Synthetic Anti-IL3 Receptor Antibodies As Therapeutics to Block Innate Imatinib Resistance in Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is maintained by a minor population of leukemic stem cells (LSCs) that exhibit innate resistance to tyrosine kinase inhibitors (TKIs) targeting BCR-ABL. Innate resistance can be induced by cytokines and growth factors secreted by bone marrow stromal cells that protect CML-LSCs from TKIs, resulting in minimal residual disease. Developing therapies that can be combined with TKIs to eradicate TKI-insensitive CML-LSCs, is critical for disrupting innate TKI resistance and preventing disease relapse.

We previously showed that interleukin-3 (IL-3) reduces the sensitivity of CML cells to BCR-ABL TKIs. Based on this, we hypothesized that inhibiting IL-3 receptor (IL-3R) would reduce the innate resistance of CML cells to BCR-ABL TKIs. We generated IL-3R inhibitors using synthetic human antibody technology. We constructed combinatorial libraries of human antibody antigen binding fragments (Fabs) using a single-framework synthetic Fab based on the optimized human 4D5 Fab framework that is derived from one of the most frequently used light chain (Vκ1-1) and heavy chain (VH3-23) germline sequences. We generated the Fab library by randomizing amino acids in the Fab heavy-chain complementarity regions (CDRs) and the L3 CDR of the light chain. In CDRs H1 and H2, solvent-accessible positions were softly randomized with binary degenerate codons that encode equal proportions of two amino acids, mostly tyrosine and serine. CDRs L3 and H3 were randomized using codon mixes that encoded pre-defined proportions of nine amino acids (Y (25%), S (20%), G (20%), A (10%), F (5%), W (5%), H (5%), P (5%) or V (5%)). CDR H3 contain different lengths ranging form 1 to 17 amino acids and CDR L3 contained different lengths ranging form 3 to 7 amino acids. Oligonucleotide directed mutagenesis was used to generate a combinatorial Fab library of 10¹⁰ members, where CDRs H1-3 and CDR L3 of the 4D5 Fab were replaced with the above amino acid combinations. The Fab library was expressed in E. coli as a fusion to the PIII coat protein of M13 filamentous phage. The Fab phage library was screened for interactions against the ectodomain of murine IL-3R (aa17-331). After four rounds of phage display selections, we analyzed CDR-H3 sequences in the Fab phage pool using the Ion Torrent Next Generation sequencing (NGS) platform. We chose the most abundant Fab to further mature by “softly” randomizing CDRs H1-3 and L3, where each codon was mutated to contain 70% of the wild-type nucleotide and 10% of each of the other three nucleotides. After three rounds of phage display selection, we analyzed the Fab phage pools by NGS and chose the most abundant Fab to convert to the IgG1 format. Dissociation constants for the IgGs generated from the original selection and the affinity maturation were 1.3x10^-8 nM and 1.1x10^-9 nM, respectively. The specificity of IL-3R antibodies was demonstrated by showing that they bound to HEK293 cells transiently transfected with IL-3R receptor and not to untransfected HEK293 cells.

To investigate IL-3R antibody effects on IL-3 mediated innate TKI resistance, we compared its activity with imatinib against TonB210, a murine hematopoietic cell line with inducible BCR-ABL expression, in response to IL-3. Imatinib did not suppressed BCR-ABL(+) Ton-B210 growth, and IL-3 protected against imatinib growth suppression. IL-3R antibody suppressed the ability of IL-3 to block imatinib activity of BCR-ABL(+) TonB210 cells. We then compared effects of IL-3R antibody as a single agent, and in combination with imatinib, on the growth of CML-LSCs derived from an established murine retroviraltransduction/transplantation model of CML blast crisis, in response to IL-3. The presence of IL-3 reduced cytotoxicity, apoptosis induction, and colony formation by imatinib. Treatment with IL-3R antibody reduced the protective effect of IL-3 in these assays.

In summary, our findings demonstrate that synthetic human antibodies against IL-3R can effectively block IL-3 mediated innate TKI resistance in in vitro assays and suggest that IL-3R antibodies may be effective as a co-drug to complement TKIs in CML treatment by disrupting the innate resistance of CML-LSCs. Interestingly, the human IL3-R antibodies isolated against murine IL-3R by phage display also recognize human IL-3R, suggesting they recognize a conserved epitope and that they may also be useful in developing human therapies.