2. CD47-Blocking Antibodies

To date, several studies have been done using several anti-CD47 antibodies in various cancer studies with insightful mechanisms of action. CD47 expression become an interesting research area when cancer cells were known to have an increased CD47 expression as a survival mechanism and the blockade of CD47 resulted in increased phagocytosis by macrophages and suppression of many tumors.

Due to the increased surface expression of CD47 on cancer cells, anti-CD47 can selectively target a macrophages mediate phagocytosis and clearance. It is important to understand that blocking an antiphagocytic molecule such as CD47 may not be enough for phagocytosis. The expression of several pro-phagocytic ligands or molecules on the surface of stressed cells, tumor, and infected cells is important to facilitate phagocytosis. Pro-phagocytic molecules such as phosphatidylserine, asialoglycoproteins and migration of calreticulin from the endoplasmic reticulum to cytoplasmic membrane occur on tumor and infected cells unlike in healthy cells [68,69,70]. Considering the fact that healthy cells does not or express these pro-phagocytic molecules at low level, therefore the administration of anti-CD47 may not cause phagocytosis or apoptosis on healthy cells.

Several different types of anti-CD47 antibodies such as MIAP301, MIAP410, Hu5F9-G4, CC-90002, SRF231, B6H12.2, ALX148 has been use in during cancer and infection [12,15,25,71,72]. The two most known clones of anti-mouse CD47-blocking antibodies are MIAP410 and MIAP301. The MIAP410 treatment in virus infected mice is reported to result to an increased proliferation and activation of innate and adaptive immune cells. This antibody has been reported to have both antiviral and anti-tumor in two distinct viral infections and several tumors. The Hu5F9-G4 is a humanized anti-CD47 that disrupts the CD47-SIRPa interaction thereby promoting macrophage mediated phagocytosis and elimination of several human tumors both in vivo and in vitro. Due to the unselected inhibition of CD47, the administration of anti-CD47 antibody leads to a mild anemia as a result of increased red blood cells attack by phagocytic cells [71,73,74]. The CC-90,002 is also a humanized anti-CD47 Abs that is reported to be under clinical trial for the treatment of both solid and hematologic malignancies [75]. Another humanized antibody (SRF 231) is also reported to be tested on pre-clinical trial for malignancies [15]. The B6H12.2 CD47 blocking antibody has been use to suppress non-Hodgkin lymphoma tumor [72,76]. The ALX148 is a more modified version of CD47 blocking antibody by fusing the inactivated human IgG1 Fc with the modified SIRPa D1 domain. This antibody has been reported to promote macrophages and dendritic cells activation, increased inflammatory cytokine production and increased antitumor T cells effector function. Addition to its effective antitumor function, the antibody does not cause anemia in an in vivo setting [77]. All these CD47-blocking antibodies (both mouse and humanized) unanimously show a similar immunological effect resulting in increased phagocytosis and activation of antigen presenting cells, increased proliferation and activation of cytotoxic CD8 T cells during tumor and viral infections.

3. CD47 Blockade and APCs Activation During Infection

As an inhibitory protein, both the genetic knockout and blockade of CD47 results in increased activation of antigen presenting cells (APCs) during cancer and infection [74,78]. Several studies reveal that the blockade of CD47 as an antiphagocytic ligand increase the phagocytic activity and thereby enhancing antigen presentation to T cells [10,52,79,80]. Therefore, treatment with anti-CD47 antibody increases the capacity of the APCs in bridging innate and adaptive immune response leading to enhanced potency of T and B cells immunity. However, there are growing evidences that the blockade of CD47 ligation can induce cancer cell apoptosis [81]. In addition to above-mentioned effect, the blockade of CD47 enhances interferon-I response and this was strongly associated with upregulation of STING pathway in APCs in tumor [82,83,84]. Demeure et al. suggests that the disruption of ligation of CD47 during pathogen invasion may increase inflammatory cytokine response by dendritic cells [85]. Though not well studied, CD47 was identified as an interferon stimulated gene and as a host defense mechanism upon IFNa stimulation [86]. With such an immunotherapeutic effect of anti-CD47 antibody, one could speculate that the treatment of the anti-CD47 may also increase the interferon response and APCs activation during infection.

Our recent study provides evidence that the administration of anti-CD47 leads to increased macrophages proliferation, infiltration and uptake of virus and/or virus infected cells by macrophages during LCMV infection. The treatment of anti-CD47 enhances the activation of antigen presenting cells as indicated by surface expression of activation markers such CD86 and CD80 on macrophages and dendritic cells. Cham shown that in vivo administration of anti-CD47 in wild type during LCMV infection increases et al. APCs activation as illustrated on Figure 2. To further determine whether macrophages and/or dendritic cells are responsible for the antibody mediated increased activation of adaptive immune response, we depleted the macrophages using clodronate. The depletion of CD11b⁺ macrophages followed by anti-CD47 treatment can still enhance T cells response. On the contrary, the depletion of dendritic cells using the CD11c-DTR mice followed by anti-CD47 treatment shows impaired T cells response and persistence of LCMV virus replication in serum and all organs. This indicates that macrophages may an essential innate immune cell mediating the anti-CD47 phenotype at an early time point. However, dendritic cells and not macrophages were the most crucial cells in bridging and activation of the robust CD8 T cells immune response. In vitro, dendritic cells—CD8Tcells co-culture with anti-CD47 treatment reveals that blockade of CD47 in vitro can enhance dendritic cells activation in priming of CD8 T cells. This increased APC activation status in absence of CD47-signaling amplifies T cell activation and cytotoxicity during viral infection [12]. It is tempting to speculate that with such an increased APC activation, this could enhance neutralizing antibody response in a similar manner.

Figure 2. Treatment of isotype control to mice following a viral infection leads to limited change in the splenic architecture, normal phagocytosis, normal antigen presentation and therefore resulting to a normal activation and cytotoxicity of T cells. On the other hand, the administration of anti-CD47 antibody result to a splenomegaly, increases phagocytosis of infected cells, increase antigen presentation, enhances T cell activation and function and faster viral control.

4. Conclusions

As highlighted above, both the genetic knockout and blockade of CD47 as an immunosuppressive protein leads to an elevated innate and adaptive immune responses. The administration of anti-CD47 antibody increases the phagocytic activities of virus infected cells, increases antigen processing and presentation by APCs and this enhances activation capacity of antiviral CD8 T cells. Therefore, the anti-CD47 antibody increases the activation of both innate and adaptive immune response in two distinct viral infections. With such an immune-therapeutic effect of anti-CD47 blockade on both cancer and viral infection, this antibody would be a unique target in virus-associated cancers such human papillomavirus, hepatitis B and hepatitis C virus, Epstein–Barr virus, human T-lymphotropic virus, Kaposi’s sarcoma-associated herpesvirus (KSHV) and (re)emerging infections. The anti-CD47 is currently under clinical trial for cancer and can be amenable as an immunotherapeutic target for infectious diseases.