# Cathepsins: Novel opportunities for antibody therapeutics in cancer

Timothée David, Pénélope Desroys Du Roure, Aude Mallavialle, Valérie Laurent-Matha, Pascal Roger, Séverine Guiu, Thierry Chardès, Emmanuelle Liaudet-Coopman

► To cite this version:

Timothée David, Pénélope Desroys Du Roure, Aude Mallavialle, Valérie Laurent-Matha, Pascal Roger, et al.. Cathepsins: Novel opportunities for antibody therapeutics in cancer. British Journal of Pharmacology, In press, 10.1111/bph.17437. hal-04903563

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1 Cathepsins: novel opportunities for antibody therapeutics in cancer

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3 Timothée David¹*, Pénélope Desroys du Roure¹*, Aude Mallavialle¹, Valérie Laurent-Matha¹, Pascal

4 Roger¹,², Séverine Guiu¹,³, Thierry Chardès**¹,⁴, Emmanuelle Liaudet-Coopman**#¹

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6 ¹IRCM, INSERM U1194, University of Montpellier, ICM, Montpellier, France.

7 ²Department of Pathology, CHU Nîmes, Nîmes, France.

8 ³Department of Medical Oncology, ICM, Montpellier, France.

9 ⁴CNRS, Centre National de la Recherche Scientifique, Paris, France

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11 #Corresponding author: E Liaudet-Coopman

12 IRCM - Inserm U1194

13 208, rue des Apothicaires

14 F-34298 Montpellier Cedex 5, France

15 E-mail: emmanuelle.liaudet-coopman@inserm.fr

16 *T David and P Desroys du Roure contributed equally to this work

17 **T Chardès and E Liaudet-Coopman contributed equally to this work

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# 1 List of Abbreviations

2 ADCC: antibody-dependent cellular cytotoxicity; cDC: conventional dendritic cells; DC: dendritic cells;

3 ECM: Extra-Cellular Matrix; Fab: Fragment antigen-binding; Fc: fragment crystallizable; Ig:

4 Immunoglobulin; IL-10: Interleukin-10; IL-15: Interleukin-15; mAbs: monoclonal antibodies; MDSC:

5 myeloid-derived suppressor cells; NK: natural killer cell; PDX: Patient-Derived Xenografts; RER:

6 rough endoplasmic reticulum; TAM: tumour-associated macrophages; TNBC: Triple-negative breast

7 cancer; scFv: single-chain variable fragment.

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ABSTRACT

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Cathepsins, the most abundant lysosomal proteases, have key functions in cell maintenance and homeostasis. They are overexpressed and hypersecreted in cancer and associated with poor prognosis.

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Secreted cathepsins display pro-tumour activities in the tumour microenvironment and thus, represent interesting molecular targets in oncology. Recently, several antibody-based cancer therapies have targeted the pro-tumour activity of the extracellular cathepsin pool, altering several cancer hallmarks, but not the intracellular cathepsin levels that are often crucial for cell homeostasis. In this mini-review, we describe advances in antibodies against extracellular cathepsins in cancer, and their effect on the proteolytic cascade, matrix remodelling, proliferation, and modulation of the anti-cancer immune response. We also discuss the add-on value of combination strategies (anti-cathepsin antibodies with chemotherapy and/or biologics) that make of anti-cathepsin antibodies a new opportunity for disease management.

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**Keywords:** cathepsins, antibody-based therapy, tumour microenvironment, proteolysis, ECM, cell signalling, immunomodulation, chemotherapeutic drugs, biologics.

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# 1. CATHEPSINS IN THE TUMOUR MICROENVIRONMENT AS TARGETS FOR ANTIBODY-BASED THERAPY

