Genetic Engineering of Antibodies

• Lecture 16 focuses on genetic engineering of antibodies

Acquired Immunity/Immune Memory

• Memory B cells and Killer T cells are activated via acquired immunity

Antibody Structure

• Antibodies consist of 2 polypeptide chains, encoded by 2 genes
• Heavy and light chains are held together by disulfide bonds
• Each chain contains a constant and a variable region
• A region at the end of each chain binds to antigens, known as the antigen-binding site (ABS)
• The sequence of ABS varies among antibodies
• Heavy and light chains are encoded by two different genes

Polyclonal Antibodies

• An antigen is injected into an animal to produce antibodies
• The immune system reacts by finding B cells that make antibodies to bind the antigen
• These B cells multiply and secrete antigen-specific antibodies
• Each B cell makes a unique antibody
• Many different antibodies from B cells recognise the same antigen, forming a polyclonal mixture
• Polyclonal antibodies can be extracted from blood and used in vitro for diagnostics

Monoclonal Antibodies (mAbs)

• Monoclonal antibodies are used for therapeutic applications due to safety
• Monoclonal antibodies are well-characterized and understood molecules for human injection
• Production is closely regulated by authorities like FDA, EMA, and HPRA
• Monoclonal antibody production isolates single B cells

mAb Production

• The process is similar to polyclonal antibody production
• Instead of harvesting blood, the animal is sacrificed and the spleen is taken
• B cells are extracted from the spleen and fused with a special tumor cell line (myeloma cells)
• Fusion is needed, as B cells die after being put into culture dishes Hybridoma cells are produced via this, giving them an immortal lifespan while producing only one type of antibody
• Then, individual hybridoma cells are isolated into clones, allowed to expand, and tested for monoclonal antibody production
• The most effective monoclonal antibody that binds to the antigen is selected for use
• The method was developed by Milstein and Kohler in 1975, who won the Nobel Prize, but did not patent the technology

Large-Scale Antibody Production

• Hybridomas are not efficient at producing monoclonal antibodies
• Serum is needed
• High densities are not achieved in culture
• Some monoclonal antibody-based therapies need over 1 tonne of protein annually for global supply (Botox example needs 1g)
• A lot of cells are needed, but hybridoma cells are not easy to scale up
• As such, another cell line is used: CHO

Molecular Biology of Antibody Production

• Monoclonal antibody genes are cloned from hybridoma and B cells
• Unlike insulin production, two genes are needed to make an antibody: heavy and light chains
• Each gene is amplified using DNA extracted from the hybridoma or B cell via PCR
• It is then ligated into a plasmid and transformed into E. coli
• The E. coli is grown and the plasmid is extracted, now containing the monoclonal antibody genes
• The E. coli bacteria is not used to create the monoclonal antibody protein - CHO cells are used
• Bacteria are used as a tool to clone the gene

CHO Cell Production

• The expression vector (plasmid) is amplified in E. coli in the previous step
• Bacteria are lysed, and the plasmid is harvested and purified (~1-2mg)
• This plasmid is introduced into the CHO cells
• As mammalian cells are too fragile, heat shock cannot be used
• Cationic lipid-based reagent is added to transfect the cells
• This plasmid goes into the nucleus, where its genes are transcribed and translated

Transient vs Stable Expression

• Once inside CHO cells, the plasmid serves as a template to express the monoclonal antibody
• Transient expression occurs if the plasmid is eventually lost as the cells divide
• Stable expression occurs when a plasmid accidentally incorporates into the genomes of CHO cells to create a recombinant CHO cell
• The cell line will stably express the product due to permanent insertion
• Like bacteria, CHO cells that stably integrate the plasmid are selected by either adding cytotoxic drugs or removing critical nutrients (compensation via a gene on the plasmid)
• The stable recombinant CHO cells are grown at large scales to produce and secrete significant amounts of monoclonal antibodies

Cell Line Development Workflow

• Requires roughly 4 - 6 weeks

CHO Cell Heterogeneity

• Within a population, cells can have quite different characteristics
• Even after cloning a single cell, daughter cells diverge genotypically and phenotypically
• Heterogeneity aids the screening process of transfected individual cells in a CLD lab, as the cells find the traits
• A subset of good clones diverge after further generations in culture, known as instability, requiring focused research to overcome

Engineering Desirable Cell Characteristics

• Focus on cell growth, protein production, and new moieties

Hosts

• Increase expression of genes that promote cell growth and protein secretion
• Reduce expression of genes that promote apoptosis, antibody fucosylation and contaminants via knockdown using RNAi or knockout using CRISPR

CRISPR

• Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) was discovered by Doudna/Charpentier, winning the 2020 Nobel Prize in Chemistry
• It is a bacterial adaptive immune system
• Pieces of virus (phage) genome is stored in the bacterial genome
• When a future infection happens, a stored piece of viral DNA can be used as a guide
• Guides are required (genome enzymes that chop the virus’s genome and prevent replication)
• The CRISPR are transcribed from the bacterial genome as short RNAs (sRNA)

CRISPR-Cas9 usage

• A system comprising of:
  • Cas9 nuclease
  • Single guide RNA (sgRNA)
    • Target-specific
    • Cas9 guide
• Targets DNA for cutting by Cas9
  • DNA is repaired, but mistakes can be made
    • Mistakes equal mutations and knockouts (KO)
    • It can also be used to insert new genes
• Advanced applications are available
  • Dead cas9 with other functions bolted on, e.g. demethylase activity
  • Gene therapy for future study

GFP Gene in CHO cells

• Green Fluorescent Protein (GFP) is inserted into the genome of CHO cells
• When exposed to a certain wavelength of light, cells appear green and easily express expression
• A CRISPR-Cas9 plasmid contains the complementary sequence sgRNA
• When transfected into a CHO cell the sgRNA directs Cas9 enzymes to the GFP sequence to cleave DNA
• Repair processes will then activate the cells
• The repair is done correctly most of the time
• Occasionally insertion or deletion and mistakes happen (indel)
  • This influences gene codons
  • Any gene or sequence can be targeted in this manner

CRISPR example of improving therapeutic product quality

• CRISPR engineering reduces product heterogeneity
• Alpha-1-Antitrypsin deficiency can lead to COPD and liver disease
• Very distinct homogenous N-glycan are attached to protein
• Recombinant versions made in CHO cells have heterogenous N-glycans
• This protein is treated with material derived from blood donors on patients that are lacking

TEN Genes

• TEN genes are knocked out simultaneously via use of CRISPR
• These code enzymes to build/attach various forms of N-glycan to proteins
• Researchers eliminated 95% of unwanted sugars to create a more homogeneous compound
• Characteristics for CHO cells were unaffected
• This means safer products, lower dose amounts required, and lower yields needed for process to supply market

Summary

• The immune system's B cells can create specific antibodies to recognise antigens
• Inoculation of animals with a target protein will then make Her2 receptor cells present on a cancer cell
• This produces different B cells to link to a target (polyclonal)
• The B cells extracted fuse with tumour cells to make them immortal
  • Each hybridoma produces one type of antibody (monoclonal mAb)
• Next, extract and amplify the genes from the hybridoma that encodes heavy and light antibody chains
• E. coli and CHO cells are used for transfer of plasmids
• There is an ability to produce a biopharmaceutical drug
• As a result, a producer cell can be genetically engineered to have more attractive traits using targeted engineering strategies