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Biotechnology > Mammalian Cell Expression of Recombinant Antibodies > Flashcards

Mammalian Cell Expression of Recombinant Antibodies Flashcards

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1
Q

Why do we need mammalian expression systems?

A

Post-translational modifications E.g. antibodies Fc region glycosylation

Protein folding:
Primary - Peptide bonds
Secondary - Beta pleated sheets/Alpha helix
Tertiary (conformational structure - decides biological activity): Di-sulphide bonds (between cysteines), Hydrophobicity
Quaternary (some proteins, not all), Globular - joining of protein units E.g. antibodies, Heavy chains (x2), Light chains (x2)

Location of protein creation, Eukaryotic - separation of different parts of process
Nucleus 
Cytoplasm 
Quality control  - ER
Glycosylation - ER + Golgi body
Di-sulphide bonds - ER
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2
Q

Major Mammalian Cell Lines

A

Chinese Hamster Ovary (CHO) cells
Human Embryonic Kidney (HEK) cells
Murine - Sp2/0 OR NSO
HeLa

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3
Q

Characteristics of Chinese Hamster Ovary (CHO) cells

A

Quite durable
Can survive variations in conditions
No batch-to-batch variation (or at least v.low)
Easily adapted to suspension/serum-free conditions
Serum introduces unwanted proteins
E.g. endotoxins
Yields - 2-6g/L
Human pathogens cannot replicate in CHO cells
Glycosylation patterns need to be carefully monitored
Why? Avoid anaphylactic response

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4
Q

What are Chinese Hamster Ovary (CHO) cells used for?

A 
Monoclonal antibodies
Cytokines
Enzymes
Hormones 
Clotting factors...etc
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5
Q

Characteristics of Human Embryonic Kidney (HEK) cells

A

Made using adenovirus (retrovirus???) fragments
Yield - 1g/L (LOWER)
Very close match to other human cells

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6
Q

What are Human Embryonic Kidney (HEK) cell used for?

A

Used for clotting factors

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7
Q

What are murine cell lines used for?

A

Make mAbs (monoclonal antibodies) - humanize everything but key binding part

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8
Q

Vector Systems (Basic)

A

Origin of replication (ORI)

Selectable markers

Antibiotic resistance

Promoter (e.g. CMV used as is a strong promoter)

Multiple cloning sites - increases expression

Termination site

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9
Q

Types of vector system (other than basic)

A

Two-Vector System

Bicistronic Vector

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10
Q

Bicistronic Vector

A

Two genes in one vector

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11
Q

Two-Vector System

A

Two vectors in one cell

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12
Q

How do you make a bicistronic vector?

A

Internal ribosome entry site (IRES), initiated translation independently of the 5’ cap

start-gene1-IRES-gene2-termination

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13
Q

Purification methods

A

Affinity Chromatography

Size exclusion

Ion Exchange Chromatography

Hydrophobic Interaction Chromatography

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14
Q

Affinity Chromatography

A

E.g. nickel-ion affinity chromatography

Column and tags (e.g. His-tags)
Binding at X pH
Elution at X pH
Wash off non-bound proteins/molecules

A good general first step

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15
Q

Size Exclusion

A

A further way to filter (no usually the first step)
Filtration through a gel

E.g. 150 kDa, smaller than get excluded

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16
Q

Ion Exchange Chromatography

A

Cation & anion

Resin with modified charged functional groups
Selecting for cations-> negative
Selecting for anions-> positive

Based on isoelectric point
pH give protein a neutral charge at X point
Use that pH point to elute

Flow through
Bind to resin and flow through attached

Bind and elute

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17
Q

What are polyclonal antibodies?

A

From hyperimmunization animals

Uses adjuvant - elevate immune response

Made by different B lymphocytes

Bind to different parts of the antigen - multiple paratopes target multiple epitopes

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18
Q

What are monoclonal antibodies?

A

Secreted by single B lymphocyte clone - select for cell that produces antibody of interest

One antigen binding site (paratope), recognises a single epitope (antigen binding site on antigen)

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19
Q

What produces monoclonal antibodies?

