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1891 A Novel Engineered Single-Chain Antibody Fragment for Targeting Pediatric Philadelphia Chromosome-like Acute Lymphoblastic Leukemia

Program: Oral and Poster Abstracts
Session: 605. Molecular Pharmacology, Drug Resistance—Lymphoid and Other Diseases: Poster II
Hematology Disease Topics & Pathways:
Leukemia, ALL, Diseases, Biological Processes, Technology and Procedures, Lymphoid Malignancies, imaging, flow cytometry, molecular interactions
Sunday, December 6, 2020, 7:00 AM-3:30 PM

Sara M.A. Mohamed1,2*, Keith Sia, PhD2*, Karl-Heinz Friedrich, PhD3*, Andreas Wohlmann, Dr3*, Savvas Savvides, PhD4*, Peter Schofield, PhD5*, Daniel Christ, PhD5,6*, Maria Kavallaris, PhD2,7*, Richard B. Lock, PhD8 and Narges Bayat, PhD2*

1Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt
2Children's Cancer Institute, School of Women’s and Children’s Health, UNSW Sydney, Sydney, NSW, Australia
3Institute of Biochemistry II, Jena University Hospital, Jena, Germany
4VIB-UGent Centre for Inflammation Research, Gent, Belgium
5Garvan Institute of Medical Research, Sydney, NSW, Australia
6St. Vincent’s Clinical School, Faculty of Medicine, UNSW Sydney, Sydeny, Australia
7Australian Centre for Nanomedicine, ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, UNSW Sydney, Sydney, Australia
8Children's Cancer Inst. Australia Lowy Cancer Research Centre, Univ N.S.W., Randwick, NSW, Australia

Introduction: Philadelphia-like acute lymphoblastic leukaemia (Ph-like ALL) is a high-risk ALL subtype characterized by an inferior survival rate and chemotherapeutic drug resistance (Tasian et al, Blood 130: 2064-2072, 2017). Around 50% of Ph-like ALL cases harbour gene rearrangements leading to the overexpression of cytokine receptor-like factor 2 (CRLF2) (Loh et al, Blood 121: 485-488, 2013). CRLF2 (also known as thymic stromal lymphopoietin receptor, TSLPR) heterodimerizes with the interleukin-7 receptor alpha (IL-7Rα) subunit to form the functional TSLPR. Upon TSLP binding, CRLF2 activates the JAK/STAT signalling pathway, leading to enhanced proliferation and survival of leukemia cells resulting in poor outcomes in patients (Harvey et al, Blood 115: 5312-5321, 2010). The extracellular overexpression of CRLF2 and association with poor prognosis suggest the translational value of designing precision-based therapeutics targeting CRLF2 in Ph-like ALL. Although conventional immunotherapy using full-sized antibodies is a promising treatment strategy that can improve treatment efficiency and minimize off-target toxicity, their clinical translation is challenging due to the high production cost and large size affecting targeting efficiency (Holliger et al, Nat Biotech 23: 1126-1136, 2005). Herein, we validated a novel single-chain variable fragment against CRLF2 (CRLF2-ScFv) for targeting Ph-like ALL cells.

Methods: A CRLF2-rearranged Ph-like ALL cell line (MHH-CALL-4) was lentivirally transduced with a CRLF2-targeting shRNA driven by an inducible promoter, enabling the inducible knockdown of CRLF2. CRLF2 expression in MHH-CALL-4 cells after shRNA induction (KD-CALL-4) was evaluated using fluorescence-activated cell sorting (FACS). The cellular association of the CRLF2-ScFv was determined in MHH-CALL-4 and KD-CALL-4 at 4° and 37°C using an indirect immunofluorescence assay. Confocal laser scanning microscopy was used to visualize and compare the cellular association of CRLF2-ScFv in MHH-CALL-4 and KD-CALL-4. The cellular association of CRLF2-ScFv was also investigated ex vivo using a small panel of Ph-like and non-Ph-like ALL patient-derived xenografts (PDXs), representing similar immunophenotype and genetic characteristics to their original disease subtypes (Liem et al, Blood 103: 3905-3914, 2004), and peripheral blood mononuclear cells (PBMCs) to investigate the non-selective association. CRLF2 expression in MHH-CALL-4 and Ph-like ALL PDX cells was quantified using indirect immunofluorescence assay. The downstream impact of CRLF2-ScFv on STAT5 phosphorylation (pSTAT5) was determined by FACS either with or without TSLP stimulation. The statistical significance was tested using Unpaired unequal variances t-test or one-way ANOVA followed by multiple comparisons test. Statistical significance was considered when P ≤ 0.05. All experiments were performed in triplicate.

Results: KD-CALL-4 showed a 75% reduction in CRLF2 expression compared with MHH-CALL-4 cells (P = 0.0009). CRLF2-ScFv exhibited a 94% higher association with MHH-CALL-4 compared with KD-CALL-4 cells at 37°C (P = 0.0013). The association of CRLF2-ScFv with MHH-CALL-4 cells was reduced by 75% at the non-proliferating state of cells at 4°C compared to 37°C (P = 0.006). Orthogonally viewed confocal microscopy images showed 82% higher intracellular uptake of CRLF2-ScFv in MHH-CALL-4 compared to KD-CALL-4 cells (P = 0.0003). CRLF2-ScFv showed >80% higher association with a Ph-like PDX sample compared with a control CRLF2low PDX and PBMCs (P < 0.001). Of note, a Ph-like ALL PDX exhibited only one-third of the association with CRLF2-ScFv compared with MHH-CALL-4 cells (P = 0.04), corresponding to the significant difference in CRLF2 surface expression (P = 0.01). CRLF2-ScFv significantly reduced pSTAT5 expression in MHH-CALL-4 cells (P = 0.05) and prevented TSLP-induced STAT5 phosphorylation (P = 0.01), suggesting competition with the TSLP binding site.

Conclusion: CRLF2-ScFv is a selective targeting moiety for CRLF2 with a significant internalization potential and receptor antagonistic effect, highlighting the therapeutic implications for targeting Ph-like ALL.

Keywords: CRLF2, ScFv, STAT5 phosphorylation, Patient-Derived Xenografts.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH