Background CD24 is a small GPI-anchored, highly glycosylated membrane protein, which interacting with Siglec-10 on TAMs (Tumor Associated Macrophages) acting as ‘don’t eat me’ signals, and helping tumor cells avoid being engulfed by macrophages. CD24 is widely detected in many solid tumors.1 We have developed a unique humanized anti-CD24 antibody, KH-801, that depletes tumor cells not only by the Fc-mediated immune effector function, but also through blocking the CD24/Siglec10 axis signals in vivo and in vitro preclinical models.
Methods Cell-based binding to human T cell, human B cell , human granulocyte and a panel of tumor cells was evaluated by flow cytometry. Mice M2 TAMs elimination and T cell infiltration in vivo were evaluated by flow cytometry after anatomy. The ability of KH-801 in enhancing phagocytosis of tumor cells was evaluated using human monocyte-derived M2 macrophage by live-cell microscopy and flow cytometry. in vivo efficacy of KH-801 was test in wild-type C57BL/6 and C57BL/6JSmoc-Siglecgtm1(hSIGLEC10)/Smoc bearing MC38 murine syngeneic colorectal cancer cells stable expressing human CD24. Toxicokinetics parameters of KH801 were evaluated in the one-month multiple-dose toxicity study in cynomolgus monkeys.
Results KH-801 selectively binds to tumor cells compared to CD24 positive blood cells such as granulocytes, B cells and T cells, which may improve the therapeutic window of KH-801 in clinic (figure 1). KH-801 significantly promotes human monocyte-derived M2 macrophage phagocytosis of tumor cells with a single-digit nM activity. KH-801 Fc variant 2 without binding to FcγRs, still promote phagocytosis of Monocyte-derived M2 macrophages (figure 2). The results indicate that KH-801, a IgG1 isotype antibody, promotes phagocytosis of tumor cells not only through the Fc-mediated immune effector function, but also the blocking of CD24/Siglec10 axis signals. Besides, KH-801 promotes T cell infiltration into the tumor tissues and M2 TAMs elimination, that may transform cold tumors into hot tumors, and increase the anti-tumor potential for combination with other checkpoint inhibitors (figure 3). Moreover, dose-dependent anti-tumor efficacy has been demonstrated in multiple mouse tumor xenograft models (figure 4). Additionly, the differentiated epitope makes KH-801 exhibit good tolerance in monkey non-GLP toxicity studyv (table 1).
Conclusions KH801 displays potent tumor growth inhibition activity through not only phagocytosis enhancement by blocking of CD24/Siglec10 axis signals, ADCC and ADCP to kill tumor cells directly, but also regulating tumor microenvironment by enhancing M2 TAMs elimination and T cell infiltration. The results show that KH-801 has potential treatment value in a variety of solid tumors.
Barkal AA, Brewer RE, Markovic M, Kowarsky M, Barkal SA, Zaro BW, Krishnan V, Hatakeyama J, Dorigo O, Barkal LJ, Weissman IL. CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy. Nature. 2019 Aug;572(7769):392–396.
Abstract 503 Figure 1
KH-801 Preferentially Binds Tumor Cells with Stronger Binding Activity. Binding curves were constructed in which the concentration of KH-801 used was plotted against the FACS signals (MFI, or mean fluorescent intensity) obtained. (A) In comparison with competitor antibody, KH-801 owns more powerful binding potency to cancer cells. (B) KH-801 has weaker binding affinity to human granulocyte, B cell and T cell than CD24 positive tumor cells. (C) In comparison with competitor antibody, KH-801 owns stronger binding potency to monkey granulocytes and weaker binding affinity to human granulocytes.
Abstract 503 Figure 2
KH-801 Promotes M2-like Cell Phagocytosis of Tumor Cells by Blocking the CD24/SIGLEC10 Axis Signals. (A) Representative images from live-cell microscopy showed phagocytosis of HT29, Nalm6 and A549 cells by human monocyte-derived M2 macrophages. pHrodo-red+tumor cells was treated with KH-801 (right) or IgG control (left) at t=0&5 h with two experimental replicates. (B) Monocyte-derived M2 macrophages cocultured with MCF7 cells was induced by KH-801 and its Fc variants. Curves of phagocytosis were constructed in which the concentration of antibodies used was plotted against the phagocytic index obtained.
Abstract 503 Figure 3
KH-801 Modulates T cells and M2 Macrophages in Tumor Microenviroment. (A,B) Female C57BL/6 mice bearing MC38-hCD24 tumors were administered PBS or KH-801 at the dose of 3 mpk biw. Tumor volumes and body weights were periodically measured. (C,D) Representative flow cytometry dot plots of the CD45 positive cells in the tumor tissues from animals, present T cell infiltration (CD3-APC/P4) into tumors after one dose treatment with KH-801 compared PBS control. (E,F) Representative flow cytometry dot plots of the F4/80 positive cells in the tumor tissues from animals, present that M2 macrophage cells (CD206-APC/P5) have been changed or eliminated after one dose treatment with KH-801 compared PBS control. Data represent mean ± SEM(n = 3), where * P<0.05 versus vehicle-control using a one-way ANOVA at the endpoint.
Abstract 503 Figure 4
KH-801 Shows Strong In vivo Anti-tumor efficacy. (A) Anti-tumor efficacy of KH-801 was tested in female wild-type C57BL/6 mice bearing MC38-hCD24 tumors, when average tumor volume reached approximately 100mm3 after inoculated for 12days. Mice were administered PBS or KH-801 with different doses (10mpk, 3mpk, 1mpk) biw and tumor volume measured periodically. Tumors were dissected and weighed at the last day. (B) Anti-tumor efficacy of KH-801 was tested in female C57BL/6JSmoc-Siglecgtm1 (hSIGLEC10)/Smoc bearing MC38-hCD24 tumors, when average tumor volume reached approximately 120mm3 after inoculated for 10 days. Mice were administered PBS or KH-801 with different doses (10mpk, 3mpk, 1mpk) biw and tumor volumes measured periodically. Tumors were dissected and weighed at the last day. (C) Tumor growth inhibition rates(TGI) of each model were summarized. Data represent mean ± SEM(n = 8), where * P<0.05 versus vehicle-control using a one-way ANOVA at the endpoint.
Abstract 503 Table 1
KH-801 Exhibits Good Tolerance in Monkey Non-GLP Toxicity Study. A.B. Toxicokinetics parameters of KH801 were evaluated in the one-month multiple-dose toxicity study in cynomolgus monkeys. KH801 was intravenously administered at the doses of 50 mg/kg, 100 mg/kg and 200 mg/kg once weekly for three weeks (total of 4 doses). The blood samples were collected after the first and third doses. The accumulation coefficient of male and female animals was 1.03–1.66, and there was no accumulation effect at the doses of 50 mg/kg, 100 mg/kg and 200 mg/kg.
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