A

Produced by hybridoma (cells) or recombinant antibody technology (vectors)

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20
Q

Why use polyclonal antibodies?

A

Fast

Inexpensive

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21
Q

Why not use polyclonal antibodies?

A

Cannot engineer, don’t know gene, just produced by immunized animals

Finite - whatever is produced by the animal

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22
Q

What are hybridoma cells?

A

Injecting a specific antigen into a mouse, collecting an antibody-producing cell from the mouse’s spleen, and fusing the antibody-producing cell with a tumour cell called a myeloma cell.

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23
Q

Antibody fragments

A 
Fv
scFv 
dsFv
scAb
Fab - variable heavy and variable light, constant heavy and constant light domains (one size of antibody) = monovalent
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24
Q

Fv

A

Fragment of antibody Variable regions

Variable heavy and variable light, without constant domain (NOT STABLE)

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25
scFv
Single-Chain Fragment of antibody Variable regions FV +linker (single-chain (sc) linker = cellulose)
26
dsFv
FV +disulphide bonds (double chain (ds) linker)
27
scAb
scFV + human constant light domain (stability and detection) Secondary antibody binds to constant light domain (draw two elements closer together) Assay detection improvement
28
Fab
Variable heavy and variable light, constant heavy and constant light domains (one size of antibody) = monovalent
29
Epitope types
``` LINEAR CONFORMATIONAL IMMUNO-DOMINANT IMMUNOGENIC NEUTRALISING ```
30
LINEAR epitope
RECOGNISES AMINO ACID SEQUENCE | CAN BIND WHETHER PROTEIN DENATURED OR NOT (BASED ONLY ON PRIMARY STRUCTURE)
31
CONFORMATIONAL epitope
RECOGNISES TERTIARY STRUCTURE | ABILITY TO BIND LOST WITH DENATURATION
32
IMMUNOGENIC
PRODUCE STRONG IMMUNE RESPONSE
33
NEUTRALISING
BINDING STOPS ACTIVITY (NOTHING ELSE REQUIRED)
34
EPITOPE
Antigen receptor (where the antibody binds)
35
PARATOPE
Antibody component binding site | collection of CDRs - variable regions
36
What can you do to improve antibody affinity?
``` Change size (make smaller) Improve stability Subtype switching Species switching Isotype switching Reformatting ```
37
Why change the subtype of an antibody?
Think about immune system recruitment IgG types 1, 2, 3, 4...etc, respond to different types of infection
38
Why would you carry out species switching with an antibody?
To reduce immunogenicity | e.g. humanise mouse antibodies
39
Why would you reformat an antibody?
Don’t want full IgG molecule Don’t want/need full molecule Just need binding site/particular ligand
40
Shortcomings of mouse mAbs in therapy
Glycans in Fc part considered “foreign” by human immune system
41
In vitro technologies developed to generate fully human antibodies
Phage display | Ribosome display
42
How does phage display work?
Display protein on surface of filamentous phage recombinant protein (usually scFV) + P3 gene for filamentous display
43
What is bio-panning?
How to get BACTERIOPHAGE DISPLAYING ANTIBODY (contain gene encoding for that antibody fragment)
44
How does bio-panning work?
Antibody library of bacterial cells NOT bacteriophages YET (e.g. E.coli containing the vector (e.g. pHEN)) +infect with helper phage N13KO7 Replicates inside bacterial cell and is released with phage particles displaying the antibody fragment on its surface Then use ELISA as appropriate to isolate bacteriophages
45
Differences between library types
Niave = IgM of B cells - first level of response (because it is not responding to anything) IgM must be affinity matured to IgG, which adds time (~6 months) Synthetic - DO I NEED TO KNOW THIS??? Immunised = IgG of B cells - second level of response
46
What is a single-domain antibody?
Single domain = only heavy chain (does not req. Light chain)
47
Benefits of a single-domain antibody
Allows for rapid tissue penetration Good expression in bacterial and yeast systems (cheaper & more stable (temp./pH)) Can target additional/novel epitopes (antigens) E.g. shark (IgNARs) and camel (HcAbs)
48
What is ribosome display?
