A novel complement-fixing IgM antibody targeting GPC1 as a useful immunotherapeutic strategy for the treatment of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a survival rate of less than 10% at 5 years and represents a major unmet medical need [1]. Unfortunately, the majority of PDAC patients are diagnosed when surgery is not possible and the only therapeutic option remains chemotherapy [2]. Gemcitabine is the most commonly used agent; its administration has also been suggested in combination with albumin-linked paclitaxel [3]. FOLFIRINOX (5-Fluorouracil, Leucovorin, Irinotecan, and Oxaliplatin), as an alternative chemotherapeutic combination strategy, has been shown to be effective in metastatic disease [3]. Nevertheless, the use of chemotherapeutic agents has generally shown only a modest improvement in survival, but is often associated with severe toxicity events [3].

The ability to distinguish cancer cells from healthy tissues is the main goal of immunotherapeutics, which selectively kill tumor cells and reduce toxicity events through targeted activation of the immune system [2, 3]. To this end, monoclonal antibodies (mAbs) have been approved for several types of solid cancers. Antibody-based immunotherapy for the treatment of PDAC, targeting different tumor-associated antigens (TAA), has been proposed, but its efficacy has thus far proven limited [4]. Problems to overcome with this therapeutic approach are the insufficient activation of the immune system but also the immunosuppressive state of the PDAC microenvironment as well as its high content of desmoplastic tissue, leading to impaired drug delivery [4, 5].

One of the mechanisms of action exploited by therapeutic mAbs is complement-dependent cytotoxicity (CDC), which involves activation of the classical pathway of the complement system (CS). The CS can kill cancer cells directly, but can also recruit effector cells of the immune system that contribute to the killing of cancer cells via antibody-dependent cytotoxicity (ADCC) or phagocytosis [6, 7]. The CS has a clear advantage over cytotoxic cells as a defense system, as it consists of soluble molecules that can easily reach and diffuse into the tumor mass; it may be particularly important in the context of PDAC desmoplastic tissue. In addition, the components of CS are readily available as a first line of defense as they are locally synthesized by many cell types, such as macrophages, fibroblasts, and endothelial cells. Several neoplastic cells have also been shown to synthesize and secrete components of CS [8]. Direct killing of tumor cells by the membrane attack complex is one of the mechanisms used by CS to control tumor growth. However, CS can also exert its antitumor activity through additional non-cytotoxic effects. For example, C3b deposited on tumor cells promotes the binding and activation of effector immune cells, including phagocytes and natural killer (NK) cells expressing complement receptor 3 (CR3 -CD11b-CD18), resulting in complement-dependent cell cytotoxicity (CDCC) [7].

Although the IgG isotype represents the majority of mAbs approved for cancer immunotherapy and their activity is usually also associated with activation of the CS, the IgM isotype may be a better alternative due to its higher avidity for the target and because it is the most efficient CS activator [9, 10]. Indeed, the multimeric IgM exploits the proximity of multiple Fc that can efficiently bind and activate C1, the first component of the classical pathway of the CS cascade [6, 7, 9], which eventually induces CDC, recruits inflammatory cells such as macrophages and NK cells and also causes CDCC [7, 11, 12]. In this context, it is of interest that several mAbs of the IgM isotype have been investigated in recent phase I clinical trials and showed promising antitumor activity [13‐17].

Among the various TAA, one of the possible candidates is glypican-1 (GPC1), which is highly expressed in PDAC tumor tissues and is not expressed or is expressed at very low levels in normal pancreatic tissue and in chronic pancreatitis [18‐20]. GPC1 is expressed in the embryo, where it is essential for development, but its expression is very limited in most adult tissues [21, 22]. GPC1 is a cell surface proteoglycan composed of the first 23 amino acids representing the secretory signaling peptide, the N-terminal region localized between amino acids 24 and 474, and the C-terminal region localized between amino acids 475 and 530, and has a total molecular weight of 62 kDa [23]. The GPI anchor linked with the C-terminal region is essential for the binding to the cell membrane [23].

As for the functional aspect, GPC1 could represent an interesting TAA to target with immunotherapeutics, also because it is associated with several growth factors such as fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), heparin-binding EGF-like growth factor (HB-EGF) and transforming growth factor-β (TGF-β), which are involved in cancer cell proliferation, angiogenesis and metastasis [24]. Considering the potential impact on interactions with the tumor microenvironment, the expression of GPC1 has also been observed in cancer-associated fibroblasts (CAF) involved in stroma formation, which is associated with an immunosuppressive state that supports cancer progression [24].

