Monoclonal Antibodies in Researchand as Agents in Medicine for

Diagnosis and Therapy…Antibody Engineering

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: Slide 33

What Can We Make it Do For Us?

To here August 26th

From 447 Intro Slide 34

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.

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: Slide 63

Making Monoclonal Antibodies

Now Immortal!

From Antibody: Slide 64

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 can’t reproduce.

How Do We get an Immortalized Line Producing a Single Monoclonal Antibody?

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.Select out the fused hybrid cell line making the antibody that you want.

See Figure 4-21, Kuby, 4th Edition)

Making Nucleotides for DNA Synthesis

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.

See Figure 4-22, Kuby 4th Edition

Single Hybridoma Clone by Limiting Dilution (getting one clone or less per well)

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 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)

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)

Microfilaments 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

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

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

MAbinSLECD4 is a T-Cell Antigen

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

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

Arm

Head

SeeMets

Using Antibodies to “Address” Therapeutic Attack:

Immunotoxins

Re-addressing Diphtheria Toxin to Cancer Cells

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This is five-item matching question

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

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

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 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

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

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

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?

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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Manufacturing personalized therapeutic vaccines for B-cell lymphomas

“Using Tobacco to Treat Cancer”

Science, August 22, 2008

pp. 1052- 1053