Post on 16-Feb-2017
Biotechnological Applications
Università Del Piemonte OrientaleMasters Degree in Medical Biotechnologies
Steven R. Ellis, Ph.D.Professor, Department of Biochemistry
Assistant Dean of Basic Science Education, School of MedicineUniversity of Louisville
May 18 – May 29, 2015
Science Magazine’s Breakthrough of the Year 2013
immunoglobulinmembrane
attack complex
molar????
MHC/antigen
T cell receptor
Lunedi Martedi Mercoledi Giovedi Venerdi
9:00 immunoglubulins / B cells I
innate immune system
recombinant DNA technology
I
Glycoproteins and
proteoglycans I
take home exam
10:00 immunoglubulins / B cells II
DNA sequencing technology
recombinant DNA technology
II
Glycoproteins and
proteoglycans II
take home exam
11:00 Introduction/monoclonal antibodies
therapeutic antibodies II –
rituximab/cancer
biotechnological advancements –
further humanization
Anti-angiogenesis - bevacizumab
Phage display and other screening
technologies
12:00 therapeutic antibodies I –
rituximab/cancer
therapeutic antibodies
clinical trials
angiogenesis Fully human antibodies - adalimumab
Antibody engineering via constant
regions
Week One
ElectronicSoftChalk
Face to Face
mode of instruction
Therapeutic Antibodies
Rituximab – 1997, first approved clinical use for a monoclonal antibody
Paul Ehrlich – 1906, magic bullet to cure human diseases – it should be possible to create a compound that selectively targets and destroys a disease causing organism
Milstein and Köhlor – 1975, hybridoma technology – the ability to create a unlimited amount of a highly specific antibody to a particular antigen, “monoclonal antibodies”
• growth in the number of monoclonal antibodies entering clinical trials through 2008
• A number of these antibodies are approved for use in humans and generate billions of dollars in annual sales
Continuous cultures of fused cells secreting antibody of predefined specificity
G. Köhler and C. Milstein 1975 Nature 256, 495-497
The Nobel Prize in Physiology or Medicine 1984Niels K. Jerne, Georges J.F. Köhlor and César Milstein"for theories concerning the specificity in development and control of the immune system and the discovery of the principle for production of monoclonal antibodies"
Classic Paper
Myeloma Cells
Multiple myeloma cells are capable of producing antibodies but they arise naturally and are not programmed to make antibodies against specified antigens
Spleen Structure and Function
B lymphocytes in the spleen are used in creating hybridomas for monoclonal antibody
production
HAT Selection for Fused Hybridoma Cells
Which compounds are NOT critical components of HAT selection media?
A. TyrosineB. HypoxanthineC. Hyaluronic acidD. AdenineE. ThymidineF. Aminopterin
N5,N10-methylene tetrahydrofolate dihydrofolatedUMP dTMP+ +thymidylate
synthaseThe thymidylate synthase reaction is the only reaction in the human body where a tetrahydrofolate derivative is oxidized to dihydrofolate
NADPH
NADP+THF
serine
glycine
serine hydroxymethyl transferase dihydrofolate
reductase
Thymidine
thymidylate kinase
aminopterin
CH2 CH3 oxidized
N5,N10-methylene tetrahydrofolate
N5 –methyl tetrahydrofolate
N10-formyl tetrahydrofolate
methionine +
dTMP + DHF
purines +
DHFR
AMPT
AMPT
THF
THF
Inhibitors of DHFR Disrupt Folic Acid Metabolism in Cells Making TMP
ribose-5-phosphate
pRpp
5-phosphoribosylamine
IMP
AMP GMP
ADP GDP
ATP GTP
two steps require folic acid derivatives
hypoxanthine + pRpp IMP + PPi