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Cathepsins are classified, based on their structure and catalytic type, into serine (cathepsins A and G), aspartic (cathepsins D and E), and cysteine cathepsins (cathepsins B, C, F, H, K, L, O, S, V, W, and X). Cathepsins, the most abundant lysosomal proteases, are mainly found in acidic endo/lysosomal compartments where they play a key role particularly in intracellular protein degradation, energy metabolism, and immune responses (Yadati, Houben, Bitorina, & Shiri-Sverdlov, 2020). In many tumour types, cathepsins are overexpressed, associated with poor prognosis, and involved in many processes, including invasion, migration, proliferation, and angiogenesis, immune evasion, apoptosis, and metastasis (Kang et al., 2020; Mansouri et al., 2020; Olson & Joyce, 2015; Rot, Hrovatin, Bokalj, Lavrih, & Turk, 2024; Wilkinson et al., 2019). There are several endogenous inhibitors, such as cystatins (Ochieng & Chaudhuri, 2010) and serpins (Maas & de Maat, 2021), that block the proteolytic activity of the pool of cathepsins overexpressed in tumours, with the exception of aspartic cathepsins. However, the limited quantities of these inhibitors lead to an imbalance in favour of dysregulated proteolysis. In cancer cells, cathepsin precursors are frequently hypersecreted in the extracellular space (Capony et al., 1989; Frosch, Berquin, Emmert-Buck, Moin, & Sloane, 1999; Troen, Ascherman, Atlas, & Gottesman, 1988) and reviewed by (Olson & Joyce, 2015; Yadati et al., 2020). Secreted cathepsins may play a crucial role in some immune cell types, particularly macrophages, that can modulate the tumour microenvironment (TME) (Gocheva et al., 2010; Ruiz-Blazquez et al., 2024). Secreted cathepsins can be partly tethered to the cell surface through interaction with cell surface receptors, such as mannose-6-phosphate/IGF-II receptor, low-density lipoprotein receptor-related protein 1 (LRP1), annexin 2 tetramer, integrin αvβ3, or through association with caveola site (Akkari et al., 2014; Beaujouin et al., 2010; Cavallo-Medved et al., 2003; Fusek & Vetvicka, 1994; Laurent-Matha et al., 1998; Lechner et al., 2006; Mai, Waisman, & Sloane, 2000). Secreted pro-cathepsins become proteolytically-active only after proteolytic removal of the pro-domain. This can be achieved autocatalytically or through the action of other proteases (Quraishi & Storer, 2001), and is promoted at acidic pH, such as the acidic tumour extracellular environment, or through pro-cathepsin interaction with negatively charged surfaces,
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including glycosaminoglycans and cell membrane components (Caglic, Pungercar, Pejler, Turk, & Turk, 2007; Zebrakovska et al., 2011). It is not entirely clear why cathepsins are often secreted as zymogens, but one plausible explanation is that the high expression level of cathepsins, and possibly other endolysosomal proteins, could saturate the conventional trafficking of lysosomal proteins via the mannose-6-phosphate receptor pathway, which in turn leads to aberrant trafficking and/or secretion of pro-cathepsins. Cathepsins can also be secreted in the active form by malignant and immune cells (Akkari et al., 2014; Reddy, Zhang, & Weiss, 1995; Rozhin, Sameni, Ziegler, & Sloane, 1994).

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The extracellular space around a developing tumour exposes cathepsins to many potential substrates, including Extra-Cellular Matrix (ECM) proteins, soluble proteins secreted by tumour and stromal cells, and the extracellular domains of transmembrane and membrane-associated proteins. In the TME, cathepsins can cleave and/or degrade various ECM and matricellular proteins, cytokines, growth factors, proteases and their inhibitors. They also regulate cell adhesion, migration and endocytosis, interfere with cytokine/chemokine signalling involved in the antitumour immune response, and promote cell invasion and extravasation, angiogenesis and metastasis (Pislar, Perisic Nanut, & Kos, 2015; Yadati et al., 2020). Besides their catalytic functions, cathepsins bind to cell surface receptors (Beaujouin et al., 2010; Laurent-Matha et al., 1998; Pranjol, Gutowski, Hannemann, & Whatmore, 2018) and affect cell signalling in the tumour stroma (Akkari et al., 2014; Masson et al., 2010; Pislar et al., 2015; Pranjol et al., 2018; Radisky, 2024).

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In this mini-review, we mainly focused on advances in antibody-based therapies against extracellular cathepsins to normalize the ECM architecture, reduce tumour cell proliferation/migration and tumour angiogenesis, and bypass tumour immunosuppression. We also discuss the interest of combining anti-cathepsin antibodies with chemotherapy and immunotherapy. Finally, we present new challenges for targeting intracellular cathepsins with intrabodies, antibody derivatives and mimetics in cancer.

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## 2. RECENT PRE-CLINICAL TRIALS ON ANTI-CATHEPSIN ANTIBODIES

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Designing a highly selective inhibitor for a given cathepsin is a challenge because it involves creating a molecule that binds specifically to the target cathepsin without cross-reacting with other closely related members of the cathepsin family. In addition, the relatively broad substrate specificity

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and cellular expression of cathepsins increase the full range of potential off-target effects and the risk of toxicity. Anti-cathepsin antibodies, due to their exquisite specificity, are suitable molecules to overcome these problems, which have been mainly observed with small molecules. Indeed, some clinical trials using small-molecule inhibitors of individual cathepsins showed lack of efficacy and side effects in autoimmune and neurological diseases, osteoporosis, metastatic bone disease and breast cancer (Brown et al., 2020; Kramer, Turk, & Turk, 2017; Petruzzella et al., 2024; Stone, McCrea, Witter, Zajic, & Stoch, 2019). This is probably explained by the low bioavailability, insufficient affinity and low selectivity of such small chemical drugs. Recently, inhibitory peptides selective to cysteine cathepsin-S, -B, -K, or -L were conjugated to anti-receptor antibodies (anti-CD22, anti-CD19, anti-HER2 (human epidermal growth factor receptor 2), or anti-SIGLEC-15 ((sialic acid-binding Ig-like lectin 15)) for intracellular cell-type-specific delivery of covalent inhibitors blocking the intracellular pool of cysteine cathepsins (B cell lymphoma, breast cancer, osteoclasts or cancer cells) (Petruzzella et al., 2024). In this case, antibodies are used as trojan horse, unless they bind to a membrane receptor that mediates their endocytosis and favours intracellular delivery of cathepsin-specific inhibitory peptides. Another strategy could be the development of antibodies directly targeting cathepsins, who mainly affect the extracellular pool of the cathepsins, therefore overcoming the toxicity of cell-permeable chemical drugs or intracellularly delivered inhibitory peptides (Mijanovic et al., 2021).

In cancer research, extracellular cathepsins are considered primary targets for antibody-based therapies, due to their protumour activity, with the aim of normalizing the tumour stroma and ECM extracellular proteome, reducing tumour cell proliferation/migration and tumour angiogenesis, and restoring the antitumour immune response (Ashraf et al., 2019; Burden et al., 2009; Olson & Joyce, 2015; Petushkova, Savvateeva, Korolev, & Zamyatnin, 2019; Radisky, 2024). However, cathepsin-specific antibodies have not been tested in clinical trials yet. Several studies showed that the catalytic activity of several mature cathepsins can be inhibited by their pro-peptides (Fox, de Miguel, Mort, & Storer, 1992; Fusek, Mares, Vagner, Voburka, & Baudys, 1991; Guay, Falgueyret, Ducret, Percival, & Mancini, 2000). Recombinant cathepsin-S, -L, -K and -B pro-peptides, alone (Guay et al., 2000) or fused with antibodies used in the clinic (Burden et al., 2008; Dai, Cheng, & Zhang, 2020; Shi & Zhang, 2020;
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Yu, Kao, Dai, Nasertorabi, & Zhang, 2024), inhibit the proteolytic activity of their parent mature enzyme
in the nanomolar range (Table 1).

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Targeting secreted cathepsins, particularly the aspartic cathepsin-D, with fully specific
antibodies was first proposed in the late 1990s (Table 1). These antibodies act mainly by inhibiting
cathepsin proteolytic activity and therefore by (i) remodelling the matrix architecture, (ii) inhibiting
tumour cell angiogenesis, invasion, and proliferation, (iii) modulating the anti-tumour immunity, and
(iv) improving drug combinations in various diseases (Figure 1). Mouse monoclonal antibodies (mAbs)
against the whole (aa 1-44) or part (aa 38-44 and aa 27-44) of human cathepsin-D pro-peptide inhibited
*in vitro* proliferation and *in vivo* growth of human prostate, breast and lung cancer cells (Vetvicka,
Vetvickova, & Fusek, 1999, 2000). These antibodies do not inhibit the proteolytic activity of the
enzyme, but might compete with pro-cathepsin-D for the binding to its mitogenic cell surface receptor
(Table 1). Two murine anti-human cathepsin-D mAbs, D8F5 and M2E8, partially inhibited cathepsin-
D proteolytic activity on endothelial extracellular matrices at pH 5 (1,000-fold excess of antibody over
pro-cathepsin-D) (Freiss, Vignon, & Rochefort, 1988; Garcia et al., 1985) (Table 1). However, their
therapeutic effects were not assessed *in vivo*. Conversely, our novel fully-human anti-cathepsin-D
antibody F1, selected from a human scFv expression library by phage display, displayed strong
therapeutic antitumour efficacy (Ashraf et al., 2019) (Table 1). The F1 antibody localized at the tumour
site, inhibited triple-negative breast cancer (TNBC) tumour growth (MDA-MB-231
(RRID:CVCL_0062) and Patient-Derived Xenografts (PDX)) and prevented the recruitment of myeloid
M2-like tumour-associated macrophages (TAM) and myeloid-derived suppressor cells. This was
associated with natural killer (NK) cell activation through *interleukin-15* (IL-15) upregulation,
*granzyme B* and *perforin* induction, and *IFN-γ* release (Ashraf et al., 2019) (Figure S1). Therefore,
cathepsin-D is a prime target for anti-tumour therapies with antibodies that also have
immunomodulatory anti-tumour activities (Ashraf et al., 2019). In agreement, in immunocompetent
C57BL/6 (RRID:MGI:2159769) mice grafted with a murine TNBC model (E0771
(RRID:CVCL_GR23)) (David et al., 2023), the surrogate anti-cathepsin-D mouse IgG2a antibody F1
and its improved version F1M1 with aglycosylated Fab inhibited tumour growth (David et al., 2023)
(Figure S2). In this highly immunogenic mouse model, F1 and F1M1 remodelled the tumour immune
and cytokine landscape by triggering the innate antitumour immunity via NK cell activation and M2-TAM depletion, and by inducing the adaptive antitumour immunity, via activation of antigen-presenting cell functions (M1-polarized CD86⁺ TAMs and conventional CD86⁺ type 1 dendritic cells), resulting in a reduction of PD-L1⁺ tolerogenic T cells and the LAG3 and TIGIT exhaustion markers (David et al., 2023) (Figure S2). Next, F1M1 was Fc-optimized (by introducing the Fc mutations S239D, H268F, S324T and I332E) to increase its affinity for Fcγ receptors and its Fc-dependent cytotoxic activity (F1M1-Fc⁺ antibody) (Desroys du Roure et al., 2024) (Figure S3). *In vitro*, F1M1-Fc⁺ potently activated NK cells, with increased CD107a cell surface expression and intracellular IFN-γ production, and triggered antibody-dependent cellular cytotoxicity (ADCC) in TNBC cells and cancer-associated fibroblasts (CAFs). F1M1-Fc⁺ showed enhanced *in vivo* antitumour potency in TNBC models (MDA-MB-231, SUM159 (RRID:CVCL_5423) and PDXs) and triggered NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts in nude mice. Following F1M1-Fc⁺ treatment, the expression levels of CD107a (at the cell surface) and intracellular granzyme B significantly increased in NK cells within MDA-MB-231 cell xenografts. Remarkably, F1M1-Fc⁺ induced the tumour recruitment of immature CD27⁻CD11b⁻ and mature stage 1 CD27⁺CD11b⁻ NK cells undergoing activation. NK cell depletion with the anti-asialo GM1 antibody impaired F1M1-Fc⁺ therapeutic efficacy, showing their key role in its antitumour activity. Of note, F1M1-Fc⁺ capacity to trigger ADCC in CAFs highlights its dual action in tumour and stromal cells to modulate the overall tumour architecture. Finally, in combination therapy, F1M1-Fc⁺ improved the therapeutic index of paclitaxel and enzalutamide in TNBC (Desroys du Roure et al., 2024) (Figure S3), paving the way for future combination strategies or targeted delivery of chemotherapies using cathepsin-D-specific antibodies, as initially proposed by others for mouse antibody-drug conjugates (Table 1). Overall, these results showed that immunotherapy with fully-human anti-cathepsin-D antibodies affects cancer cells, CAFs, immune cells and cytokines in the TME.

