23ME-00610, a genetically informed, first-in-class antibody targeting CD200R1 to enhance antitumor T cell function

Abstract

Immune checkpoint inhibition (ICI) has revolutionized cancer treatment; however, only a subset of patients benefit long term. Therefore, methods for identification of novel checkpoint targets and development of therapeutic interventions against them remain a critical challenge. Analysis of human genetics has the potential to inform more successful drug target discovery. We used genome-wide association studies of the 23andMe genetic and health survey database to identify an immuno-oncology signature in which genetic variants are associated with opposing effects on risk for cancer and immune diseases. This signature identified multiple pathway genes mapping to the immune checkpoint comprising CD200, its receptor CD200R1, and the downstream adapter protein DOK2. We confirmed that CD200R1 is elevated on tumor-infiltrating immune cells isolated from cancer patients compared to the matching peripheral blood mononuclear cells. We developed a humanized, effectorless IgG1 antibody (23ME-00610) that bound human CD200R1 with high affinity (K_D <0.1 nM), blocked CD200 binding, and inhibited recruitment of DOK2. 23ME-00610 induced T-cell cytokine production and enhanced T cell-mediated tumor cell killing in vitro. Blockade of the CD200:CD200R1 immune checkpoint inhibited tumor growth and engaged immune activation pathways in an S91 tumor cell model of melanoma in mice.

Keywords: 23ME-00610; CD200; CD200R1; cancer immunotherapy; immune checkpoint.

Figure 1.

Identification of the immune checkpoint protein CD200R1 as a novel immuno-oncology target. GWAS analysis was performed using genetic and phenotypic information in the 23andMe database. Phenotypic data are derived from medical surveys that capture the cancer- and immune-related diseases of participants. a) Heatmap corresponding to the -Log(p value) of the GWAS (capped at -10 and 10 for visualization) multiplied by the sign of the effect (-1 or 1 indicated in blue or red) for a variant rs3087243(A/G) mapping to CTLA4 via eQTL, showing opposing effects on cancer (violet) and immune diseases (pink), thereby defining an I/O genetic signature. The color (blue to red) corresponds with the direction of the effect. b) Heatmaps for the 3 variants (rs140763487, rs1131199, rs34215892) corresponding to CD200R1, CD200, and DOK2, respectively. Test alleles for which the direction of effect is specified are shown in bold. c) Evidence supporting variant to gene mapping.

Figure 2.

CD200R1 is increased on tumor-infiltrating lymphocytes.a) Expression of CD200R1 correlates positively with expression of immune-cell markers CD45, CD4, CD8, and CD11B in renal clear cell carcinoma; b) Expression of CD200R1 is enriched in exhausted TIL cell types from melanoma patients previously treated with anti-CTLA4 and anti-PD-1 checkpoint inhibitors (right panel, orange data points); c) Plot of percent of cells that express CD200R1 in all samples. d) CD200R1 expression on TILs from different tumor types (Breast, Kidney Clear Cell [KCC], Melanoma, Ovarian [OC], and Endometrial Adenocarcinoma [EMAD]) was compared to matched PBMCs from the same patient. A ratio paired t-test was used to determine statistical significance. * p<0.05, ** p<0.01, *** p<0.001. Healthy PBMCs from 1 donor were also profiled (black dot) as a reference. Fluorescent background was recorded with an isotype-matched control (gray dot).

Figure 3.