The high expression of GPC1 on the surface of PDAC cells, its involvement in tumor progression and its role in the immunosuppressive tumor microenvironment prompted the development of a novel anti-GPC1 IgM mAb (AT101) for the treatment of PDAC patients. Unlike other antibodies against the same target, we focused on an epitope as close to the cell membrane as possible to maximize the activation of CS on tumor cells. Indeed, it has already been shown that several factors may play an important role in promoting more efficient CS by therapeutic mAbs, including the proximity of the target epitopes to the cell surface [25]. In the present study, we demonstrated that AT101 can be readily produced and purified; it specifically recognizes PDAC cells, both in-vitro and in a subcutaneous mouse PDAC xenograft model in athymic nude mice. AT101 leads to potent local activation of CS on the cell surface, causing lysis of cancer cells and recruitment of NK cells and macrophages to the tumor microenvironment, ultimately leading to a significant reduction in PDAC tumor growth and increasing survival of all treated mice.

In the present study, a novel anti-GPC1 antibody (i.e. AT101) was characterized for the treatment of PDAC. We demonstrated that AT101 is able to: (i) specifically recognize GPC1-expressing cells; ii) specifically accumulate in the tumor microenvironment of a PDAC xenograft mouse model; iii) elicit a very strong immune response; iv) control tumor growth in the xenograft mouse model of PDAC.

PDAC represents one of the most malignant tumor types. In most patients, surgery, the only curative option, is not suitable because the disease is already in an advanced stage at the time of diagnosis [32‐35]. At this stage, the therapeutic proposal remains the use of chemotherapy, which unfortunately is not very efficient, due to chemoresistance events, and because it is unable to converge the cytotoxic effect only on the tumor cells, causing frequent and severe side effects [2, 3, 35, 36]. In this scenario, the use of immunotherapeutic agents, such as mAbs, could ensure that antitumor activity is concentrated on tumor cells, reducing the side effects, generally caused by conventional therapy [37, 38]. In the context of PDAC the introduction of mAbs has not provided benefits for the patients. This failure could be ascribable to the immunosuppressive tumor microenvironment and to the desmoplasia condition responsible of an impairment in drug delivery [4, 5] but it could also depend from the target and the type of antibody that was respectively chose and developed. In the present study, GPC1 was defined as a specific PDAC TAA with the following characteristics: (i) localization on the cell surface [23]; (ii) increased expression in PDAC cells compared to low or absent expression in chronic pancreatitis or normal tissues [18‐20]; (iii) interaction with various growth factors involved in cell proliferation, angiogenesis and metastasis [18‐21, 39, 40]; (iv) the possibility of modulating the tumor microenvironment and reducing the state of desmoplasia, thanks to its expression on CAF [24]. Moreover, the correct epitope on TAA and the mechanism of action activated by mAbs remain important aspects to be considered [7, 8, 25]. In this context, the results of the present study show that targeting GPC1 using an epitope close to the cell membrane with a specific IgM could be a promising therapeutic option for the treatment of PDAC patients.

Here, the well-established hybridoma technique was used for the development of anti-GPC1 [41]. This technique offers the possibility of obtaining a large amount of mAbs with a high degree of specificity and sensitivity, and it is the method by which most FDA-approved mAbs have been obtained [41]. The choice of the region of GPC1 used for immunization of mice was based on several factors, including high sequence homology among members of the glypican family. In this context, it is noteworthy that the C-terminal region is the region with the lowest homology [29, 30]. Another factor is that the close localization of the target region to the cell membrane facilitates the activation of the CS by the antibody [25]. For these reasons, a small portion of the C-terminal region was used for the immunization to increase specificity exclusively for that GPC1 epitope. The decision to develop a murine IgM was also based on the aim to activate the murine CS in the human-mouse model developed to test therapeutic activity.

Hybridoma technology also made it possible to develop a method for the production and purification of AT101 and to guarantee a protein capable of targeting the GPC1 protein; these results were demonstrated by ELISA, flow cytometry and IF, confirming the specificity of AT101 for its target.

In the present study, a subcutaneous mouse model was created in athymic nude mice. Nude mice are characterized by an impairment of the adaptive immune response, but innate immunity (CS, NK cells, neutrophils, and macrophages) remains active. Therefore, the use of nude mice allows the characterization of the immune response to antibody-based immunotherapy, such as the use of AT101 [42, 43]. A subcutaneous model allows easy measurement of the size of tumor masses and facilitates the analysis of accumulation in the tumor microenvironment [44]. In addition, the subcutaneous model generally had a sufficient amount of functional vessels allowing the transport of a molecule, such as IgM, within the tumor mass [44, 45]. Here, the presence of tumor vessels was investigated by determining the expression of VWF, a typical marker for endothelial cells. This result is important because the distribution of IgM is normally restricted to the bloodstream, but increased permeability caused by an inflammatory process or described in tumor vessels allows selective accumulation in these microenvironments [46, 47]. On the contrary, IgG is normally distributed from the blood to all biological fluids [46, 47]. Thus, the use of IgM can also become a mechanism to improve the specificity of the therapeutic approach due to the selective permeability of the tumor microenvironment.