Cathepsin potential role in modulating the immune system was also underlined by the anti-serine cathepsin-G scFv-Fc antibody LKAb (Han et al., 2016) (Table 1). LKAb, selected from a combinatorial antibody library and expressed in the scFv-Fc format, induced *in vitro* CD14/NFAT-mediated myeloid
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M2 macrophage differentiation, and p38, AKT and ERK activation in mouse bone marrow cells (Han et al., 2016).

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Moreover, the anti-cysteine cathepsin-S antibody Fsn0503 and derivatives (Table 1) induced NK cell-mediated destruction of colorectal and pancreatic cancer cells (Kwok et al., 2011). The mouse mAb Fsn0503 and the humanized Fsn0503h (Table 1) inhibited tumour cell invasion and proliferation through blockade of extracellular cathepsin-S-mediated proteolysis and ECM degradation, and reduced angiogenesis, leading to the *in vivo* inhibition of colorectal cancer cell xenograft growth and neovascularization (Burden et al., 2009; Burden et al., 2012; Kwok et al., 2011; Vazquez et al., 2015). The combination of Fsn0503 with anti-VEGF therapy induced a synergistic anti-tumour effect (Ward et al., 2010) and with irinotecan improved anti-tumour efficacy in colorectal cancer (Burden et al., 2012) (Table 1). These results emphasize the potential of anti-cathepsin antibodies in combination to increase anti-tumour efficacy. Moreover, the improved affinity of Fsn0503h for cathepsin-S, by engineering from a novel library of antibody variants (WO/2020/084298) (Table 1), underlines the anti-cathepsin antibody versatility for drug design.

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Moreover, several antibodies against cysteine cathepsin-B inhibited its proteolytic activity, ECM degradation and cell invasion (Table 1). The mouse mAb 2A2, selected by hybridoma technology, binds to an epitope near the active site of cathepsin-B. This antibody inhibited cathepsin-B proteolytic activity, and decreased invasion of RAS-transformed MCF-10A (RRID:CVCL_0598) human breast epithelial cells, at the concentration of 500 nM (Fan et al., 2002; Premzl, Zavasnik-Bergant, Turk, & Kos, 2003). The authors mentioned that 2A2 could impair the activity of both the intracellular and extracellular pools of cathepsin-B because it may be internalized by cancer cells. The 2A2 epitope was localized in the EPGYSP sequence, between amino acid residues 133 and 138 of cathepsin-B (Mirkovic et al., 2009). The 2A2 antibody inhibited proteolysis of protein substrates, casein and collagen IV, at the concentration of ~700nM. Interestingly, 2A2 induced cathepsin-B dissociation from its endogenous inhibitor cystatin C (Mirkovic et al., 2009).

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Compared with small-molecule inhibitors, mAbs are attractive because they offer the required specificity. However, the search for inhibitory antibodies that act at concentrations within the low nanomolar range is often challenging, mainly due to the lack of a function-based selection method.

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Recently, a highly efficient selection method using synthetic human Fab antibody libraries was developed to generate cathepsin-B inhibitory antibodies by co-expressing, in the periplasmic space of *Escherichia coli*, a Fab antibody clone, cathepsin-B and β-lactamase modified by insertion of a cathepsin-B cleavable peptide sequence (Lopez et al., 2019). During the functional selection, inhibitory antibodies prevented cathepsin-B-mediated cleavage of the modified β-lactamase, thereby allowing cells to survive in the presence of ampicillin. Using this method, the authors could isolate the Fabs CBA3, CBA2, and CBA1 and showed that they inhibit cathepsin-B proteolytic activity on the synthetic peptide substrate KLHFSKQ (Ki of 91 nM, 120nM, and 210 nM, respectively) (Lopez et al., 2019).

Other anti-cathepsin antibodies have been used only for diagnostic purposes or detection in physiological fluids, for instance the anti-cathepsin-B mouse mAbs 3E4, 4H9 and 8D7 that bind to the VVAGIPR, EPGYSP, and DIMAEIYKNG sequences of cathepsin-B, respectively, as determined with the SPOT method (Weber et al., 2015).

Intrabodies, antibody derivatives and mimetics have been developed for intracellular delivery in order to target the activity of intracellular cathepsins. The 3D8 scFv, which interacts with an epitope localized on the 156-197 amino acid sequence of cathepsin-L was ectopically expressed as a fusion protein in the Rough Endoplasmic Reticulum (RER) through a RER signal (peptide SEKDEL). This scFv inhibited pro-cathepsin-L secretion from melanoma cells (Guillaume-Rousselet, Jean, & Frade, 2002), and reduced *in vitro* invasion and *in vivo* angiogenesis and increased apoptosis of melanoma cells (Rousselet et al., 2004) (Table 1). Among non-Ig structures, shark-derived variable new antigen receptors (vNARs) targeting cathepsin-S inhibited its catalytic activity and prevented the activation of pro-cathepsin-S-to the mature enzyme (Smyth et al., 2023) (Table 1). When intracellularly expressed using an intrabody format, anti-cathepsin-S vNARs reduced glioblastoma cell invasion. These molecules are attractive for therapy due to their small size, remarkable stability and unusual structure (i.e. protruding extended loops that bind to difficult-to-access cryptic epitopes, such as enzymatic cavities). However, the main challenges for developing intrabodies against cathepsins as therapeutics are their intracellular delivery mode, their efficacy and cell type selectivity. Lastly, the ankyrin repeat protein DARPin 8h6, selected by ribosome display (Kramer, Renko, et al., 2017), inhibited cathepsin-
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B proteolytic activity and reduced tumour cell invasion. Altogether, these findings demonstrate that cathepsins are targets for antibody-based therapies in cancer (Table 1).

### 3. CONCLUDING REMARKS

Cathepsins are tumour antigens that are abnormally released by cancer cells and TAMs in the TME, affecting the ECM proteome. Preclinical trials have shown the therapeutic effects of cathepsin-specific antibodies in cancer and stromal cells to re-normalize the ECM architecture, inhibit cancer cell proliferation and invasion, reduce tumour angiogenesis, circumvent tumour immunosuppression, and prevent tumour progression and metastasis.

### 4. NOMENCLATURE OF TARGETS AND LIGANDS

Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY http://www.guidetopharmacology.org, and are permanently archived in the Concise Guide to PHARMACOLOGY 2021/22 (Alexander et al., 2021).

### AUTHOR CONTRIBUTIONS

ELC, TC, AM prepared the manuscript. TD, PDR, AM, VLM, PR, SG, TC, ELC proof-read the manuscript.TC, and ELC finalized the manuscript.

### ACKNOWLEDGMENTS

This work was supported by a public grant overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" programme (reference: LabEx MabImprove ANR-10-LABX-53-01) and of the "France 2030" programme (reference: PEPR-BBIT ACCREDIA ANR-22-PEBI-0009), University of Montpellier, Inserm Transfert, Région Occitanie, Ligue Nationale contre le Cancer, the associations Ligue Régionale du Gard, Ligue Régionale de l'Herault, and Ligue Régionale de la Charente Maritime.
1 CONFLICTS OF INTEREST STATEMENT

2 No potential conflicts of interest were disclosed by all authors.

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4 DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

5 This Declaration acknowledges that this paper adheres to the principles for transparent reporting and
6 scientific rigour of preclinical research as stated in the *BJP* guidelines for *Natural Products Research*,
7 *Design and Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation*, and as
8 recommended by funding agencies, publishers and other organisations engaged with supporting
9 research.

