Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

366 17 Immunotoxins and Recombinant Immunotoxins in Cancer Therapy
Phage-display technology can be used not only to create new scFv antibodies, but
also improve the properties of existing scFvs. Improvements in antibody stability,
expression and binding affinity can be achieved by using a combination of strategies
including random and directed mutagenesis of CDR regions, DNA shuffling,
and error-prone PCR [175, 176]. These mutagenesis strategies combined with the
powerful selection methods available to screen antibody phage-display libraries can
yield scFv molecules with significantly improved properties for clinical applications.
For example, phage display was used to improve antibody affinity by mimicking somatic
hypermutation in vitro [177]. In vivo affinity maturation of antibodies involves
mutation of hot spots in the DNA encoding the variable regions. This information
was used to develop a strategy to improve antibody affinity in vitro using phage-display
technology. The anti-mesothelin scFv, SS(scFv), was used to identify DNA sequences
in the variable regions that are naturally prone to hypermutations. In a
few selected hot spot regions encoding non-conserved amino acids, random mutations
were introduced to make libraries with a size requirement between 10 3 and
10 4 independent clones. Panning of the hot spot libraries yielded several mutants
with a 15- to 55-fold increase in affinity compared with a single clone with a 4-fold
increased affinity from a library in which mutagenesis was done outside the hot
spots (Tab. 17.2). This is an example of a powerful phage-display-based strategy that
should be generally applicable for the rapid isolation of higher-affinity mutants of
Fvs, Fabs and other recombinant antibodies from antibody phage libraries that are
smaller in size.
In another example, random CDR mutagenesis to obtain mutants of MR1(Fv)±
PE38, a single-chain recombinant immunotoxin that targets a mutant form of the
EGF receptor, EGFRvIII, that is frequently over-expressed in malignant glioblastomas,
was performed [178](Tab. 17.2). Initially, nine residues of heavy chain CDR3
were randomly mutagenized and several mutants with increased binding affinity
were isolated. All mutations were in regions which correspond to a DNA hot spot.
The mutant MR1Fvs with an increased affinity for EGFRvIII had an increased activity
when converted to recombinant immunotoxins. A specific region of the variable
region of the antibody light chain CDR3 was mutagenized that corresponded to a
hot spot, and a mutant antibody with an additional increase in affinity and cytotoxic
activity was isolated. These studies further show that targeting hot spots in the
CDRs of Fvs is an effective approach to obtaining Fvs with increased affinity.
17.8
Improving the Therapeutic Window of Recombinant Immunotoxins:
The Balance of Toxicity, Immunogenicity and Efficacy
Although some of the problems, including design, large-scale production and stability,
associated with the initial recombinant immunotoxins have been solved, other
fundamental problems need to be addressed that are relevant to much of the immunotherapy
field. Specificity, toxicity and immunogenicity are major factors that will
determine the usefulness and success of recombinant immunotoxins.