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. 2020 Jun 6;23(6):101250. doi: 10.1016/j.isci.2020.101250

A Structurally Simple Vaccine Candidate Reduces Progression and Dissemination of Triple-Negative Breast Cancer

Amedeo Amedei 1, Fatemeh Asadzadeh 2,3, Francesco Papi 4, Maria Giuliana Vannucchi 1, Veronica Ferrucci 2,3, Iris A Bermejo 5, Marco Fragai 4,6, Carolina Vieira De Almeida 1, Linda Cerofolini 6,7, Stefano Giuntini 4,5, Mauro Bombaci 8, Elisa Pesce 8, Elena Niccolai 1, Francesca Natali 9, Eleonora Guarini 10, Frank Gabel 11, Chiara Traini 1, Stefano Catarinicchia 1, Federica Ricci 1, Lorenzo Orzalesi 1, Francesco Berti 12, Francisco Corzana 6, Massimo Zollo 2,3,∗, Renata Grifantini 8,∗∗, Cristina Nativi 4,13,∗∗∗
PMCID: PMC7322362  PMID: 32629615

Summary

The Tn antigen is a well-known tumor-associated carbohydrate determinant, often incorporated in glycopeptides to develop cancer vaccines. Herein, four copies of a conformationally constrained mimetic of the antigen TnThr (GalNAc-Thr) were conjugated to the adjuvant CRM197, a protein licensed for human use. The resulting vaccine candidate, mime[4]CRM elicited a robust immune response in a triple-negative breast cancer mouse model, correlated with high frequency of CD4+ T cells and low frequency of M2-type macrophages, which reduces tumor progression and lung metastasis growth. Mime[4]CRM-mediated activation of human dendritic cells is reported, and the proliferation of mime[4]CRM-specific T cells, in cancer tissue and peripheral blood of patients with breast cancer, is demonstrated. The locked conformation of the TnThr mimetic and a proper presentation on the surface of CRM197 may explain the binding of the conjugate to the anti-Tn antibody Tn218 and its efficacy to fight cancer cells in mice.

Subject Areas: Medical Biochemistry, Immunology, Cancer

Graphical Abstract

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Highlights

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    Structurally simple vaccine candidate reduces BC tumor size and delays lung metastasis

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    TnThr mimetic, fused to CRM197 adjuvant, is able to elicit T and B immune responses

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    TnThr mimetic-based vaccine candidate able to activate human DCs

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    The vaccine candidate recruits T helper CD4+ in the tumor microenvironment

Medical Biochemistry; Immunology; Cancer

Introduction

Cancer immunotherapy is nowadays a consolidated strategy, and therapeutic cancer vaccines are a matter of vast research (Bray and Soerjomataram, 2015). The use of cancer vaccines is an active, specific immunotherapy strategy to present non-self-antigens to the immune system, eliciting an antitumor immune response and leading to tumor cells' killing (Floudas et al., 2019). Cell surface-exposed carbohydrates deserve special attention, because several cancer cells can be differentiated from normal cells by the presentation on their surface of non-common glycosylation motifs (tumor-associated carbohydrate antigens [TACAs]) (Wei et al., 2018). In fact, although poorly immunogenic and usually T cell independent, carbohydrate antigens, if properly presented to the immune system, can be recognized as non-self and induce an effective immune response (Buskas et al., 2004, Danishefsky et al., 2015, Gilewski et al., 2001, Helling et al., 1995). Indeed, it has been reported about some patients with cancer able to induce natural auto-antibodies directed against native TACAs and with an improved survival rate (Livingston et al., 1994, Osinaga et al., 1996).

In the last two decades great attention has been caught by the MUC1 antigen, α-Tn (or Tn, Figure 1). The Tn is a cryptic antigen masked in normal human cells but exposed in more than 90% primary adenocarcinomas, due to the incomplete synthesis of the oligosaccharide portions, which are physiologically O-glycosylated to the MUC1 backbone. Tn antigen has been detected at early tumor stage, and its expression is associated with tumor invasiveness and metastasis (Itzkowitz et al., 1992). In breast cancer (BC) a correlation among the high level of Tn antigen expression in the primary tumor, tumor size, and a poor prognosis is known, since 20 years (Springer, 1997, Tsuchiya et al., 1999). Worldwide, BC is the most common cancer in women (20% of all cases). Among the different BC kinds, the triple-negative BC (TNBC) is the most difficult to treat for it is very aggressive, prone to metastasize, and resistant to the current anti-BC therapies (Koboldt et al., 2012).

Figure 1.

