Abstract 3248: B7-H3, an immune checkpoint protein is overexpressed in AML and the blocking monoclonal antibodies enhance NK cell-mediated apoptosis in AML cells

Abstract

Acute myeloid leukemia (AML) is the most common and aggressive acute leukemia found in adults. Immune checkpoint inhibition has led to important clinical advances in cancer therapy in recent years due to superior cure rates compared with standard therapy. We hypothesize that B7-H3 (CD276) an immune checkpoint protein is overexpressed in AML cells and targeting B7-H3 activates immune cells against AML cells. We analyzed B7-H3 expression in peripheral blood (PB) and bone marrow (BM) mononuclear cells from AML patients (n=65) and healthy donors (n=10) at MD Anderson Cancer Center. Cell surface expression analysis by flow cytometry revealed that the cells of ~60% of the patients were positive for B7-H3 and its expression was 2- to 3-fold higher in AML cells than in healthy donor cells. B7-H3 expression is relatively higher in CD34+ AML cells than in CD34- AML cells (p<0.01). In contrast, no difference was observed between CD34+ and CD34- cells from healthy donors. The Cancer Genome Atlas RNA sequencing data revealed that patients with high B7-H3 expression had significantly lower overall and disease-free survival durations than did patients with low B7-H3 expression (p=0.024). To investigate the role of B7-H3 in immunomodulation, we stably knocked down B7-H3 in OCI-AML3 and co-cultured them with or without human PB-derived NK cells at a 2:1 ratio and measured apoptosis induction in AML cells by annexin-v binding approach. We found that knockdown of B7-H3 induced NK cell-mediated apoptosis in AML cells 3-fold compared to control AML cells. These data indicate that inhibition of B7-H3 in AML cells enhances NK cell-mediated apoptosis in AML cells. To target B7-H3, we have generated four monoclonal antibodies: B1, B2, B3 and B4 (codenamed to protect IP). To investigate whether these novel anti-B7-H3 monoclonal antibodies are able to block B7-H3 immunomodulatory function and activate NK cells, we performed a co-culture experiment with GFP-expressing OCI-AML3 cells and PB-derived NK cells in the presence or absence of anti-B7-H3 antibodies. Apoptosis induction was measured by real-time annexin-v binding using IncuCyte live cell imaging system. The addition of anti-B7-H3 monoclonal antibodies, B1, B2, B3 and B4 at 25μg/ml enhanced NK cell-induced apoptosis 3-fold in OCI-AML3 cells. These data indicate that anti-B7-H3 antibodies block the immunomodulatory function of B7-H3 and induce NK cell-mediated apoptosis in AML cells. In vivo testing of these antibodies against AML-PDX models is currently ongoing. In conclusion, we found that B7-H3 is overexpressed in AML cells and its expression is associated with bad prognosis in AML patients. Knockdown or antibody-mediated blocking of B7-H3 enhanced NK cell-induced apoptosis in AML cells. These data indicated that B7-H3 is a novel immune-checkpoint protein in AML and patients could potentially benefit from anti-B7-H3 therapies. Citation Format: Stanley Ly, Bin Yuan, Sabrina Grimm, Michael Andreeff, Hans-Jörg Bühring, Venkata Lokesh Battula. B7-H3, an immune checkpoint protein is overexpressed in AML and the blocking monoclonal antibodies enhance NK cell-mediated apoptosis in AML cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3248.

Evidence supporting a role for the immune checkpoint protein B7-H3 in NK cell-mediated cytotoxicity against AML

“…To block the immunomodulatory function of B7-H3, we tested 3 novel anti-B7–H3 murine mAbs, T-1A5, HEK5-1B3, and 58B1, 34 and measured their binding affinities using flow cytometry in AML patient samples and cell lines. As expected, B7-H3 expression was significantly higher in AML patient samples than the control samples for all 3 antibodies tested ( P ≤ .02) ( Figure 2G-I ).…”