CCRL2 As a Novel Target in Acute Erythroid Leukemia

Background:

C-C chemokine receptor-like 2 (CCRL2) is normally expressed in differentiated myeloid cells and encodes for an atypical chemokine receptor involved in inflammation activation. We have found that CCRL2 receptor is overexpressed on the surface of acute erythroid leukemia (AEL) cell lines and primary samples compared to healthy stem and progenitor cells.

Aim:

To assess the efficacy of our newly developed antibody-drug conjugate (ADC) targeting CCRL2 in AEL.

Methods:

The anti-CCRL2 and isotope control (IgG2A) ADCs were developed by conjugating commercially available monoclonal anti-CCRL2 and IgG2A antibodies with SG3249, an SG3199 linker-drug molecule. This was done by reducing monoclonal antibodies using 5x molar excess Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) followed by TCEP removal using Zeba Spin columns. Subsequently, antibodies were reacted with 5x molar excess SG3249 in 10% DMSO at room temperature. Excess unreacted drug linkers were subsequently removed by buffer exchange into PBS using Zeba Spin columns. ADC drug conjugation was analyzed by hydrophobic interaction chromatography (HIC). The activity of the ADCs was measured by MTT assay, apoptosis assay by 7AAD staining, methylcellulose-based clonogenicity assays in CCRL2 wild-type (WT) and knocked down (KD) TF-1, F36P and doxycycline-inducible CCRL2 TF-1 cells. Luciferase+ TF-1 xenografts in NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice were treated with one intravenous dose of 0.75 mg/kg and growth was measured by bioluminescence signal. Mice survival was assessed by Kaplan-Meier analysis. CD34+ and mononuclear cells (MNCs) from 3 healthy controls and MNCs from 3 AEL patients were treated with 40 ng/ml ADCs for 5 days and activity was measured by apoptosis assay by 7AAD staining, differentiation assessment by CD34, CD38, CD71, CD235a, and CD117 staining and methylcellulose-based clonogenicity assays.

Results:

HIC confirmed antibody-drug conjugation and absence of free linker-drug. MTT assays with increasing doses of ADCs showed significantly higher toxicity in TF1/F36P WT cells treated with the anti-CCRL2 ADC compared to KD cells or cells treated with IgG2A (p=0.000146 for TF-1 and p=0.00315 for F36P). Treatment with anti-CCRL2 ADC induced high levels of apoptosis in both TF1 (p=0.0004) and F36P (p= 3.0058E-05) WT but not in KD cells. Anti-CCRL2 ADC treatment (10 ng/ml and 20ng/ml) suppressed clonogenicity in both TF-1 and F36P cells in a dose-dependent effect (p=0.0038 for TF-1 and p=0.0003 for F36P). Doxycycline treatment added to doxy-inducible CCRL2 TF-1 cells significantly increased the cytotoxicity based on an MTT assay with different doses of anti-CCRL2 ADC (p= 0.00798). Treatment with anti-CCRL2 ADC suppressed the leukemic growth of TF-1 xenografts and improved the survival of NSG mice engrafted with TF-1 cells(p=0.058). Treatment of MNCs from AEL patients with 40 ng/ml anti-CCRL2 ADC for 5 days significantly increased apoptosis(p=0.0006), decreased the percentage of CD71+CD117+(p= 0.0017) and CD71+CCRL2+(p=0.0014) cells and suppressed their clonogenicity (p= 0.0027). On the contrary, no increase of apoptosis, induction of differentiation or suppression of clonogenicity was observed in CD34+ cells from healthy controls treated with 100 ng/ml anti-CCRL2 ADC was observed. Finally, treatment of MNCs from healthy controls with 100 ng/ml anti-CCRL2 ADC did not alter the percentage of CD71+CD235a-, CD71+CD235a+ and CD71-CD235a+ cells suggesting no significant impact on healthy erythroid and megakaryocytic progenitors.

Conclusion:

Anti-CCRL2 ADC induces apoptosis and suppresses the clonogenicity of AEL cells showing no toxicity against healthy hematopoietic cells. CCRL2 is a promising target in AEL and the anti-CCRL2 ADC shows single-agent activity in this disease and can be potentially combined with currently available therapies for further improvement of its efficacy.