Figure 1

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Structure of Tn Antigen, TnSer, TnThr, and TnThr Mimetic, 1

Structurally simple, the Tn determinant (α-O-GalNAc, linked to the MUC-1 peptide through a residue of Ser or Thr) has been the TACA selected for the development of fully or semi-synthetic cancer vaccines (Cai et al., 2012, Lakshminarayanan et al., 2012, Renaudet et al., 2008, Yin et al., 2017). Recently, relevant insights have been reported on the non-equivalence of Ser versus Thr MUC1 O-glycosylation residue (TnSer versus TnThr, Figure 1) (Grinstead et al., 2002). Noteworthy, TnThr is rather rigid in solution when compared with the more flexible TnSer; consequently in TnThr the sugar portion assumes a different spatial arrangement compared with TnSer (Mazal et al., 2013, Brister et al., 2014, Martínez-Sáez et al., 2015, Martínez-Sáez et al., 2017). This difference is mirrored in their bond state with a lower binding affinity of TnSer with respect to TnThr for anti-MUC1 SM3 antibody. Taking advantage from these findings, non-natural determinants have been proposed to obtain more antigenic structures (Compañón et al., 2019, Fernández et al., 2016, Somovilla et al., 2017). About that, few years ago we proposed a “locked” mimetic of the TnThr antigen, namely, 1 (Figure 1), which is stable and able to elicit a specific immune response in vivo (Richichi et al., 2014). In this study, we report on the synthesis, characterization, and immunological evaluation of CRM197 (Cross Reactive Material 197) glycoconjugates presenting residues of mimetic 1, as candidate vaccine to treat non-responsive TNBC.

CRM197 decorated with only four residues of 1, named mime[4]CRM, is able to properly activate human dendritic cells (DCs) and, of note, the administration of mime[4]CRM to a TNBC animal model not only produced tumor size reduction but also interfered in the lung metastasis' development. To the best of our knowledge, although specific antibodies or immune checkpoint inhibitors (passive immunotherapy) against TNBC have been approved or are in clinical trial (Force et al., 2019), no example of cancer vaccine for the active immunotherapy of TNBC is currently approved or under advanced clinical development (Vikas et al., 2018, Zeichner, 2012). In this panorama, the results herein reported represent a novelty in the non-native TACA-based vaccines research.

Results and Discussion

Synthesis and Characterization of Compounds 2, 3, and Mime[4]CRM

In the design of glycoconjugate vaccines, important issues shall be taken into account, in particular (1) due to the typical weak binding interactions between lectins (i.e., macrophage galactose lectins [MGLs], Dectin-1, or DC-SIGN on DCs) and single glycans, a multivalent presentation of individual carbohydrate antigens linked to carriers (generally immunogenic proteins, peptides or synthetic scaffolds) is assembled to augment the binding interaction and trigger a robust recognition event and (2) to elicit TACA-specific IgG antibodies, vaccine constructs also include Toll-like ligands (Toll-like receptor) (Li and Guo, 2018, Toyokuni et al., 1994, van Duin et al., 2006) or a T helper peptide, as internal adjuvant (Renaudet et al., 2008). The outcome is the assembly of demanding constructs presenting immunodominant protein carriers, which often fail in inducing TACA-specific antibodies, eliciting undesired auto-immunity. In keeping these issues and capitalizing on the encouraging results obtained with TnThr mimetic 1 (Fallarini et al., 2017, Gracia et al., 2018, Manuelli et al., 2014, Richichi et al., 2014), we synthesized the differently activated derivatives 2 and 3, from 4 as starting material (Scheme 1), to decorate the clinically validated carrier-adjuvant protein CRM197 under mild conditions. The acetyl derivative 4, obtained as reported (Ardá et al., 2015), was reacted with the mono Boc-protected 1,6-diaminohexane, in the presence of 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate and N-methylmorpholine (NMM), in dimethylformamide (DMF), to afford derivative 5 (85%). The acetyl protecting groups were removed by treating 5 with a solution of ammonia in methanol (6, >90%), whereas the Boc protecting group was cleaved with trifluoroacetic acid. The trifluoroacetic salt 7 was then reacted with para-nitrophenyl adipates and NMM in DMF to afford compound 2 (88%, over two steps) (Scheme 1). The reaction of the activated para-nitrophenyl derivative 2 with N-hydroxysuccinimide produced, under mild conditions, the corresponding derivative 3 (40%) (Scheme 1). More efficiently, compound 3 was also prepared by reacting 7 at room temperature with adipic acid activated as bis-succinimide and NMM, in DMF (51%, over two steps).

Scheme 1.

Scheme 1

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Synthesis of Activated Derivatives 2 and 3

CRM197 is a genetically detoxified mutant of diphtheria toxin, obtained by a single amino acid substitution (G52E) (Malito et al., 2012) and characterized by a lower immunogenicity and immunostimulatory effect compared with diphtheria toxin (Bröker et al., 2011). Currently, CRM197 is one of the most effective carrier proteins, largely found in human conjugate vaccines, licensed to treat bacterial infections (Pecetta et al., 2016, Shinefield, 2010). This non-toxic protein is a poor B cell-mediated immunogen, also enhancing a carbohydrate-specific plasma response (Pecetta et al., 2016). In this study, the mimetic 1 was first reacted with a non-immunogenic aliphatic chain (Danishefsky et al., 2015), presenting amidic linkages and ending with a carboxylic residue activated as para-nitrophenyl adipate (compound 2 Scheme 1), or succinimidyl adipate (compound 3, Scheme 1), for the subsequent conjugation to the carrier protein. According to published conditions (Tontini et al., 2013), CRM197 was treated with 2 or 3, and, as expected, a different loading of the protein was yielded. Upon conjugation with 2, up to four synthetic antigens were coupled to CRM197 (mime[4]CRM), whereas conjugation with 3 allowed to vary the loading of the protein and up to 19 or 34 synthetic glycans were grafted to CRM197 (mime[19]CRM, mime[34]CRM) (Figure 2 and Supplemental Information, Data S8–S11).

Figure 2.

Figure 2

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CRM197 Decorated with 4, 19 and 34 Residues of Mimetic 1 or with Residues of Glucose

CRM197 decorated with 4 residues (mime[4]CRM), 19 residues (mime[19]CRM), or 34 residues (mime[34]CRM) of TnThr mimetic 1, and CRM197 decorated with glucose residues (Glc-CRM, used as control, see Supplemental Information, Data S12).

1D 1H-nuclear magnetic resonance (NMR) spectra were recorded on non-functionalized CRM197 (Figure S1A), on its glycoconjugates mime[4]CRM (Figure S1B) and mime[19]CRM (Figure S1C). Significantly, although CRM197 decorated with four residues preserves its original folded structure, the protein conjugated with 19 synthetic glycans is largely unfolded. The dispersion of the NMR signals in the regions of the amide protons and methyl protons provided the main indicators for the folding state of proteins. In the spectrum of mime[4]CRM, these resonances are well separated, with some signals in the region between 0.5 and −1 ppm, whereas in mime[19]CRM the signals have narrow chemical shift dispersion without resonance lines of the protein below 0.5 ppm. On the other hand, small-angle X-ray scattering spectra recorded on mime[19]CRM sample confirmed that this glycoconjugate is aggregated in solution (see Figure S2).

Molecular Dynamics Simulation on Mime[4]CRM

We then performed molecular dynamics (MD) simulations on mimetic 1. These calculations showed clear evidence of the conformational restriction imposed by the additional rings to the mimetic (root-mean-square deviation [heavy atoms] = 0.84 Å, Figure 3A) and highlighted the orientation of the amino acid with respect to the sugar moiety relative to natural TnThr. Being aware that a protruding of the TnThr mimetic residues linked to the protein is required for any immune recognition, as an example, we performed also 0.5-μs MD simulations on mime[4]CRM in explicit water (Figure 3B). The four unnatural counterparts were conjugated to the most exposed lysine residues displayed on the surface of the protein. According to these simulations, the tertiary structure of CRM197 is not significantly altered upon the chemical modifications and all TnThr mimetic residues are exposed to the solvent (Figure 3), which is considered essential for the efficacy of the vaccine.

Figure 3.

Figure 3

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Molecular Dinamic Simulation on Mimetic 1and mime[4]CRM

(A) Representative conformer of TnThr (as a diamide derivative) in water solution, together with the structural ensemble derived from 0.5-μs MD simulation of mimetic 1.

(B) Representative snapshot derived from 0.5-μs MD simulations of CRM197 (PDB CRM197: 4AE0) upon chemical modification with 4 molecules of mimetic 1. A root-mean-square deviation value of 4.7 ± 1.1 Å (protein backbone), relative to the starting structure, was derived from the MD simulations. CRM197 is shown as ribbons, and the unnatural residues are represented as sticks.

Binding of Mime[4]CRM to anti-Tn Antibody Tn218

The affinity shown by the conjugate mime[4]CRM to the anti-Tn monoclonal antibody Tn218 (Gibadullin et al., 2017, Wua et al., 2005) was determined by SPR (Surface Plasmon Resonance) assays (Figure 4 and Supplemental Information). The isolated mimetic 1 exhibited an affinity comparable to that of the natural Tn (Figure S3), with a KD = 0.0160 M and 0.0125 M respectively, whereas the multipresentation of the TnThr mimetic 1 on the surface of CRM197 yielded a KD = 1.15 ×10−5 M. This result attested the accessibility of 1 to the antibody and showed a binding improvement by presenting multiple copies of the TnThr mimetic on the surface of the protein (Figure 4).

