LAIR-1 agonism as a therapy for acute myeloid leukemia

Effective eradication of leukemic stem cells (LSCs) remains the greatest challenge in treating acute myeloid leukemia (AML). The immune receptor LAIR-1 has been shown to regulate LSC survival; however, the therapeutic potential of this pathway remains unexplored. We developed a therapeutic LAIR-1 agonist antibody, NC525, that induced cell death of LSCs, but not healthy hematopoietic stem cells in vitro, and killed LSCs and AML blasts in both cell- and patient-derived xenograft models. We showed that LAIR-1 agonism drives a unique apoptotic signaling program in leukemic cells that was enhanced in the presence of collagen. NC525 also significantly improved the activity of azacitidine and venetoclax to establish LAIR-1 targeting as a therapeutic strategy for AML that may synergize with standard-of-care therapies.

. Supplemental Table S3.Patient samples used in ex vivo whole blood killing assays or PBMC signaling studies.Headings denote the assay which utilized the respective sample, patient diagnosis, sex, race, age, and treatment history (if available).N/A = not available/unknown.S4.Leukemic cell lines tested for LAIR1 expression.Summary and description of AML cell lines tested for LAIR-1 expression.Headings denote cell type, morphology, FAB classification, molecular mutation, and disease diagnosis of source patient.NA = not applicable/unknown.S5.Significantly differential gene expression in CDX mouse bone marrow after NC525 treatment.

Supplemental Table
Significantly different gene expression in MV4-11 cells recovered at day 27 post-challenge from the bone marrow of CDX mice.p-values and log2 fold changes, with an adjusted p-value < 0.05 and absolute log2 fold change > 1, define differentially expressed genes between 3 isotype-treated verses 3 NC525-treated CDX mice.
concentration (log µg/mL) H LAIR-1 ligand blockade Activation of H LAIR-1 reporter cells Coated Iso + 10 ug/mL sol.col Coated col + 10 ug/mL sol.NC525 Coated col + 10 ug/mL sol.Iso Coated Iso + 10 ug/mL sol.NC525 Coated NC525 + 10 ug/mL sol.Binding profile of NC525 to human (H) LAIR-1 + cells or (B) mouse (M) LAIR-1 + cells.(C) Schematic of human LAIR-1 reporter cell line UT-140.UT-140 cells that express GFP under the NFAT promoter were transduced with human LAIR-1 fused to the zeta chain of CD3.Upon engagement of the LAIR-1 extracellular domain (ECD), signal transduction activates GFP fluorescence.(D) Profile of NC525 blockade of collagen-1 binding to LAIR-1, measured by UT-140 reporter cell activation.(E) Activation profiles of UT-140 LAIR-1 reporter cells by NC525, collagen, or isotype (Iso) under the indicated coated or soluble (sol) treatment conditions.(F) (left) Western blot and (right) quantification (pixel density normalized to histone H3) for phosphorylated SHP-1 in healthy donor blood monocytes under the indicated NC525, complement C1q, or isotype treatment conditions.Each line represents an individual donor.(G) MV4-11 cell lysis after coincubation with healthy donor peripheral blood mononuclear cells (PBMCs) at an 80:1 ratio in the presence of 10 µg/mL soluble NC525 or isotype control.Target cell lysis is plotted against mAb concentration.For graphs AD, G, isotype treatment is indicated by gray circles; NC525 treatment is indicated by red squares. of NC525-mediated AML cell death ex vivo.(A) Representative flow cytometry gating and scatterplots of (B) primary live, dead, or apoptotic total blood leukocytes or (C) CD45 Lo SSC Lo blast cells after ex vivo treatment with 10 ug/mL isotype control or NC525 in the presence of collagen.and collagen induced phosphorylation signaling.(A) Human phosphokinase array dot blots and (B) human phospho-immunoreceptor array dot blots with the respective keys for AML patient PBMCs treated with 10 µg/mL isotype control, LAIR-1 agonist mAb NC525, 50 µg/mL collagen-1 plus isotype control, or collagen-1 plus NC525.NC525 does not induce cytotoxic signaling in healthy cells.Reverse phase protein microarray of mTOR or caspase-7 activation in (A) AML donor or (B) healthy donor PBMCs cultured in 50 ug/mL coated collagen-1 and treated with 10 ug/mL isotype (gray) or NC525 (red) for 5 minutes.(C) Immunophosphoarray of CD34 + enriched cord blood cells (CBCs) treated with 10 ug/mL isotype (gray) or NC525 (red) and anti-IgG for 30 minutes.N = 3 technical replicates per condition.Error bars represent standard error of mean.P values determined by Student's T test.
. LAIR-1 expression on AML cell lines.Histograms of LAIR-1 cell surface expression (blue) on the indicated AML cell lines relative to isotype control staining (red).
gene expression in CDX mouse bone marrow.Significantly downregulated (purple) or upregulated (green) genes in MV4-11 cells recovered at day 27 postchallenge from the bone marrow of CDX mice treated with NC525 compared to isotype control.Significance threshold set at P value < 0.05.Gene values and descriptions are listed in Supplemental Table antibody NC525 does not impact healthy leukocytes.(A) Schematic of the model system for defining NC525 effects on human ( H ) immune cells in vivo.(B) Cell counts of human CD45 + cells or human CD3 + cells in the spleen or bone marrow of engrafted mice treated with vehicle (gray) or 10 mg/kg NC525.N = 7 mice per group.P values determined by Student's T test.(C) Model schematic and leukemic growth of MV4-11 challenged mice engrafted with human PBMCs.N = 68 mice.(D) Percent of human CD3 + T cells, CD4 + T cells, CD8 + T cells, CD20 + B cells, and the degree of CD25 expression on CD3 + T cells recovered from the spleen or (E) bone marrow of PBMC engrafted CDX mice after treatment with 10 mg/kg isotype (gray) or NC525 (red).Bar graphs are generated from mice euthanized at day 28 postchallenge.N = 29 biological replicates.Error bars represent standard error of mean.P values determined by Student's T test.Supplemental TableS1.AML patient samples.Summary and description of AML patient BM samples tested for LAIR-1 expression.Headings denote patient/sample AML type, percent circulating blasts, karyotype, and molecular genotype as determined by sequencing (NGS).Supplemental TableS2.AML patient samples.Summary and description of AML patient BM utilized in CFU and PDX modeling.Headings denote the assay(s) which utilized the respective patient/sample, patient and sample identifiers (ID), AML type, percent circulating blasts, karyotype, and molecular genotype as determined by sequencing (NGS) and treatment history if available.