Antibodies against the multifaceted cathepsin D protein open new avenues for TNBC immunotherapy

Abstract

Triple-negative breast cancer (TNBC) is a heterogeneous breast cancer subtype characterized by aggressive clinical behavior and poor prognosis. The immune landscape associated with TNBC often reveals high immunogenicity. Therefore, immunotherapy, which has demonstrated its efficacy in different cancer types, could be a promising strategy for TNBC, given the limited therapeutic options currently available besides conventional chemotherapy. The aspartic protease cathepsin D (cath-D) is a tumor cell-associated extracellular protein with protumor activity, a marker of poor prognosis, and a target for antibody-based therapy in TNBC. This commentary provides a synopsis/narrative summary of the development of anti-cath-D antibodies in different formats, their key roles in restoring the antitumor immunity, particularly via activation of tumor-infiltrating natural killer cells, and their dual antitumor effects on cancer cells and stromal cancer-associated fibroblasts, suggesting their interest for clinical use in the light of the current clinical knowledge on TNBC.

Correspondence to Dr Emmanuelle Liaudet-Coopman; [email protected]

Introduction

The aspartic protease cathepsin D (cath-D), a marker of poor prognosis in breast cancer (BC),^{1 2} is overproduced by BC cells and hypersecreted in the tumor microenvironment.³ Overexpressed cath-D exhibits tumor-promoting activity in BC by modulating cancer and stromal cell proliferation, tumor growth and angiogenesis, tumor cell apoptosis, and metastasis formation.^4–8 Cath-D was proposed as a potential therapeutic target to enhance anticancer drug-induced apoptosis.⁹ In BC, extracellular cath-D displays protumor roles through proteolytic cleavage of stromal/tumor components and also through non-proteolytic mechanisms by binding to receptors expressed on the surface of tumor cells and stromal cells, such as cancer-associated fibroblasts (CAFs). Secreted cath-D can alter the local extracellular matrix by cleaving collagens, fibronectin, proteoglycans, matricellular proteins (eg, SPARC),¹⁰ and by releasing growth factors trapped in the extracellular matrix (eg, FGF2).¹¹ Secreted cath-D can also disrupt the antitumor immune landscape by cleaving chemokines/cytokines involved in immune cell recruitment and activation.^{12 13} In the tumor microenvironment, cath-D can trigger the proteolytic cascade by activating the cathepsin B and L precursors,¹⁴ or by degrading protease inhibitors (eg, cystatin C).¹⁵ Concerning its non-proteolytic mechanisms of action, cath-D promotes BC cell proliferation via its propeptide,⁵ triggers breast fibroblast growth by binding to the LRP1 receptor,¹⁶ and induces endothelial cell proliferation and migration via the ERK and AKT signaling pathways.¹⁷ Therefore, extracellular cath-D could be a novel molecular target in BC for antibody-based therapy to trap the abnormal pool of the secreted protease. In this commentary, we show that anti-cath-D antibody therapy can inhibit tumor growth, restore the antitumor immunity, particularly by activating tumor-infiltrating natural killer (NK) cells that target both cancer and stromal cells (CAFs), and improve the therapeutic index of chemotherapy and hormone therapy. We also discuss what needs to be investigated to complete the preclinical characterization of these antibodies in view of their clinical development.

The fully human anti-cath-D antibody F1 as a new immunomodulator in triple-negative BC

The first-in-class F1, a fully human anti-cath-D immunoglobulin G (IgG)1 originally isolated by phage display, localizes at the tumor site and inhibits the growth of MDA-MB-231 (triple-negative BC (TNBC)-derived) cell xenografts and patient-derived xenografts (figure 1A). Mechanistically, F1 activates NK cells and prevents the recruitment of M2-polarized tumor-associated macrophages (TAM) and myeloid-derived immunosuppressive populations (MDSCs) in TNBC cell xenografts in nude mice (figure 1A).¹⁸ Interestingly, the Fc-aglycosylated F1 variant, where the N297A mutation prevents binding to the Fcγ receptors, no longer induces NK cell activation, and is associated with reduced tumor growth inhibition.¹⁸ This demonstrates the essential role of the Fc part of the F1 antibody for cath-D-specific targeted therapy, possibly via NK cell activation and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). It also suggests that cath-D is a prime target for monoclonal antibody-based therapies with immunomodulatory activity.¹⁸