Figure 4.

Figure 4

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Affinity of Glycoconjugate mime[4]CRM to the Monoclonal anti-Tn Antibody Tn218

(A and B) SPR curves and fit obtained for mime[4]CRM (A) and isolated mimetic 1 (B) toward monoclonal antibody Tn218 (related to Figure S3).

Mime[4]CRM-Induced Activation of Human Dendritic Cells

DCs are central regulators of the adaptive immune response and important actors in elicitation of anti-tumoral responses. MGLs are C-type lectins exclusively expressed by human DCs and activated macrophages. A structural preference of MGLs for terminal GalNAc, including Tn antigen, has been reported (Zizzari et al., 2015, Diniz et al., 2019). We thus investigated the potential role of mime[4]CRM in DCs' activation and/or maturation, aiming to check the possible immunogenicity of this new vaccine candidate in humans. For that, DCs were differentiated from peripheral blood adherent mononuclear cells (PBMCs) of healthy donors, and the expression of markers CD83 and CD86 after stimulation for 48 h with mime[4]CRM or controls was checked (Figure 5). Flow cytometry analysis showed that mime[4]CRM induced activation and maturation of DCs as demonstrated by the increased expression of CD83 and CD86 markers. In contrast, Glc-CRM (Figure 2) and native TnThr peptide, used as controls, did not (see Supplemental Information for details). Although in-depth studies are necessary to confirm a binding interaction of MGLs with TnThr mimetic residues on CRM, these results clearly demonstrated the ability of candidate vaccine to properly activate DCs.

Figure 5.

Figure 5

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Evaluation of Human DCs' Activation

DCs isolated from three healthy donors have been stimulated with mime[4]CRM, Glc-CRM, or native Tn. Glc-CRM and native Tn showed some effect in DCs activation, whereas mime[4]CRM increased the expression of CD83 and CD86 markers. The data (median ±SD) of CD80, CD83, and CD86 expression on negative control (ctr−), positive control (ctr+, lipopolysaccharide), added with 0.2 mg/mL mime[4]CRM, with 0.2 mg/mL Glc-CRM, or with native Tn (0.05 or 0.2 mg/mL). The difference between mime[4]CRM treatment and the ctr− was assessed using paired t test; ∗p < 0.05 (see Supplemental Information for detail).

Therapeutic Effect of Mime[4]CRMIn Vivo by Using Triple-Negative Breast Cancer Transplanted Model

The unique structural features and interesting in vitro properties of mime[4]CRM tethered us to assess its potential anti-tumorigenic action against the challenging TNBCs. Compared with other BC subtypes, TNBCs are more aggressive and prone to generate “neo” antigens (including Tn antigen) (Bianchini et al., 2016). We first verified the overall lack of toxicity of mime[4]CRM both on the 4T1-Luc cell line, as a cellular model of highly metastatic TNBC (Tao et al., 2008), and in human PBMC exposed to mime[4]CRM (from 2.3 to 57.5 μg/mL). The glucose conjugate Glc-CRM (see Figure 2) (Richichi et al., 2016) or CRM197 alone was also screened as control molecule. Mime[4]CRM did not show any influence on viability and growth rate on the two cell populations over the observation period (Figure S5 and Supplemental Information). Similar data have been obtained using the human TNBC cell line MDA-MB231 (Figure S5).

The potential anti-tumorigenic action of mime[4]CRM, together with the activation of immune cells within the tumor microenvironment, was then evaluated in vivo by performing a preclinical study. For this purpose, Tn expressing (Solatycka et al., 2012) murine 4T1-luc cells (stably expressing Firefly Luciferase gene, see Supplemental Information) were implanted into the mammary fat pad gland of immunocompetent syngeneic mouse model (BALB/c mice). Of note, the use of immunocompetent mice properly allows to test the efficacy of immunomodulating compounds. Moreover, recent data showed that wild-type and huMUC1 transgenic mice produced equivalent antitumoral response against a native Tn-containing candidate vaccine (Stergiou et al., 2017).

After the tumors were established, mice were imaged (Bioluminescence Imaging, BLI, see Supplemental Information) at the time of implantation (day 0, T0) and then subcutaneously administered mime[4]CRM (n = 9) or CRM197 as vehicle for control group (n = 10) every week for 6 weeks (Figure 6A). As known, high-sensitivity BLI technique adequately allows the study of cell proliferation and migration in vivo in specific anatomical sites (Asadzadeh et al., 2017).

Figure 6.

