Download - Set to Anonymous Polling Open New Session for Anonymous Slide on Scope and Pace of the course so far
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Set to Anonymous Polling
Open New Session for Anonymous Slide on Scope and Pace of the course so far.
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This question will be set to anonymous so no one will know what any specific student says.
For the first 12 classes the pace of the course is:
1. 2. 3.
0% 0%0%
1. Too slow. We need to move faster and cover more about Immunology.
2. About right. I can keep up Ok but it is a challenge.
3. Way too fast. Slow down! We’ll figure out later what you don’t get to cover in class.
Response
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On a scale of 5 to 1 rate Thursday’s AbGenes Presentation Was5. Too slow. Get moving
4. Not especially challenging. 3. About right. I can figure out what I missed.
2. Too fast1. I’m lost!
(This is anonymous. We won’t know who you are.)
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Folder Title: ClonalAbNoTP
Version of October 20, 2015
Monoclonal Antibodies in Researchand as Agents in Medicine for
Diagnosis and Therapy
…Antibody Engineering
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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?
From 447 Intro
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Immunology in Human and Animal
Health and Disease
What Can We Make
it Do
For Us?
From 447 Intro
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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.
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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.
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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
From Antibody:
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How Do We get an Immortalized Line Producing a Single Monoclonal Antibody?
Fuse immortalized myeloma cells with antibody-producing normal cells that die off after a time.
Problems:1. Get rid of the unfused antigen-primed B-cells2. Get rid of the unfused Myeloma cells or ones that
fused with each other.3. Make sure that the myeloma cells does not know
how to make its own antibody4. Select out the fused hybrid cell line making the
antibody that you want.
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Making Monoclonal Antibodies
Now Immortal!
From Antibody
How do we get rid of the unfused cells, either myeloma of B-cells that did not find a partner?
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See Figure 4-21, Kuby, 4th Edition)
Making Nucleotides for DNA Synthesis
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Getting a Mixture of Fused and Unfused CellsMyeloma 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 without a B-cell partner can’t reproduce.
Fused B-Cells and Unfused B-cells do not clone. They are not immortal
3 Properties the Myeloma Cells Need to Have
2 Properties the B-Cells Need to Have
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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
Block option 1 (“denovo” pathway) with aminopterin, myeloma cells will die.
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See Figure 4-22, Kuby 4th Edition
Single Hybridoma Clone by Limiting Dilution (getting one clone or less per well)
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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
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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
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Step 5: Run crude mixture of interferon and contaminants through the insoluble matrix column holding bound anti-interferon antibody.
Step 6: Wash unbound contaminants off the column.
Step 7: Elute the bound interferon from the column using something that will compete for its binding sites (e.g. high salt)
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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)
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Filaments and Tubules of the CytoskeletonMicrofilaments Microtubules Intermediate Filaments
(Fig 1-20, ESB 1998)
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
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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
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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
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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
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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
To Here October 14, 2014
Treatment of Autoimmune Systemic Lupus in Mice
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Experimental Autoimmune Encephalomyelitis Treated by Suppression of T-Cell Autoimmunity Using Monoclonal Ab to V-region Gene Products
of T-Cell Receptor Gene Expression
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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
Antibody to T-Cell Receptor V-Region Gene Products in Treatment of Autoimmune Encephalomyelitis in Mice
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Immunosuppression of T-Cell Responses in Transplantion Medicine
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Clinical Applications of Monoclonals in Autoimmunityand Transplantation Medicine
ZenapaxTargeted to IL-2 Receptor alpha subunit on activated T-Cells (Anti-TAC)Modulates acute kidney rejection
Orthoclone OKT3Targeted to CD3 co-receptor on activated T-cellsControls rejection in liver, heart, and kidney transplants
RemicadeTargets Tumor Necrosis Factor mediator of inflammationTreatment 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
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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 Growthe.g. Avastin monoclonal antibody attacking tumor vascularization
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Imaging on Metastatic Colon Carcinoma with Radioactive-Iodine-Labelled Monoclonal Ab to A33 AgLloyd Old, Scientific American, August, 1996, p. 138)
Arm
Head
SeeMets
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Using Antibodies to “Address” Therapeutic Attack:
Immunotoxins
Re-addressing Diphtheria Toxin to Cancer Cells
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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
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Biotechnology and Clinical Applications of Monoclonalsin 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
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Biotechnology and Clinical Applications of Monoclonalsin 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)
ErbituxAntibody to Epidermal Growth Factor Receptor
MAbCarcinoma
To Here, October 16, 2014
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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?
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B-lymphoma cell’s antibody is used as the immunogen
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Designates also on following table for cancer monoclonals
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Monoclonal Antibodies for Cancer Treatment.
p. 645
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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 antigen we feel like using
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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
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Antibody Engineering: Human-Made Antibodiesand 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
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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
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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.
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To Get to Molecular VisualizationsProduced for Kuby Immunology
http://bcs.whfreeman.com/immunology6e/
Or search “Kuby Immunology”, Click on “Kuby Immunology 6e” , go to Student Resources
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Manufacturing personalized therapeutic vaccines for B-cell lymphomas
“Using Tobacco to Treat Cancer”
Science, August 22, 2008
pp. 1052- 1053
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