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11 DATA AVAILABILITY STATEMENT

12 N/A. Review

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14 ORCID

15 Timothée David: https://orcid.org/0000-0003-0581-2406

16 Pénélope Desloys du Roure: https://orcid.org/0009-0000-0848-5450

17 Aude Mallavialle: https://orcid.org/0000-0002-9380-6788

18 Valérie Laurent-Matha: https://orcid.org/0000-0002-3277-7397

19 Pascal Roger: https://orcid.org/0000-0002-4040-5892

20 Séverine Guiu: https://orcid.org/0000-0002-4901-6295

21 Thierry Chardès: https://orcid.org/0000-0002-1836-7439

22 Emmanuelle Liaudet-Coopman: https://orcid.org/0000-0001-9313-9690

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**Figure 1. Cellular, molecular and tissue processes affected by antibodies targeting cathepsins.**

Antibodies targeting cathepsins can modulate proteolytic activity, matrix remodelling, tumour angiogenesis, invasion and proliferation, and can bypass tumour immunosuppression, leading to inhibition of tumour progression and metastasis. Moreover, anti-cathepsin antibody-based therapy can enhance the therapeutic effects of chemotherapies and biologics. Created with BioRender.com.

**Antibodies targeting cathepsins**

<figure><img src="image_2.png" /></figure>

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# 1 Table 1. Cathepsin-specific antibodies<sup>a</sup>.

