Transcript ClonalAbNoTP

Monoclonal Antibodies in Research
and as Agents in Medicine for
Diagnosis and Therapy
…Antibody Engineering
Folder Title: ClonalAbNoTP
Version of October 14, 2014
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?
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.
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
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:
1. Get rid of the unfused antigen-primed B-cells
2. 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.
Making Nucleotides for DNA Synthesis
See Figure 4-21, Kuby, 4th Edition)
Mutant Myeloma Cell Line Chosen as the
Immortalized Partner because it can use only option 1,
the De novo pathway
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)
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 antiinterferon monoclonal onto an
insoluble support
Step 5: Run crude
mixture of interferon
and contaminants
through the insoluble
matrix column
holding bound antiinterferon 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)
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
Anti-actin Antibody
with bound rhodamine
(red) fluorescent label
Anti-tubulin antibody with
bound fluorescein (green)
label
(Fig 1-20, ESB 1998)
Intermediate Filaments
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 nonimmunized (“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
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
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
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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 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
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
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
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Manufacturing personalized therapeutic vaccines for
B-cell lymphomas
“Using Tobacco to Treat Cancer”
Science, August 22, 2008
pp. 1052- 1053