Is targeting of CD47-SIRP (cid:1) enough for treating hematopoietic malignancy?

With interest we have read the report by Chao et al,1 which is the most recent of a pioneering and impressive series of publications by the same group of researchers over the past 2 years,2-6 in which the interaction between the broadly expressed CD47 surface molecule and the myeloid inhibitory receptor SIRP is implicated as a potential therapeutic target in a variety of hematopoietic malignancies. Chao et al use xenotransplantation models in which human NHL cells are engrafted into immunocompromised mice, and they show that lymphoma dissemination is inhibited by antibodies directed against human CD47 that block interactions with SIRP , but not by nonblocking anti-CD47 antibodies. In similarly designed studies with AML3 and ALL5 they had already demonstrated prominent tumor cell elimination with this blocking anti-CD47 antibody, and they had also previously shown that anti-CD47 treatment synergizes with the therapeutic anti-CD20 antibody rituximab in NHL.2 Furthermore, their findings show that macrophages are essential in the process of leukemic cell elimination. They suggest that targeting of CD47-SIRP interactions, both in the absence as well as in the presence of a cancer therapeutic antibody such as rituximab, could facilitate the eradication of tumor cells by promoting their phagocytic clearance by macrophages.6 One problem that hampers a straightforward interpretation of the in vivo findings of these studies is the use of intact IgG antibodies. For instance, the anti-CD47 antibodies may not only disrupt CD47-SIRP interactions but may instead, or at the same time, opsonize the tumor cells for antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent phagocytosis. In fact, our recent findings7 with the same monoclonal anti-CD47 antibody (B6H12) that is used in the studies described above show that intact B6H12 induces direct ADCC of neutrophils toward breast cancer cells. The same is true for ADCC by monocytes, as indicated by the cytotoxicity against Jurkat lymphoma cells (Figure 1). Clearly, direct ADCC cannot be simply excluded by using a control nonblocking anti-CD47 antibody, because this may actually have a very different capacity to induce antibody-dependent effector functions, even although it may have the same isotype. In fact, intact antibodies against CD47 might also trigger ADCC toward the healthy cells of the host, which would obviously create a highly undesirable condition. One way to really exclude this direct ADCC is to use these antibodies “stripped” of their Fc regions, that is, as B6H12 F(ab )2 fragments. Obviously, these do not directly induce antibody effector function, but the F(ab )2 fragments do in fact synergize with anti-CD3 antibodies against lymphoma cells (Figure 1) or with trastuzumab in the context of Her2/Neu-positive breast cancer cells.7 This suggest that at least under these in vitro conditions the targeting of CD47-SIRP interactions only enhances tumor cell killing when an appropriate cancer therapeutic antibody such as trastuzumab or rituximab is present. Would this mechanism also apply in vivo? This is clearly something that needs to be sorted out in more detail in future experiments. However, our own findings in a fully immunocompetent syngeneic model of metastatic melanoma are promising and suggest that animals that carry a defect in the SIRP inhibitory signaling capacity do not show enhanced elimination of tumor cells in the absence of therapeutic antibody, while in the presence of therapeutic antibody a strongly potentiated effect is observed.7 Another open question is whether CD47 on tumor cells could actually be efficiently targeted at all in the context of human cancer, even if it was done with the appropriate agents. The problem is that CD47 is very broadly expressed among both hematopoietic and nonhematopoietic cells, and this could seriously compromise efficient targeting of the cancer cell CD47 molecules. Instead, we propose to target SIRP . Blocking antibodies against human SIRP are available now7 and these would be promising agents to test further in clinical trials. The final issue is whether the targeting of CD47 and/or SIRP per se would not cause autoimmune disease. This subject clearly needs careful attention. In fact, there is perhaps already some comforting evidence, at least from animal experiments. First, mice that lack CD47 or have mutant SIRP apparently do not display overt autoimmune symptoms,8,9 and second, autoimmunity is not readily induced on injection of blocking antibodies against CD47 Figure 1. Antibody-dependent cellular cytotoxicity of human monocytes, precultured with GM-CSF for 18 hours, toward Jurkat acute T leukemia cells is enhanced by blocking anti-CD47 F(ab )2, but intact anti-CD47 IgG induces cytotoxicity alone. Cytotoxicity was measured by the release of 51Cr from loaded target cells. (A) Surface expression of CD3 (using CLB-T3/4.2a mAb) and CD47 (using B6H12 mAb) and on Jurkat cells as evaluated by flow cytometry. (B) ADCC of human monocytes toward Jurkat cells after preincubation with mouse IgG2a T3/4.2a anti-CD3 (20 g/mL) and/or B6H12 (10 g/mL) anti-CD47 B6H12 F(ab )2 or intact IgG. Values shown are means SD of (n 3) independent experiments. Note that intact anti-CD47 IgG alone opsonizes and induces monocyte-mediated cytotoxicity in Jurkat cells, while F(ab )2 act only in concert with anti-CD3. P values of statistically significant differences, as determined by Student t test, are indicated.


