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Anti-GD2 synergizes with CD47 blockade to mediate tumor eradication

Nature Medicine volume 28, pages 333–344 (2022)Cite this article

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Abstract

The disialoganglioside GD2 is overexpressed on several solid tumors, and monoclonal antibodies targeting GD2 have substantially improved outcomes for children with high-risk neuroblastoma. However, approximately 40% of patients with neuroblastoma still relapse, and anti-GD2 has not mediated significant clinical activity in any other GD2+ malignancy. Macrophages are important mediators of anti-tumor immunity, but tumors resist macrophage phagocytosis through expression of the checkpoint molecule CD47, a so-called ‘Don’t eat me’ signal. In this study, we establish potent synergy for the combination of anti-GD2 and anti-CD47 in syngeneic and xenograft mouse models of neuroblastoma, where the combination eradicates tumors, as well as osteosarcoma and small-cell lung cancer, where the combination significantly reduces tumor burden and extends survival. This synergy is driven by two GD2-specific factors that reorient the balance of macrophage activity. Ligation of GD2 on tumor cells (a) causes upregulation of surface calreticulin, a pro-phagocytic ‘Eat me’ signal that primes cells for removal and (b) interrupts the interaction of GD2 with its newly identified ligand, the inhibitory immunoreceptor Siglec-7. This work credentials the combination of anti-GD2 and anti-CD47 for clinical translation and suggests that CD47 blockade will be most efficacious in combination with monoclonal antibodies that alter additional pro- and anti-phagocytic signals within the tumor microenvironment.

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Fig. 1: Anti-CD47 and anti-GD2 synergize to mediate anti-tumor activity in xenograft models of neuroblastoma.
Fig. 2: CD47 blockade licenses TAMs to enhance the activity of anti-GD2 in a syngeneic model of neuroblastoma.
Fig. 3: Absence of neurotoxicity related to combined anti-GD2 and anti-CD47 treatment.
Fig. 4: Anti-GD2 blocks the interaction of GD2 with its ligand Siglec-7, an inhibitory immune receptor.
Fig. 5: Anti-GD2 antibody primes neuroblastoma cells for removal by the immune system.
Fig. 6: Anti-CD47 and anti-GD2 elicit anti-tumor responses in models of osteosarcoma via TAM activation.

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Data availability

All statistics for the main and extended data figures are provided in the Supplementary Information. Source data for Figs. 1–3 and 5 and 6 and Extended Data Figs. 1–2 and 4–9 are supplied. The unprocessed gel blot image for Extended Data Fig. 3 is also provided. Source data are provided with this paper.

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Acknowledgements

This work was supported by an Alex’s Lemonade Stand ‘A’ Award (R.G.M.) and National Institutes of Health P01 CA217959 (C.L.M., W.A.W. and R.G.M.). R.G.M. is the Taube Distinguished Scholar for Pediatric Immunotherapy at Stanford University School of Medicine. J.T. is supported by German Cancer Aid (Deutsche Krebshilfe) grant number P-91650709. This work was supported by the National Cancer Institute (R01-CA227942 to C.R.B., F30-CA232541 to B.A.H.S., U01-CA217864 to W.A.W. and U01-CA213273 and R35-CA231997 to J.S.), the American Cancer Society (postdoctoral fellowship to G.L.C.) and the Virginia and D. K. Ludwig Fund for Cancer Research (I.W., and M. Monje). B.A.H.S. is supported by the Stanford School of Medicine Medical Scientist Training Program (T32-GM007365). M.H.L. is supported by a Blavatnik Family Fellowship. P.L.L. is supported by the Stanford Bio-X Bowes Fellowship and the Stanford Medical Scientist Training Program. A.C.G. is supported by the Waxman Family Research Fund. We thank the Stanford Neuropathology Department for their help with immunohistochemistry. We thank K. Stegmaier and N. Mabe for providing the SY5Y-GD2-high cell line as well as expertise and advice. All cartoons were created with BioRender.com.

