Currently approved immune checkpoint inhibitor therapies targeting the PD-1 and CTLA-4 receptor pathways are powerful treatment options for certain cancers; however, most patients across cancer types still fail to respond. Consequently, there is interest in discovering and blocking alternative pathways that mediate immune suppression. One such mechanism is an upregulation of sialoglycans in malignancy, which has been recently shown to inhibit immune cell activation through multiple mechanisms and therefore represents a targetable glycoimmune checkpoint. Since these glycans are not canonically druggable, we designed an αHER2 antibody–sialidase conjugate that potently and selectively strips diverse sialoglycans from breast cancer cells. In syngeneic breast cancer models, desialylation enhanced immune cell infiltration and activation and prolonged the survival of mice, an effect that was dependent on expression of the Siglec-E checkpoint receptor found on tumor-infiltrating myeloid cells. Thus, antibody–sialidase conjugates represent a promising modality for glycoimmune checkpoint therapy.
This is a preview of subscription content, access via your institution
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
All data shown in this manuscript are provided either as source data files or in Supplementary Dataset 1. Any unique materials presented in the manuscript may be available from the authors upon reasonable request and through a materials transfer agreement.
Schadendorf, D. et al. Pooled analysis of long-term survival data from phase II and phase III trials of ipilimumab in unresectable or metastatic melanoma. J. Clin. Oncol. 33, 1889–1894 (2015).
Ribas, A. et al. Association of pembrolizumab with tumor response and survival among patients with advanced melanoma. J. Am. Med. Assoc. 315, 1600–1609 (2016).
Sharma, P., Hu-Lieskovan, S., Wargo, J. A. & Ribas, A. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell 168, 707–723 (2017).
Marin-Acevedo, J. A. et al. Next generation of immune checkpoint therapy in cancer: new developments and challenges. J. Hematol. Oncol. 11, 39 (2018).
Li, C.-W. et al. Eradication of triple-negative breast cancer cells by targeting glycosylated PD-L1. Cancer Cell 33, 187–201 (2018).
Boligan, K. F., Mesa, C., Fernandez, L. E. & von Gunten, S. Cancer intelligence acquired (CIA): tumor glycosylation and sialylation codes dismantling antitumor defense. Cell. Mol. Life Sci. 72, 1231–1248 (2015).
Varki, A., Kannagi, R., Toole, B. & Stanley, P. in Essentials of Glycobiology (Cold Spring Harbor Laboratory Press, 2017).
Cohen, M. et al. Sialylation of 3-methylcholanthrene–induced fibrosarcoma determines antitumor immune responses during immunoediting. J. Immunol. 185, 5869–5878 (2010).
Perdicchio, M. et al. Tumor sialylation impedes T cell mediated anti-tumor responses while promoting tumor associated-regulatory T cells. Oncotarget 7, 8771–8782 (2016).
Stanczak, M. A. et al. Self-associated molecular patterns mediate cancer immune evasion by engaging Siglecs on T cells. J. Clin. Invest. 128, 4912–4923 (2018).
Varki, A. & Gagneux, P. Multifarious roles of sialic acids in immunity. Ann. NY Acad. Sci. 1253, 16–36 (2012).
Swindall, A. F. & Bellis, S. L. Sialylation of the Fas death receptor by ST6Gal-I provides protection against Fas-mediated apoptosis in colon carcinoma cells. J. Biol. Chem. 286, 22982–22990 (2011).
Feng, M. et al. Programmed cell removal by calreticulin in tissue homeostasis and cancer. Nat. Commun. 9, 3194 (2018).
Lübbers, J., Rodríguez, E. & van Kooyk, Y. Modulation of immune tolerance via Siglec-Sialic acid interactions. Front. Immunol. 9, 2807 (2018).
Macauley, M. S., Crocker, P. R. & Paulson, J. C. Siglec-mediated regulation of immune cell function in disease. Nat. Rev. Immunol. 14, 653–666 (2014).
Paulson, J. C., Macauley, M. S. & Kawasaki, N. Siglecs as sensors of self in innate and adaptive immune responses. Ann. NY Acad. Sci. 1253, 37–48 (2012).