guanine + pRpp GMP + PPi
hypoxanthine/guanine phosphoribosyl transferase
(HGPRT)
Salvage pathway
De Novo and Salvage Pathways for Purine Synthesis
HAT Selection (Continued)
Cells can survive in HAT media as long as they have functional thymidylate kinase and hypoxanthine/guanine phosphoribosyltransferase
aminopterin
purines
dTMP
hypoxanthine
HGPRT
thymidine
TK
• myeloma cells are defective in the salvage gene hypoxanthine/guanine phosphoribosyl transferase (HGPRT) and therefore cannot grow on HAT media
• normal B lymphocytes have short half lives and cannot be grown for extended periods in culture
Hybridoma Selection
• T.C. standard tissue culture media
In the hybridomas • normal B cells provide the HGPRT
cells myeloma cells lack• myeloma cells provide the ability
to grow indefinitely in culture that normal B cells lack
complementation
High Resolution Electrophoresis (Isoelectric focusing)
4
5
6
7
8
9
10
Stab
le p
H Gr
adie
nt
+++
++
+
---
--
-
++
++
-
-
-+-
-+
-
++
-+
-
-++
-+
-
-
+ + +
Isoelectric point
time time
COO -
C HH3+ N
H
COOH
C HH3+ N
H
COO -
C HH2 N
H
below pH 2 pH 2 - 10 above pH 10
diffusion
diffusion
reacquisition of net charge
reacquisition of net charge
High Resolution Electrophoresis (SDS-PAGE)
SDS binds to proteins in a ratio of ~ 1 SDS per 2 amino acids
βME reduces disulfide bonds
proteins have equal charge to mass ratios
separates according to size
Network of pores created by polyacrylamide
CH3
OS
O -
O
O -
CH2
CH2
SH
OH
SDS sodium dodecyl sulfate
β-mercaptoethanol
SS
heat with SDS and mercaptoethanol
SH
SH
+
smaller
larger
Highest Resolution Electrophoresis
(Two Dimensional Gel Electrophoresis)
Proteomics
basic acidic
IEF
large
small
SDS-
PAGE
normal diseased
P P H H H H HM M P
Characterization of Secreted Proteins from Hybridoma Cell Lines
New species representing hybrids of light and heavy chains produced from genes in parental cells indicating cells had fused
• Added radiolabeled lysine to hybridoma cells
• Analyzed proteins secreted from hybridoma cells by IEF
• P, parental cells• M, mixture of
parental cells• H, hybridoma
Both parental cells express IgG
P1: H2L2
P2: H2L2
H: H2L2 , H2L2, H2L2,
H2L2
grow hybridoma cells in soft agar
add sheep red blood cells
add complement
Plaque Assay for Antibodies Against Sheep Red Blood Cells
actual data from Köhler and Milstein
• To facilitate our studies we used a myeloma parental line which itself produced an Ig. (thus a mixture of IgG’s were produced)
• Variants hybridomas where one of the parental chains is no longer produced seem fairly common. Therefore selection of lines where only the specific antibody is produced seems reasonably simple.
• Alternatively, non-producing variants of myeloma lines could be used for fusion.
• Such cells can be grown in vitro in massive cultures to provide a specific antibody.
• Such cultures could be valuable for medical and industrial use.
Conclusions by Köhler and Milstein (1975)
Rituximab – 1997, first approved clinical use for a monoclonal antibody
Properties of an Ideal Tumor-Associated Antigen (TAA)
• Expression on all tumor cells including cancer stem cell
• Expression on tumor cell surface
• Functions in tumor cell survival
• Lack of expression on normal tissues
• No current TAAs display all of these ideal characteristics!
Overview of Rituximab: Structure, Target, and Mechanisms of Action
Maloney DG. N Engl J Med 2012;366:2008-2016.
I now know what the molar was!
