L12 Making humanised mAbs Flashcards
Learning outcomes: To explain why rodent antibodies are limited in human therapy. To explain somatic recombination. Understand how rodent antibodies are made more human. Outline how human antibodies can be produced in transgenic animals
What response is seen in patients given rodent antibodies?
Human anti-mouse antibody (HAMA) response.
What could happen if mouse antibody given to human?
The antibody could be immunogenic itself, cause immune response that would neutrialise and clear it, (at least, could be toxic).
What are the problems with producing human hybridomas for mAbs?
Lack of human myeloma fusion partner. Cannot immunise and isolate B cells from a human. Unethical and probably wouldn’t work anyway.
What four ways can human mAbs be produced?
Transformation of B cells using EBV. Virus drives replication, but not permanently immortal.
Interspecies hybridoma. Use mouse myeloma with human lymphocyte, but not stable.
In vitro immunisation. - B cells stimulated against toxin.
Genetic engineering of mouse - produce chimeric Ab or fully human Ab by removal of mouse Ig genes and addition of human.
What three gene segments undergo recombination for the heavy chain?
V, D, and J rearrange and mutate to make the Fab region. C region forms constant domain.
What two processes give rise to specificity of Fab?
Gene rearrangement of V J and D, and RNA processing.
Which segments undergo rearrangement for the light chain?
Does this occur before or after heavy?
V, J and C. (constant)
Occurs after heavy chain.
What are the two classes of light chain?
kappa and lambda.
Which class of antibody is produced first?
IgM produced first.
How is a B cell initially activated?
Binding antigen via the mIgM (membrane bound IgM)
What cell stimulates class switching?
Th cells, release cytokines which change IgM to G, A or E.
Changes constant region of heavy chain by splicing. (RNA)
When a B cell proliferates, as well as plasma cells, what other cell is generated? What is its purpose?
Memory B cells also produced. Stored in spleen.
Convey long-term resistance so that if antigen is encountered again, rapid Ig response can be seen.
This is immunity.
How are existing hybridoma cells humanised?
GE, mouse segments of constant domain of Ig gene removed, human is added in vector.
Promoter, leader and variable domain from mouse antibody gene (Fab)
Constant domain exons from human antibody gene (Fc)
Inserted into plasmid vector, then expression system (ie myeloma cell line)
What is CDR grafting?
Grafting of mouse CDR regions onto a human antibody.
How are 100% humanised antibodies produced by mice? Using Mouse ES cells and HAC
Silence expression of mouse Ig genes in a mouse ES cell. HAC with human Ig genes added. This ES cell added to blastocyst.
Mouse grown in utero.
Chimeric mouse produced, interbreed these mice.
Mice generated that are full knockdown of mouse Ig, and only express human Ig.
Can then be immunised with antigen and produce 100% human antibody.