Figure 6

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Description of the Vaccine Therapy Using Mime[4]CRM in Transplanted Mammary BC Cells and Their Rate of Proliferation In Vivo

(A) Representative scheme for the in vivo preclinical trial showing 4T1-Luc cells' injection into the mammary fat pad of n = 19 syngeneic BALB/c mice (at T0), and the weekly subcutaneous administration of mime[4]CRM (17 mg/kg/weekly) started after 7 days from the time of cell implantation. CRM197 was administered to the control group.

(B) Representative BLI images of mice orthotopically transplanted using 4T1-Luc cells implanted (n = 10 control mice, n = 9 treated mice). Mice were imaged every 7 days via in vivo BLI to monitor tumor growth from time of implantation (T0) to 28 days after tumor implantation.

(C) Quantification of photon emission (p/s) from the region of interest (ROI) in mice treated with mime[4]CRM or with CRM197 as vehicle. The differences in total flux (photons/seconds, P/S) between the two groups of mice indicated a statistically significant reduction of tumor growth (14 days of treatment: 3.7-fold reduction; 28 days of treatment: 2-fold reduction) as measured by luminescence signal emission from tumorigenic 4T1-LUC cells after 1 week and 3 weeks from tumor injection (∗p < 0.01, ∗∗p < 0.05, see Supplemental Information for detail). The values are expressed as mean ± SD.

(D) Evaluation of drug toxicity using mice body weight in mime[4]CRM-treated and control mice. The values are expressed as mean ± SD. No significant differences were observed between the two groups (related to Figure S5).

Mice were imaged every 7 days to monitor tumor growth in vivo (Figure 6B and Supplemental Information for dissection of the tumor growth from T0 to T42). Mice were then sacrificed at T28, T42, and T54. The obtained results clearly showed that the trend of total flux was significantly reduced after a week (p < 0.01) and also after 3 weeks (p < 0.05) of treatment (T14 and T28 post-implantation, respectively) in the group of mice that received mime[4]CRM compared with the vehicle group (Figure 6C), thus demonstrating an anti-tumorigenic action in vivo of mime[4]CRM. Remarkably, the reduction in the primary tumor growth between the two groups of mice was still visible after 4 weeks of treatment (i.e., after T28), even though it did not reach statistical significance (data not shown). This was likely due to an untimely interruption of the immunization schedule, along with a substantial tumor burden activation typical of highly aggressive 4T1 cells.

The absence of body and organs' weight loss (Figures 6D and S6) in treated mice confirmed that there was no significant acute toxicity induced by mime[4]CRM, in line with our in vitro data.

The immune cells (both lymphoid and myeloid cell populations) infiltrating 4T1 mammary tumor sections from mice (two mice, #9 and #19) treated for 3 weeks (T28 post-implantation, Figure 6A) with CRM197 and mime[4]CRM, respectively, were investigated by immunofluorescence analyses, by using antibodies directed against CD4 (T lymphocytes), TCR (T lymphocytes), CD11c (DCs), PD1 (immunosuppressive marker mostly expressed on T cells surface), FOXP3 (T regulatory cells [Tregs]), CD68 (granulocytes), F4/80 (macrophages), and CD163 (pro-tumorigenic M2 macrophages) (Figure 7). These markers allowed to investigate the role of mime[4]CRM in triggering in vivo immune cells exerting inhibitory effects or, conversely, promote tumor spread (i.e., CD4+FOXP3+PD1+, immunosuppressive regulatory T lymphocytes; CD68+ F4/80+ CD163+ M2-type macrophages) (Aras and Zaidi, 2017).

Figure 7.

Figure 7

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Representative Immunofluorescence (IF) Staining of Primary Mammary Tumor Sections from Transplanted Mice Treated with Mime[4]CRM or with CRM197 as a Vehicle

(A–F) IF double staining with (A) T cell receptor (TCR, marker of T cells, red) and CD4 (expressed by both T helper and dendritic cells, green), (B) CD11c (marker of DCs, red) and CD4 (green), (C) programmed cell death protein 1 (PD1, marker of immunosuppressive immune cells, red) and CD4 (green), (D) FOXP3 (marker of regulatory T cells, Tregs, red) and CD4 (green), (E) CD163 (specific marker of M2-polarized macrophages, red) and CD68 (marker of macrophages, green), and (F) CD163 (red) and F4/80 (marker of macrophages, green). DAPI was used to stain the nuclei (blue) in yellow or in pink, the overlays indicating (A) CD4+TCR+ (T lymphocytes), (B) CD4+ CD11c+ (DCs), (C) CD4+ PD1+ (immunosuppressive lymphocytes), (D) CD4+ FOXP3+ (T regulatory cells), and (E and F) CD68+ CD163+ and F4/80+ CD163+ cells (M2 macrophages). Magnification 63×. (a–f) Graphs showing the percentage of positive cells counted in fluorescence staining by using ImageJ software (n = 3 different sections for each tumor were screened within the tumor mass and images and cell positivity were counted). Mice treated with mime[4]CRM, instead of CRM197, showed a statistically significant increase of T helper lymphocytes (CD4+ TCR+, ∗p < 0.03; a) and DCs (CD4+ CD11c+, ∗p < 0.04, b) and a reduction of immunosuppressive lymphocytes (CD4+ PD1+, ∗p < 0.04, c), T regulatory cells (CD4+FOXP3+, ∗p < 0.01, d), and M2 tumor-associated macrophages (TAMs; i.e., CD68+ CD163+, ∗p < 0.003 and F4/80+ CD163+ cells ∗∗p < 0.007, e and f). The values are expressed as mean ± SD. ∗p < 0.05; ∗∗p < 0.007. See Supplemental Information for detail.