Is targeting of CD47-SIRP␣ enough for treating hematopoietic malignancy?
With interest we have read the report by Chao et al, 1 which is the most recent of a pioneering and impressive series of publications by the same group of researchers over the past 2 years, [2][3][4][5][6] in which the interaction between the broadly expressed CD47 surface molecule and the myeloid inhibitory receptor SIRP␣ is implicated as a potential therapeutic target in a variety of hematopoietic malignancies. Chao et al use xenotransplantation models in which human NHL cells are engrafted into immunocompromised mice, and they show that lymphoma dissemination is inhibited by antibodies directed against human CD47 that block interactions with SIRP␣, but not by nonblocking anti-CD47 antibodies. In similarly designed studies with AML 3 and ALL 5 they had already demonstrated prominent tumor cell elimination with this blocking anti-CD47 antibody, and they had also previously shown that anti-CD47 treatment synergizes with the therapeutic anti-CD20 antibody rituximab in NHL. 2 Furthermore, their findings show that macrophages are essential in the process of leukemic cell elimination. They suggest that targeting of CD47-SIRP␣ interactions, both in the absence as well as in the presence of a cancer therapeutic antibody such as rituximab, could facilitate the eradication of tumor cells by promoting their phagocytic clearance by macrophages. 6 One problem that hampers a straightforward interpretation of the in vivo findings of these studies is the use of intact IgG antibodies. For instance, the anti-CD47 antibodies may not only disrupt CD47-SIRP␣ interactions but may instead, or at the same time, opsonize the tumor cells for antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent phagocytosis. In fact, our recent findings 7 with the same monoclonal anti-CD47 antibody (B6H12) that is used in the studies described above show that intact B6H12 induces direct ADCC of neutrophils toward breast cancer cells. The same is true for ADCC by monocytes, as indicated by the cytotoxicity against Jurkat lymphoma cells ( Figure 1). Clearly, direct ADCC cannot be simply excluded by using a control nonblocking anti-CD47 antibody, because this may actually have a very different capacity to induce antibody-dependent effector functions, even although it may have the same isotype. In fact, intact antibodies against CD47 might also trigger ADCC toward the healthy cells of the host, which would obviously create a highly undesirable condition. One way to really exclude this direct ADCC is to use these antibodies "stripped" of their Fc regions, that is, as B6H12 F(abЈ)2 fragments. Obviously, these do not directly induce antibody effector function, but the F(abЈ)2 fragments do in fact synergize with anti-CD3 antibodies against lymphoma cells (Figure 1) or with trastuzumab in the context of Her2/Neu-positive breast cancer cells. 7 This suggest that at least under these in vitro conditions the targeting of CD47-SIRP␣ interactions only enhances tumor cell killing when an appropriate cancer therapeutic antibody such as trastuzumab or rituximab is present. Would this mechanism also apply in vivo? This is clearly something that needs to be sorted out in more detail in future experiments. However, our own findings in a fully immunocompetent syngeneic model of metastatic melanoma are promising and suggest that animals that carry a defect in the SIRP␣ inhibitory signaling capacity do not show enhanced elimination of tumor cells in the absence of therapeutic antibody, while in the presence of therapeutic antibody a strongly potentiated effect is observed. 7 Another open question is whether CD47 on tumor cells could actually be efficiently targeted at all in the context of human cancer, even if it was done with the appropriate agents. The problem is that CD47 is very broadly expressed among both hematopoietic and nonhematopoietic cells, and this could seriously compromise efficient targeting of the cancer cell CD47 molecules. Instead, we propose to target SIRP␣. Blocking antibodies against human SIRP␣ are available now 7 and these would be promising agents to test further in clinical trials.