Riley, J. L. PD-1 signaling in primary T cells. Immunol. Rev. 229, 114–125 (2009).
Chemnitz, J. M., Parry, R. V., Nichols, K. E., June, C. H. & Riley, J. L. SHP-1 and SHP-2 associate with immunoreceptor tyrosine-based switch motif of programmed death 1 upon primary human T cell stimulation, but only receptor ligation prevents T cell activation. J. Immunol. 173, 945–954 (2004).
Läubli, H. et al. Engagement of myelomonocytic Siglecs by tumor-associated ligands modulates the innate immune response to cancer. Proc. Natl Acad. Sci. USA 111, 14211–14216 (2014).
Hudak, J. E., Canham, S. M. & Bertozzi, C. R. Glycocalyx engineering reveals a Siglec-based mechanism for NK cell immunoevasion. Nat. Chem. Biol. 10, 69–75 (2014).
Büll, C. et al. Sialic acid blockade suppresses tumor growth by enhancing T-cell-mediated tumor immunity. Cancer Res. 78, 3574–3588 (2018).
Xiao, H., Woods, E. C., Vukojicic, P. & Bertozzi, C. R. Precision glycocalyx editing as a strategy for cancer immunotherapy. Proc. Natl Acad. Sci. USA 113, 10304–10309 (2016).
Thobhani, S., Ember, B., Siriwardena, A. & Boons, G.-J. Multivalency and the mode of action of bacterial sialidases. J. Am. Chem. Soc. 125, 7154–7155 (2003).
Watson, J. N. et al. Use of conformationally restricted pyridinium-d-N-acetylneuraminides to probe specificity in bacterial and viral sialidases. Biochem. Cell Biol. 83, 115–122 (2005).
Minami, A. et al. Catalytic preference of Salmonella typhimurium LT2 sialidase for N-acetylneuraminic acid residues over N-glycolylneuraminic acid residues. FEBS Open Bio. 3, 231–236 (2013).
Shepard, H. M., Jin, P., Slamon, D. J., Pirot, Z. & Maneval, D. C. in Therapeutic Antibodies (eds. Chernajovsky, Y. & Nissim, A.) 183–219 (Springer, 2008).
Agarwal, P., van der Weijden, J., Sletten, E. M., Rabuka, D. & Bertozzi, C. R. A Pictet–Spengler ligation for protein chemical modification. Proc. Natl Acad. Sci. USA 110, 46–51 (2013).
Agarwal, P. et al. Hydrazino-Pictet–Spengler ligation as a biocompatible method for the generation of stable protein conjugates. Bioconjug. Chem. 24, 846–851 (2013).
Barfield, R. M. & Rabuka, D. Noncanonical Amino Acids. (ed. Lemke, E. A.) 3–16 (Humana Press, 2018).
Drake, P. M. et al. Aldehyde tag coupled with HIPS chemistry enables the production of ADCs conjugated site-specifically to different antibody regions with distinct in vivo efficacy and PK outcomes. Bioconjug. Chem. 25, 1331–1341 (2014).
Maclaren, A., Levin, N., Lowman, H. & Trikha, M. Trph-222, a novel anti-CD22 antibody drug conjugate (ADC), has significant anti-tumor activity in NHL xenografts and is well tolerated in non-human primates. Blood 130, 4105 (2017).
Baskin, J. M. & Bertozzi, C. R. Copper-free click chemistry: bioorthogonal reagents for tagging azides. Aldrichimica Acta 43, 15–23 (2010).
Rockwell, S. C., Kallman, R. F. & Fajardo, L. F. Characteristics of a serially transplanted mouse mammary tumor and its tissue-culture-adapted derivative. J. Natl Cancer Inst. 49, 735–749 (1972).
Amico, L. et al. A novel anti-HER2 anthracycline-based antibody-drug conjugate induces adaptive anti-tumor immunity and potentiates PD-1 blockade in breast cancer. J. Immunother. Cancer 7, 16 (2019).
Wu, H. et al. Development of motavizumab, an ultra-potent antibody for the prevention of respiratory syncytial virus infection in the upper and lower respiratory tract. J. Mol. Biol. 368, 652–665 (2007).