RNA fragments expressed on the surface of ribosome Limited to single-chain display - usually scFv (same as phage) No bacterial transformation Very large libraries
49
How does ribosome display work?
``` Antibody library (of vectors in bacterial cells) Select for best from that set ``` ``` Incorporate into ribosome display vector PCR + display vector -> mutagenesis Transcribed to mRNA (in vitro) +ribosomal complex -> protein expressed on surface of ribosomes ``` Selection - for affinity Dissociate complex - take the mRNA for that binder Reverse transcribe to cDNA Put back in to ribosomal display vector Several cycles of selection and mutagenesis possible
50
How to make a transgenic mouse?
XenaMouse - embryonic stem cells altered Human heavy and light loci for antibody production -> mice that will produce human antibodies in response to immunization
51
What is a biosimilar?
Binds to the same epitope (antigen) Similar efficacy, safety and immunogenicity Different structure of antibody
52
What are next generation antibody therapeutics?
Antibody drug conjugates Bispecifics CAR-T therapy (personalised medicine)
53
Antibody Drug Conjugates
Specific monoclonal antibody with highly potent cytotoxic agents attached by biodegradable linker To deliver cytotoxic agent(s) Why use an antibody? -> Increases specificity of targeting
54
Bispecifics
One antibody, two distinct binding arms Bind to different antigens or epitopes on the same antigen E.g. re-target T cells to kill tumour cells 1 - binds to cancer cell 2 - binds to recruit cytotoxic T cell
55
CAR-T therapy
PERSONALISED MEDICINE Chimeric antigen receptor - T (cell) therapy - Type of adoptive cell therapy (ACT) - Chimeric antigen receptor - antibody - Engineer T cells from patients T cells clear tumour cells/cancer cells - Make antibody to help T cells to find cancer cells - Insert antibody gene into T cell - T cells now express chimeric antibody on the surface - bind to specific targets on tumour cells Put modified T cells back into patient
56
What is ELISA?
Enzyme-Linked Immunosorbent Assay
57
How does ELISA work?
Antibody immobilised on solid surface - direct antigen on surface binds to antibody (of interest & linked to enzyme) -> enzyme activity - indirect antigen on surface binds to antibody (of interest) then another antibody (linked to enzyme binds) -> enzyme activity
58
What is affinity?
The strength of the antigen-antibody bonding
59
What is avidity?
The overall stability of the antibody-antigen complex
60
What type of bonding is between antigen-antibody?
non-covalent | e.g. hydrogen bonds, van der Waals (gen.) /ionic bonds/hydrophobic
61
What does chimeric mean in the context of chimeric antibody?
Variable light and variable heavy regions are different in some way (e.g. species switching) to the constant regions. e.g. ximab
62
What is a humanised mouse antibody?
zumab variable heavy and variable light regions have been altered to be humanised (mouse interspersed with human sections - particular areas of immunogenicity)
63
How might you maintain the phenotype(displayed)-genotype(DNA) connection?
Ribosome display Phage display Yeast Surface Display
64
Post bio-panning formatting
Phage display scFV (usually) and so these are then reformatted (once harvested) into mAb/scAb and Fab as appropriate.
65
Construction of a phage display antibody library
Select B cells that produce antibodies - extract all RNA | Use primers to locate variable regions (light chain and heavy change)
66
What is a CDR?
Complementarity-determining regions (CDRs) = part of the variable chains in immunoglobulins (antibodies) = where antibodies bind to their specific antigen
67
What does ADME properties mean?
Absorption, Distribution, Metabolism, and Excretion properties of the drug molecule
68
What are ADCs?
antibody drug conjugates | - application for antibody technology
69
What to ADCs do?
Mode of delivery of cytotoxic agents
70
How might you get mAbs across the Blood-Brain Barrier?
Bi-specific antibody that can “piggy-back” on an existing transportation system into the brain One arm binding engage with receptor-mediated transcytosis (type of cellular transport) Second arm binds therapeutic target

Biotechnology (5 decks)

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