Phagocytosis is an essential process utilized by an organism for pathogen or apoptotic cell clearance (Poon et al., 2014). CD47 is a cell surface glycoprotein with a variety of functions including regulation of phagocytosis through binding to the macrophage and dendritic cell specific protein signal regulatory protein alpha (SIRPα) (Oldenborg, 2013). Binding of SIRPα to CD47 essentially sends a ‘don't eat me’ message to macrophages by initiating signaling to inhibit phagocytosis (Murata et al., 2014).

Increased expression of CD47 is proposed to be a mechanism through which cancer cells evade immune detection and phagocytosis. CD47 expression is increased in several cancer types including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), primary effusion lymphoma, multiple myeloma, leiomyosarcoma, and bladder cancer, and targeting of CD47 on cancer cells with an anti-CD47 blocking antibody can promote phagocytosis by macrophages in vitro (Chan et al., 2009; Jaiswal et al., 2009; Majeti et al., 2009; Chao et al., 2010a; Edris et al., 2012). Further, treatment with an anti-CD47 blocking antibody synergized with rituximab treatment to promote phagocytosis in vitro and to eliminate cancer cells in an in vivo xenograft model of non-Hodgkin lymphoma (Chao et al., 2010b). This is supported in two syngeneic murine tumor models, in melanoma and squamous cell carcinoma, where irradiation combined with antisense suppression of CD47 delayed tumor growth (Maxhimer et al., 2009). Willingham et al., 2012 further extend these results to demonstrate that CD47 expression increases in a variety of human solid tumor types and that blocking the SIRPα/CD47 interaction with an anti-CD47 antibody can promote phagocytosis of solid tumor cells in vitro and reduce growth of solid tumors in vivo. While it is not clear if SIRPα signaling is involved in the antitumor activity of an anti-CD47 antibody, these results indicate that anti-CD47 antibody therapy may be an effective treatment for a variety of solid tumor types (Soto-Pantoja et al., 2012).

In Figures 6B, 6C, and Table S4, the safety and efficacy of anti-CD47 antibody treatment are tested in immune competent mice using a syngeneic breast cancer model. MT1A2 mouse mammary cancer cells were implanted in the mammary fat pads of FVB mice and IgG control or anti-CD47 antibody treatment commenced upon detection of palpable tumors. Tumor growth was measured by gross weight and analyzed by immunohistochemistry. Willingham et al., 2012 showed that anti-CD47 antibody treatment reduced tumor growth and increased lymphocytic infiltration to the tumor site without unacceptable toxicity, thus demonstrating that anti-CD47 therapy is effective in reducing solid tumor growth in immune competent hosts. This key experiment demonstrates that CD47 is a therapeutic target for solid tumors and follows similar reports from the same laboratory that also demonstrated that anti-CD47 antibody treatment reduced growth of primary human cancer xenografts of several hematopoietic cancers and of solid leiomyosarcoma tumors (Jaiswal et al., 2009; Majeti et al., 2009; Chao et al., 2010a; Edris et al., 2012). Subsequent reports extended these results to multiple myeloma and primary effusion lymphoma models (Kim et al., 2012; Goto et al., 2014). These experiments will be replicated in Protocol 1.

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Thank you for sending your work entitled “Registered report: The CD47-signal regulated protein alpha interaction is a therapeutic target for human solid tumors” for consideration at eLife. Your article has been favorably evaluated by Tadatsugu Taniguchi (Senior editor), a Reviewing editor, and four reviewers, one of whom is a biostatistician.

The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission. There was considerable discussion about the proposed replication. One of the reviewers asked for studies with human patient xenograft tumors to be included for a comprehensive and representative reproduction of the original study, but the other reviewers and the editors favor strict reproduction. The key experiment from the prior publication that suggests therapeutic potential for an anti-CD47-based strategy is the syngeneic experiment in an immune-competent host, where the potential toxicity of the antibody against the host could be assessed. This experiment seems to be the most critical to reproduce as it most closely mimics a therapeutic scenario. Therefore we support the original proposed design, with comments on the proposal and revision requests following.

Overview:

Chroscinski et al. propose to replicate the experiments from Figure 6B, 6C, and Table S4 of Willingham et al, to verify the safety and efficacy of anti-CD47 antibody treatment in immune competent mice utilizing a syngeneic breast cancer model, and to assess the effects on tumor growth and lymphocytic infiltration. The tumor model consists of MT1A2 tumor cells injected into the mammary fat pad of FVB mice. The proposed protocol is detailed and carefully considered. Tumors will be weighed after 30 days, and embedded in paraffin and processed for histological analysis of lymphocytic infiltration. Toxicity will be determined five days after tumor cell injection by complete blood cell counts.

The authors appropriately discuss the relevant background for the study, and have also thoroughly considered all parameters of the proposed experiments, for which they have also consulted with the original authors. Author consultation allowed for the inclusion of key parameters including multiple details associated with proper media conditions for the tumor cells.

Notable differences between this proposed work and the previous study include the exclusion of anti-CD47 clone MIAP301, which is appropriate given the lack of a statistically significant effect of this clone in the previous study, and toxicity in this replicate study will be conducted on the same mice for which efficacy is also being considered, which appears to be preferable to the previous work that used a different mouse strain (BALB/c). Blood analysis will also be performed with the same dose of antibody used to determine efficacy, which also seems preferable. The number of mice is appropriate, as the authors seek to derive data from the same number of animals as in the original study (n=5 per group final). Finally the tumor cell line will also be subjected to STR profiling to verify its identity.

Overall the authors propose to replicate what amounts to the key findings of Willingham et al, in a carefully considered and complete proposal.