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Author notes

  1. These authors contributed equally: Marie Menard, Benjamin A. H. Smith, Miles H. Linde.

Authors and Affiliations

  1. Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA

    Johanna Theruvath, Garry L. Coles, Guillermo Nicolas Dalton, Molly Thomas Radosevich, Shaurya Dhingra, Aidan Tousley, John Lattin, Peng Xu, Jing Huang, Andy He, Michelle Monje, Heike E. Daldrup-Link, Julien Sage, Crystal L. Mackall & Robbie G. Majzner

  2. Departments of Neurology, Pediatrics, and Neurological Surgery, Brain Tumor Research Center, University of California, San Francisco, San Francisco, CA, USA

    Marie Menard, Nicole Nasholm & William A. Weiss

  3. ChEM-H Institute, Stanford University, Stanford, CA, USA

    Benjamin A. H. Smith & Carolyn R. Bertozzi

  4. Department of Chemical & Systems Biology, Stanford University, Stanford, CA, USA

    Benjamin A. H. Smith & Carolyn R. Bertozzi

  5. Immunology Graduate Program, Stanford University School of Medicine, Stanford, CA, USA

    Miles H. Linde & John L. Silberstein

  6. Institute for Stem Cell Biology and Regenerative Medicine, Stanford, CA, USA

    Miles H. Linde, Allison Banuelos, Amira Barkal, Rachel E. Brewer, Kristopher D. Marjon, Michelle Monje, Heike E. Daldrup-Link, Irving L. Weissman & Ravindra Majeti

  7. Stanford Cancer Institute, Stanford University School of Medicine, Stanford, CA, USA

    Miles H. Linde, Garry L. Coles, Elena Sotillo, Allison Banuelos, Sabine Heitzeneder, Amira Barkal, Rachel E. Brewer, Kristopher D. Marjon, Michelle Monje, Irving L. Weissman, Julien Sage, Ravindra Majeti, Crystal L. Mackall & Robbie G. Majzner

  8. Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Miles H. Linde, Ravindra Majeti & Crystal L. Mackall

  9. Department of Radiology, Stanford University School of Medicine, Stanford, CA, USA

    Wei Wu, Louise Kiru & Heike E. Daldrup-Link

  10. British Columbia Cancer Agency, Vancouver, BC, Canada

    Alberto Delaidelli & Poul H. Sorensen

  11. Department of Bioengineering, Stanford University Schools of Engineering and Medicine, Stanford, CA, USA

    John L. Silberstein & Jennifer R. Cochran

  12. Department of Neurology and Neurological Sciences, Stanford University, Stanford, CA, USA

    Anna C. Geraghty & Michelle Monje

  13. ALX Oncology, Burlingame, CA, USA

    Tracy C. Kuo, Emma R. B. Sangalang & Jaume Pons

  14. Ludwig Center for Cancer Stem Cell Research and Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Amira Barkal, Rachel E. Brewer, Kristopher D. Marjon, Michelle Monje, Irving L. Weissman & Ravindra Majeti

  15. Stanford Medical Scientist Training Program, Stanford University, Stanford, CA, USA

    Amira Barkal & Payton L. Marshall

  16. Department of Comparative Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Jose G. Vilches-Moure

  17. Chinese Academy of Medical Sciences (CAMS) Oxford Institute (COI), University of Oxford, Oxford, UK

    Ricardo Fernandes

  18. Nuffield Department of Medicine, University of Oxford, Oxford, UK

    Ricardo Fernandes

  19. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

    Michelle Monje & Irving L. Weissman

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  1. Johanna Theruvath
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  2. Marie Menard
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J.T., M. Menard, B.A.H.S., M.H.L., G.L.C., G.N.D., W.W., L.K., A.D., E.S., J.L.S., A.C.G., A.B., M.T.R., S.D., S.H., A.T., J.L., P.X., J.H., N.N., A.H., A.B., R.B., K.D.M., J.G.V.-M., P.L.M., R.F. and R.G.M. designed experiments, performed experiments and/or performed data analysis. M. Monje, J.R.C., P.H.S., H.E.D.-L., I.L.W., J.S., R.M., C.R.B., W.A.W., C.L.M. and R.G.M. designed experiments and/or oversaw the research. T.C.K., E.R.B.S. and J.P. generated and provided ALX301. J.T., E.S. and R.G.M. wrote the manuscript. All authors reviewed the manuscript.

Corresponding author

Correspondence to Robbie G. Majzner.