Figure 1. Antibody targeting the multifaceted cathepsin D protein as a novel clinical opportunity for TNBC immunotherapy. (A) Immunotherapy of TNBC with the F1 anti-cath-D targeting antibody. 18 F1 antitumor response is triggered through NK cell activation via IL15 upregulation, associated with granzyme B and perforin production, and IFN[gamma] release. F1 also prevents the recruitment of M2-TAMs and MDSCs, a specific effect associated with a less immunosuppressive tumor microenvironment highlighted by TGF-[beta] decrease. (B) Anti-cath-D immunotherapy with F1 and F1M1 triggers both innate and adaptive antitumor immunity in an immunocompetent mouse model of TNBC 19 . The F1 and F1M1 antibodies in the mouse IgG2a format promote the innate antitumor immunity by activating NK cells and preventing the recruitment of immunosuppressive M2-TAMs in the tumor microenvironment of C57BL/6 mice harboring E0771 cell grafts. They also enhance activation of antitumor antigen-presenting cells (CD86+ M1-polarized TAMs and cDC1 cells), supporting the reduction of PD-L1+ tolerogenic CD4+ and CD8+ T cells and exhaustion markers, including LAG3 and TIGIT, in the tumor microenvironment. (C) Generation of the Fc-engineered F1M1-Fc+ anti-cath-D antibody as a novel therapeutic opportunity for TNBC. 23 F1M1-Fc+ enhances in vitro NK cell activation and ADCC of both TNBC cells and CAFs. F1M1-Fc+ triggers tumor-infiltrating NK cell recruitment and cytotoxic activity in TNBC. In combination therapies, F1M1-Fc+ improves the therapeutic efficacy of paclitaxel and enzalutamide in TNBC. F1M1-Fc+ enhances in vitro NK cell activation and ADCC of both TNBC cells and CAFs. F1M1-Fc+ triggers tumor-infiltrating NK cell recruitment and cytotoxic activity in TNBC. In combination therapies, F1M1-Fc+ improves the therapeutic efficacy of paclitaxel and enzalutamide in TNBC. ADCC, antibody-dependent cellular cytotoxicity; CAFs, cancer-associated fibroblasts; cath-D, cathepsin D; cDC1, conventional type 1 dendritic cells; GzmB, granzyme B; IFN[gamma], interferon gamma; IgG, immunoglobulin G; IL15, interleukin-15; MDSCs, myeloid-derived immunosuppressive populations; MHC, major histocompatibility complex; NK, natural killer; PD-L1, programmed death-ligand 1; TAMs, tumor-associated macrophages; TNBC, triple-negative breast cancer.

F1M1 immunomodulatory activity in an immunocompetent mouse model of TNBC

To explore the overall impact of anti-cath-D antibodies on the immune landscape of TNBC, we next investigated the therapeutic effect and immunomodulatory activity of the anti-cath-D murine IgG2a antibody F1 and of its improved version F1M1 with aglycosylated Fab in an immunocompetent mouse model of TNBC (C57BL/6 mice harboring E0771 cell grafts).¹⁹ This was possible because F1 and F1M1 cross-react with mouse cath-D. This study showed that both F1 and F1M1 inhibit tumor growth in the highly-immunogenic E0771 mouse model. Both antibodies promoted the innate antitumor immunity by preventing the recruitment of immunosuppressive M2-TAMs and by activating NK cells in the tumor microenvironment (figure 1B). This translated into a reduction of programmed death-ligand 1 (PD-L1)⁺ tolerogenic T cells and of exhaustion markers, including LAG3 and TIGIT, in the tumor microenvironment, possibly locally supported by enhanced activation of antigen-presenting cell (M1-polarized CD86⁺ TAMs and CD86⁺ conventional type 1 dendritic, cDC1, cells) functions and upregulation of major histocompatibility complex class 1 molecules, β2-microglobulin and FcγRs (figure 1B). Thus, F1 and F1M1 remodel the tumor immune landscape by triggering both innate and adaptive antitumor immunity in an immunocompetent mouse model of TNBC.¹⁹ Although few data are available on cath-D roles in antitumor immunity, it has been shown that cath-D promotes M2-TAM polarization through TGFBI-CCL20 signaling in TNBC in vivo.²⁰ To further validate F1M1 efficacy in preclinical settings, it would be important to include other immunogenic TNBC mouse models. Unfortunately, the 4T1 TNBC mouse cell line does not secrete the cath-D precursor.¹⁹ If EMT-6 cells secrete the cath-D precursor, the EMT-6 TNBC model in Balb/c mice could be an interesting alternative. This model has already been used to study the efficacy of anti-immune checkpoint antibodies (against programmed cell death protein-1 (PD1), PDL1, cytotoxic T-lymphocytes-associated protein 4) and chemotherapy (carboplatin) and radiotherapy, either alone or in combination.²¹