Kelly, R. L. et al. High throughput cross-interaction measures for human IgG1 antibodies correlate with clearance rates in mice. MAbs 7, 770–777 (2015).
Armour, K. L., Clark, M. R., Hadley, A. G. & Williamson, L. M. Recombinant human IgG molecules lacking Fcγ receptor I binding and monocyte triggering activities. Eur. J. Immunol. 29, 2613–2624 (1999).
Shields, R. L. et al. High resolution mapping of the binding site on human IgG1 for FcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants with improved binding to the FcγR. J. Biol. Chem. 276, 6591–6604 (2001).
Li, J. et al. Desialylation is a mechanism of Fc-independent platelet clearance and a therapeutic target in immune thrombocytopenia. Nat. Commun. 6, 7737 (2015).
Tribulatti, M. V., Mucci, J., van Rooijen, N., Leguizamón, M. S. & Campetella, O. The trans-sialidase from Trypanosoma cruzi induces thrombocytopenia during acute Chagas’ disease by reducing the platelet sialic acid contents. Infect. Immun. 73, 201–207 (2005).
Assi, H., Ibrahimi, S., Machiorlatti, M., Vesely, S. K. & Asch, A. S. Thrombocytopenia is a biomarker for response in patients treated with anti PD-1/PDL-1 therapy. Blood 132, 1138 (2018).
Peng, G.-L. et al. CD8+ cytotoxic and FoxP3+ regulatory T lymphocytes serve as prognostic factors in breast cancer. Am. J. Transl. Res. 11, 5039–5053 (2019).
Laoui, D. et al. Tumor-associated macrophages in breast cancer: distinct subsets, distinct functions. Int. J. Dev. Biol. 55, 861–867 (2011).
Uchiyama, S. et al. Dual actions of group B Streptococcus capsular sialic acid provide resistance to platelet-mediated antimicrobial killing. Proc. Natl Acad. Sci. USA 116, 7465–7470 (2019).
Sedlacek, H. H. & Seiler, F. R. Immunotherapy of neoplastic diseases with neuraminidase: contradictions, new aspects, and revised concepts. Cancer Immunol. Immunother. 5, 153–163 (1978).
Barkal, A. A. et al. CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy. Nature 572, 392–396 (2019).
Haas, Q. et al. Siglec-9 regulates an effector memory CD8þ T-cell subset that congregates in the melanoma tumor microenvironment. Cancer Immunol. Res. 7, 707–718 (2019).
Jandus, C. et al. Interactions between Siglec-7/9 receptors and ligands influence NK cell–dependent tumor immunosurveillance. J. Clin. Invest. 124, 1810–1820 (2014).
Wang, J. et al. Siglec-15 as an immune suppressor and potential target for normalization cancer immunotherapy. Nat. Med. 25, 656–666 (2019).
Tsui, C. K. et al. CRISPR-Cas9 screens identify regulators of antibody–drug conjugate toxicity. Nat. Chem. Biol. 15, 949–958 (2019).
Christensen, S. & Egebjerg, J. Cloning, expression and characterization of a sialidase gene from Arthrobacter ureafaciens. Biotechnol. Appl. Biochem. 41, 225–231 (2005).
Vimr, E. R., Lawrisuk, L., Galen, J. & Kaper, J. B. Cloning and expression of the Vibrio cholerae neuraminidase gene nanH in Escherichia coli. J. Bacteriol. 170, 1495–1504 (1988).
Barouch, D. H. et al. A human T-cell leukemia virus type 1 regulatory element enhances the immunogenicity of human immunodeficiency virus type 1 DNA vaccines in mice and nonhuman primates. J. Virol. 79, 8828–8834 (2005).
Angata, T., Nycholat, C. M. & Macauley, M. S. Therapeutic targeting of Siglecs using antibody- and glycan-based approaches. Trends Pharmacol. Sci. 36, 645–660 (2015).
Ikehara, Y., Ikehara, S. K. & Paulson, J. C. Negative regulation of T cell receptor signaling by Siglec-7 (p70/AIRM) and Siglec-9. J. Biol. Chem. 279, 43117–43125 (2004).
Crespo, H. J., Lau, J. T. Y. & Videira, P. A. Dendritic cells: a spot on sialic acid. Front. Immunol. 4, 491 (2013).
Munday, J., Floyd, H. & Crocker, P. R. Sialic acid binding receptors (Siglecs) expressed by macrophages. J. Leukoc. Biol. 66, 705–711 (1999).
von Gunten, S. & Bochner, B. S. Basic and clinical immunology of Siglecs. Ann. NY Acad. Sci. 1143, 61–82 (2008).
Nguyen, K. A. et al. Role of Siglec-7 in apoptosis in human platelets. PLoS ONE 9, e106239 (2014).
Toubai, T. et al. Siglec-G represses DAMP-mediated effects on T cells. JCI insight 2, e92293 (2017).
We thank S. Banik, C.J. Cambier and S. Wisnovsky for critical reading of this manuscript, T. McLaughlin and the Stanford University Mass Spectrometry facility for intact protein characterization and high-resolution mass spectrometry analysis and the Stanford Shared FACS Facility for use of flow cytometry instruments. We are grateful to A. Zippelius (University of Basel) for providing the HER2+ EMT6 cell line, D. Rabuka (Catalent Pharma Solutions) for providing SMARTag trastuzumab, M. Appel (Stanford University) for generating the pET28-MBP-tev-tb-FGE plasmid, as well as C.C. Angelakos for assistance with and usage of the Leica cryostat. We thank S. Christensen (University of Copenhagen) for the AU54pET9d* plasmid, E. Vimr (University of Illinois Urbana-Champaign) for the plasmid pCVD364 and J. Kohler (UT Southwestern) for the pGEX-Neu2 construct. This work was supported in part by the Goldschmidt-Jacobson Foundation (H.L.), the Promedica Foundation (M.A.S) and a Swiss National Science Foundation grant (no. 310030_184720/1) (H.L.), as well as a grant from the National Institutes of Health (no. NIH CA227942 to C.R.B.) Researchers were also supported by the National Science Foundation Graduate Research Fellowship (M.A.G., G.A. and P.A.W.) and the Stanford ChEM-H Chemistry/Biology Interface Predoctoral Training Program (M.A.G. and P.A.W.) as well as the Swiss Government Excellence Scholarship for Foreign Scholars and Artists (N.R.M.), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (N.R.M), an NIH F32 Postdoctoral Fellowship (S.A.M.), the Stanford Undergraduate Summer Research Program in Chemistry funded through Stanford VPUE/UAR (J.T.T.) and the US National Institutes of Health Predoctoral Fellowship F31CA200544 (E.C.W.).
M.A.G., H.X., E.C.W. and C.R.B. are inventors of the patent filed by Stanford University (international publication number WO2018006034A1) titled ‘Conjugates for targeted cell-surface editing’ published on January 4, 2018 and licensed by Palleon Pharmaceuticals on 06/27/2017. C.R.B. is a cofounder and Scientific Advisory Board member of Palleon Pharmaceuticals, Enable Bioscience, Redwood Biosciences (a subsidiary of Catalent) and InterVenn Biosciences, and a member of the Board of Directors of Eli Lilly & Company. H.L. received research and traveling support from Bristol-Myers Squibb. H.L. received traveling support from Merck Sharp Dome and Roche. H.L. is a member of the Scientific Advisory Board of Palleon Pharmaceuticals.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 The PD-1 receptor and eight members of the human Siglec family have a homologous ITIM-ITSM cytosolic sequence motif and are expressed on a variety of immune cells.
Both PD-1 and Siglecs contain an N-terminal binding domain to their ligands (PD-L1/2 and sialoglycans, respectively), a helical single pass through the cell membrane, and an 82-126 amino acid cytosolic C-terminus containing an ITIM motif, a 12-18 amino acid spacer, and an ITSM or ITSM-like motif. Siglecs are expressed on a variety of immune cells; cell types that are Siglec+ or have Siglec+ subsets reported in humans and mice are indicated above for each Siglec receptor. T = T cell, B = B cell, MDSCs = myeloid-derived suppressor cells, My = Myeloid-precursor, Mon = Monocyte, Mi = Microglia, Neu = Neutrophil, Mac = Macrophage, Troph = Trophoblast, NK = Natural killer cell, Eos = Eosinophil, Bas = Basophil, DC=Dendritic cell, Mast = Mast cell, Pt = platelet. Siglec protein sequences are from Uniprot with added cell expression information from multiple sources10,14,15,46,54,55,56,57,58,59,60.
Extended Data Fig. 2 ST sialidase is a small, stable sialidase that enhances NK cell-mediated ADCC towards breast cancer cells and cleaves Siglec-7 and -9 ligands at high concentrations.
a, PAGE-SDS reducing gel of six recombinantly expressed and purified sialidases: Neu2, Neu3, Vibrio cholerae sialidase, Salmonella typhimurium NanH, Clostridium perfringens NanH, Arthrobacter ureafaciens NanH b, Molecular weights and specific activities (in μmol substrate converted per min per mg enzyme) of sialidases determined by in vitro activity assays with the fluorogenic substrate 4-MUNANA (n = 3 experimental replicates; mean ± SD). c, IL2-activated NK cell-mediated ADCC assay against target BT-20 breast cancer cells treated with 10 nM trastuzumab (Tras) and 2 μM sialidases at E/T = 4, percent cytotoxicity was detected by LDH release after 4 h. Figure shows mean ± SD from n = 3 experimental replicates against the same biological NK cell donor. Statistical analysis by one way ANOVA with Tukey’s multiple comparisons adjusted p-values. d, In vitro activity assay of freshly expressed sialidase compared with sialidase stored in PBS at 4 °C for 2 years reveals that significant ST sialidase activity is preserved (n = 3 experimental replicates fit to a linear regression ± SD). e-g, Siglec ligand depletion by ST sialidase cleavage is effective across many breast cancer cell lines. ST sialidase (2 μM) was incubated with nine different breast cancer cell lines for 1 h and the removal of Siglec ligands compared to PBS-treated cells was assessed by staining with Siglec-9-Fc (e) or Siglec-7-Fc (f) and anti-human-488 secondary antibody (control: secondary only). Gating is shown on the right, first selecting the cell population (FSC-A/SSC), then gating on single cells (FSC-A/FSC-H). Representative images from n = 2 experimental replicates of the n = 9 biological cell line replicates each with >2,000 cells are shown. The mean and median (± SD) percent decrease in Siglec ligand-binding fluorescent signal upon ST treatment of the n = 9 cell lines are quantified in g.
Extended Data Fig. 3 T-Sia 2 enhances NK cell-mediated and gamma-delta T cell-mediated ADCC in vitro.
a, Representative flow cytometry plots assessing purity of isolated primary NK cells. For n = 3 independent biological replicates of NK cell isolation, cells were >95% CD3− and had both CD16high and CD16low populations. b, NK cell-surface expression of some sialic acid-binding receptors from a representative NK donor. Isolated NK cells in all n = 3 biological replicates were positive for Siglec-7, had mixed expression of Siglec-9, and had low-to-moderate unimodal expression of NKG2D. c, NK cell-mediated ADCC of BT-20 target cells treated with PBS, 20 nM free ST sialidase, 10 nM trastuzumab, or 10 nM T-Sia 2 (mean ± SD). Plots from BT-20 cells alone (left) and from incubation with NK cells are each from n = 3 experimental replicates, E/T = 4, no IL-2 activation, detecting LDH release after 4 h; statistical analysis by two-tailed t tests. d, Plotting the NK cell-mediated cytotoxicity of trastuzumab and T-Sia 2-treated cells from (c) shows that desialylation enhances ADCC significantly across the n = 3 biological NK cell donor replicates. Mean ± SD, analyzed by a paired two-tailed t test. e, Representative gating showing the purity of isolated primary γδ T cells. Consistent with n = 3 biological replicates, γδ T Cells are somewhat impure (only ~85% are γδ+ and CD3+) and there are apparent sub-populations. f, A representative histogram (from n = 3 biological replicates) of a typical donor demonstrates that these cells are negative for Siglec-7 and -9 expression, although they also have a moderate unimodal expression of NKG2D. g, Mean ± SD from γδ T cell-mediated ADCC assays targeting HER2+ EMT6 cells treated with PBS, 20 nM free ST sialidase, 10 nM trastuzumab, or 10 nM T-Sia 2. Results are shown from n = 3 independent experimental replicates on EMT6 cells alone (left) and from incubation with IL-2 activated γδ T cells isolated from n = 3 distinct biological donors, E/T = 1, detecting LDH release after 8 h; statistical analysis by two-tailed t tests. h, Comparing trastuzumab vs T-Sia 2 treatment across n = 3 biological NK donors from (g), shows significant enhancement of cytotoxicity by desialylation. Mean ± SD, statistical analysis by a paired two-tailed t test. i, An IL-2 activated γδ T cell-mediated ADCC assay targeting SK-BR-3 cells incubated with PBS, trastuzumab (10 nM) or T-Sia 2 (10 nM), (n = 3), E/T = 3, detecting LDH release after 6 h; (Mean ± SD), statistical analysis by two-tailed t test.
Extended Data Fig. 4 T-Sia 2 treatment desialylates EMT6 tumors and delays tumor growth compared to trastuzumab- or PBS-treatment.
a, Mean ± SD tumor volume over time for HER2+ EMT6 tumor-bearing mice described in Fig. 4b, treated with PBS (n = 5), trastuzumab (10 mg/kg injection, n = 6), T-Sia 2 (10 mg/kg injection, n = 6), and T-Sia 2 (15 mg/kg injection, n = 6). RM two-way ANOVA with adjusted p-values from Tukey’s multiple comparisons between groups shown. b, Mouse weight from all of the mice in (a) measured 5x over the course of 24 days of treatment and tumor growth. Ordinary two-way ANOVA with Dunnet’s multiple comparisons to PBS mice, multiplicity-adjusted p-values for T-Sia (15 mg/kg) compared to PBS are shown at three time points, (mean ± SD). c, Lectin flow cytometry staining with fluorescent PNA to detect exposed galactose of extracted tumor cells from mice in Fig. 4b upon their sacrifice 13–23 days after final administration of PBS, trastuzumab, or T-Sia 2, (geometric mean (gMFI) ± SD), normalized to ConA fluorescence (a control that binds to mannose on cells), n = 4 (trastuzumab, PBS) n = 9 (T-Sia 2). Ordinary one-way ANOVA with adjusted p-values shown from Tukey’s multiple comparisons.
Extended Data Fig. 5 T-Sia 2 accumulates in the HER2+ EMT6 tumors in mice and can desialylate tumor cells at a single injection concentration as low as 0.2 mg/kg.
Mice bearing HER2+ EMT6 subcutaneous tumors on their left flanks were imaged using non-invasive in vivo fluorescence imaging at a, 48 h, and b, 4 days after IR800-labeled T-Sia 2 was injected IP into n = 3 mice per dose: 500 pmol, 100 pmol, 20 pmol, and PBS control. All mice are shown above. c, Representative photographs overlaid with fluorescent images of the tumor, spleen, heart, liver, and kidneys removed 4 days after conjugate injection, showing where fluorescently-labeled T-Sia 2 was localized. These images are representative of n = 3 biological mouse replicates. d, Maximum radiant efficiency was quantified for the n = 3 replicate mice per group and normalized to the average of the PBS mouse organ control. Mean ± SD are displayed with p values from a two-way ANOVA with Dunnet’s multiple comparisons to the respective control organ of mice injected with PBS. e, N = 3 tumors taken from each mouse group were pooled, resuspended, and single tumor cells were analyzed for lectin staining by flow cytometry. Although the SNA ligands in vivo were insensitive to T-Sia 2 desialylation at these doses, dramatic desialylation was apparent by detecting with the more sensitive reagents PNA (detecting exposed galactosyl (β-1,3) N-acetylgalactosamine) and MALII (detecting α2,3 linked sialic acids). The x-axis is displayed on a biexponential scale, y-axis is cell count normalized to mode. For MALII, SNA, and PNA staining n > 2,600 cells for all histograms except the 500 pmol T-Sia 2-dosed-mice histograms only represent n = 827 and n = 1,809, and n = 234 cells, respectively, as the treated tumors were very small. These tumor desialylation results are from one independent mouse experiment with n = 3 biological replicates (mouse tumors); and are consistent with other experiments in this work showing mouse tumor desialylation with SNA, PNA, and MAL II (Fig. 5c, and Extended Data Figs. 4c, 6, and 7a). Color bars are in units of radiant efficiency.