Revisions to the proposal and questions to consider:

1) Methods: In the original report two antibodies reactive against CD47, with different isotypes were used; one isotype control [not specified which) was used as a control. Why is only one antibody used in replicate here?

2) Methods: In an immunocompetent mouse, tumor can be cleared via CMC, ADCC, opsonization, phagocytosis, etc, mediated by a number of different effectors. In the proposed experiment, the test antibody might mediate these mechanisms in addition to blocking the CD47-SIRPa interaction, with a similar or partial therapeutic result. Using an isotype control, therefore, does not yield an interpretable result unless the isotype antibody binds to the same target cell (optimally CD47) with similar affinity, but does not block the interaction with SIRPa. Such a control antibody was used in some of the original experiments in vitro, (PNAS, Figure 3A-C), but apparently the antibody later became “unavailable” and hence was not included in subsequent in vitro or any animal experiments. Alternatively, use of the same CD47 antibody with a D265A Heavy Chain mutation to eliminate FcR binding would be a useful contriol. Without such controls, at the completion of this replicate, a conclusion that the mechanism is via blockade of the interaction of CD47 with SIRPa, (as in title) is not possible.

3) Methods: Injection of such large and frequent antibody doses, and directly into the tumor bed, is an unusual administration plan. Use of an IV or IP administration route would be advised in addition as a control group.

4) How will injections into the orthotropic site, but not the tumor itself be achieved? It is not clear what Willingham did from reading the paper.

5) Methods: Step 5, How are mice randomized? Please state method.

1) Methods: In the original report two antibodies reactive against CD47, with different isotypes were used; one isotype control [not specified which) was used as a control. Why is only one antibody used in replicate here?

We included only the MIAP410 clone because of the larger effect compared to the MIAP301 clone, which the reviewers have also commented on. Also, the authors shared with us the isotype control used in the original experiment, which was a mouse IgG isotype control (the same control antibody used in this replication). The MIAP301 clone is a rat IgG antibody and thus the isotype control antibody was not matched to the MIAP301 clone host species, but was for the MIAP410 clone.

2) Methods: In an immunocompetent mouse, tumor can be cleared via CMC, ADCC, opsonization, phagocytosis, etc, mediated by a number of different effectors. In the proposed experiment, the test antibody might mediate these mechanisms in addition to blocking the CD47-SIRPa interaction, with a similar or partial therapeutic result. Using an isotype control, therefore, does not yield an interpretable result unless the isotype antibody binds to the same target cell (optimally CD47) with similar affinity, but does not block the interaction with SIRPa. Such a control antibody was used in some of the original experiments in vitro, (PNAS, Figure 3A-C), but apparently the antibody later became “unavailable” and hence was not included in subsequent in vitro or any animal experiments. Alternatively, use of the same CD47 antibody with a D265A Heavy Chain mutation to eliminate FcR binding would be a useful contriol. Without such controls, at the completion of this replicate, a conclusion that the mechanism is via blockade of the interaction of CD47 with SIRPa, (as in title) is not possible.

We agree adding a control antibody to eliminate FcR binding would be an informative approach to determine if the mechanism is via blockage of the interaction of CD47 with SIRPa, however, as the reviewers have already described, Willingham and colleagues did not use this approach. The Reproducibility Project: Cancer Biology aims to perform direct replications using the same methodology reported in the original paper. We agree this additional antibody control would be useful, but is beyond the scope of this project. As such, we will restrict our analysis to the experiments being replicated and will not include discussion of the other in vitro experiments (Figure 3A-C; Willingham et al., 2012) that are not being replicated in this study.

3) Methods: Injection of such large and frequent antibody doses, and directly into the tumor bed, is an unusual administration plan. Use of an IV or IP administration route would be advised in addition as a control group.

We agree adding an additional administration route would be of interest to test if the effect is dependent on the route of antibody delivery, however, as the reviewers have already described, Willingham and colleagues did not use this approach. The Reproducibility Project: Cancer Biology aims to perform direct replications using the same methodology reported in the original paper. We are attempting to identify a balance of breadth of sampling for general inference with sensible investment of resources on replication projects to determine to what extent the included experiments are reproducible. Thus, while the use of an additional administration route would be of interest, it would be a conceptual replication.

4) How will injections into the orthotropic site, but not the tumor itself be achieved? It is not clear what Willingham did from reading the paper.

We have contacted the original authors who have provided additional details for how these injections were performed. The antibody was injected into the mammary fat pad next to the tumor with an approximate distance of 2 mm to the tumor. We have updated the manuscript to address this point.

5) Methods: Step 5, How are mice randomized? Please state method.

We have updated the manuscript to describe this process.

Author details

1. Denise Chroscinski

   Noble Life Sciences, Gaithersburg, United States

   Contribution

   DC, Drafting or revising the article

   Competing interests

   DC: This is a Science Exchange Associated lab.

2. Nimet Maherali

   Harvard Stem Cell Institute, Cambridge, United States

   Contribution

   NM, Drafting or revising the article

   Competing interests

   No competing interests declared.

3. Erin Griner

   University of Virginia, Charlottesville, United States

   Contribution

   EG, Drafting or revising the article

   Competing interests

   No competing interests declared.

4. Reproducibility Project: Cancer Biology

   Contribution

   RP:CB, Conception and design, Drafting or revising the article

   For correspondence

   tim@cos.io

   Competing interests

   RP:CB: EI, FT and JL are employed by and hold shares in Science Exchange Inc.

   1. Elizabeth Iorns, Science Exchange, Palo Alto, California
   2. William Gunn, Mendeley, London, United Kingdom
   3. Fraser Tan, Science Exchange, Palo Alto, California
   4. Joelle Lomax, Science Exchange, Palo Alto, California
   5. Timothy Errington, Center for Open Science, Charlottesville, Virginia

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