The engineering of antibodies and antibody fragments for affinity maturation, stability, and other biophysical characteristics is a common aspect of therapeutic development. Maturation of antibodies in B cells during the adaptive immune response is the result of a process called somatic hypermutation (SHM), in which the activation-induced cytidine deaminase (AID) acts to introduce mutations into immunoglobulin (Ig) genes. Iterative selection and clonal expansion of B cells containing affinity-enhancing mutations drive an increase in the overall affinity of antibodies. Here we describe the use of SHM coupled with mammalian cell surface display for the maturation of antibodies in vitro and the complementarity of these methods with the mining of immune lineages using next-generation sequencing (NGS).

Tyrosine kinase inhibitors (TKIs) induce molecular remission in the majority of patients with chronic myelogenous leukemia (CML), but the persistence of CML stem cells hinders cure and necessitates indefinite TKI therapy. We report that CML stem cells upregulate the expression of pleiotrophin (PTN) and require cell-autonomous PTN signaling for CML pathogenesis in BCR/ABL+ mice. Constitutive PTN deletion substantially reduced the numbers of CML stem cells capable of initiating CML in vivo. Hematopoietic cell-specific deletion of PTN suppressed CML development in BCR/ABL+ mice, suggesting that cell-autonomous PTN signaling was necessary for CML disease evolution. Mechanistically, PTN promoted CML stem cell survival and TKI resistance via induction of Jun and the unfolded protein response. Human CML cells were also dependent on cell-autonomous PTN signaling, and anti-PTN antibody suppressed human CML colony formation and CML repopulation in vivo. Our results suggest that targeted inhibition of PTN has therapeutic potential to eradicate CML stem cells.