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

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. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-023-04745-9.

1 Additional methods

mAb purification
The hybridoma supernatant was collected twice weekly, tested for productivity and specificity and finally the AT101 was purified.Purification was performed using Hitrap IgM Purification HP affinity chromatography (Cytiva, United Kingdom).The supernatant was filtered with a 0.45 µm filter before purification.The following buffers were used for purification: binding buffer 20 mM sodium phosphate, 0.8 M (NH4)2SO4, pH 7.5; elution buffer 20 mM sodium phosphate, pH 7.5; regeneration buffer 20 mM sodium phosphate, pH 7.5 with 30% isopropanol.The column was washed with 5 column volumes of each buffer and equilibrated with 5 column volumes of the binding buffer.The sample was added to the column with a syringe.The unbound sample was washed with 15 column volumes of the binding buffer.AT101 was eluted with 12 column volumes of elution buffer.Finally, the column was regenerated with 7 column volumes of regeneration buffer.To replace the elution buffer, dialysis was performed in PBS using a cellulose dialysis membrane with a cut-off of 14000 Da (Merck, Germany).

ELISA
To confirm binding of the anti-GPC1 antibody AT101, wells (Corning-Costar, MA, USA) were first coated with GPC1 (100 ng/well, LSBio, Seattle, WA, USA) overnight (O/N) at 4°C.At the end of incubation, wells were blocked with 2% milk in PBS (Euroclone S.p.A, Italy) for 30 minutes at room temperature (RT).Then, 500 ng of AT101 were added to the wells and incubated for 1 hour at RT. Serum from mice immunized with GPC1 (1:100, Takis S.r.l., Italy) was used as positive control.The negative control (CTRL-) was performed by only using the goat anti-mouse IgG/IgM antibody conjugated with alkaline phosphatase (AP) without AT101.To remove the excess of unbound antibody, three washes with PBS-TWEEN20 0.1% and three washes with PBS were performed.To detect binding of AT101 to GPC1, a goat anti-mouse IgG/IgM antibody conjugated to alkaline phosphatase (AP) was used (Sigma-Aldrich, Italy, Cat.No. SAB3700987).Bound antibodies were detected by adding the phosphatase substrate para-nitrophenyl phosphate (pNPP, Sigma-Aldrich, Italy) to the wells.Absorbance was measured at 405 nm using the Tecan M200 Infinite® Pro Microplate Reader (Tecan Life Sciences, Switzerland).During data elaboration the negative control was subtracted from the value of absorbance.

Immunofluorescence
Cells were seeded at a density of 25000 cells over Deckglaser cover glasses 18 mm (Marienfeld, Germany) and cultured o/n.The day after, cells were fixed with paraformaldehyde solution 4% in phosphate buffer solution (PBS-PFA) (Sigma-Aldrich, Italy) and washed with 0.1 M of glycine (Sigma-Aldrich, Italy) and then with PBS without calcium and magnesium (Euroclone S.p.A., Italy).
After fixation, non-specific binding sites were blocked with PBS Bovine Serum Albumine (BSA) 5% (Sigma-Aldrich, Italy) for 1 h at RT.After the blocking, cells were incubated with primary antibodies diluted in PBS BSA 1% for 1 h at RT. Cells were washed twice with PBS and incubated with the secondary antibody for 1 h at RT. Cells were then washed in PBS and incubated for 5 minutes with DAPI (Sigma-Aldrich, Italy) and washed with PBS, with distilled water and mounted using the FluorSave™ mounting medium (Merck, Germany) to Superfrost® Microscope Slides (Thermo Scientific, Thermo Fisher Scientific, Italy).Images were acquired using the Nikon Eclipse Ti-U microscope (Nikon Europe B.V.).
Organs and BXPC3 tumors from xenograft murine models were embedded into Killik O.C.T. (OCT) medium (Bio-Optica S.p.A., Italy) and maintained at -80 °C.A cryostat (Thermo Electron Corporation, MA, USA) was utilized to cut organs and tumors into slices of 7 µm, slices were subsequently mounted into SuperFrost® Plus (Fisher Scientific, Thermo Fisher Scientific, Italy) glass slides and stored at -80 °C.For immunofluorescence (IF), samples were fixed for 10 minutes with cold PBS-PFA, washed with PBS and blocked using a solution of 2% BSA, 0.25% casein from bovine milk (Sigma-Aldrich, Italy), and 0.1% gelatine from cold water fish skin (Sigma-Aldrich, Italy) in PBS for 1 h at RT. Slices were then incubated with primary antibodies diluted into blocking solution for 1 h at RT, then washed with blocking solution and incubated with secondary antibodies in blocking solution for 1 h at RT.Samples were further washed in PBS and incubated with DAPI 5 min at RT.Samples were washed twice with PBS and then with distilled water.Samples were mounted using FluorSave™ on high precision microscope cover glasses (Marienfeld, Germany).Images were acquired using the Nikon Eclipse Ti-U microscope.