Ethics declarations

Competing interests

R.G.M. and C.L.M. are founders of, hold equity in and receive consulting fees from Syncopation Life Sciences. C.L.M. is a founder of, holds equity in and receives consulting fees from Lyell Immunopharma. R.G.M. and E.S. are consultants for Lyell Immunopharma. R.G.M. is a consultant for NKarta, Illumina Radiopharmaceuticals, GammaDelta Therapeutics, Aptorum Group and Zai Labs. J.T. is a consultant for Dorian Therapeutics. C.L.M. has also received consulting fees from NeoImmune Tech, Nektar Therapeutics and Apricity Health and royalties from Juno Therapeutics for CD22-CAR. W.A.W. is a founder of, holds equity in and receives consulting fees from StemSynergy Therapeutics. R.M. is on the Board of Directors of BeyondSpring and the Scientific Advisory Boards of Coherus BioSciences, Kodikaz Therapeutic Solutions and Zenshine Pharmaceuticals. R.M. and I.W. are inventors on several patents related to CD47 cancer immunotherapy that are licensed to Gilead Sciences. J.P. is an employee and shareholder of ALX Oncology, and T.C.K. and E.R.B.S. are shareholders of ALX Oncology. J.S. receives research funding from Stemcentrx/Abbvie and Pfizer and licensed a patent to Forty Seven/Gilead on the use of CD47 blocking strategies in SCLC (with I.W.). C.R.B. is a co-founder of Redwood Biosciences (a subsidiary of Catalent), Enable Biosciences, Palleon Pharmaceuticals, InterVenn Bio, Lycia Therapeutics and OliLux Biosciences and is a member of the Board of Directors of Eli Lilly. J.R.C. is a co-founder and equity holder of xCella Biosciences, Combangio and Trapeze Therapeutics. All other authors declare no competing interests.

Peer review information

Nature Medicine thanks Leonid Metelitsa, John Anderson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Editor recognition statement Saheli Sadanand was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 anti-GD2 and anti-CD47 synergize to promote tumor cell phagocytosis.

a-b, Representative Images of Incucyte based live cell phagocytosis assay. a, pHrodo red labeled KCNR (top) or CHLA255 (bottom) neuroblastoma cells were co-cultured with human blood derived macrophages in the presence of anti-GD2 mAb, anti-CD47 mAb or dual treatment. Red indicative of tumor cell phagocytosis. Images were obtained after 24 hours of co-culture. b, Magnified image of cells shown in a. c-d, Quantification of phagocytosis normalized to the phagocytosis in the untreated control for each cell line, c, KCNR (Untreated vs. GD2+CD47 p = 2.9E-14, GD2 vs. GD2+CD47 p = 5.4681E-10, CD47 vs. GD2+CD47 p = 1.13E-12) d, CHLA255 (Untreated vs. GD2+CD47 p = 3.3E-14, GD2 vs. GD2+CD47 p = 2.128E-12, CD47 vs. GD2+CD47 p = 3.3E-14). Data are mean values +/− s.e.m. of five experimental replicates. Experiment was performed one time. e, Flow-based phagocytosis assay of KCNR neuroblastoma cells co-cultured with human blood derived macrophages in the presence of different concentrations of anti-GD2 mAb, anti-CD47 mAb or dual treatment. % phagocytosing macrophages are reported. Data are mean values +/− s.d. of three experimental replicates. Experiment was performed one time. Statistical comparisons performed with one-way ANOVA with multiple comparisons correction, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns P>0.05.

Source data

Extended Data Fig. 2 Anti-CD47 and anti-GD2 synergize to mediate significant anti-tumor activity in orthotopic models of neuroblastoma.

One million KCNR neuroblastoma cells expressing GFP-luciferase were implanted into the renal capsule and treated four days later with IgG control, anti-G2 mAb, anti-CD47 (magrolimab, Hu5F9-G4) mAb or dual anti-GD2/anti-CD47 (Hu5F9-G4) every other day for three doses as in Fig. 1c. a, Quantification of tumor progression for each individual mouse as measured by flux values acquired via bioluminescence (BLI) photometry. b, BLI images of representative mice from each treatment group at different time points. Experiment was performed one time. N = 4 mice for IgG, and N = 5 for anti-GD2 per group and those groups are the same as in Fig. 1c–f. N=3 for anti-CD47 and anti-CD47 + anti-GD2 c, One million CHLA255 neuroblastoma cells expressing GFP-luciferase cells were implanted into the renal capsule in NSG mice. Four days later, mice were injected with IgG control, anti-GD2 mAb, anti-CD47 mAb or dual anti-GD2/anti-CD47 treatment every other day for three doses. d, Quantification of tumor progression for each individual mouse as measured by flux values acquired via BLI photometry. e, BLI images of representative mice from each treatment group shown in d at different time points. Red cross indicates deceased mouse. f, Survival curves for mice bearing tumors shown in d. d-f, Representative data from three independent experiments with N = 5 mice per group. All survival curves were compared using the Log-rank test (two-tailed).

