Immunotherapy of triple-negative breast cancer with cathepsin D-targeting antibodies

Abstract

Background: Triple-negative breast cancer (TNBC) treatment is currently restricted to chemotherapy. Hence, tumor-specific molecular targets and/or alternative therapeutic strategies for TNBC are urgently needed. Immunotherapy is emerging as an exciting treatment option for TNBC patients. The aspartic protease cathepsin D (cath-D), a marker of poor prognosis in breast cancer (BC), is overproduced and hypersecreted by human BC cells. This study explores whether cath-D is a tumor cell-associated extracellular biomarker and a potent target for antibody-based therapy in TNBC.

Methods: Cath-D prognostic value and localization was evaluated by transcriptomics, proteomics and immunohistochemistry in TNBC. First-in-class anti-cath-D human scFv fragments binding to both human and mouse cath-D were generated using phage display and cloned in the human IgG1 λ format (F1 and E2). Anti-cath-D antibody biodistribution, antitumor efficacy and in vivo underlying mechanisms were investigated in TNBC MDA-MB-231 tumor xenografts in nude mice. Antitumor effect was further assessed in TNBC patient-derived xenografts (PDXs).

Results: High CTSD mRNA levels correlated with shorter recurrence-free survival in TNBC, and extracellular cath-D was detected in the tumor microenvironment, but not in matched normal breast stroma. Anti-cath-D F1 and E2 antibodies accumulated in TNBC MDA-MB-231 tumor xenografts, inhibited tumor growth and improved mice survival without apparent toxicity. The Fc function of F1, the best antibody candidate, was essential for maximal tumor inhibition in the MDA-MB-231 model. Mechanistically, F1 antitumor response was triggered through natural killer cell activation via IL-15 upregulation, associated with granzyme B and perforin production, and the release of antitumor IFNγ cytokine. The F1 antibody also prevented the tumor recruitment of immunosuppressive tumor-associated macrophages M2 and myeloid-derived suppressor cells, a specific effect associated with a less immunosuppressive tumor microenvironment highlighted by TGFβ decrease. Finally, the antibody F1 inhibited tumor growth of two TNBC PDXs, isolated from patients resistant or not to neo-adjuvant chemotherapy.

Conclusion: Cath-D is a tumor-specific extracellular target in TNBC suitable for antibody-based therapy. Immunomodulatory antibody-based strategy against cath-D is a promising immunotherapy to treat patients with TNBC.

Keywords: Human antibody-based therapy; Immunomodulation; Phage display; Protease; TNBC; Tumor microenvironment.

Fig. 1

Cath-D is eligible for antibody-mediated targeted therapy in TNBC. a Kaplan-Meier curves of recurrence-free survival according CTSD mRNA expression in TNBC. n = 255 patients with TNBC; HR = 1.65 [1.08–2.53], P = 0.019, log-rank test. b Detection of extracellular and cell-surface cath-D in TNBC by proteomics. A TNBC biopsy and its paired normal breast tissue were biotinylated followed by protein digestion. The corresponding glycoprotein fractions containing proteins that are located either in the extracellular matrix or at the exterior face of the plasma membrane were analyzed by proteomics. c Detection of extracellular cath-D in a TNBC TMA. Cath-D was monitored in a TMA by IHC using a monoclonal anti-human cath-D (C-5; sc-377,127) antibody. Scale bar, 50 μm (left panel). Higher magnifications of the boxed regions showing extracellular cath-D (right panel). Scale bar, 10 μm. d Quantification of extracellular cath-D in a TNBC TMA. n = 123 samples. e Detection of membrane-associated cath-D at the cancer cell surface in a TNBC TMA. Scale bar, 50 μm (left panel). Higher magnifications of the boxed regions showing perimembranous cath-D (right panel). Scale bar, 20 μm. f Quantification of membrane-associated cath-D at the cancer cell surface in a TNBC TMA. n = 123 samples. g Detection of cath-D in normal breast tissue. Scale bar, 50 μm (left panel). Higher magnifications of the boxed regions (right panel). Scale bar, 20 μm. h Quantification of extracellular and membrane-associated cath-D in a normal breast TMA. n = 50 samples