<table><caption>TABLE 1. Cathepsin-specific antibodies<sup>a</sup></caption><thead><tr><th>Target</th><th>Antibody</th><th>Isotype</th><th>pH dependence<sup>b</sup></th><th>Binding parameter<sup>c</sup></th><th>Characteristics and biological functions</th><th>References/company</th><th>Patents</th></tr></thead><tbody><tr><td rowspan="2">Cathepsin D</td><td>M2E8</td><td>mIgG1</td><td>Affected at pH 4.7</td><td>ND</td><td>Inhibition of ECM degradation and proteolytic activity of proCathD</td><td>Garcia et al. Cancer Res 1985</td><td></td></tr><tr><td>D8F5</td><td>mIgG1</td><td>Not affected</td><td>ND</td><td>D8F5, but not M2E8, prevented proCathD binding and endocytosis<br>D8F5, but not M2E8, inhibited proliferation of CathD transfectants</td><td>Freiss et al. Cancer Res. 1988<br>Glondu et al. Oncogene 2001</td><td></td></tr><tr><td rowspan="2">Ab against<br>44aa-pro-CathD</td><td></td><td>mIgG1</td><td>ND</td><td>ND</td><td>Inhibition of proliferation of the breast cancer cell line MDA-MB231<br>Inhibition of LncAP prostate tumour growth in vivo<br>Inhibition of MDA-MB231 cell tumour growth in vivo<br>Inhibition of proliferation and invasion of the keratinocyte cell line HaCaT<br>Inhibition of proliferation of the lung cancer cell line NCI-H23</td><td>Vetvicka et al. Breast Cancer Res Treat 1999<br>Vetvicka et al. Prostate 2000<br>Vetvicka et al. Cancer Gene Ther 2002<br>Vashista et al. Eur Cell Biol 2007<br>Vetvicka et al. Anticancer Res 2011</td><td></td></tr><tr><td>F1</td><td>mIgG1</td><td>Not affected<br>(pH range 5.5-7.5)</td><td>0.2nM</td><td>Inhibition of MDA-MB231 cell and TNBC PDX tumour growth in vivo<br>Prevention of immunosuppressive M2-like TAM and MDSC recruitment<br>induction of NK cell activation through IL-15 upregulation<br>induction of granulocyte and perforin production and release of IFN-γ</td><td>Ashraf et al. JTC 2019</td><td>WO/2016/188911<br>WO/2020/127411</td></tr><tr><td>F1M1</td><td>mIgG1</td><td>Not affected<br>(pH range 5.5-7.5)</td><td>0.04-0.07nM</td><td>Two-point mutations in VL-CDR3 and VH-FR3 of F1<br>NK cell activation and ADCC induction on both TNBC cells and CAFs<br>Inhibition of MDA-MB231 cell tumour growth in vivo</td><td>Desroys du Roure et al. JTC 2024</td><td>EP23305789.2</td></tr><tr><td>Surrogate F1M1</td><td>mIgG2a</td><td>Not affected<br>(pH range 5.5-7.5)</td><td>0.9nM</td><td>Mouse IgG2a reformatting of F1M1<br>In vivo tumour growth inhibition of the EQ771 TNBC mouse model<br>Prevention of immunosuppressive M2-like TAMs<br>Activation of M1-like TAMs, CD1 and NK cells<br>Reduction of the T-cell exhaustion markers PD1L, TIGIT and LAG3</td><td>David et al. Brit J Pharmacol 2023</td><td>EP23305789.2</td></tr><tr><td>F1M1-Fc+</td><td>mIgG1</td><td>Not affected<br>(pH range 5.5-7.5)</td><td>0.048nM</td><td>S239D H268F S234T B32E Fc mutations of F1M1<br>Increase of NK cell activation and ADCC induction on both TNBC cells and CAFs<br>Inhibition of MDA-MB231, SUM159 cell and PDX TNBC tumour growth in vivo<br>Improved the therapeutic efficacy of enzalutamide and paclitaxel</td><td>Desroys du Roure et al. JTC 2024</td><td>EP23305789.2</td></tr><tr><td>IMB101 ADC<br>IMB102 ADC</td><td>mIgG1<br>mIgG1</td><td>ND<br>ND</td><td>8.5-63nM<br>1.0-4.0nM</td><td>DM1-stable ADC-inhibition of cell viability (MDA-MB231, MCF7)<br>DM4-labile ADC-inhibition of cell viability (MDA-MB231, MCF7, LNCaP)</td><td>ImmunoBiochem Corp</td><td>WO/2016/145536</td></tr><tr><td>Cathepsin S</td><td>rCatSPP-Fc</td><td>mIgG2b</td><td>ND</td><td>8.9nM (kI)</td><td>Fusion of Caths pro-peptide (Q17-R113) with mouse IgG2b Fc (CH2-CH3)<br>Inhibition of Caths proteolytic activity<br>Inhibition of tumour cell invasion (MDA-MB231)</td><td>Burden et al. Mol. Cancer Ther. 2008</td><td></td></tr><tr><td>Her-CTC-CTSSpp<br>Syn-LC-CTSSpp</td><td>mIgG1<br>Fab</td><td>ND<br>ND</td><td>6.03nM (kI)<br>2.12nM (kI)</td><td>2.8nM</td><td>Fusion of the Caths pro-peptide Q17-R113 in place of W99-M107 CDR3 herceptin<br>Fusion of the Caths pro-peptide Q17-R113 into the N-terminus of synagis LC<br>Inhibition of Caths proteolytic activity</td><td>Yu et al. Arch Biochem Biophys 2024</td><td></td></tr><tr><td>Fsn0503 (Ie11)</td><td>mIgG1</td><td>Affected at pH 4.9</td><td>2.8nM</td><td>Inhibition of tumour cell invasion (HCT116, MDA-MB231, PC3)<br>Blockade of Cath5-mediated proteolysis<br>Blockade of HUVEC angiogenesis and reduction of tumour angiogenesis<br>Inhibition of ECM degradation/proteolysis (U251MG cells, HUVEC)<br>Synergistic combination with anti-VEGF Ab to inhibit HUVEC angiogenesis<br>The Fsn0503/irinotecan combination better inhibited HCT16 tumour cell growth<br>Cath5 was upregulated by chemotherapy (HCT116, LoVo, Colo205)<br>Irinotecan efficiency enhanced by the combination with Fsn0503 in HCT116 cells</td><td>Burden et al. Clin Cancer Res 2009<br>Ward et al. PLoS ONE 2010<br>Burden et al. Biochimie 2012</td><td>WO/2006/109045<br>WO/2008/044076<br>WO/2010/094981<br>WO/2007/128987</td></tr><tr><td>Fsn0503h<br>(humanized<br>format)</td><td>mIgG1</td><td>ND</td><td>3.0-5.4nM</td><td>Induction of NK cell-targeted killing of LoVo and Colo205 cancer cells<br>PK profile with terminal half-life of 274hr (11.4 days)<br>Inhibition of tumour cell invasion and proliferation (MDA-MB361, Colo205)<br>Reduction of growth of Colo205 colorectal cancer cell xenografts in vivo</td><td>Kwok et al. Mol. Cancer 2011<br>Vasquez et al. Biochimie 2015</td><td>WO/2010/094981</td></tr><tr><td>Fsn0503h variants</td><td>mIgG1</td><td>ND</td><td>up to 0.35nM</td><td>Improved affinity of Fsn0503h variants derived from a novel antibody library</td><td>Fusion Antibodies Ltd</td><td>WO/2020/084298</td></tr><tr><td>clone 3, clone 5<br>intrabodies</td><td>vNAR</td><td>Not affected<br>(pH range 4.0-7.2)</td><td>344-183nM</td><td>Inhibited activation of pro-Caths to the mature enzyme<br>Intracellularly reduced invasion of U251 glioblastoma cells</td><td>Smyth et al. Front Pharmacol 2023<br>Elasmogen Ltd</td><td></td></tr><tr><td>Cathepsin L</td><td>Syn-LCNT-CTSLpp</td><td>mIgG1</td><td>ND</td><td>5.4nM<br>0.09nM (kI)</td><td>Fusion of Caths pro-peptide T18-Y112 into the N-terminus of synagis LC<br>Inhibition of Caths proteolytic activity</td><td>Shi et al. Protein Sci 2020</td><td></td></tr><tr><td>3D8</td><td>mIgG1<br>mScFv</td><td>ND<br>ND</td><td>6nM<br>50nM</td><td>Reduction of growth of melanoma cells<br>Inhibition of proCathL secretion by melanoma cells</td><td>Rousselet et al. Cancer Res 2004<br>Rousselet et al. Biochem. J. 2002</td><td>WO/2004/020574</td></tr><tr><td>C716</td><td>mIgG</td><td>ND</td><td>ND</td><td>Inhibition of proliferation of the hepatocarcinoma cell line 5MMC-7721</td><td>Guangzhou Daiwei Tech. Co. Ltd</td><td>CN115806823</td></tr><tr><td>Cathepsin B</td><td>CT58 IgG</td><td>mIgG1</td><td>ND</td><td>191nM (kI)</td><td>Fusion of the Cath 8 pro-peptide R18-L87 in place of W99-M107 CDR3 herceptin<br>Inhibition of Caths proteolytic activity</td><td>Dai et al. Biochem. 2020</td><td>WO/2017/173359</td></tr><tr><td>2A2</td><td>mIgG2a</td><td>Not affected<br>(pH range 4.5-7.1)</td><td>2.7-4.7nM</td><td>Inhibition of Caths proteolytic activity and cell invasion (MCF10A)<br>Dissociation of the Caths/cystatin 8 complex - Lysosomal internalization<br>Inhibition of ECM degradation</td><td>Fan et al. Biol. Chem. 2002<br>Premzi et al. Exp. Cell. Res. 2003<br>Mirkovic et al. FEBS J. 2009</td><td>WO/2002/094881</td></tr><tr><td>CBA3<br>CBA2<br>CBAL</td><td>hFab<br>hFab<br>hFab</td><td>ND</td><td>16nM<br>200nM<br>300nM</td><td>Functional enzymatic selection in E. coli bacteria<br>co-expressing Fab plasmids, CathB and synthetic peptide substrates<br>Inhibition of Caths proteolytic activity</td><td>Lopez et al. Proc Natl Acad Sci USA 2019</td><td>US2019628509</td></tr><tr><td>DARPin Bh6</td><td>ND</td><td>ND</td><td>0.031nM<br>0.034nM (kI)</td><td>Selection by ribosome display<br>Inhibition of tumour cell invasion - Tumour cell imaging<br>Inhibition of CathB proteolytic activity</td><td>Kramer et al. Theranostics 2017</td><td></td></tr><tr><td>3E4<br>4H9<br>8D7</td><td>ND</td><td>ND</td><td>ND</td><td>Binding to Cath B by western blotting (3E4) and immunofluorescence (4H9, 8D7)<br>EpiTOPs identified by spot</td><td>Weber et al. Biol. Chem. 2015</td><td></td></tr><tr><td>Cathepsin G</td><td>LKAb</td><td>hScFv-Fc</td><td>ND</td><td>ND</td><td>ScFv selection from a library<br>Induction of CD14/NFAT-mediated M2 macrophage differentiation<br>Activation of p38, AKT and ERK in mouse bone marrow cells<br>Induction of IL-10 production in vivo<br>In vivo reduction of SLE autoimmunity</td><td>Han et al. FASEB J. 2016</td><td>WO/2017/048629</td></tr></tbody></table>

