Hu8F4, a humanized monoclonal antibody that recognizes PR1/HLA-A2 complex, is a first-in-class T cell receptor (TCR) - mimic antibody is currently being tested in a phase 1 trial for AML. We previously reported that activity of Hu8F4 primarily depends on its binding to Fcγ receptors (FcγR) on effector cells. Additionally, we identified antibody-dependent cellular phagocytosis (ADCP) as a principal mechanism of Hu8F4 anti-AML activity in vivo.
Leukemia cells overexpress the “don't eat me” membrane protein CD47. CD47 on AML binds SIRPα on macrophages and neutrophils to inhibit phagocytosis. Blocking this interaction can allow antibodies to exert powerful phagocytic activity. Importantly, the role CD47 may play in the anti-tumor activity of TCR-mimic antibodies has not yet been investigated.
To examine the relationship between CD47 and Hu8F4 activity, we used both in vitro ADCP assays and in vivo NSG mouse models. CD47 blockade was studied as a potential strategy to enhance Hu8F4 activity.
To characterize the in vitro phagocytosis, we used an HLA-A2 transfected leukemia cell line, U937 (U937-A2), and an endogenously expressing HLA-A2 cell line THP1, as targets. NSG mouse bone marrow derived macrophages (BMDM) were used as effectors. At an initial E:T=1, Hu8F4 alone (10 mg/ml) was insufficient to prevent growth of U937-A2 and THP1. However, addition of neutralizing anti-CD47 F(ab') 2 antibody significantly slowed leukemia growth and eliminated both target cells by day 5. To further confirm the critical role of CD47 in Hu8F4-mediated ADCP, we created CRISPR/Cas9 CD47 KO U937-A2 and THP1 cells lines. Complete phagocytosis of both CD47KO cell lines was only observed in the presence, but not the absence of Hu8F4.
To test if CD47 blockade plays a role in Hu8F4 anti-AML activity in vivo, we injected NGS mice with U937-A2 (5x10^3). Mice were treated with Hu8F4 +/- anti-CD47 F(ab') 2 antibody at 1mg/kg (three times per week for a total of 10 doses starting on day 5, n=5 per group). Control mice, treated with PBS, succumbed to leukemia by day 28. Both Hu8F4 and anti-CD47 F(ab') 2 single-agent treatment slowed leukemia growth, while the combination of Hu8F4 and anti-CD47 F(ab') 2 completely prevented outgrowth of the aggressive U937-A2 cells ( Figure 1), indicating that blocking CD47 significantly increased the activity of Hu8F4 in vivo. Interestingly, CD47 KO U937-A2 did not engraft in NSG mice as expected, (n=5), confirming that CD47 expression is critically important for cell line engraftment in NSG. HuCD47 is known to bind to NSG mouse SIRPα with high affinity providing protection to the human cells from phagocytosis by mouse macrophages.
Growth of CD47 KO THP1 in NSG mice was delayed compared to parental THP1, median survival of untreated mice increased from 58 days for THP1 to 99 days for CD47 KO THP1 (p=0.0017, n=5). To facilitate THP1 cell growth in vivo, we used NSGS mice which allows for faster growth of human myeloid cells. Treatment with Hu8F4 for 7 weeks (1 mg/kg three times per week; started on day 2), significantly reduced THP1 leukemia growth and fully eliminated CD47 KO THP1 cells, confirming the role of CD47 in the activity of Hu8F4 ( Figure 2).
Finally, to test whether CD47 is also important in Hu8F4-mediated phagocytosis by human effector cells, we generated human macrophages (Mφ) from CD14+ monocytes from healthy donors. We incubated fluorescently labeled human Mφ with CFSE-labelled target cells (U937-A2 or THP1), that had been pre-treated with Hu8F4 or isotype control antibody (Herceptin), at E:T=10, with or without anti-CD47 F(ab') 2. After 3 hours, Hu8F4-mediated phagocytosis, defined as the mean percentage of double-positive target cells, increased from mean 9.7% to 12.3% (n=4, p=0.199) for U937-A2, and from 12.25% to 17.58% (n=4, p=0.005) for THP1 when anti-CD47 F(ab') 2 blocking antibody was added. Anti-CD47 F(ab') 2 alone and in the presence of control Herceptin did not induce phagocytosis.
Our data shows that the presence of CD47 on leukemia cells can significantly limit the activity of Hu8F4 both in vitro and in vivo. Importantly, combining Hu8F4 with CD47 blockade significantly increased the Hu8F4 activity and together eliminated AML. The role of other phagocytosis checkpoints, such as SIRPα and LILRB-1 also warrant further investigation.
Disclosures
No relevant conflicts of interest to declare.
