BIOTECHNOLOGICAL APPLICATIONS 1

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