Labelling with a near-infrared fluorescent dye, such as Cy5.5, has been used extensively for biodistribution studies of antibodies and antibody fragments [11, 48, 49]. In-vivo characterization of AT101 began with Cy5.5 labelling and injection into tumor-bearing mice to study its biodistribution compared to unspecific murine IgM. AT101 and the unspecific IgM showed a different accumulation profile at the tumor level. Unspecific IgM accumulated in the tumor microenvironment with a pick 48 h after their intravenous administration, demonstrating the possibility that this antibody isotype can migrate through tumor vessels. AT101 showed higher accumulation in the BXPC3 tumor mass compared to unspecific IgM, with a peak at 72 h. These data were also confirmed ex-vivo at the end of the study (96 h) and can be linked to the specificity of AT101 for the GPC1 protein expressed in the BXPC3 tumor masses, allowing for durable localization at the tumor site.

Despite the benefits of developing anti-tumor treatments that can take advantage of CS in controlling tumor growth, the involvement of CS mediated function in mAb action is often not yet fully considered. Indeed, it must be taken into account that CS consists of soluble molecules that can easily reach the tumor site and diffuse into the tumor mass [11, 14, 22, 23]. In addition, the components of CS are synthesized locally by various cell types, including macrophages, and they often act as a first line of defense against cancer cells [11]. In this context, however, the presence of IgM not provides information about their binding to cancer cells or about their ability to activate the CS. Only bound IgM (immune complexes) modify their Fc, enable the binding of C1 and activate the classical pathway of the CS [50]. In the present study, C1q, C3 and C9 were detected by IF only in the tumor mass of animals treated with AT101 and not with non-specific IgM, that have no specificity for tumor cells. The presence of C9 indicates full activation of the cascade and formation of the membrane attack complex, which can cause direct death of tumor cells [6]. This result is usually due to lysis of the cell, as documented in this study by hematoxylin eosin staining, where a strong purple-blue staining (hematoxylin) was observed in the AT101-treated mice, which was not documented in the tumor microenvironment of the animals treated with unspecific IgM. The activation of the CS normally alters the microenvironment by producing anaphylatoxins such as C3a and C5a, which are able to activate the endothelial cells of the tumor vessels and recruit leukocytes from the bloodstream [7]. The massive presence of NK cells and macrophages in the tumor sections of mice treated with AT101 is a typical result of this phenomenon [7, 11, 12], which demonstrated the great potential of this anti-GPC1 mAb in coordinating an immune response. This was confirmed by in-vivo experiments in which treatment of mice bearing BXPC3 tumors with AT101 was very effective in controlling tumor growth and prolonging survival.

A crucial step for the possibility of clinical application of AT101 is humanization. The ultimate goal of the humanization process is to obtain a humanized mAb with high antigen binding activity and a minimal immunogenicity that prolongs half-life. On the other hand, biophysical properties such as stability and expression yield are important for commercial purposes. From a technical point of view, complementarity-determining regions (CDR)-grafting humanization, in which the whole mouse CDRs are grafted onto an acceptor human framework, is the most commonly used technique [51]. To avoid reducing the potential immunogenicity of framework residues due to somatic mutations containing T cell effector epitopes, the use of frameworks based on human germline sequences or consensus sequences as acceptor human frameworks was introduced [52‐54]. Another approach to reduce the potential immunogenicity of non-human CDRs is the use of specificity-determining residues (SDRs), i.e. the minimum CDR residues required for antigen-binding activity, grafted onto the human germline framework [55‐57].

Usually, the development and clinical use of therapeutic mAbs mainly focused on humanized or fully human mAbs of the IgG class [9, 10], although several mAbs of the IgM isotype have been investigated in phase I clinical trials [13‐17]. Results of the present study suggest that the therapeutic potential of humanized IgM is to be considered for the specific features of this class, i.e. their high avidity and the effective complement activation, that, in other monoclonal antibody models showed therapeutic advantages at least in a pre-clinical phase [9]. On the other hand, whether the creation of an IgG3, capable of recognizing the AT101 epitope would have given a better result than IgM remains to be considered. Moreover, further studies are needed to evaluate if a CAR-T recognizing the AT101 epitope could demonstrate a higher therapeutic potential than a mAb either of the IgM or the IgG class.

In the present study we showed a novel therapeutic strategy based on the use of AT101, an IgM specific for an epitope of GPC1 close to PDAC cell surface and able to selectively activate the CS and recruit effector cells in the tumor microenvironment, allowing to reduce tumor mass growth and improve survival in all treated mice. AT101 could also provide a platform for other mAb-based approaches, such as antibody–drug conjugates (ADC), drug-loaded nanoparticles or chimeric antigen receptor (CAR) based cell therapies.