The data obtained showed increased levels of both T cells (CD4+TCR+) and peripheral DCs expressing CD4+ (CD4+CD11c+) in mice treated with mime[4]CRM (Figures 7A and 7B). This suggests that the candidate vaccine is able to modulate the recruitment of both antigen-presenting cells (APCs, i.e., DCs) and T helper CD4+ in the tumor microenvironment of the treated mice. Furthermore, the tumor sections from the same treated mouse also showed a significant reduction of CD4+ T cells expressing the immunosuppressive marker PD1 (PD1+CD4+, Figure 7C) and the transcriptional factor FOXP3 (Figure 7D), thus indicating a reduction in the quote of immunosuppressive Tregs in the TNBC microenvironment.

Of interest, in the mime[4]CRM-treated mouse's sections, data also showed a significant reduction in the pro-tumorigenic M2-polarized tumor-associated macrophages (TAMs; i.e., CD68+ CD163+; F4/80+ CD163+; Figures 7E and 7F). Moreover, the reduction of immunosuppressive T lymphocytes (i.e., PD1+ CD4+) was also shown in the tumor tissues from mice after 42 days from tumor cell implantation (T42), thus suggesting a long-lasting effect of mime[4]CRM in inhibiting immunosuppressive markers (e.g., PD1) in the tumor microenvironment of TNBC (data not shown). In contrast, the decrease of Tregs (FOXP3+ CD4+) in the same mime[4]CRM-treated mice was not substantially observed, thus inferring a loss of regulation of conventional T lymphocytes at T42 (results did not reach a statistical significance; data not shown).

Altogether, immunofluorescence staining indicates that mime[4]CRM vaccine has a clear potential role in modulating the recruitment and phenotype of CD4+ T cells and, as a consequence, on the polarization status of TAMs in the TNBC tumor microenvironment.

As 4T1 cells, used in this study as a cellular model of TNBC, are highly metastatic, the presence of lung metastases was evaluated by performing in vivo analyses 42 days after 4T1 TNBC cells were implanted. At T42, our in vivo BLI analyses indicated the presence of lung metastases in seven of nine controls and four of eight mime[4]CRM-treated mice (Figures 8A and 8B). In contrast, as determined through ex vivo BLI imaging, the presence of metastatic foci was detected in eight of nine control mice (seven mice at T42 and one mouse at T54, Figure 8B), and in four of eight mime[4]CRM-treated mice (one mouse at T42 and three mice at T54, Figure 8A). Therefore, although we cannot exclude the presence of lung micro-metastases at T42 in the mime[4]CRM-treated group of mice because they might not be detectable with the in vivo BLI technology, data showed a clear effect of our candidate vaccine in modulating lung metastasis development.

Figure 8.

Figure 8

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In Vivo and Ex Vivo Analyses to Evaluate the Presence of Lung Metastases

(A and B) In vivo BLI analyses showed only 50% of the mime[4]CRM-treated mice (four of eight animals) developed lung metastases (A), whereas the presence of metastatic foci was found in seven of nine control mice (i.e.,78%) at T42 (B). Ex vivo BLI analyses proved the presence of metastatic foci in eight of nine control mice (seven mice at T42 and one mouse at T54, B lower panel) and in four of eight mime[4]CRM-treated mice (one mouse at T42 and three mice at T54, A lower panel).

To investigate the immunoediting of mice treated with mime[4]CRM compared with control mice, the composition of immune cell infiltrate was also analyzed at T54 (i.e., 2 weeks after the last treatment) in the lung (Figure S6) and in BC (Figure S7) tissue, by H&E staining and by immunohistochemistry (two mice and two sections/mouse) using antibodies against CD4, CD8, and FOXP3 markers. In BC, the results showed a statistically significant increase of CD4-positive cells in mime[4]CRM-treated compared with CRM197-treated mice, whereas no significant differences were seen for CD8 and FOXP3 markers between the two groups (see Supplemental Information and Figure S7).