Extended Data Fig. 6 Representative lectin immunohistochemistry of tumor and organ slices show desialylation that can be detected in all examined organs.
Sialoglycan degradation is detectable by fluorescence microscopy of stained tumor and organ slices from the mice in Extended Data Fig. 5 treated with 500 pmol T-Sia 2. Staining with Maackia Amurensis Lectin II (MALII, top images), which preferentially binds sialic acid in an (α-2,3) linkage, reveals a dramatic decrease in fluorescent signal from binding (α-2,3) sialic acid in the tumor, comparable to positive control tissue slices treated with a sialidase cocktail ex. vivo. In the heart, kidney, and spleen, a moderate decrease in fluorescence can also be perceived. Staining with Peanut Agglutinin (PNA, middle images), which binds preferentially to exposed galactose upon desialylation, is increased in the tumor tissue upon desialylation, this effect is also quite apparent in the heart, spleen, and liver, kidney. Staining with Sambucus nigra (Elderberry Bark) lectin (SNA), which preferentially binds sialic acid in an (α-2,6) linkage, was not performed on the T-Sia 2 samples as there was consistently little to no difference between tumor staining in the PBS control mice and in tumor slices treated ex vivo with a high concentration of ST and VC sialidase, indicating that SNA signal in the tumor tissue in the mouse is either background or represents signal from glycans that are not readily cleavable by ST or VC sialidase, this finding is in agreement with tumor lectin staining by flow cytometry (Extended Data Figs. 5 and 7). All images are representative of at least n = 3 organ images per mouse taken from n = 3 different mice injected with 500 pmol T-Sia 2.
For flow cytometry analysis of sialoglycan degradation, a single 20 pmol injection of T-Sia 2, Isotype-Sia, or PBS control was injected IP into mice with subcutaneous HER2+ EMT6 cells. After four days, tumors were harvested. a, Staining with Sambucus nigra (Elderberry Bark) lectin (SNA), which preferentially binds sialic acid in an (α-2,6) linkage, did not show significant desialylation changes between these samples, n = 3 mice, geometric mean ± SD. Right: gating strategy: dead cells and tumor debris were gated out (V450-A-/SSC-A), followed by gating on size (FSC-A/SSC-A - *blue cells depict where HER2+ stained cells fall in the gate). Finally, single cells are gated with FSC-A/FSC-H. b, Growth curves of the HER2+ EMT6 tumors of mice treated with T-Sia 2 (10 mg/kg) were consistent between the first tumor growth mouse experiment (shown in red, depicted in Fig. 4c, n = 6 mice) and the second independent experiment (blue, depicted in Fig. 5d, n = 7 mice); data are reported as mean ± SD. c. Average (mean + SD) tumor growth of all the mice in Fig. 5d plotted together until the first mouse death (day 19). Mice were injected IP with PBS (n = 6), 10 mg/kg trastuzumab (tras) (n = 6), T-Sia-LOF (n = 6), Isotype-Sia (n = 6), T-FcX-Sia (n = 7), or T-Sia 2 (n = 7). A two-way ANOVA was performed with adjusted p-values from Tukey’s multiple comparisons. d, Lectin stain with PNA of exposed galactose from extracted tumor cells taken from the mice in Fig. 5d, e after mouse sacrifice. Geometric mean ± SD of PNA/ConA, (n = 5: PBS, Tras, T-Sia-LOF, T-FcX-Sia; n = 6: Isotype-Sia).
