Monoclonal Antibodies in Research and as Agents in Medicine for Diagnosis and Therapy…Antibody Engineering Folder: ClonalAb Antibodies are produced by antigen-selection of unique B-cell clones. Antigen binding causes activation, proliferation, and differentiation of the selected clones. As end-product plasma cells, these selected B-cell clones produce and secrete their unique antibodies. Antigen-activation usually produces a mixture of antibodies because a number of B-cell clones with cross-reactive antibody-receptors are activated. It is possible artificially to select in the laboratory a unique single B-cell clone, and to get it to produce its unique antibody. Such an antibody is called a "monoclonal antibody".

Immunology in Human and Animal Health and Disease From 447 Intro: Slide 33 Immunology in Human and Animal Health and Disease Why do we want to know about Immunology? What does it tells us about ourselves and about biology? What can it do for us? As a tool in biomedical research? As a diagnostic and therapeutic modality in clinical and veterinary medicine? What can it do to us, as a source of pathology? 447Intro: InHealth Allergic or hypersensitivity responses such as asthma, anaphylaxis (acute hypersensitivity to bee stings and other allergens), rheumatoid arthritis, reaction to blood-group antigens, poison ivy (delayed-type hypersensitivity) are examples of pathological effects on the host generated by an inappropriate or over-reactive immune response.

Immunology in Human and Animal Health and Disease From 447 Intro Slide 34 Immunology in Human and Animal Health and Disease What Can We Make it Do For Us? To here August 26th

Passive Polyclonal Antibody Therapy (See Clinical Focus, p. 98. Edition 6) Passive transfer of pooled immunoglobulin from multiple donors to a recipient. (e.g. treatment of measles, diphteria, hepatitis) Antiserum contains polyclonal antibodies to infectious organisms, toxins, or for treating immunodeficiency. Patient may receive more than 15 g of immunoglobulin protein in multiple treatments. Potential transfer of viral pathogens. Potential sensitization to allogeneic antibody sequences.

Potential Advantages of Monoclonal Antibodies Single matched genetic source of antibody. Prevention of allogeneic immune response to mixed sequences in non-antigen-binding regions. Clone producing the antibody can be amplified to produce unlimited quantities of monoclonal as a pharmaceutical source. Cloned source cell line can be preserved indefinitely in an ultra-freezer and revived when needed. Antibody coding information can be engineered to precise sequential specifications to produce desired target binding and to avoid adverse immunological reactions.

Humanized monoclonal Ab From Antibody: Slide 63 Therapeutic Monoclonal antibody would use antibody matched as isotype and matched in the framework allotype in V-regions (i.e matched as close as possible to the patient). Then graft in the CDR’s from mouse to get the antigenic specificity that is needed. Foreign idiotopes could go unrecognized and be non-immunogenic. Matched human isotype and human allotype. Graft in mouse CDR’s Humanized monoclonal Ab

Making Monoclonal Antibodies From Antibody: Slide 64 Making Monoclonal Antibodies Now Immortal!

Getting a Mixture of Fused and Unfused Cells Myeloma cells cannot use the salvage pathway to reuse DNA precursors (nucleotides). Must make DNA precusors from scratch using DeNovo pathway. Lack the enzyme Hypoxanthine-guanine phosphoribosyl transferase (HGPRT negative). B-cells can use both the salvage pathway and the DeNovo pathway. Therefore poison the DeNovo pathway with aminopterin. Myeloma cells can’t reproduce.

How Do We get an Immortalized Line Producing a Single Monoclonal Antibody? Problems: Get rid of the unfused antigen-primed B-cells Get rid of the unfused Myeloma cells or ones that fused with each other. Select out the fused hybrid cell line making the antibody that you want.