Of note, H&E staining showed a cell infiltrate in the lung of the groups of treated mice (Figures S6B, S6F, and S6G) compared with untreated mice (Figure S6A). However, it is important to note that in CRM197-treated mice the organ presented numerous metastatic cell masses (Figure S6B), whereas mime[4]CRM-treated mice showed just a single metastatic cell mass (Figure S6G). The composition of the immune cell infiltrate in CRM197-treated mice consisted of several CD4-positive cells diffused in the entire organ, although more numerous in the metastatic masses (Figure S6C), whereas the FOXP3- and CD8-positive cells were confined to the metastatic mass (Figures S6D and S6E). Conversely, in mime[4]CRM-treated mouse carrying the metastatic mass, there were numerous and dispersed CD4-positive cells (Figure S6H), but very few FOXP3-positive cells limited to the metastatic mass (Figure S6J).

Furthermore, we investigated the potential ability of mime[4]CRM to decrease Prune-1 protein levels in the treated mice. This protein had previously been reported to be overexpressed in 4T1 cells (Virgilio et al., 2012) and positively correlated to advanced stage in metastatic BC (D'Angelo et al., 2004, Zollo et al., 2005). However, immunofluorescence analyses performed on primary tumor sections derived from mime[4]CRM-treated and vehicle mice did not show differences in Prune-1 levels (data not shown), thus suggesting that the ability of the vaccine mime[4]CRM to reduce tumor outgrowth does not involve the down-regulation of Prune-1 levels in vivo, confirming its action on the modulation of immune infiltrating cells by activating the lymphocytes component.

Cumulative survival analyses between the two groups of mice revealed that seven of nine (77.7%) controls and two of eight treated (25%) mice died at T42, thus indicating a clear trend of therapeutic benefit in mime[4]CRM-treated versus control mice in the observed period (long rank test p < 0.083, data not shown).

Anti-TnThr Mimetic 1-Specific Antibody Response Elicited by mime[4]CRM Immunization

Results so far described show that mice engrafted with 4T1 cancer cells and immunized with mime[4]CRM in a therapeutic experimental setting have a reduced tumor burden and, remarkably, impaired distal dissemination. The antibody response introduced by the vaccine was analyzed by measuring the IgG serum titers elicited upon immunization against the glycan portion of mime[4]CRM. To detect mimetic 1-specific antibodies, mice sera collected during immunization (T28 and T42) were tested by ELISA on plates coated with the hexapeptide Ala-Pro-Asp-H2NSer-Arg-Pro 8 (as negative control, see Supplemental Information) or with the glycosylated Ala-Pro-Asp-HNSer(mimetic 1)-Arg-Pro hexapeptide (9, see Supplemental Information). Immunization with mime[4]CRM induced predominantly IgG (almost exclusively IgG1, whereas IgG2a was barely detectable), and to a lower lever IgM (mean titer 1:1,350 and 1:600 respectively), compared with control mice (mean titer 1:600 and 1:100, respectively) (see Figure S8). Sera were then tested for binding on the surface of 4T1 cancer cells by flow cytometry. Notably, mice immunized with mime[4]CRM elicited antibodies that can better recognize native Tn antigen expressed by BC cells than control mice (approximately 3-fold increase in mean fluorescence intensity, see Figure S9).

A preliminary analysis of serum cytokines revealed a higher level of anti-tumor Th17 cytokines after immunization with mime[4]CRM (Figure S10). This result was not unexpected, because the ability of Tn glycosylation in inducing interleukin (IL)-17 responses was recently described (Freire et al., 2011). Th17 cells producing IL-17 have been detected in patients with ovarian cancer (Kryczek et al., 2009) and in mouse tumor models (Kryczek et al., 2007). Although relevance of IL17+ T lymphocytes in anti-cancer immunity is still controversial, it may contribute to protective tumor immunity by recruiting effector cells to the tumor microenvironment (Amedei et al., 2013, Kryczek et al., 2009, Martin-Orozco et al., 2009). In our study, the presence of IL-17 in serum of immunized mice is an additional evidence of mime[4]CRM immunomodulatory activity on T cells.

Isolation of Mime[4]CRM-Specific T Cells

Capitalizing on these notable results, 12 patients with BC were enrolled to assess the presence of mime[4]CRM-specific T cells, as in the peripheral blood as in the tumor tissues. In detail, the PBMCs were cultured in the presence of medium alone or with mime[4]CRM. The presence of mime[4]CRM-specific T cells was documented in 8 (67%) of the 12 patients. Thus, we isolated mime[4]CRM-specific T cells, by cloning the tumor-infiltrating cells of the same patients with BC. We obtained a total of 90 T cell clones and 15 (17%) that were specific for mime[4]CRM. Of note, evaluating the profile of cellular markers of the intra-tumoral mime[4]CRM-specific T cells, we found that 69% were CD4+, whereas 31% were CD8+ (see Figure S11 and Supplemental Information). This last evidence demonstrates the ability of mime[4]CRM to stimulate T cells in patients with BC. A compelling hypothesis is that this might be due to an activation of Tn-specific T cell response spontaneously elicited in cancer tissues.

