytic activity. - Similarities of the binding of an Ab to its Ag and an enzyme to its substrate: noncovalent interactions, high specificity, high affinity - Ab does not alter the Ag, whereas the enzyme catalyzes a chemical change in its substrate.

23 A central goal of catalytic Ab research is the derivation of a battery of abzymes that cut peptide bonds at specific amino acid residues, much as restriction enzymes cut DNA at specific sites.

24 Ab Genes and Ab Engineering

25 Problems of mouse mab for clinical uses: 1. Human anti-mouse Ab 2. Formation of immune complexes

26 Human mab 1. Human hybridoma Human B cells x human myeloma cells 2. Human B cells transformed by Epstein-Barr virus (EBV) 3. Humanized mab 4. Human Ab constructed from Ig-gene libraries

27 Production of chimeric mouse-human mab or transfectoma

28 1. Less immunogenic 2. Fc retains the biological effector functions of human Ab.

29 Chimeric and hybrid mab engineered by recombinant DNA technology or bispecific Ab

30 mab Constructed from Ig-gene Libraries

31 Therapy for Non-Hodgkin s Lymphoma by a Genetically Engineered Ab

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33 SCID-human Mouse

34 Mice with Human Ig Loci

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36 The End