Issue #71: The monoclonal antibody story part 2: One cell, one antibody and a felicitous fusion

Written by Nobel Laureate Professor Peter Doherty

Any antigen-specific (adaptive) immune response is polyclonal, with a spectrum of cell-surface receptor molecules attaching, perhaps in slightly different configurations, to the same molecular structure (#18, #20, #34 and #40). With COVID-19, at least so far as the B cell lineage is concerned, the structure of interest is the receptor binding domain (RBD) on the SARS-CoV-2 spike protein that attaches to the ACE2 molecule on the surface of epithelial cells. Block that with virus specific immunoglobulin (Ig, antibody), and the virus can’t get into cells and start reproducing itself.

Way back (1958), a young Australian immunologist Gustav (Gus) Nossal working at  Melbourne‘s Walter and Eliza Hall Institute of Medical Research (WEHI) did an extraordinarily elegant experiment to show that, in an immune response, ‘one cell makes one antibody’. That, together with the clonal selection theory of acquired immunity proposed independently a year earlier by FM (Sir Macfarlane) Burnet, the then Director of the WEHI, and David Talmage of the University of Colorado is central to our understanding of antigen-specific, adaptive immunity. What we have in a monoclonal antibody (mAb) is the single Ig product of one immune B lymphocyte/plasma cell that has been ‘immortalised’.  

As mentioned last week (#70), the discovery of how to make mAb-producing cell lines earned Cesar Milstein (#70) and his young associate Georges Kohler a Nobel Prize which, in 1984, they shared with veteran immunologist Niels Jerne. Jerne was included for his theoretical contributions to understanding antibody responses. The story of how Cesar and George got to their breakthrough follows.

Last week (#70) I summarised, based on the story told in Cesar’s Nobel Lecture how his Cambridge University research group added a cell culture approach to their protein chemistry expertise as they probed the genetic basis of antibody diversity (#18). Basically, they ‘picked’ mutant clones from antibody producing myelomas (tumours), then studied the molecular consequences.  

At that time, another powerful mechanism for looking at genetic effects was to fuse different types of cells together and analyse how the genomes interacted. This approach had been pioneered at Oxford University by Australian-educated medical scientist and cell biologist Henry Harris, who succeeded his mentor, Sir Howard Florey (shared the 1945 Nobel with Alexander Fleming and Ernst Chain for penicillin) as Director of the Sir William Dunn School of Pathology, a job Sir Henry held for 31 years. An innovative laboratory researcher, Harris pioneered the use of an inactivated mouse parainfluenza type 1 virus, Sendai virus (SV), to make a ‘chimera’ of two different cell types.

Parainfluenza viruses, like the two human CoVs circulating prior to the year 2000, cause colds and croup. Unlike the spike RBD/ACE-2 interaction used by SARS-CoV-2 to infect, the ‘paraflus’ express a surface fusion protein that ’joins’ the viral and cell outer membranes, allowing the viral RNA to invade. Using SV that had been inactivated to destroy its RNA Harris found, for example, that fusing a normal cell and a cancer cell together led to the suppression of the malignant, cancer ‘phenotype’.

Seeing the potential of SV-fusion for probing the antibody diversity question, young Australian agricultural science graduate and microbial geneticist RGH (Dick) Cotton, who had recently joined the Milstein laboratory, set out to fuse two different myeloma lines together. What Dick found was the that the allelic exclusion (using the Ig genes from only one parent) characteristic of normally antibody producing cells did not apply to these hybridomas. The reason: that die was already cast, the myelomas are cancerous forms of the plasma cell ‘factories’ (#18, #21) that have already rearranged their antibody genes and are pumping-out Ig molecules.

Having completed his short-term contract, Dick Cotton returned to Australia in 1974 and later (1986, with pediatrician David Danks) founded what is now the Murdoch Children’s Research Institute. A major figure for decades in Australian medical genetics and a wonderful colleague at the University of Melbourne, Dick just missed out on a Nobel Prize.

Dick’s replacement in the Milstein laboratory was Georges Kohler from the then world-leading (now sadly defunct), Basel Institute for Immunology, headed by founding Director Niels Jerne. Georges did the next obvious experiment and fused a mouse myeloma line (MOPC 21) with immune cells from a mouse that had been immunised to make antibodies to sheep red blood cells (SRBC). Using a technique known colloquially as the Jerne/Cunningham ‘plaque assay’ – the cells were plated on a ‘lawn’ of SRBCs to find those making specific antibodies – it was easy to detect (and isolate) the responding plasma cells. We’ve met Jerne, while New Zealander AJ (Alastair) Cunningham graduated the year ahead of me at the University of Queensland Veterinary School. Intellectually driven, Al completed an ANU PhD, published with Gus Nossal, and was a great colleague at the JCSMR, Canberra, when Rolf and I did the experiments that led to our Nobel award (#31, #32).

Returning to the Milstein laboratory, Georges used the Jerne/Cunningham plaque assay to show that, by fusing a myeloma and an immune B cell, he had generated hyridoma clones that were pumping out SRBC-specific antibodies. Without that being their primary intent, Kohler and Milstein had discovered how to make mAbs that would bind to any antigen of interest. As we’ll discuss over the next couple of weeks, that began a revolution with implications across biology and into therapeutics.