Source data

Extended Data Fig. 3 Expression and affinity of ALX301, an engineered molecule capable of blocking murine CD47.

a, Schematic of ALX301: Murine IgG1 containing a N297A mutation was fused to a mutated SIRPα capable of binding murine CD47 with enhanced affinity. b, Purified ALX301 was detected on 4-20% Tris-glycine gel in non-reducing (NR) and reducing (R) buffer. ALX301 runs slightly larger than the expected 76.16 kDa (NR) and 38.038 kDa (R). c, ALX301 was immobilized on GLC sensor chip (Bio-rad). Recombinant mouse CD47 protein was injected as analyte over the chip at 5 concentrations, 3-fold dilution (100nM, 33.3nM, 11.1nM, 3.7nM, 1.2nM). Using Langmuir binding model for curve fitting, the binding of ALX301 to mouse SIRPα was determined to be 3.41nM. Association rate (ka), dissociation rate (kd), affinity (KD). b-c were performed one time.

Extended Data Fig. 4 Absence of toxicity of anti-GD2 and anti-CD47 treated mice in a model of metastatic neuroblastoma.

a, One million CHLA255 neuroblastoma cell were injected in the tail vein of the mice. After 4 days mice were treated intraperitoneally with PBS (control), anti-GD2, anti-CD47 or the combination of anti-GD2 and anti-CD47 3x/week for a total of 7 doses. b, Weight was serially recorded. c-e, CatWalk gait analysis was performed at indicated time points and c, swing speed, d, stride length and e, regularity index are reported from mice treated as in a. Data shown are mean values +/− s.e.m. n = 5 mice per group. Experiment was performed one time. f, Hematoxylin and eosin (H&E) stained sections of all organs examined were within normal limits as assessed by brightfield microscopy. Shown are sections of cerebrum, cerebellum, peripheral nerve, heart, liver, kidney, spleen, and lung of animals from mice treated as in a. Magnification: 40x, Scale bars: 20𝝁m.

Source data

Extended Data Fig. 5 Lack of synergy of anti-B7-H3 with anti-CD47 in xenograft models of neuroblastoma or osteosarcoma.

a, Graphs show flow cytometry-based quantification of phagocytosis of neuroblastoma cell lines co-cultured with human blood derived macrophages in the presence of anti-CD47 mAb, anti-B7-H3 mAb, or dual treatment, compared with untreated control; results normalized to the phagocytosis in the untreated control for each cell line and blood donor. Shown are mean values +/− s.d. of three experimental replicates. Statistical comparisons performed with one-way ANOVA with multiple comparisons correction (KCNR: control vs. anti-B7-H3 p = 0.9643, anti-CD47 vs. anti-B7-H3 and anti-CD47 p = 0.9976; CHLA255: control vs. anti-B7-H3 p>0.9999, anti-CD47 vs. anti-B7-H3 and anti-CD47 p = 0.8519). Representative data from at least three experiments with three different blood donors. Inset: B7-H3 expression as assessed by flow cytometry on respective cell lines. b, One million CHLA255 neuroblastoma cells were engrafted into NSG mice by tail-vein injection. On D+4, mice were treated with IgG control, anti-CD47 mAb, anti-B7-H3 mAb, or anti-B7-H3+anti-CD47 every other day for three doses. Quantification of tumor progression for each individual mouse as measured by flux values acquired via BLI photometry. c, BLI images of representative mice from each treatment group shown in b at one time point. d, Survival curves for mice bearing tumors shown in b. N = 5 mice per group, performed one time. Survival curves were compared using the Log-rank test (two-tailed). e, Graph shows flow cytometry-based quantification of phagocytosis of osteosarcoma cell lines co-cultured with human blood derived macrophages; results normalized to the phagocytosis in the untreated control for each cell line and blood donor. Shown are mean values +/− s.d. of three experimental replicates. Statistical comparisons performed as in a. Representative data from at least three experiments with three different blood donors. Inset: B7-H3 expression as assessed by flow cytometry on respective cell lines. f, Survival curves for mice that received hind leg injection of 1e6 MG63.3 cells and treated on D+7 with indicated antibodies 3x/week for four weeks. N = 5 mice per group, performed one time. Survival curves were compared using the Log-rank test (two-tailed). *P < 0.05, ns P>0.05.