Fc-engineering of F1M1 for future clinical development in TNBC

Based on our previous studies suggesting that F1 and F1M1 can activate tumor-infiltrating NK cells,^{18 19} we protein-engineered the Fc part of the F1M1 IgG1 to enhance NK cell activation and potentiate its antitumor efficacy in TNBC, as previously demonstrated.^{22 23} As Fc dependence is often a prerequisite to ensure antibody efficacy in clinical settings, deciphering NK cell activation after F1M1 administration was an essential step for its future clinical development. The Fc-optimized F1M1-Fc⁺ antibody (derived from F1M1 by introducing the S239D, H268F, S324T, and I332E mutations) displays an enhanced affinity for CD16a/FcγRIIIa regardless of 158F/V allotypes. In vitro, among the different anti-cath-D F1-derived antibodies, F1M1-Fc⁺ most potently activated NK cells, with increased CD107a cell surface expression and intracellular interferon gamma production (figure 1C), and also triggered ADCC of TNBC cells and stromal CAFs. This dual cytotoxic effect in cancer and stromal cells following cath-D targeting was never described before (figure 1C). F1M1-Fc⁺ showed improved antitumor potency compared with F1M1, and triggered NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts in nude mice (figure 1C).²³ Following the F1M1-Fc⁺ treatment, the expression of CD107a (at the cell surface) and intracellular granzyme B was significantly increased in NK cells that infiltrated MDA-MB-231 cell xenografts (figure 1C). Remarkably, F1M1-Fc⁺ induced the tumor recruitment of immature CD27⁻CD11b⁻ and mature stage 1 CD27⁺CD11b⁻ NK cells undergoing activation, as indicated by the concomitant Eomes upregulation (figure 1C). Moreover, NK cell depletion, by treatment with the anti-asialo GM1 antibody, impaired the F1M1-Fc⁺ effect in nude mice harboring MDA-MB-231 cell xenografts, demonstrating the key role of tumor-infiltrating NK cells in F1M1-Fc⁺ antitumor activity. Recent studies and clinical trials underlined that NK cell-based immunotherapy can awaken the innate anticancer response, particularly against tumor metastases.²⁴ One hypothesis is that F1M1-Fc⁺ targets NK cells to primary tumors and metastases, sustaining dormancy as demonstrated in TNBC.²⁵ Importantly, F1M1-Fc⁺ ability to trigger ADCC in CAFs highlights its dual action in tumor and stromal cells to modulate the whole tumor architecture.

Combinatorial strategies with optimized anti-cath-D antibodies to tackle the TNBC microenvironment

In the clinic, antibody-based therapies are usually combined with chemotherapy, hormone therapy and/or other targeted therapies for TNBC management. In metastatic TNBC, the immune checkpoint inhibitor (ICI) pembrolizumab (anti-PD-L1 antibody) in association with chemotherapy shows improved overall and progression-free survival compared with chemotherapy alone.²⁶ Moreover, the androgen receptor (AR) inhibitor enzalutamide shows clinical activity and is well tolerated in patients with early and advanced AR⁺ TNBC.²⁷ Therefore, we assessed the added value of the F1M1-Fc⁺ lead antibody in combination with chemotherapy and hormonotherapy. F1M1-Fc⁺ improved paclitaxel and enzalutamide efficacy,²³ suggesting its clinical relevance in combination therapies (figure 1C). Comparison of the therapeutic efficacy of the anti-cath-D antibodies in the highly-immunogenic TNBC E0771 and the immune-excluded BC TUBO cell graft models suggests that their antitumor activity is immune cell-mediated.¹⁹ Therefore, they could be efficient in combination with ICI in “hot” tumors.¹⁹ The highly-immunogenic E0771 cell-based TNBC mouse model could be a tool of choice to assess the combination of F1M1 with anti-PD1 and anti-PD-L1 ICIs because E0771 cells express these checkpoint markers.²⁸

Conclusions and future challenges

Classically, antibody-based therapy targets a receptor overexpressed by cancer cells (eg, EGFR by cetuximab) or by stromal cells (eg, FAP by sibrotuzumab). Overall, in preclinical models of TNBC, anti-cath-D antibody-based therapy inhibits tumor growth, triggers the antitumor innate and adaptive immunity, and has a dual role on both cancer cells and CAFs through ADCC mechanisms, suggesting diverse effects on the whole tumor architecture.^{18 19 23} This could open new therapeutic avenues for designing smarter antibodies that target a soluble protumorous antigen specifically hypersecreted within the tumor and capable of interacting with cancer cells and also tumor stroma cells.

