Registered report: The CD47-signal regulated protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors

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.