In conclusion, the results reported in this study demonstrated a therapeutic efficacy of the glycoconjugate mime[4]CRM in inhibiting tumor growth in mice. The efficacy, likely correlated with a higher frequency of CD4+ T cells and a lower frequency of M2-type macrophages, was assessed in mice inoculated with 4T1-Luc cells and treated with mime[4]CRM. Specific IgGs were elicited in mice immunized with mime[4]CRM, which recognized native Tn antigen on BC cells better than sera from control mice. Mime[4]CRM properly activated human DCs and was able to modulate the recruitment of both APCs and T helper CD4+ in the tumor microenvironment of treated mice. In addition, the isolation from patients with BC of tumor-infiltrating lymphocytes specific for mime[4]CRM highlighted the intrinsic immunogenicity of the candidate vaccine and its ability to stimulate a specific immune response. These results are unprecedented for a TACA mimetic-based construct. Mice in vivo and human ex vivo immunogenicity of mime[4]CRM, characterized by a carbohydrate determinant quite different from that of the native TACA, could be of great interest for the design of structurally innovative cancer vaccines.

Limitations of the Study

The study provided preclinical data demonstrating the efficacy of mime[4]CRM as cancer vaccine for TNBC. The predictive value of the proposed TNBC mouse cancer model for mime[4]CRM as human vaccine deserves further investigations. In particular, efforts should be focused to assess mime[4]CRM efficiency in the stimulation of the Tn-specific T and B human lymphocytes naturally induced in oncologic patients affected by different Tn-positive cancers.

Resource Availability

Lead Contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Cristina Nativi (cristina.nativi@unifi.it).

Materials Availability

All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

Authors thank Dr. F.P. Pennino (University of Naples) for his initial contribution to the animal settings, Dr. X. Ferhati, G. Salerno, and Ms. A. Mazzara (University of Florence) for initial contribution to CRM glycosylation. We thank MIUR-Italy (Progetto Dipartimenti di Eccellenza 2018–2022 allocated to Dept. of Chemistry and Prin 2015), CISM (University of Florence) for MALDI-MS, CEINGE for supporting the use of mice facility and animal regulation study, PON 01–02388/1 2007–2013, and POR Rete delle Tecnologie in Campania Movie. I.A.B. thanks the Asociación Española Contra el Cáncer en La Rioja for a grant. F.C. was supported by Ministerio de Ciencia, Innovación y Universidades (project RTI2018-099592-B-C21).

Author Contributions

A.A. designed and analyzed DCs tests and human ex vivo tests; A.A. and M.G.V. designed and analyzed the IHC tests; F.A. prepared and performed the in vivo tests; F.A., V.F., and M.Z. analyzed the in vivo data and performed quantitative immunofluorescence analysis; F.C. designed and analyzed MD and SPR assays; I.A.B. performed MD and SPR experiments, F.P. performed chemical syntheses; F.P. performed peptides' synthesis and functionalization; F.P. and S.G. performed CRM glycosylation; L.C. performed NMR studies; M.F. designed and analyzed the NMR experiments; M.B. and E.P. performed the serum in vitro tests; E.N. and C.V.D.A. performed DCs, E.N. and F.R. performed human ex vivo experiments, F.N., E.G., and F.G. designed and run SAXS experiments; C.T. and S.C. prepared and performed IHC assays; L.O. prepared human tissues samples, F.B., M.F., and C.N. designed and analyzed the CRM glycosylation experiments; M.Z. designed the in vivo experiments; R.G. designed and analyzed the serum in vitro tests; R.G. and C.N. co-wrote the paper; C.N. directed the project. A.A. and F.A. are co-first authors. All authors discussed the results and commented on the paper.

Declaration of Interests

F.B. is an employee of the GSK group of companies. All the other authors declare no competing financial interests.

Published: June 26, 2020

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2020.101250.

Contributor Information

Massimo Zollo, Email: massimo.zollo@unina.it.

Renata Grifantini, Email: grifantini@ingm.org.

Cristina Nativi, Email: cristina.nativi@unifi.it.

Supplemental Information

Document S1. Transparent Methods, Figures S1–S11, Tables S1–S3, and Data S1–S12
mmc1.pdf (8MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent Methods, Figures S1–S11, Tables S1–S3, and Data S1–S12
mmc1.pdf (8MB, pdf)

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