Mammalian Cell Expression of Recombinant Antibodies Flashcards
Why do we need mammalian expression systems?
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
Major Mammalian Cell Lines
Chinese Hamster Ovary (CHO) cells
Human Embryonic Kidney (HEK) cells
Murine - Sp2/0 OR NSO
HeLa
Characteristics of Chinese Hamster Ovary (CHO) cells
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
What are Chinese Hamster Ovary (CHO) cells used for?
Monoclonal antibodies Cytokines Enzymes Hormones Clotting factors...etc
Characteristics of Human Embryonic Kidney (HEK) cells
Made using adenovirus (retrovirus???) fragments
Yield - 1g/L (LOWER)
Very close match to other human cells
What are Human Embryonic Kidney (HEK) cell used for?
Used for clotting factors
What are murine cell lines used for?
Make mAbs (monoclonal antibodies) - humanize everything but key binding part
Vector Systems (Basic)
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
Types of vector system (other than basic)
Two-Vector System
Bicistronic Vector
Bicistronic Vector
Two genes in one vector
Two-Vector System
Two vectors in one cell
How do you make a bicistronic vector?
Internal ribosome entry site (IRES), initiated translation independently of the 5’ cap
start-gene1-IRES-gene2-termination
Purification methods
Affinity Chromatography
Size exclusion
Ion Exchange Chromatography
Hydrophobic Interaction Chromatography
Affinity Chromatography
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
Size Exclusion
A further way to filter (no usually the first step)
Filtration through a gel
E.g. 150 kDa, smaller than get excluded
Ion Exchange Chromatography
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
What are polyclonal antibodies?
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
What are monoclonal antibodies?
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)
What produces monoclonal antibodies?
Produced by hybridoma (cells) or recombinant antibody technology (vectors)
Why use polyclonal antibodies?
Fast
Inexpensive
Why not use polyclonal antibodies?
Cannot engineer, don’t know gene, just produced by immunized animals
Finite - whatever is produced by the animal
What are hybridoma cells?
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.
Antibody fragments
Fv scFv dsFv scAb Fab - variable heavy and variable light, constant heavy and constant light domains (one size of antibody) = monovalent
Fv
Fragment of antibody Variable regions
Variable heavy and variable light, without constant domain (NOT STABLE)
scFv
Single-Chain Fragment of antibody Variable regions
FV +linker (single-chain (sc) linker = cellulose)
dsFv
FV +disulphide bonds (double chain (ds) linker)
scAb
scFV + human constant light domain (stability and detection)
Secondary antibody binds to constant light domain (draw two elements closer together)
Assay detection improvement
Fab
Variable heavy and variable light, constant heavy and constant light domains (one size of antibody) = monovalent
Epitope types
LINEAR CONFORMATIONAL IMMUNO-DOMINANT IMMUNOGENIC NEUTRALISING
LINEAR epitope
RECOGNISES AMINO ACID SEQUENCE
CAN BIND WHETHER PROTEIN DENATURED OR NOT (BASED ONLY ON PRIMARY STRUCTURE)
CONFORMATIONAL epitope
RECOGNISES TERTIARY STRUCTURE
ABILITY TO BIND LOST WITH DENATURATION
IMMUNOGENIC
PRODUCE STRONG IMMUNE RESPONSE
NEUTRALISING
BINDING STOPS ACTIVITY (NOTHING ELSE REQUIRED)
EPITOPE
Antigen receptor (where the antibody binds)
PARATOPE
Antibody component binding site
collection of CDRs - variable regions
What can you do to improve antibody affinity?
Change size (make smaller) Improve stability Subtype switching Species switching Isotype switching Reformatting
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
Why would you carry out species switching with an antibody?
To reduce immunogenicity
e.g. humanise mouse antibodies
Why would you reformat an antibody?
Don’t want full IgG molecule
Don’t want/need full molecule
Just need binding site/particular ligand
Shortcomings of mouse mAbs in therapy
Glycans in Fc part considered “foreign” by human immune system
In vitro technologies developed to generate fully human antibodies
Phage display
Ribosome display
How does phage display work?
Display protein on surface of filamentous phage
recombinant protein (usually scFV) + P3 gene for filamentous display
What is bio-panning?
How to get BACTERIOPHAGE DISPLAYING ANTIBODY (contain gene encoding for that antibody fragment)
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
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
What is a single-domain antibody?
Single domain = only heavy chain (does not req. Light chain)
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)
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
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
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
What is a biosimilar?
Binds to the same epitope (antigen)
Similar efficacy, safety and immunogenicity
Different structure of antibody
What are next generation antibody therapeutics?
Antibody drug conjugates
Bispecifics
CAR-T therapy (personalised medicine)
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
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
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
What is ELISA?
Enzyme-Linked Immunosorbent Assay
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
What is affinity?
The strength of the antigen-antibody bonding
What is avidity?
The overall stability of the antibody-antigen complex
What type of bonding is between antigen-antibody?
non-covalent
e.g. hydrogen bonds, van der Waals (gen.) /ionic bonds/hydrophobic
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
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)
How might you maintain the phenotype(displayed)-genotype(DNA) connection?
Ribosome display
Phage display
Yeast Surface Display
Post bio-panning formatting
Phage display scFV (usually) and so these are then reformatted (once harvested) into mAb/scAb and Fab as appropriate.
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)
What is a CDR?
Complementarity-determining regions (CDRs)
= part of the variable chains in immunoglobulins (antibodies)
= where antibodies bind to their specific antigen
What does ADME properties mean?
Absorption, Distribution, Metabolism, and Excretion properties of the drug molecule
What are ADCs?
antibody drug conjugates
- application for antibody technology
What to ADCs do?
Mode of delivery of cytotoxic agents
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