<footnote><sup>a</sup> Abbreviations: mIgG1, murine IgG1; ND, not done; CathD, cathepsin D; mIgG1, human IgG1; PDX, patient's derived xenograft; TNBC, triple negative breast cancer; TAM, tumour-associated macrophages; MDSC, myeloid-derived suppressor cells; NK, Natural Killer; IL-15, Interleukin 15; IFN-γ, interferon gamma; VL, variable light chain; CDR3, complementarity-determining region 3; VH, variable heavy chain; FR3, framework region 3; ADCC, antibody-dependent cell cytotoxicity; CAF, cancer-associated fibroblasts; mIgG2a, murine IgG2a; CD1, conventional type 1 dendritic cells; ADC, antibody-drug conjugate; DAR, drug to antibody ratio; Ki, inhibitor constant; Cath5, cathepsin S; CH2, constant heavy chain domain 2; CH3, constant heavy chain domain 3; LC, light chain; HUVEC, human umbilical vein endothelial cells; ECM, extracellular matrix; VEGF, vascular endothelial growth factor; PK, pharmacokinetic; VNAR, variable new antigen receptor; CathL, cathepsin L; mScFv, murine single-chain fragment variable; hScFv-Fc, human single chain fragment variable-fragment crystallisable; IL10, interleukin 10; SLE, systemic lupus erythematosus; CathB, cathepsin B; hFab, human fragment antigen binding; DARPin, designed ankyrin repeat protein inhibitor.</footnote>

<footnote><sup>b</sup> When available, the pH range tested in the study is added.</footnote>

<footnote><sup>c</sup> The binding parameter measured is sometimes the inhibitory constant (kI), when available (as indicated between brackets). If this information was not indicated in the study, the binding parameter measured is the dissociation constant (kD).</footnote>
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<figure><img src="image_3.png" /><figcaption>Anti-cathepsin-D F1 human IgG1 antibody modulates immune cell activation and recruitment in tumors</figcaption></figure>

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