Source data

Extended Data Fig. 6 Upregulation of ‘Eat me’ signal calreticulin and induction of cell death by ligation with anti-GD2 mAb on neuroblastoma cell lines.

a, Flow cytometric analysis of the levels of expression of GD2 on the surface of neuroblastoma cell lines CHLA255 and KCNR and their GD2-KO (B4GALNT1 KO) versions. b, Flow-based quantification of cell viability. Neuroblastoma cell lines and their GD2-KO versions were incubated with anti-GD2 mAb for the indicated times at 37 degrees and stained with DAPI. Percent of DAPI- populations were normalized to the untreated control for each cell line. Shown are mean values +/− s.d. of 6 experimental replicates. (CHLA255: 3hr p = 4.17E-08, 6hr p = 6.5044E-09, 12hr p = 1.2983E-11, 24hr p = 1.9094E-07, 48hr p = 7E-15, 72hr p = 1.2397E-08; CHLA255 GD2 KO: 3hr p = 0.3013, 6hr p = 0.506, 12hr p = 0.4125, 24hr p = 0.983, 48hr p = 0.4263, 72hr p = 0.8453; KCNR: 3hr p = 3.75E-10, 6hr p = 7.0881E-07, 12hr p = 1.1406E-07, 24hr p = 4.2986E-08, 48hr p = 2.6777E-06, 72hr p = 2.9544E-06; KCNR GD2 KO: 3hr p = 0.0059, 6hr p = 0.0083, 12hr p = 0.3223, 24hr p = 0.8197, 48hr p = 0.1443, 72hr p = 0.1002). c, Graph shows flow cytometric quantification of the expression of calreticulin (∆MFI) on the surface of live neuroblastoma cell lines treated as in b. Shown are mean values +/− s.d. of 3 experimental replicates. CHLA255: 3hr p = 6.57E-05, 6hr p = 0.0004, 12hr p = 0.0002, 24hr p = 9.2150E-06, 48hr p = 0.0167, 72hr p = 0.027; CHLA255 GD2 KO: 3hr p = 0.4701, 6hr p = 0.9563, 12hr p = 0.9024, 24hr p = 0.7235, 48hr p = 0.6415, 72hr p = 0.4069; KCNR: 3hr p = 7.41E-07, 6hr p = 7.4552E-07, 12hr p = 0.0038, 24hr p = 0.0024, 48hr p = 0.0019, 72hr p = 0.0005; KCNR GD2 KO: 3hr p = 0.671, 6hr p = 0.9445, 12hr p = 0.023, 24hr p = 0.2359, 48hr p = 0.0426, 72hr p = 0.8656. ∆MFI was calculated as the difference between the MFI in the PE channel of the calreticulin stained sample and the MFI in the PE channel of an isotype stained sample from the same experimental condition. The full time-course experiment was performed twice and the twelve-hour timepoint shown is identical to Fig. 4h. b-c, Statistical comparisons performed with the unpaired t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns P>0.05.

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Extended Data Fig. 7 Anti-CD47 and anti-GD2 synergize to mediate significant anti-tumor activity in xenograft model of small cell lung cancer (SCLC).

a, NCI-H69 SCLC were engrafted on both flanks of NSG mice. Quantification of tumor growth for each individual tumor was assessed by caliper measurement. b, Survival curves for mice bearing tumors shown in a. Survival curves were compared using the Log-rank test (two-tailed). Representative of two independent experiments with n = 5 mice per group.

Source data

Extended Data Fig. 8 Anti-GD2 and anti-CD47 treatment leads to an increase in macrophage infiltration and reduction of M2 macrophages.