Flow cytometry
For flow cytometry analysis, 300000 cells were blocked using PBS plus 2% BSA and then incubated with primary antibody diluted in the same blocking solution for 1 hour at RT under shaking.Cells were then washed twice with PBS plus 2% BSA and incubated with secondary antibody diluted in the same blocking solution for 1 hour at RT.The samples were washed and then resuspended into PBS plus 2% BSA and 1% PFA.Samples were acquired using BD FACSCanto™II Cell Analyzer (Becton, Dickinson and Company, NJ, USA).Primary antibodies employed were: anti-GPC1 (Thermo Fisher Scientific, Italy, Cat.No. PA5-28055); AT101 25 µg/ml.Secondary antibodies employed were: anti-mouse IgM 488 conjugated (Bethyl, Fortis Life Science, MA, USA, Cat.No. A90-201D2) diluted 1:250; anti-rabbit IgG 488 conjugated (Bethyl, Fortis Life Science, MA, USA, Cat.No. A120-212D2) diluted 1:100.Data were analyzed using BD FACSDiva Software.

Labeling with cyanine 5.5
For the in-vivo biodistribution study, AT101 was conjugated with cyanine 5.5 (Cy5.5)(Cytiva, United Kingdom, Cat.No. PA25501), a dye that produces an intense signal in the near infrared region of the spectrum.One mg of AT101 was added to the Cy5.5 vial and the reaction was incubated for 30 minutes at RT, with additional mixing every 10 minutes.The labelled AT101 was separated from the excess free Cy5.5 by dialysis in PBS using a cellulose dialysis membrane with a cutoff of 14000 Da (Merck, Germany).Quantification of Cy5.5 was performed using a spectrophotometer (Cary 100 UV-VIS, Agilent) according to the guidelines in the protocol "Amersham fluorolink Cy5.5 monofunctional dye PA25501".

Hematoxylin Eosin staining
The tumor masses were collected and embedded in OCT.The frozen organs were then cut with the cryostat as described in the section "Immunofluorescence". Tumor slices were fixed in cold PFA-PBS for 10 minutes, washed in distilled water for 1 minute and stained with hematoxylin solution (Merck, Germany) for 3 minutes.Before eosin staining (Bio-Optica, Italy) for 45 seconds, the slices were washed in distilled water for 1 minute and incubated with 95% ethanol for 1 minute.
Subsequently, the slices were dehydrated by serial incubation with ethanol 95% for 1 minute and by 2 incubations with ethanol 100%.Finally, the slices were incubated with xylene for 2 minutes and the cover glasses were mounted with Entellan™ (Merck, Germany).For IF, the sections were treated as described in the section "Immunofluorescence".

Statistical analyses
Statistical analyses were performed in RStudio (v4.0.5).The unpaired Student's t-test was used to calculate statistical difference between the means of two data sets.Survival function estimates between treatment groups were calculated with the Kaplan-Meier estimator using the package "survival"(v3.2-10);survival curves were plotted by Kaplan-Meier curves using the package "survminer"(v0.4.9).The log-rank test was used to calculate the p-values of the survival difference between the treatment groups using the package "survival" (v3.2-10).The comparison between the two groups (tumor growth curves) was performed using the 1-way analysis of variance test.P < 0.05 was considered statistically significant.  .Immunofluorescence analysis to evaluate GPC1 expression in BXPC3 and Jurkat using AT101 and the commercial anti-GPC1 antibody as positive control.In red the signal related to GPC1 protein is reported and in blue the signal related to the nuclei is reported.Scale bar: 25 µm.

Figure A1
Figure A1.Immunofluorescence analysis to evaluate GPC1 expression in BXPC3 and Jurkat using AT101 and the commercial anti-GPC1 antibody as positive control.In red the signal related to GPC1 protein is reported and in blue the signal related to the nuclei is reported.Scale bar: 25 µm.FigureA2.Hematoxylin eosin staining to evaluate the possible presence of tissue damage.Purple blue refers to the nuclei, pink refers to the cytoplasms and the extracellular matrix.Scale bar: 100 µm.

Figure A2 .
Figure A1.Immunofluorescence analysis to evaluate GPC1 expression in BXPC3 and Jurkat using AT101 and the commercial anti-GPC1 antibody as positive control.In red the signal related to GPC1 protein is reported and in blue the signal related to the nuclei is reported.Scale bar: 25 µm.FigureA2.Hematoxylin eosin staining to evaluate the possible presence of tissue damage.Purple blue refers to the nuclei, pink refers to the cytoplasms and the extracellular matrix.Scale bar: 100 µm.

Table 1 .
Primers employed for sequencing of VL gene

Table 2 .
Primers employed for sequencing of VH gene

Table 3 .
Aminoacid sequences of VL and VH chains of AT101