HER2+ B16D5 cells cultured in vitro bound to commercially available mouse Siglec-Fc proteins (Siglecs -E and -F-Fc). The Siglec ligands detected on the B16D5 cells were sensitive to 2 μM ST sialidase treatment for 1 h. y-axis shows cell count normalized to mode, x-axis is on a bi-exponential scale, >13,500 cells per histogram. PBS control-treated cells are shown in outlined dark blue with, ST sialidase-treated cells are colored teal, secondary only control stains are shown in gray. Gating is shown below, cells were selected by SSC-A/FSC-A gating, followed by selection of single cells by linear gating on FSC-A vs FSC-H. Data are representative of n = 3 independently performed experiments on HER2+ B16D5 cells.
Extended Data Fig. 9 HER2+ B16D5 tumor growth curves of wt and Sig-E-/- C57BL/6 mice treated with T-Sia 2 or the loss of function T-Sia-LOF.
a, Tumor growth curves of (left): wt mouse experiment 1 with n = 6 mice per group, and (right): wt mouse experiment 2 with n = 5 mice per group. b, Average tumor growth of wt and Sig-E-/- mice treated with either T-Sia 2 or T-Sia-LOF inactive control. Data shown are mean + SD until the date of the first mouse death (day 15), analyzed by RM two way ANOVA with from Tukey’s multiple comparisons test. Sample sizes are n = 11 for wt mice (T-Sia 2 and T-Sia-LOF) and n = 8 for Sig-E-/- mice (T-Sia 2 and T-Sia-LOF) c, Individual tumor growth curves with Siglec-E deficient mice (left): mouse experiment 1 (n = 6 mice per group), and (right): experiment 2 (n = 2 mice per group). d, Survival curves based on euthanasia criteria with HER2+ B16D5 tumors from Fig. 6a,b demonstrate that knocking out Siglec-E in this mouse tumor model benefits mouse survival. For T-Sia LOF-treated mice, the Sig-E-/- mice have prolonged survival compared to wt mice. For T-Sia 2−treated mice, sialoglycan degradation of the wt mice extends the survival to an outcome similar to the Sig-E-/- mice. P-values are shown from a log-rank (Mantel-Cox) test, n = 11 wt mice treated with T-Sia 2 or T-Sia-LOF, n = 8 for Sig-E-/- treated with T-Sia 2 or T-Sia-LOF.
Extended Data Fig. 10 B16D5 tumors in C57Bl/6 mice have significant infiltration of Siglec-E+ immune cells, which can largely be attributed to Siglec-E expressing CD11b+ leukocytes.
Representative histograms of tumor infiltrating leukocytes gated as shown in Supplementary Fig. 22, with Siglec-E expression on the biexponential x-axis and cell count normalized to mode (n = 10 for wt mice, n = 12 for Siglec-E deficient mice). A fluorescence minus one control (FMO, negative control that lacks the Siglec-E antibody) for each sample is shown as a black outline. Total leukocytes (defined as CD45+ cells) show a detectable increased Siglec-E expression in wild-type mice (blue) compared to Siglec-E-/- mouse controls (red). Investigating further, T cells, B cells, and NK cells (represented by CD3+, CD19+, and NK1.1+ leukocyte populations did not show significant Siglec-E expression compared to Sig-E-/- control cells, however, myeloid (CD11b+) cells, which include macrophages, granulocytes, certain dendritic cells, and monocytes, had significant anti Siglec-E signal and represent the vast majority of Siglec-E+ leukocytes in these tumors.
Statistical source data
Statistical source data
Statistical source data
Statistical source data
Statistical source data
Statistical source data
Statistical source data
Statistical source data
Statistical source data
Statistical source data
Statistical source data
About this article
Cite this article
Gray, M.A., Stanczak, M.A., Mantuano, N.R. et al. Targeted glycan degradation potentiates the anticancer immune response in vivo. Nat Chem Biol 16, 1376–1384 (2020). https://doi.org/10.1038/s41589-020-0622-x
This article is cited by
Nature Communications (2023)
Nature Chemistry (2023)
Structural insights into Siglec-15 reveal glycosylation dependency for its interaction with T cells through integrin CD11b
Nature Communications (2023)
Nature Biotechnology (2023)
Whole-cell tumor vaccines desialylated to uncover tumor antigenic Gal/GalNAc epitopes elicit anti-tumor immunity
Journal of Translational Medicine (2022)