23ME-00610 potently binds CD200R1, blocks CD200 binding, and inhibits downstream signaling.a) Binding of 23ME-00610 to immobilized human CD200R1 on a CAP chip was evaluated by SPR. A representative sensorgram is shown from 1 of 3 independent experiments using a representative isoform of human CD200R1 (CD200R1-iso4-Alt); b) Donor PBMCs were incubated with serial dilutions of biotinylated 23ME-00610 or 200 nM of biotinylated isotype-matched control (open symbols). Serial dilutions were analyzed by flow cytometry in biological replicates. The median MFI ± SD are graphed. c) U937 cells engineered to express CD200R1 were pre-incubated with varying concentrations of biotinylated CD200-Fc (2 to 800 nM) followed by incubation with serial dilutions of 23ME-00610 at 4°C for 30 minutes. MFI values of streptavidin detection are depicted from 1 of 3 independent experiments; d) Jurkat cells were engineered to express CD200R1 and DOK2, each modified at the C-terminus with a separate ß-galactosidase enzyme fragment. When co-cultured with HEK293T cells expressing CD200, this reporter cell line induced a chemiluminescent signal reflective of CD200R1 signaling. Serial dilutions of 23ME-00610 were added to the co-culture to evaluate blocking of CD200-induced recruitment of DOK2 to CD200R1. HEK293T cells with no CD200 endogenous expression were used to determine the baseline of signal. Luminescent values from 1 of 6 independent experiments are shown as mean +/- SD (N=3 replicates for each concentration).

Figure 4.

23ME-00610 rescues the immunosuppressive activity of CD200 in T cells.a) Human pan-T cells isolated from 4 healthy donors were chronically stimulated with phytohemagglutinin and IL-2 for 7 days. Cells were harvested, rested of stimulants, and primed with human IL-4 for 24 hours. Cells were treated with 50 nM isotype control (Iso) or 23ME-00610 (‘610) in plates coated with anti-CD3 and CD200-Fc or anti-lysozyme-matched Fc control (Control Coat, Ctrl) for 24 hours. Mean IL-2 secretion levels were graphed for each donor (diamond, circle, square and triangle); b) Cells were stimulated as before, but IFNγ levels were measured 72 hours post-treatment with 50 nM isotype control (Iso) or 23ME-00610 in plates coated with anti-CD3 and CD200-Fc or anti-lysozyme-matched Fc control. A representative dose-response curve for 23ME-00610 rescue of IFNγ secretion is plotted for the mean of technical quadruplicate measurements ± SEM.

Figure 5.

23ME-00610 enhances IFNγ secretion from cancer patient PBMCs. PBMCs from 9 cancer patients were incubated with 100 nM of 23ME-00610 or isotype control. Cells were stimulated with SEB. IFNγ levels were determined by ELISA. Mean biologic triplicates were normalized to isotype control. The unpaired t-test was applied to determine statistical significance between 23ME‑00610 and Isotype control treatment groups. * p<0.05, ** p<0.005, **** p<0.00005. Each donor was tested once. Data are presented as mean +/- standard error.

Figure 6.

23ME-00610 Enhances PBMC-Mediated Tumor-Cell Killing.Killing of a tumor cell line that endogenously expresses CD200, COV-644-GFP, by PBMCs was evaluated upon treatment with 23ME-00610 or isotype control. a) CD200R1+ PBMCs were primed with SEB and treated with a titration of 23ME-00610. COV644-GFP cells were co-cultured with PBMCs for 120 hours and GFP signal was monitored as a readout of tumor-cell number. Tumor-cell killing was observed as a decrease in total GFP signal at the end of the experiment relative to the GFP signal observed at the beginning of the experiment. The change in GFP signal over time relative to isotype, in the killing phase, was used to calculate the EC50. b) A representative dose-response curve of tumor-cell killing relative to isotype control; data are represented as the mean ± standard error (N = 4 replicates for each concentration) using PBMCs from one donor.

Figure 7.

Inhibition of the CD200:CD200R1 immune checkpoint elicits an antitumor response in the S91 melanoma mouse model.a) CD200 cell-surface staining in S91 tumors were analyzed by flow cytometry (Black, Isotype control; Red, CD200). b) S91 cancer cells were subcutaneously injected into DBA/2 mice and were treated when tumor volume reached 100 mm3 with 20 mg/kg of anti-CD200 (OX90) or Isotype control antibodies twice weekly (n=15/group). Plots shown are mean tumor volume ± SEM (left), and individual animal tumor measurements (middle - isotype treated mice, right – OX90-treated mice). Analysis of variance (ANOVA) was used to establish statistical significance; * p<0.05; ** p<0.01; *** p≤0.001. c) Pathway enrichment analysis of DEGs indicated immune system activation in OX90 treated samples. Enrichment of immune-relate pathways are shown with normalized enrichment score (NES) and adjusted p-values.