CD20 is an Excellent Target for a Therapeutic Antibody to Treat B Cell Hyperproliferative Disorders
CD = Clusters of Differentiation (Classification Determinant) antigens
Early progenitors do NOT express CD20, so B cell-
mediated adaptive immunity could be restored after therapy
Characteristics of CD20
• Not shed from B cell surface, no serum CD20 to compete with binding• Does not internalize after binding antibody
• Accessible, stimulates immune effector mechanisms that result in tumor lysis
• Mouse-monoclonals against CD20 showed some efficacy in human clinical trials
• Required for B lymphocyte proliferation• Appears to play a role in Ca2+ transport
• 35 kDa integral membrane protein of the cell surface, spanning the plasma membrane 4 times
Structure
Function
Properties useful as a target for a therapeutic antibody
Mice immunized weekly with a human lymphoblastoid cell line (SB)
Spleens harvested from mice expressing high titers of anti-CD20
Hybridomas formed from spleen lymphocytes and a mouse myeloma cell line SP2/0
125I-antiCD20(murine) + hybridoma culture supernatant + SB cells
recover SB cells, wash, monitor radioactivity
hybridomas secreting anti-CD20 should inhibit 125I-
binding
Screening Hybridomas for Specific Monoclonal Antibodies (circa 1994)
Reff et al (1994) Blood 83:435-445
Modern Approaches to Antibody Screening
ELISA Enzyme-Linked ImmunoSorbant Assay
luminol
horseradish peroxidase
made by hybridomas
Mice immunized weekly with a human lymphoblastoid cell line (SB)
Spleens harvested from mice expressing high titers of anti-CD20
Hybridomas formed from spleen lymphocytes and a mouse myeloma cell line SP2/0
125I-antiCD20(murine) + hybridoma culture supernatant + SB cells
recover SB cells, wash, monitor radioactivity
hybridomas secreting anti-CD20 should inhibit 125I-
binding
Screening Hybridomas for Specific Monoclonal Antibodies (circa 1994)
Reff et al (1994) Blood 83:435-445
Eukaryotic Expression Vector used for the Expression of the Heavy and Light Chains of a Monoclonal
Antibody Against CD20antibiotic selection in E. coli
ampicillin
The AMP Gene Encoding β-Lactamase is used as a Selectable Marker in Bacterial Cells
β-lactamase cleaves this bond
ampicillin
Eukaryotic Expression Vector used for the Expression of the Heavy and Light Chains of a Monoclonal
Antibody Against CD20
neomycin resistance, selectable marker in mammalian cells
Eukaryotic Expression Vector used for the Expression of the Heavy and Light Chains of a Monoclonal
Antibody Against CD20SV40 origin
of replication
Eukaryotic Expression Vector used for the Expression of the Heavy and Light Chains of a Monoclonal
Antibody Against CD20
dihydrofolate reductase
grown in the present of methotrexate, will increase
gene dosage
Eukaryotic Expression Vector used for the Expression of the Heavy and Light Chains of a Monoclonal
Antibody Against CD20light chain
CMV promoter
consensus signal peptide for
human immunoglobulins
mouse light chain
variable region
human light chain constant region
PCR amplified from selected hybridoma
Creation of Mouse/Human Hybrid Monoclonal Antibodies (Light Chains)
BGH polyadenylation
signal
similar construct used for heavy chain
Antibody Purification
Protein A from Staphylococcus aureus binds up to 5 molecules of IgG through the Fc domain. This is the opposite orientation an IgG would normally bind to a pathogen surface. The interaction of Protein A with IgG in this manner disrupts opsinization
Prot A Prot A Prot A
serum
IgG
flow through- other stuff purified IgG
washloadelute – low pH elution buffer
(pH2.8)
Antibody Characteristics: Scatchard Plot of Equilibrium Binding Data
B/Lf = -B/Kd + RT/Kd
Plot B/Lf versus B
RT/Kd
C2B82B8 (murine)
Antibody Characteristics: Competitive Binding Assay
Ability of different monoclonoal antibodies to inhibit the binding of 125I-CD20 (murine) to SB lymphoblastoid cells
C2B82B8 (murine)
Antibody Function: Directing Complement Binding to Antigens
Antibody + lymphoblastoid cells + fluorescently-labeled human complement
FACS (fluorescence-activated cell sorting)
analysis
murine monoclonal Ab
humanized monoclonal Ab
Antibody Function: Orchestrating Cell Lysis
load SB cells with 51Cr
mix with antibody and a source of human
complement
monitor 51Cr release
murine monoclonal Ab
humanized monoclonal Ab
load SB cells with 51Crmix with antibody and
effector cells (peripheral mononuclear cells)
monitor 51Cr release
humanized monoclonal Ab
Antibody Function: Depleting B Lymphocytes in a Non-Human Primate Model
• Infuse Clonus monkeys with humanized Ab
• Injections weekly for 4 weeks
• Assess levels of B lymphocytes 36 days after last injection
Antibody Function: Selectivity
• Infuse Clonus monkeys with humanized Ab
• Injections weekly for 4 weeks
• Assess levels of T lymphocytes 36 days after last injection