In parallel to the development of anti-cath-D antibodies, we analyzed cath-D expression in TNBC tissue microarrays to identify which patients could benefit from this novel targeted therapy in order to develop personalized medicine. AR⁺/cath-D⁺ co-expression identifies a subgroup of TNBC with a poor prognosis that could potentially be eligible for combination therapy with an anti-cath-D antibody.²⁹ Importantly, a new classification of TNBC using immunohistochemistry has been developed and can be easily used by hospitals, compared with genetic and genomic analyses that are difficult to use in routine practice, to stratify patients for selecting the best treatment combinations.³⁰ As anti-cath-D antibodies can improve chemotherapy and hormone therapy in TNBC²³ and have immunomodulatory activity, it could be interesting to test them in combination with ICIs.

Regarding the future positioning of anti-cath-D antibody-based therapy, naked antibodies could be used to overcome (or reduce) ICI-induced toxicity, as a second-line treatment after anti-PD-1/anti-PD-L1 antibodies in patients with metastatic TNBC, or in combination to synergize ICI effects for enhancing the patients’ immunological antitumor defenses. They could also be used in patients with PD-L1-negative (<1% PD-L1⁺ immune cells) metastatic TNBC for whom ICI-based immunotherapy is not indicated. Anti-cath-D antibodies could be used in combination with antiandrogens in patients with hormone-dependent (AR⁺/cath-D⁺) metastatic TNBC, or as a second-line treatment in (AR⁺/cath-D⁺) metastatic TNBC that has become resistant to hormone therapy.

Cath-D is upregulated, and abnormally secreted in the tumor microenvironment of many cancer types, including ovarian, pancreatic, melanoma, squamous cell carcinoma, hepatocarcinoma, prostate, glioblastoma and colorectal cancer, and displays tumor-promoting roles. Therefore, anti-cath-D therapy could also be tested in other tumor types, particularly the most aggressive cancers for which there is no effective targeted therapy and/or with high infiltrating stroma, particularly pancreatic cancer and prostate cancer. This strategy of using antibodies to target protumor proteins that are specifically hypersecreted in the tumor microenvironment and modulate tumor architecture, and therefore with a triple impact on cancer, stromal, and immune cells, could be relevant also for other hypersecreted proteins.

Ethics statements

Patient consent for publication

Not applicable.

Ethics approval

Mouse experiments were performed in compliance with the French regulations and ethical guidelines for experimental animal studies in an accredited establishment (Agreement No. #31135 2021042212479661). The study approval for PDXs was previously published. For immunohistochemistry, triple-negative breast cancer (TNBC) biopsy samples were provided by the biological resource center (Biobank number BB-0033-00059) after approval by the Montpellier Cancer Institute Institutional Review Board, following the French national Ethics and Legal dispositions for patients’ information and consent. For TNBC cytosols, patient samples were processed according to the French Public Health Code (law n°2004-800, articles L. 1243-4 and R. 1243-61), and the biological resources center has been authorized (authorization number: AC-2008-700; Val d’Aurelle, ICM, Montpellier) to deliver human samples for scientific research. All patients were informed before surgery that their surgical specimens might be used for research purposes. For human primary natural killer cell isolation and expansion, this work benefited from umbilical cord blood units and John De Vos’ expertise (head of the Biological Resource Center Collection of the University Hospital of Montpellier, http://www.chumontpellier.fr/en/platforms; BIOBANQUES Identifier: BB-0033-00031).

Footnote

PDdR and TD contributed equally.

Contributors PDdR, TD, AM, TC, and EL-C prepared the manuscript. PDdR, TD, AM, VL-M, PR, SG, TC, and EL-C proofread and finalized the manuscript. PDdR and TD contributed equally to this work. EL-C acted as guarantor.

Funding This work was supported by a public grant overseen by the French National Research Agency (ANR) as part of the "Investissements d’Avenir" program (reference: LabEx MabImprove ANR-10-LABX-53-01), ACCelerating Research for Early Development of Innovative Antibodies—ACCREDIA (ANR-22-PEBI-0009), University of Montpellier, Inserm Transfert, Région Occitanie, the association "Ligue Régionale du Gard CD30", "Ligue Régionale de l’Herault CD34", and "Ligue Régionale de la Charente Maritime CD17". This publication is based on work from COST Action ProteoCure, CA20113, supported by COST (European Cooperation in Science and Technology).

Competing interests No, there are no competing interests.

Provenance and peer review Not commissioned; externally peer reviewed.

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