Making Nucleotides for DNA Synthesis Folder: ClonalAb The mixed hybridoma clones have to be separated if one is to get a single clone producing a unique monoclonal antibody. The separation is done by adding to the culture medium a poison, aminopterin, that blocks a crucial pathway to the synthesis of precursors needed to made DNA. The normal plasma cell clones have an optional pathway to the synthesis of the essential DNA precursors, the "salvage pathway". The myeloma is chosen because it is immortalized, and also because it genetically lacks the alternative pathway to the synthesis of the essential DNA precursors, the "salvage pathway". The normal plasma cells, and the hybridomas that they form can survive in a medium containing aminopterin because the plasma cell component of the hybridoma can use the salvage pathway alternative. Unfused myeloma cells cannot survive. See Figure 4-21, Kuby, 4th Edition)

Normal B-Cell can use both pathways Mutant Myeloma Cell Line Chosen as the Immortalized Partner because it can use only option 1, the De novo pathway Folder: ClonalAb This slide summarizse the points made up to here. Normal B-Cell can use both pathways Block option 1 (“denovo” pathway) with aminopterin, myeloma cells will die.

Single Hybridoma Clone by Limiting Dilution (getting one clone or less per well) Folder: ClonalAb The surviving mixed hydridoma clones are grown in tiny "micro-titer" wells in cell culture where each well is seeded with a single starting cell. Thus one ends up with thousands of wells, each with its unique hybridoma clone. The next step is to find out which, if any, of the thousands of clones is producing the unique antibody that the experimenter wants to get. This is done by seeing which well or wells have in them an antibody that will bind to the antigen that one wants a monoclonal antibody for. Any wells identified as containing a hybridoma clone that is making the desired antibody, are used to seed much largr growth systems such as big cell culture flasks, or large growth chambers. The selected unique clones can also be expanded by growing them in the peritoneal cavity (the abdominal cavity) of mice. Fluid produced in the abdominal cavity, "ascites fluid" contains large amounts of the desired monoclonal antibdy. See Figure 4-22, Kuby 4th Edition

Selected Research Applications of Monoclonal Antibodies Isolation and Purification of Immunogens from Complex Mixtures: Example: Isolation of Pure Interferon In Situ Labelling of Cell Organelles Example: Fluorescent Labelling of Cytoskeletal Elements See Essential Cell Biology, Alberts et al, 1998 ed. Figure 1-20, The Cytoskeleton

First Step: Make the Mixed Hybridomas 2nd Step: Select out the hybridoma making an antibody that binds interferon. 3rd Step: Make a bunch of Anti-INF antibody. 4th Step: Stick the anti-interferon monoclonal onto an insoluble support

Step 5: Run crude mixture of interferon and contaminants through the insoluble matrix column holding bound anti-interferon antibody. Step 7: Elute the bound interferon from the column using something that will compete for its binding sites (e.g. high salt) Step 6: Wash unbound contaminants off the column.

Selected Research Applications of Monoclonal Antibodies Isolation and Purification of Immunogens from Complex Mixtures: Example: Isolation of Pure Interferon In Situ Labelling of Cell Organelles Example: Fluorescent Labeling of Cytoskeletal Elements See Essential Cell Biology, Alberts et al, 1998 ed. Figure 1-20, The Cytoskeleton (Putting different colored “light bulbs” onto specific antigenic determinants in intact fixed tissue samples)

Filaments and Tubules of the Cytoskeleton Microfilaments Microtubules Intermediate Filaments Anti-actin Antibody with bound rhodamine (red) fluorescent label Anti-tubulin antibody with bound fluorescein (green) label Anti-vimentin? Antibody with bound blue fluorescent label (Fig 1-20, ESB 1998)

Clinical Applications of Antibodies in Therapy: Polyclonal Antibodies (see Clinical Focus, Passive Antibody Therapy, p. 98, 6th Edition) Anti-toxin antibodies passively transferred from immunized donor to non-immunized (“naive”) recipient. (See Slide 2: Passive Antibody Therapy) (e.g. anti-tetanus toxoid, anti-diphtheria toxin). Pooled plasma donations of polyclonal IgG for immunodeficiency or for protection prior to pathogen exposure. Medical Problems and Limitations of Polyclonal Antisera Infection risks from pooled donor sources. Large gram-sized doses of pooled IgG Potential Allergic reactions to Antibodies from multiple donor sources Monoclonal antibodies can circumvent (avoid) these clinical problems