a, Flow cytometric analysis of M2 macrophages (defined as CD163+ and CD206+, gated on CD45+, CD11b+, F4/80 + macrophages) in osteosarcoma tumors treated as described in Fig. 6, pooled results from two independent experiments. Shown are mean values +/− s.em. of 10 experimental replicates. Control vs. GD2+CD47 p = 0.0007, CD47 vs. GD2+CD47 p = 0.0738, GD2 vs. GD2+CD47 p = 0.8869. b-f, CHLA255 neuroblastoma tumor cells were engrafted in the flank and allowed to grow for 19 days before initiation of treatment with IgG control, anti-GD2 mAb, anti-CD47 mAb or dual anti-GD2/anti-CD47. After one week of treatment, tumors were harvested for immunohistochemistry (IHC) and flow cytometric analysis. b, Representative IHC images showing detection of macrophages via staining with anti-F4/80 on tumors harvested from mice treated with indicated mAbs. c, Quantification of percent of positive F4/80 staining obtained from IHC analysis. Shown are mean values +/− s.e.m. of 4-5 biologic replicates. Control vs. GD2+CD47 p = 0.0281, CD47 vs. GD2+CD47 p = 0.0415, GD2 vs. GD2+CD47 p = 0.2804. d, Quantification of percent of positive iNOS staining obtained from IHC analysis. Shown are mean values +/− s.e.m. of 4-5 biologic replicates. Control vs. GD2+CD47 p = 0.2401, CD47 vs. GD2+CD47 p = 0.413, GD2 vs. GD2+CD47 p = 0.7487. e-f, Flow cytometric analysis of M2 macrophages (defined as CD163+ and CD206+, gated on CD45+, CD11b+, F4/80 + macrophages) in CHLA255 tumors. e, Representative flow plots and f, quantification of M2 macrophages. Shown are mean values +/− s.e.m. of 4-5 biologic replicates. Control vs. GD2+CD47 p = 0.0218, CD47 vs. GD2+CD47 p = 0.9999, GD2 vs. GD2+CD47 p = 0.7102. b-f, Experiment was performed one time. All statistical comparisons were made between groups with the one-way ANOVA with correction for multiple comparisons correction, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns P>0.05.

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Extended Data Fig. 9 GD2 antigen density may determine response to anti-GD2/anti-CD47.

a, Expression levels of CD47 mRNA in samples from three different datasets of primary tumors from high-risk, stage 4 neuroblastoma stratified by MYCN status (amplified vs non-amplified), age at diagnosis, and ploidy (where available). b, Expression levels of CD163, CD86, and SIGLEC7, together with CD47 and MYCN in samples from the same databases as in a grouped based on MYCN amplification status. c-d, Expression levels of CD47 and MYC family genes in samples from osteosarcoma c or SCLC d primary tumors. Samples are stratified based on high or low expression of any of the three MYC family genes: MYC, MYCL or MYCN. Dot plots represent log2 (a-c) or TPM (d) expression values. Error bars represent median ± s.d. Differences in gene expression levels between groups were calculated using the Mann-Whitney U test (unpaired, two-sided). e, Flow cytometric analysis of the expression of GD2 (top) and CD47 (bottom) on tumor cell lines used in in vivo models. f, Flow cytometric analysis of the expression of GD2 (top) and CD47 (bottom) on isogenic SH-SY5Y neuroblastoma GD2-low and GD2-high cell lines. g, Graphs show flow cytometry-based quantification of phagocytosis of SY5Y-GD2-low and SY5Y-GD2-high neuroblastoma cell lines co-cultured with human blood derived macrophages in the presence of anti-GD2 mAb, anti-CD47 mAb or dual treatment, compared with untreated control; results normalized to the phagocytosis in the untreated (UT) control for each cell line and blood donor. Shown are mean values +/− s.d. of three experimental replicates. SY5Y-GD2-high-UT vs. SY5Y-GD2-low-UT p>0.9999, SY5Y-GD2-high-GD2-treated vs. SY5Y-GD2-low-GD2-treated p = 5.1941E-07, SY5Y-GD2-high-CD47-treated vs. SY5Y-GD2-low-CD47-treated p = 0.5091, SY5Y-GD2-high-GD2+CD47-treated vs. SY5Y-GD2-low-GD2+CD47-treated p = 9.8567E-07. Representative data from two experiments performed with two different blood donors. Statistical comparisons performed with one-way ANOVA with multiple comparisons correction, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns P>0.05.

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Supplementary information

41591_2021_1625_MOESM1_ESM.pdf

Supplementary Information Representative gating strategies for flow cytometry. a, Representative gating strategy for macrophage phagocytosis assay. b, Representative gating strategy for Ly6G depletion experiments. c, Representative gating strategy for M2 macrophage phenotyping experiments.

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Theruvath, J., Menard, M., Smith, B.A.H. et al. Anti-GD2 synergizes with CD47 blockade to mediate tumor eradication. Nat Med 28, 333–344 (2022). https://doi.org/10.1038/s41591-021-01625-x

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