Fig. 2

Characterization of the anti-cath-D F1 and E2 antibodies. a Binding of F1 and E2 to pro-cath-D secreted from MDA-MB-231 cells. Sandwich ELISA in which pro-cath-D from conditioned medium of MDA-MB-231 cells was added to wells pre-coated with anti-pro-cath-D M2E8 mouse monoclonal antibody in the presence of increasing concentrations of F1 (left panel) or E2 (right Panel). Binding of F1 and E2 to pro-cath-D was revealed with an anti-human Fc HRP-conjugated antibody. The EC₅₀ values are shown. b Binding of F1 and E2 to pro-cath-D secreted from MDA-MB-231 cells at acidic pH. Sandwich ELISA was performed as described in (a) but at different pH values (7.5–5.5). c Molecular docking of the scFv F1 and E2. Ribbon representation of the scFv F1 (magenta) and scFv E2 (green) interface with the contact surface of mature cath-D (upper panels). Docking model in which the space-filled view of protruding L1 CDR inserts into cath-D catalytic site (bottom panels). d Competitive ELISA. Sandwich ELISA was performed as described in (a) with 1 nM F1 or E2 and increasing concentrations of scFv F1, E2 or IR (negative scFv). e Immunoprecipitation of GST-cath-D isoforms with F1 and E2. GST-cath-D isoforms were immunoprecipitated with F1, E2, or E12, and detected by immunoblotting using the relevant antibodies (left panels). Mr, relative molecular mass (kDa). Schematic representation of the human 52-kDa pro-cath-D sequence (right panel). The 4-kDa cath-D pro-fragment, 14-kDa light, and 34-kDa heavy mature chains are indicated. The intermediate 48-kDa form (not shown) corresponds to the non-cleaved 14 + 34 kDa chains. The catalytic aspartate 33 and 231 (red) are shown

Fig. 3

The anti-cath-D F1 and E2 antibodies accumulate in MDA-MB-231 tumor xenografts, reduce tumor growth in vivo and improve survival. a SPECT/CT analysis. MDA-MB-231 cells were xenografted subcutaneously in the right flank of nude mice. When tumor volume reached 150 mm³, mice received one intra-peritoneal injection of ¹⁷⁷Lu- F1 or ¹⁷⁷Lu- E2. Representative SPECT/CT images at 24, 48 and 72 h post-injection of ¹⁷⁷Lu- F1 (left panels) and ¹⁷⁷Lu- E2 (right panels). * shows tumors. b Tumor growth. MDA-MB-231 cells were subcutaneously injected in nude mice. When tumor volume reached 50 mm³, mice were treated with F1 (n = 6) or E2 (n = 6) (15 mg/kg), or NaCl (CTRL; n = 8) three times per week for 32 days. Mice were sacrificed when tumor volume reached 2000 mm³. Tumor volume (in mm³) is shown as the mean ± SEM. ***, P < 0.001 for F1; ** P = 0.002 for E2 (mixed-effects ML regression test). c Mean tumor volumes at day 55. ***, P = 0.0005 for F1; **, P = 0.0026 for E2 (t test); mean ± SEM. d Kaplan-Meier survival analysis. ***, P = 0.0005 for F1; **, P = 0.0016 for E2

Fig. 4

Anti-cath-D antibody-based therapy prevents macrophage recruitment within MDA-MB-231 tumor xenografts. a Tumor growth. When MDA-MB-231 tumor xenografts reached a volume of 50 mm³, nude mice were treated with F1 (n = 9), E2 (n = 9), or rituximab (CTRL; n =9) (15 mg/kg) for 28 days (day 16–44). At day 44, all mice were sacrificed. ***, P = 0.001 for F1; **, P = 0.002 for E2 (mixed-effects ML regression test). b Mean tumor volume at day 44. n = 9 for CTRL; n = 9 for F1; n = 9 for E2. ***, P = 0.0001 for F1; **, P = 0.0012 for E2 (Student’s t test); mean ± SEM. c Representative images of F4/80 immunostaining in MDA-MB-231 tumor cell xenografts from CTRL- (rituximab), F1- and E2-treated mice. Scale bars, 100 μm. d Quantification of F4/80⁺ macrophages. Percentage (mean ± SEM) of positive pixels relative to the total pixels; n = 9 for rituximab (CTRL); n = 9 for F1; n = 9 for E2; ***, P < 0.0001 for F1; **, P = 0.0063 for E2 (Student’s t-test). e Linear regression analysis of F4/80⁺ macrophages and tumor volumes. R² = 0.1553; *, P = 0.0464, n = 27