Selected Clinical Applications of Monoclonal Antibodies affecting T-Cell Mediated Immune Reactions: Autoimmune Disease and Transplantation Medicine Monoclonal Antibodies in Suppression of Auto- immune Systemic Lupus Erythromastosis-like Disease in Mice Monoclonal Antibodies in Treatment of Experimental Autoimmune Encephalomyelytis (EAE): Possible counterpart to Multiple Sclerosis in Humans. Monoclonal Antibodies in Immunosuppression in Transplantation Medicine

Systemic Lupus Erythematosus Patient (Systemic autoimmune Disease). Mediated by auto-reactive T-cell clones. Attacks DNA and blood cells and other. Shut down T-cell response with anti-CD4 Monoclonal attacking activated T-cells. See Figure 16 -07, p. 530, Kuby 7th Edition

CD4 is a T-Cell Antigen MAbinSLE Anti-CD4 Monoclonal Antibody in Treatment of Autoimmune Systemic Lupus Erythematosis-like Condition in Hybrid Mice (weekly injections) (Figure 16-14, Kuby, 6th Edition, p. 420) CD4 is a T-Cell Antigen MAbinSLE

Figure 16-15, Kuby, 6th Edition, p. 421 Treatment of Experimental Autoimmune Encephalomyelitis (EAE) in Mice with MAb to Selected T-Cell Receptor Variable-Region Gene Products. Numbers are qualitative characterizations of degrees of severity. 3 = Most Severe. 0 + no symptoms

Immunosuppression of T-Cell Responses in Transplantion Medicine

Whole Body Scanning for Immune Diagnosis of Metastatic Cancers Selected Clinical Applications of Monoclonal Antibodies: Cancer Diagnosis and Immunotherapy of Cancer Whole Body Scanning for Immune Diagnosis of Metastatic Cancers See Folder Antigens, Slide "SeeMets" Antibody to A33 Antigen in Diagnosis of Colon Cancer Metastases. Antibody-Directed Delivery of Therapeutic Agent (Immunotoxins) Example: Tumor-specific Delivery of Diphtheria Toxin Antigen-Specific Attack on Neoplastic (Cancer) Cells Example: Anti-Ig Targeting of B-Cell Lymphoma Example: Anti-Breast Cancer Antibody (See Anti-HER2 "Herceptin" Later) Attacking a growth-factor receptor (HER-Neu) supporting breast cancer cell growth. Antibody-directed Attack on Host Responses Supporting Tumor Growth e.g. Avastin monoclonal antibody attacking tumor vascularization

Imaging on Metastatic Colon Carcinoma with Radioactive-Iodine-Labelled Monoclonal Ab to A33 Ag Lloyd Old, Scientific American, August, 1996, p. 138) SeeMets Arm Head

Using Antibodies to “Address” Therapeutic Attack: Immunotoxins Re-addressing Diphtheria Toxin to Cancer Cells

Please put away all notes and any devices except for your Turning Point XR Transmitter. No communication between or among students. This is five-item matching question

Limitations in Clinical Applications of Monoclonal Antibodies Mouse Monoclonals in Humans Foreign Immunogen - Quickly Cleared Potential Allergic Response Immune-Complex Disease Human Anti-mouse Ig-Ag in Kidneys Human Anti-mouse Ig-Ag in Joints Human Monoclonals: Technically Difficult to Produce No suitable Immortalized, Ab-negative, HAT-Selectable Human Line that makes Ab-secreting hybridoma with human B-cells Cannot freely immunize humans to any desired antigen MAbLimit

Solutions to Limitations in Clinical Applications of Monoclonal Antibodies: Production of Human or of Humanized Monoclonals Human Monoclonals: Some Possible Solutions Immortalize B-Cell with TransformingVirus (Epstein- Barr Virus Transformed B-Cells) Prime B-Cells in Culture with Antigen Reconstitute Immuno-Deficient Mice with Human Immune Response Cells (SCID Mice with Human Hematopoietic Tissue) Genetically Engineer Mice with Human Immunoglobulin Gene Elements (e.g. Human Fc Isotypes) MAbSolve

