Nanoscale Liposomal CD22  E12-siRNA Formulation as a Potent RNAi Therapeutic Against B-cell Precursor Acute Lymphoblastic Leukemia Fatih M. Uckun, M.D., Ph.D Professor, USC Keck School of Medicine Head, Translational Research in Leukemia and Lymphoma Children’s Center for Cancer and Blood Diseases/CHLA

DISCLOSURE STATEMENT no conflicts, patents or financial interests no corporate affiliations

Acknowledgments BioengineeringHong Ma Biochemistry Dorothea Myers, Zahide Ozer Proteomics Martha Arellano Murine Leukemia Models Anoush Shahidzadeh, Ingrid Cely, Cherish Flowers Bioinformatics Sanjive Qazi FormulationJianjun Cheng, Seang Yiv

Acute Lymphoblastic Leukemia (ALL) Most common form of childhood cancer 85-90% have B-lineage ALL Neoplastic expansion of B-cell precursors with a maturational arrest at discrete stages of B-lymphocyte ontogeny; therefore also referred to as B-precursor leukemia (BPL)

Survival of 31,695 Pediatric Patients with ALL Treated on CCG and COG Group Clinical Trials Years of Accrual , , , , , No. Patient Survival % Years From Study Entry Slide courtesy of G.H. Reaman

The Challenge With the progressive intensification of therapy, more than 80% of children with ALL achieve long term survival. To achieve this level of cure, children are exposed to very intensive therapies that have serious short and long term toxicities; and relapses occur across all risk groups; only 30% of children who relapse survive. Nearly 30% of children with ALL with “high risk” features have failed to respond to therapeutic intensification and require new therapeutic approaches for cure. Can we uncover the underlying genetic abnormalities in this resistant form of disease and how can we identify new targets for therapy? Slide courtesy of G.H. Reaman Children’s Oncology Group CureSearch

CD22 Receptor Inhibitory co-receptor of B-cells/B-cell precursors that acts as a negative regulator of multiple signal transduction pathways critical for B-cell homeostasis and survival J. Immunol. 154: (1995); Science 269: (1995); Immunity 5: (1996) The inhibitory and apoptosis-promoting signaling function of CD22 dependent on recruitment of SHP-1 to the immunoreceptor tyrosine-based inhibitory motifs (ITIMs) Cell 72: (1993); J Exp Med 183: (1996); Immunity 8: (1998); J. Biol. Chem. 274: (1999) Disruption of the LYN-CD22-SHP1 signaling network can result in development of a B-cell lymphoproliferative state as well as systemic autoimmunity Adv Immunol. 88:1-50 (2005); Nat Genet 4: (1993); Cell 83: (1995); Science 274: (1996); Nature 384: (1996) CD22  E12 caused by homozygous mutations associated with therapy refractory B-precursor leukemia in pediatric patients Proc. Natl. Acad. Sci. USA, 107: (2010); BJH 156:89-98 (2012)

P– CD22 as a Member of SIGLEC Family of Inhibitory Adhesion Receptors Y–P P–Y Shc Grb2 PLC  2 SHIP Y Lyn Syk SHP-1 PI3K Syk PLC  2 IP3 IP3R ER Ca 2+ Ca 2+ influx + – Y–P P–Y Y –P P– Vav 1,2 B cell Y YYYY ITIMITAMSH2 Ca 2+ efflux CRAC PMCA4 CD22 IgM PIP 2 BLINK/ SLP65

CD22  E12 in B-Precursor Leukemia

CD22  E12 causes BPL in Transgenic Mice

B-Precursor Leukemia Developing in CD22  E12 Tg Mice is Characterized by a Unique Transcriptome

Striking Similarities Between Human vs. Mouse CD22  E12 Signature Transcriptomes

B-precursor Leukemia Developing in CD22  E12 Tg Mice is Characterized by a Unique Phosphoprotein Expression Profile

MAPK Mutant CD22 as a Master Regulator of Growth and Therapy Resistance of Leukemic Stem Cells PI-3 kinase STATs AKTPDK Survival/ Proliferation PIP 2 PIP 3 BAD IKK MDM2 P53 inhibition Mutant CD22 PKC +

Expression of CD22  E12 Transcriptome in Relapse Clones from Pediatric BPL Patients

Effect of RNAi Knockdown of CD22  E12 Expression on Clonogenicity and Self-Renewal Rate of B-Precursor ALL Xenograft Cells B1B1 B2B2 B3B3 B4B4 C

Destroying Leukemic Stem Cells by Using Rationally- Designed Nanomedicines to Knock Down Mutant CD22 Gene Mutant CD22 Messages (“mRNA”) Mutant CD22 gene Nanomedicines to knock down mutant CD22 Cancer cell death

Liposomal Nanoformulation of CD22  E12-siRNA

Nanomedicine Candidate Delivers RNA Therapeutics into BPL Cells for Knockdown of Mutant CD22

Potency of the nanoscale liposomal CD22  E12- siRNA formulation against LSC in BPL xenograft specimens Treatment Leukemic Involvement CON4A P-Value (Fishers Exact, 2-tailed) Bone Marrow14/15 1/ Meninges14/151/ D

In vivo anti-leukemic potency of CD22  E12-siRNA liposomal nanoformulation against relapse clones from pediatric B-precursor ALL patients

PVBLG-8-based Nanoformulations of CD22  E12-siRNA Formulation platform: Yin et al., Angewandte Chemie Internatl Ed, 52: , 2013

N N N N N N N N N N N N N N N N N N N N N N N N N N N Fc region Fab region Variable Region RNA 3WJ antibody N N N N N N N 4.4Å ~ 12Å A B Protein Antibody ~11.5Å Variable Region Antibody-like pRNA-3WJ Bivalent RNA Aptamers