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Issue #70: The monoclonal antibody story 1: In the beginning, there were myelomas

16 Aug 2021

Issue #70: The monoclonal antibody story 1: In the beginning, there were myelomas

Written by Nobel Laureate Professor Peter Doherty

Throughout this series, I’ve been mentioning monoclonal antibodies, the marvellous mAbs, and promising to write about them. Recently (#68) we talked a little about the way that the mAbs have been used by structural biologists as, probing the molecular shapes and chemical interactions that allow different proteins to attach to one another, they pursue the Holy Grail of rational drug design (#69). The story of how these wonderful immunoglobulin (Ig) reagents came to be available to us is a great example of a situation where smart researchers, who were working systematically to illuminate a basic question in science, ended up doing something that was of immense practical importance.

My version of this tale begins with an Argentinian scientist, the protein chemist and immunologist Cesar Milstein who, way back in 1963, joined the massively famous Laboratory of Molecular Biology, the LMB, at Cambridge University. Short in stature and big in heart and mind, Cesar and his wife Celia were regarded with great affection and respect by those of us who were privileged to know them. Much of what I write here is taken from Cesar’s 1984 Nobel Lecture.

Cesar’s research was focused on understanding the antibody response (#18-#22), particularly with defining how the genes encoding the immunoglobulins (Igs) organise to give the extraordinary diversity of binding ‘motifs’ (patterns) that allow antibodies to recognise completely novel pathogens. What we’re interested in with COVID-19 is, of course, the spectrum of Igs (the polyclonal antibody response) with different variable (V) regions that can attach tightly to key ‘antigens’ like the receptor binding domain (RBD, for cell surface ACE2) of the SARS-CoV-2 spike protein. Now, a researcher who wanted to know about that IgV region diversity would just sequence lots of IgV genes, but that technology was yet to be developed back in the 1960’s and 1970’s.

What investigators were doing back then was to sequence masses of Ig proteins. Though the technology wasn’t there for doing that with small amounts of protein, researchers had access to naturally occurring, B-cell-lineage tumour cell lines, myelomas, that could be grown up in culture to provide a single Ig molecule by the bucket load. Cultured from randomly occurring cancers (in humans) or induced by chemical carcinogens (like methylcholanthrene) in mice, they could be maintained indefinitely in the lab. That process of continued cell division leads, as in all biological systems, to the accumulation for random mutations. As a consequence, myelomas have to be periodically subcloned (back to single cells) so they continue to make a single Ig protein. One issue was, though, that while any given myeloma clone was producing one antibody of a single type, the relevant antigen was unknown and there was no way of making them so that they were targeted to proteins of interest.

Some researchers were trying to make ‘immortalised’, antibody-producing cell lines with a known specificity by transforming responding B lymphocytes with Epstein Barr Virus (EBV). EBV is a human herpesvirus that causes infectious mononucleosis (kissing disease) in adolescents and, if we are immunosuppressed – by HIV/AIDS (before drugs were available) or by treatment with cytotoxic cancer drugs – can lead to the emergence of lethal lymphomas in us. Just about all adults have persistent EBV infections, which are normally controlled through life by our ‘immune surveillance’, CD8+ T cells (#33 #34). But, though EBV-transformed ‘LCLS’ (lymphoblastoid cell lines) can readily be made and grown in culture, they just don’t produce Ig molecules in quantities that enable any useful application.

Having masses of myeloma Igs did, however, allow the protein chemists to work out the basic structure of the antibody (Ig molecule). That achievement was recognised by a 1972 Nobel Prize for Medicine to Oxford University’s Rod Porter and the Rockefeller University’s Gerry Edelman. Much later, with the technology advancing, the genetic (DNA) basis of immunological diversity was also clarified, with the 1987 Medicine Nobel going to MIT’s Susumu Tonegawa ‘for his discovery of the genetic mechanism that produces antibody diversity’.

Back to the start of the 1970’s and Cesar Milstein in his LMB laboratory: using protein chemistry, they’d been trying to look at purified serum antibodies that bound directly to a particular antigen, and they weren’t getting far. The problem was, of course, that the antibody response is polyclonal, so the LMB group was analysing a mix of different Ig proteins. They clearly needed a new approach!

With emerging expertise in mRNA sequencing, the Milstein team decided to learn a little biology and serial passage individual myelomas in tissue culture, then develop subclones to identify Ig variants (mutants) that had arisen naturally via the process of normal, background mutation. Their first Ig region variant (detected by protein chemistry) came up after they’d looked at several thousand clones and, by number 7,000, they had a range of structural mutants (making different Igs) for further analysis. This was a breakthrough study.

In Cesar Milstein’s words: ‘We believed that this elaborate experiment provided the first evidence at the protein and nucleic acid levels of the existence of somatic mutations of mammalian cells.’ That’s a massive achievement! In the Ig context, somatic hypermutation is, of course, central to the maturation of the antibody response to give ‘best fit’ IgV antigen binding regions (#22). But this wasn’t what won Cesar a Nobel Prize. That’s where we’ll go next week. To be continued…

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