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. 2024 Jan 22:15:1343929.
doi: 10.3389/fimmu.2024.1343929. eCollection 2024.

An Fc-modified monoclonal antibody as novel treatment option for pancreatic cancer

Martina S Lutz  1   2 , Kevin Wang  1 , Gundram Jung  2   3 , Helmut R Salih  1   2 , Ilona Hagelstein  1   2
Affiliations

An Fc-modified monoclonal antibody as novel treatment option for pancreatic cancer

Martina S Lutz et al. Front Immunol. .

Abstract

Pancreatic cancer is a highly lethal disease with limited treatment options. Hence, there is a considerable medical need for novel treatment strategies. Monoclonal antibodies (mAbs) have significantly improved cancer therapy, primarily due to their ability to stimulate antibody-dependent cellular cytotoxicity (ADCC), which plays a crucial role in their therapeutic efficacy. As a result, significant effort has been focused on improving this critical function by engineering mAbs with Fc regions that have increased affinity for the Fc receptor CD16 expressed on natural killer (NK) cells, the major cell population that mediates ADCC in humans. Here we report on the preclinical characterization of a mAb directed to the target antigen B7-H3 (CD276) containing an Fc part with the amino acid substitutions S239D/I332E to increase affinity for CD16 (B7-H3-SDIE) for the treatment of pancreatic cancer. B7-H3 (CD276) is highly expressed in many tumor entities, whereas expression on healthy tissues is more limited. Our findings confirm high expression of B7-H3 on pancreatic cancer cells. Furthermore, our study shows that B7-H3-SDIE effectively activates NK cells against pancreatic cancer cells in an antigen-dependent manner, as demonstrated by the analysis of NK cell activation, degranulation and cytokine release. The activation of NK cells resulted in significant tumor cell lysis in both short-term and long-term cytotoxicity assays. In conclusion, B7-H3-SDIE constitutes a promising agent for the treatment of pancreatic cancer.

Keywords: B7-H3; NK cells; immunotherapy; pancreatic cancer; therapeutic antibody.

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Conflict of interest statement

GJ and HS are listed as inventors on the patent application “Antibodies targeting, and other modulators of, the CD276 antigen, and uses thereof,” EP3822288A1, applicant is German Cancer Research Center, Heidelberg, and Medical Faculty University of Tuebingen, Germany. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
B7-H3 Expression and antibody binding in pancreatic cancer cells. (A, B) The mRNA expression of B7-H3 was evaluated through the use of the online tool GEPIA in (A) tumor (T) and corresponding normal (N) tissues, as well as in B pancreatic cancer cases at various disease stages. (C) Analysis of B7-H3 mRNA expression compared to RPL13 mRNA in five pancreatic cancer cell lines. Combined results are shown for three independent experiments. (D) B7-H3 surface expression on specified pancreatic cancer cells was examined via flow cytometry using mAb against B7-H3 (shaded peaks) and an isotype control (open peaks). Exemplary histograms from one representative experiment of a total of three with similar results are provided. (E) B7-H3 molecules were quantified in pancreatic cancer cell lines using FACS. Results from two independent experiments are presented. (F) Pancreatic carcinoma cell lines were treated with B7-H3-SDIE or iso-SDIE at the indicated concentrations, followed by analysis with anti-human PE conjugate using flow cytometry. Data for mean fluorescence intensity (MFI) levels from a representative experiment of three with similar results are displayed. (G, H) Pancreatic cancer cells AsPC-1, MIA PaCa-2, and PANC-1 were incubated with B7-H3-SDIE at specified concentrations or iso-SDIE (10 µg/mL) for (G) 24 hours and (H) 72 hours, respectively. The cells were subsequently washed and reincubated with 1 μg/mL of B7-H3-SDIE, followed by an anti-human PE conjugate (1:200), and analyzed using flow cytometry. The relative surface expression of B7-H3 was determined by defining the MFI of cells preincubated without an antibody as 100%. Exemplary data from one representative experiment out of a total of three is presented. The results depict mean ± SD.
Figure 2
Figure 2
Effects of B7-H3-SDIE in the absence of B7-H3+ tumor cells. Effector cells of healthy donors were cultured without target cells in the presence or absence of soluble B7-H3-SDIE or the corresponding isotype control (1 μg/mL). (A, B) Activation of NK cells was determined by expression of (A) CD69 and (B) CD25 after 24 h. In the left panels, exemplary flow cytometry results obtained with one donor and in the right panel combined data with NK cells of n = 5 different donors are shown. (C) Cytokine release was analyzed using Legendplex assays for supernatants of cocultures after 4 hours. (D) Total numbers of living PBMC were analyzed by flow cytometry after 24 h. In the left panels, exemplary flow cytometry results for living cell counts obtained with one PBMC donor and in the right panel data with PBMC of n = 5 different donors are shown. Results are shown as mean ± SD.
Figure 3
Figure 3
Induction of NK cell reactivity by B7-H3-SDIE against pancreatic cancer cells. PBMC from healthy donors were cultured with pancreatic cancer cells including AsPC-1, MIA PaCa-2, and PANC-1 at 2.5:1 E:T ratio with or without the B7-H3-SDIE or corresponding isotype control (1 μg/mL). (A, B) Activation of NK cells was assessed by analyzing the expression of (A) CD69 and (B) CD25 after 24 hours. The left panels show results of flow cytometry testing conducted on a single PBMC donor along with PANC-1 cells. The right panels present data on pancreatic cancer cell lines with PBMC from four diverse donors. (C) NK cell degranulation was assessed by CD107a expression after 4 hours. The left panels display flow cytometry data obtained from one PBMC donor, while the right panels exhibit results from four independent PBMC donors with pancreatic cancer cell lines. (D, E) Cytokine release was analyzed using Legendplex assays for supernatants of cocultures after 4 hours. The release of (D) IFNγ and (E) TNF is presented for n = 4 PBMC donors, respectively. Mean ± SD is shown for the results.
Figure 4
Figure 4
Induction of target cell lysis by Fc-optimized B7-H3-SDIE antibody. PBMC from healthy donors were incubated with AsPC-1, MIA PaCa-2, and PANC-1 pancreatic cancer cells and treated with B7-H3-SDIE or control antibody iso-SDIE (1 μg/mL). (A) Lysis of pancreatic cancer cell lines was analyzed using 2-hour Europium cytotoxicity assays. Exemplary data from a single PBMC donor at various E:T ratios are presented in the left panels, while pooled data from PBMC donors (n=3-5) at an E:T ratio of 40:1 are displayed in the right panels. (B) Lysis of pancreatic cancer cell lines was evaluated using flow cytometry-based lysis assays after 72 hours, with an E:T ratio of 10:1. The left panels depict exemplary dot plots featuring one PBMC donor, while the right panel displays the combined results for each cell line with n=3-5 PBMC. (C) Using xCELLigence, cell death of pancreatic cancer cells was determined. PBMC were incubated with target cells at an E:T ratio of 10:1 for 120 hours. Exemplary data from a representative experiment of three total experiments are shown. Results are presented as mean ± SD.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This project was supported by the Deutsche Krebshilfe (70114180, 70113999), the Wilhelm Sander-Stiftung (2017.100.3), and Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy -EXC 2180 -10084-1_1. We acknowledge support from the Deutsche Forschungsgemeinschaft and the Open Access Publishing Fund of University of Tübingen.