VH Fragments

Antibody-based therapies have become a major driver for the pharmaceutical industry, addressing many medical needs and yielding $33bn of sales in 2008 with continued annual growth of >20%. This has led to an intense focus on improved platforms for the generation of antibodies, and on next generation antibody-based molecules with potentially improved, more drug-like, properties. Antibody fragments have a key role to play. Several products based on first or second generation antibody fragments (for example Fab and scFv) are marketed or in clinical development. However, VH fragments are the smallest fragments that retain target binding and affinity, and therefore have significant benefits over earlier generations of antibody fragments.

Heavy Chain Antibodies

VH fragments can currently be generated in vitro from naive human libraries, or from heavy chain antibodies that are produced naturally in llamas.

The former have the advantage of having a human origin, but have a significant disadvantage: having originated as part of a complex with a counterpart immunoglobulin domain, they frequently have suboptimal affinity, stability and solubility as separate entities.

On the other hand, the llama as a source of VH fragments has the advantage of maturation in vivo and in the absence of a partner domain, yielding high affinity, solubility and stability. However, the products must be humanised before clinical use in man.

Crescendo’s transgenic platform for VH generation combines two highly desirable characteristics of an antibody discovery platform: in vivo generation and human origin.

Crescendo Transgenic Platform

Heavy chain antibodies (HCAb, i.e. immunoglobulin which lacks light chains) occur naturally in camelids and sharks, and have been developed as a source of VH fragments, which are then humanised prior to therapeutic development. Critical to the ability of these species to secrete functional HCAb is the absence of CH1 domains from heavy chain immunoglobulin. The Crescendo transgenic mouse comprises engineered human heavy chain immunoglobulin genes (IgH), in order to produce human heavy chain antibodies in response to immunisation. This in vivo route will provide a faster more predictable route to high affinity human VH fragments than camelid or in vitro technologies.

Our approach originates from the work of a leading pioneer in the field of human antibody transgenic mice, Dr Marianne Brüggemann, and depends on generating mice devoid of endogenous murine immunoglobulin heavy and light chain expression. These mice are then crossed with transgenic mice containing a large yeast artificial chromosome (YAC) construct comprising the human heavy chain immunoglobulin (IgH) V, D and J genes linked to human or murine C genes engineered for secretion in the absence of light chains.

Crescendo is working with leading academic groups in YAC construction and transgenesis to generate a pipeline of mice towards the best-in-class human HCAb mouse. These will comprise YAC constructs with increasing numbers of human V genes linked to a murine C region.

Ribosome Display Technology

Ribosome display technology is a superior platform for in vitro molecular evolution of antibody affinity developed by Drs Mike Taussig and Mingyue He at the Babraham Institute. It has advantages based on the very high library diversity that can be achieved, and the fact that the entire process is performed rapidly in vitro. Crescendo owns IP for eukaryotic ribosome display, and has developed a robust process for optimisation of VH fragments produced from its transgenic platforms.

Click image for larger version