The final issue is whether the targeting of CD47 and/or SIRP␣ per se would not cause autoimmune disease. This subject clearly needs careful attention. In fact, there is perhaps already some comforting evidence, at least from animal experiments. First, mice that lack CD47 or have mutant SIRP␣ apparently do not display overt autoimmune symptoms, 8,9 and second, autoimmunity is not readily induced on injection of blocking antibodies against CD47

Mechanisms of targeting CD47-SIRP␣ in hematologic malignancies
Zhao et al 1 raise several issues from our prior work 2,3 characterizing the mechanism of targeting CD47 in human hematopoietic malignancies. We reported that a blocking anti-CD47 antibody can eliminate multiple human tumor types and that a major mechanism is through phagocytosis via blocking CD47-SIRP␣ ligation. Zhao et al argue that tumor elimination likely occurs through antibodydependent cellular cytotoxicity (ADCC) and that targeting CD47 may not be an effective strategy given widespread expression of CD47 in normal tissues. Here we respond to these issues.
First, Zhao et al argue that anti-CD47 antibody may not only disrupt CD47-SIRP␣ interactions but also opsonize cells for ADCC or antibody-dependent cellular phagocytosis (ADCP). They state that the distinction between these 2 mechanisms has not been clear because of the use of intact IgG anti-CD47 antibodies. While we primarily used intact anti-CD47 antibodies in our therapeutic experiments, we demonstrated in a proof-of-concept study that anti-CD47 antibody (clone B6H12) enables tumor elimination not through the Fc receptor (FcR; eg, ADCC, ADCP), but specifically through blocking CD47-SIRP␣ ligation. 3 We first used anti-CD47 F(abЈ) 2 fragments (as Zhao et al suggest), which enabled phagocytosis of lymphoma cells similarly to intact anti-CD47 antibody, demonstrating that the FcR was not required for phagocytosis. 3 Second, intact anti-CD47 antibody was incubated with macrophages deficient in the Fc␥R (thus unable to engage in FcRmediated ADCP), which still enabled phagocytosis, in contrast to rituximab, an FcR-dependent effector antibody. Third, while we found that anti-CD47 antibody did not enable human NK cellmediated ADCC of 2 lymphoma cell lines, 3 Zhao et al found that the same antibody induced ADCC of neutrophils and monocytes toward 2 cancer cell lines. 4 This difference could be because of the use of different cancer target cells or effector cells to measure ADCC.
Recently, we demonstrated that disruption of CD47-SIRP␣ led to inhibition of lymphoma dissemination. 2 Since we used intact anti-CD47 antibody in these experiments, we cannot rule out Fc-mediated effector functions. However, this was not a focus of the study given our previous work investigating this possibility. 3 Nevertheless, we agree that disruption of CD47-SIRP␣ may not be the sole mechanism and may include FcR-dependent mechanisms: ADCC, ADCP, complement-dependent cytoxicity or even direct apoptosis.
In a second inquiry, Zhao et al question whether targeting CD47 could selectively eliminate tumor cells without toxicity (including autoimmune disease). While CD47 targeting may lead to significant toxicity given its ubiquitous expression, preclinical data from our prior work suggest that anti-CD47 antibody not only has minimal toxicity but also eliminates tumor cells while sparing normal cells. 5,6 First, we showed that the anti-human CD47 antibody (clone B6H12) enabled phagocytosis of hematologic cancers but not normal cell counterparts in vitro. 3,5 Second, anti-mouse CD47 antibody administered to normal immunocompetent mice led to only a mild neutropenia with no evidence of autoimmune disease. 5 One would expect greater toxicity if anti-CD47 antibody induced cell killing through FcR-mediated effector functions. We then showed that selective targeting of tumor cells by anti-CD47 antibody was because of the presence of a prophagocytic signal, calreticulin, which is expressed on tumor but not normal cells. 6 While preclinical data are favorable, the toxicity profile of a human antibody must be tested in a clinical setting, which is ongoing in our laboratory. 7