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

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with a very low survival rate at 5 years. The use of chemotherapeutic agents results in only modest prolongation of survival and is generally associated with the occurrence of toxicity effects. Antibody-based immunotherapy has been proposed for the treatment of PDAC, but its efficacy has so far proved limited. The proteoglycan glypican-1 (GPC1) may be a useful immunotherapeutic target because it is highly expressed on the surface of PDAC cells, whereas it is not expressed or is expressed at very low levels in benign neoplastic lesions, chronic pancreatitis, and normal adult tissues. Here, we developed and characterized a specific mouse IgM antibody (AT101) targeting GPC1.

Methods: We developed a mouse monoclonal antibody of the IgM class directed against an epitope of GPC1 in close proximity to the cell membrane. For this purpose, a 46 amino acid long peptide of the C-terminal region was used to immunize mice by an in-vivo electroporation protocol followed by serum titer and hybridoma formation.

Results: The ability of AT101 to bind the GPC1 protein was demonstrated by ELISA, and by flow cytometry and immunofluorescence analysis in the GPC1-expressing "PDAC-like" BXPC3 cell line. In-vivo experiments in the BXPC3 xenograft model showed that AT101 was able to bind GPC1 on the cell surface and accumulate in the BXPC3 tumor masses. Ex-vivo analyses of BXPC3 tumor masses showed that AT101 was able to recruit immunological effectors (complement system components, NK cells, macrophages) to the tumor site and damage PDAC tumor tissue. In-vivo treatment with AT101 reduced tumor growth and prolonged survival of mice with BXPC3 tumor (p < 0.0001).

Conclusions: These results indicate that AT101, an IgM specific for an epitope of GPC1 close to PDAC cell surface, is a promising immunotherapeutic agent for GPC1-expressing PDAC, being able to selectively activate the complement system and recruit effector cells in the tumor microenvironment, thus allowing to reduce tumor mass growth and improve survival in treated mice.

Keywords: Complement System; GPC1; IgM; Immunotherapy; PDAC.

Fig. 1

In-vitro characterization of AT101. A Schematic representation of GPC1 protein and visualization of the aminoacids employed for the mouse immunization. B SDS-PAGE analysis performed in non-reducing and reducing condition to determine the IgM nature of AT101. C Elisa to assess the GPC1 binding activity of AT101, the positive control (CTRL +) was represented by serum of mice immunized with GPC1, the negative control (CTRL-) was performed by only using the goat anti-mouse IgG/IgM antibody conjugated with alkaline phosphatase (AP) without AT101 (n = 3). D Flow cytometry analysis to evaluate GPC1 expression in BXPC3 and Jurkat cells using AT101 and the commercial anti-GPC1 antibody as positive control (n = 3). ***, 0.001 < p ≤ 0.0001

Fig. 2

Ex-vivo characterization of the PDAC xenograft murine model. A, B IF to evaluate GPC1 expression using AT101 and the commercial anti-GPC1 antibody as positive control, respectively. In green the signal related to GPC1 protein and in blue the nuclei. Scale bar: 25 µm. C IF to evaluate VWF expression in order to determine the presence of vascularization. In green the signal related to VWF protein and in blue the nuclei. Scale bar: 25 µm

Fig. 3

In-vivo and ex-vivo biodistribution of AT101. A bar chart of the in-vivo biodistribution, using VIVOVISION IVIS^®Lumina, of AT101 (1 nmol of Cy5.5) in comparison with the negative control unspecific IgM (1 nmol of Cy5.5). Data are reported as average efficiency mean ± SD (n = 4). P-value was calculated using t-test. ns: ≥ 0.05; *: 0.05 < p ≤ 0.01; **: 0.01 < p ≤ 0.001. B bar chart of the ex-vivo biodistribution, using VIVOVISION IVIS®Lumina, of AT101 (1 nmol of Cy5.5) in comparison with the negative control unspecific IgM (1 nmol of Cy5.5). Data are reported as average efficiency mean ± SD (n = 4). P-value was calculated using t-test. ns: ≥ 0.05

Fig. 4

Ex-vivo evaluation of the complement system activation at tumor site. A Upper panel: IF to evaluate the accumulation of IgM in the two experimental groups (AT101 treated mice and unspecific IgM treated mice). In green the signal related to the IgM and in blue the nuclei. Scale bar: 100 µm. Lower panel: bar chart representing the quantification of fluorescence, data are expressed as normalized fluorescence (protein/nuclei). B Upper panel: IF to evaluate C1q complement protein in the two experimental groups (AT101 treated mice and unspecific IgM treated mice). In green the signal related to C1q protein and in blue the nuclei. Scale bar: 100 µm. Lower panel: bar chart representing the quantification of fluorescence, data are expressed as normalized fluorescence (protein/nuclei). C Upper panel: IF to evaluate C3 complement protein in the two experimental groups (AT101 treated mice and unspecific IgM treated mice). In green the signal related to C3 protein and in blue the nuclei. Scale bar: 100 µm. Lower panel: bar chart representing the quantification of fluorescence, data are expressed as normalized fluorescence (protein/nuclei). D Upper panel: IF to evaluate C9 complement protein in the two experimental groups (AT101 treated mice and unspecific IgM treated mice). In green the signal related to C9 protein and in blue the nuclei. Scale bar: 100 µm. Lower panel: bar chart representing the quantification of fluorescence, data are expressed as normalized fluorescence (protein/nuclei)

Fig. 5

Ex-vivo evaluation of the immunological recruitment at tumor site. A Upper panel: IF to evaluate CD14 protein in the two experimental groups (AT101 treated mice and unspecific IgM treated mice). In green the signal related to CD14 protein and in blue the nuclei. Scale bar: 100 µm. Lower panel: bar chart representing the quantification of fluorescence, data are expressed as normalized fluorescence (protein/nuclei). B Upper panel: IF to evaluate CD56 protein in the two experimental groups (AT101 treated mice and unspecific IgM treated mice). In green the signal related to CD56 protein and in blue the nuclei. Scale bar: 100 µm. Lower panel: bar chart representing the quantification of fluorescence, data are expressed as normalized fluorescence (protein/nuclei)

Fig. 6

In-vivo therapeutic efficacy of AT101. A Timeline that recapitulates the scheduling of the treatment. AT101 was administered twice a week at a dosage of 1.5 mg/kg, the last treatment was administered at day 42, the experimental endpoint was set up at day 50. B Line graph that recapitulates the tumor growth trend of the two experimental groups. In dashed line the tumor growth trend of the group of mice treated with PBS (n = 7) is reported; in continuous line the tumor growth trend of the group of mice treated with AT101 (n = 7) is reported. Data were represented as mean ± standard error (SE). C Kaplan–Meier curve of mouse survival: the survival of mice treated with AT101 was compared with the mice treated with PBS (dashed line). In dashed line the survival curve of the group of mice treated with PBS (n = 7) is reported; in continuous line the survival curve of the group of mice treated with AT101 (n = 7) is reported. P-value < 0.0001 was calculated using log rank test