Antibody Engineering: Human-Made Antibodies and Hybrid Antibody - Non-Antibody Molecules (pages 136-137, Kuby, 6th Edition) Chimeric or Hybrid Antibodies V-Regions from Mouse; C-Regions from Humans CDR Regions from Mouse; V-Framework and C-Regions from Humans (See "Rituxan" Later) Bi-Specific Antibodies (Antibody Hetero-conjugates) Example: Anti-Tumor Antigen FAb + Anti-TCR FAb Hybrid Antibody Molecules with Non-Antibody Proteins Anti-Tumor Ag FAb + Replaced Fc with Protein Toxin (Engineered Immunotoxin) Fully Humanized Antibody Protein from Non-Human Animals Transgenic Mice with Mouse Ig Coding Regions Replaced with Human VL, VH, and C-Region Genes AbEngin

Mouse V, J, and D Gene Coding; Human C-Regions Mouse CDR's; Human V-Region Framework and Human C-Region Gene Coding Examples of Antibody Engineering (Figure 5-23, Kuby Immunology, 6th Edition; p. 137) Artificial Bi-Specific Antibody; Recognizes two different antigenic determinants, Tumor Ag and TCR Epitopes MAbForms

(Chimeric Mouse CDR's with Human V-Framework and C-regions) Biotechnology and Clinical Applications of Monoclonals in Cancer Medicine: Leukemia & Lymphoma Therapy Rituxan: IDEC Pharmaceuticals Anti-CD20 Antibody Targeted to B-Cell Lymphoma (Chimeric Mouse CDR's with Human V-Framework and C-regions) Zevalin: Millennium Pharmaceuticals Anti-CD20 for B-Cell Lymphoma with Radioactive Yttrium; Non-Hodgkin’s Lymphoma Bexxar: Anti-CD20 for B-Cell Lymphoma with Radioactive Iodine Campath: Anti-CD52 for B-Cell Chronic Lymphocytic Leukemia (See page 141, Immunology, 6th Edition) MAbLeukemia

Anti-HER2/Neu Growth Factor Receptor in Breast Cancer Biotechnology and Clinical Applications of Monoclonals in Cancer Medicine: Carcinoma Therapies Herceptin: Genentech Anti-HER2/Neu Growth Factor Receptor in Breast Cancer Humanized Monoclonal Antibody (See page 141, Immunology, 6th Edition) Avastin: Antibody to Vascular-Endothelial Growth Factor Receptor (Anti-angiogenesis Therapy) Erbitux Antibody to Epidermal Growth Factor Receptor MAbCarcinoma

Anti-antibodies in B-Cell Lymphomas Monoclonal Anti-idiotype Antibodies What if a Leukemic B-Cell (Plasma Cell) Cancer is making an Antibody? Can we attack that Antibody as a Therapeutic Target? If so where on the Antibody?

Clinical Applications of Monoclonals in Autoimmunity and Transplantation Medicine Zenapax Targeted to IL-2 Receptor alpha subunit on activated T-Cells (Anti-TAC) Modulates acute kidney rejection Orthoclone OKT3 Targeted to CD3 co-receptor on activated T-cells Controls rejection in liver, heart, and kidney transplants Remicade Targets Tumor Necrosis Factor mediator of inflammation Treatment of Autoimmune Rheumatoid Arthritis and Crohn’s Disease Xolair Antibody to IgE (Antibody to an antibody) Type 1 Allergy Treatment (See page 141, Immunology, 6th Edition) MAbAuto&Trans

Please put away all notes and any devices except for your Turning Point XR Transmitter. No communication between or among students. These are real quiz questions.

To Get to Molecular Visualizations Produced for Kuby Immunology http://bcs.whfreeman.com/immunology6e/ Or search “Kuby Immunology”, Click on “Kuby Immunology 6e” , go to Student Resources

Manufacturing personalized therapeutic vaccines for B-cell lymphomas “Using Tobacco to Treat Cancer” Science, August 22, 2008 pp. 1052- 1053