Fig. 5

Anti-cath-D antibody-based therapy prevents M2-like macrophage and MDSC recruitment, and triggers antitumor response via NK cell activation in MDA-MB-231 xenografts. a Tumor growth. Nude mice bearing MDA-MB-231 tumors of 50 mm³ were treated with F1 (n = 9), F1Fc (n = 8), or rituximab (CTRL; n = 9) (15 mg/kg) for 35 days. At day 54, mice were sacrificed. *, P < 0.001 for F1 versus CTRL; P = 0.077 for F1Fc versus CTRL; P = 0.069 for F1 versus F1Fc (mixed-effects ML regression test). b Mean tumor volumes at day 54. Mean ± SEM; *, P = 0.011 for F1 versus CTRL; P = 0.231 for F1Fc versus CTRL, P = 0.189 for F1 versus F1Fc (Student’s t-test). c TAM recruitment. The percentage of F4/80⁺ CD11b⁺ TAMs was quantified by FACS and expressed relative to all CD45⁺ immune cells (n = 9 for CTRL; n = 9 for F1; n = 8 for F1Fc); *, P = 0.044 for F1 versus CTRL; P = 0.3 for F1Fc versus CTRL (Student’s t-test). d Linear regression analysis of TAM and tumor volumes. R² = 0.5425; ***, P < 0.0001; n = 26. e Quantification of CD206 mRNA expression. Total RNA was extracted from MDA-MB-231 tumor xenografts at the end of treatment, and CD206 expression analyzed by RT-qPCR and shown relative to F4/80 (n = 9 for CTRL; n = 9 for F1; n = 8 for F1Fc); P = 0.05 for F1 versus CTRL; P = 0.04 for F1Fc versus CTRL (Student’s t-test). f  MDSC recruitment. The percentage of Gr1⁺ CD11b⁺ MDSCs was quantified by FACS analysis and expressed relative to all CD45⁺ cells (n = 9 for CTRL; n = 9 for F1; n = 8 for F1Fc); **, P = 0.008 for F1 versus CTRL; P = 0.079 for F1Fc versus CTRL (Student’s t-test). g Linear regression analysis of MDSC and tumor volumes. R² = 0.23315; *, P = 0.0125; n = 26. h Quantification of TGFβ mRNA expression. Total RNA was extracted from MDA-MB-231 tumor cell xenografts at the end of treatment and TGFβ expression analyzed by RT-qPCR. Data are relative to RPS9 expression (n = 9 for CTRL; n = 9 for F1; n = 8 for F1Fc); **, P = 0.009 for F1 versus CTRL; P = 0.1 for F1Fc versus CTRL (Student’s t-test). i NK recruitment. The percentage of CD49b⁺ CD11b⁺ NK cells was quantified by FACS and expressed relative to all CD45⁺ cells (mean ± SEM; n = 9 for rituximab (CTRL); n = 9 for F1; n = 8 for F1Fc); P = 0.7 for F1 versus CTRL; P = 0.8 for F1Fc versus CTRL; P = 0.8 for F1 versus F1Fc (Student’s t-test). j Quantification of IL-15 mRNA expression. Total RNA was extracted from MDA-MB-231 tumor cell xenografts at the end of treatment and IL-15 analyzed by RT-qPCR. Data are the mean ± SEM expression level relative to RPS9 expression (n = 9 for rituximab (CTRL); n = 9 for F1; n = 8 for F1Fc); **, P = 0.0013 for F1 versus CTRL; P = 0.365 for F1Fc versus CTRL; *, P = 0.0127 for F1 versus F1Fc (Student’s t-test). k Linear regression analysis of IL-15 mRNA level and tumor volumes. R² = 0.3693; **, P = 0.0013; n = 26. l Quantification of granzyme B mRNA expression as in (j). ***, P = 0.0002 for F1 versus CTRL; **, P = 0.0011 for F1Fc versus CTRL; **, P = 0.0076 for F1 versus F1Fc (Student’s t-test). m Quantification of perforin mRNA expression as in (j). *, P = 0.033 for F1 versus CTRL; *, P = 0.0294 for F1Fc versus CTRL; P = 0.386 for F1 versus F1Fc (Student’s t-test). n Quantification of IFNγ mRNA expression as in (j). ***, P < 0.0001 for F1 versus CTRL; P = 0.0513 for F1Fc versus CTRL; **, P = 0.0078 for F1 versus F1Fc (Student’s t-test)

Fig. 6

The anti-cath-D F1 antibody inhibits tumor growth of TNBC PDXs. a Cath-D expression in TNBC PDXs. Total cath-D expression was determined in whole cytosols from five TNBC PDXs by sandwich ELISA with immobilized anti-human cath-D D7E3 antibody and anti-human cath-D M1G8 antibody coupled to HRP. Total cath-D expression was quantified also in MDA-MB-231 tumor xenografts. b Cath-D expression in TNBC biopsies. Total cath-D expression was assessed in whole cytosols from TNBC biopsies as described in (a); n = 41. c Expression of cath-D in the PDX B1995 and B3977. Cath-D expression in the PDX B1995 (left panel) and PDX B3977 (right panel) primary tumors was monitored by IHC using a monoclonal anti-human cath-D (C-5; sc-377,127) antibody. Scale bar: 25 μm. Insets, high magnification of the boxed regions. d Average passage duration for the first three passages in the five TNBC PDXs. e Therapeutic effects of F1 in mice engrafted with PDX B1995 or PDX 3977. Mice were engrafted with PDX B1995 (left panel) or PDX B3977 (right panel) and when tumor volumes reached 150 mm³, mice were treated with F1 (15 mg/kg) or NaCl (CTRL) three times per week. Mice were sacrificed when tumor volume reached 2000 mm³ and the corresponding tumor growth curves were stopped. Tumor volume (in mm³) is shown as the mean ± SEM; For PDX B1995 : n = 7 for CTRL; n = 7 for F1. ***, P < 0.001 for F1. For PDX B3977 : n = 10 for CTRL; n = 10 for F1. *, P = 0.022 for F1