The trifunctional antibody catumaxomab amplifies and shapes tumor-specific immunity when applied to gastric cancer patients in the adjuvant setting

Abstract

Background: Patients with gastric cancer benefit from perioperative chemotherapy, however, treatment is toxic and many patients will relapse. The trifunctional antibody catumaxomab targets EpCAM on tumor cells, CD3 on T cells, and the Fcγ-receptor of antigen-presenting cells. While in Europe catumaxomab is approved for treating malignant ascites, it has not been investigated in the perioperative setting and its exact immunological mode of action is unclear.

Methods: In our study, gastric cancer patients received neoadjuvant platinum-based chemotherapy, one intraoperative application of catumaxomab, and 4 postoperative doses of intraperitoneal catumaxomab. Immunomonitoring was performed in 6 patients before surgery, after completion of catumaxomab treatment, and one month later.

Results: Intraperitoneal application of catumaxomab caused an increased expression of activation markers on the patients' T cells. This was accompanied by a transient decrease in numbers of CXCR3(+) effector T cells with a T-helper (Th)-1 phenotype in the peripheral blood. All patients evidenced pre-existing EpCAM-specific CD4(+) and/or CD8(+) T cells. While these cells transiently disappeared from the blood stream after intraperitoneal application of catumaxomab, we detected increased numbers of peripheral EpCAM-specific cells and a modified EpCAM-specific T-cell repertoire 4 weeks after completion of treatment. Finally, catumaxomab also amplified humoral immunity to tumor antigens other than EpCAM.

Conclusions: Our findings suggest that catumaxomab exerts its clinical effects by (1) activating peripheral T cells, (2) redistributing effector T cells from the blood into peripheral tissues, (3) expanding and shaping of the pre-existing EpCAM-specific T-cell repertoire, and (4) spreading of anti-tumor immunity to different tumor antigens.

Keywords: EpCAM; T cells; adjuvant immunotherapy; catumaxomab; gastric cancer; tumor immunology.

Figure 1. Tumors of gastric cancer patients treated with catumaxomab show strong expression of EpCAM and pre-therapeutic infiltration by CD4⁺ and CD8⁺ T cells. (A) Within the IP-CAT-GC-03 trial, a total of 6 patients were treated at our center with intraperitoneal (i.p.) catumaxomab as shown. Blood samples (Pre, Post, EOT) for immunomonitoring were collected at three time-points as indicated by red bars. (B) Resected tumors from all 6 gastric cancer patients were analyzed by immunohistochemistry (magnification × 400). Consecutive cuts are shown from the block of one representative subject (patient GC-3). In addition to a routine H&E stain, tumor samples were analyzed for the expression of EpCAM protein as well as for the presence of CD4⁺ and CD8⁺ T cells.

Figure 2. Catumaxomab induces a mobilization of CD8⁺ T cells and NK cells and an activation of CD4⁺ and CD8⁺ T cells. (A) The catumaxomab-induced redistribution of lymphocytes subsets was analyzed in patients with gastric cancer (n = 6) shortly before surgery was performed (Pre), on day 17 after gastrectomy following infusion of the final dose of catumaxomab (Post), and 4 weeks after the last catumaxomab application (EOT) using four-color flow cytometry. Analysis was performed using a combination of a morphological lymphocyte gate and gates for CD8⁺ T cells (CD3⁺CD8⁺) and NK cells (CD3^-CD56⁺), respectively. Logarithmically plotted values depict individual changes during treatment in comparison to results at baseline (Pre). (B) Fold changes in percentages of CD4⁺ and CD8⁺ T cells expressing activation markers CD69 and HLA-DR, respectively, were evaluated for the same patients and time points. Each patient (GC-01 to GC-06) is indicated by a separate symbol as given below the figure.

Figure 3. Application of catumaxomab causes CXCR3+ Th1-type T cells to leave the peripheral blood. (A) The catumaxomab-induced redistribution of lymphocytes as characterized by the expression of different chemokine receptors was analyzed in the peripheral blood of our patients (n = 6) shortly before surgery was performed (Pre), following infusion of the final dose of catumaxomab (Post), and 4 weeks after completion of catumaxomab treatment (EOT) using flow cytometry. Dot plots demonstrating the expression of chemokine receptor CXCR3 on CD4⁺ T cells at the different time points are shown for two representative patients. (B) Results for CD4⁺ T cells being either CXCR3⁺CCR4⁻ (left) or CCR4⁺CXCR3⁻ (right) are shown for all patients (see legend of Fig. 2 regarding symbols for individual patients). Data are plotted logarithmically and depict fold changes in percentages of the given lymphocyte subset during treatment in comparison to results at baseline.

Figure 4. Application of catumaxomab results in an amplified and remodelled T-cell response against tumor antigen EpCAM. Patients with gastric cancer were analyzed before surgery was performed (Pre), following infusion of the final dose of catumaxomab (Post), and 4 weeks after completion of catumaxomab treatment (EOT). Frequencies of CD4⁺ and CD8⁺ T cells directed against 31 overlapping 20mer EpCAM peptides were determined in an ELISPOT assay following a single cycle of antigen-specific stimulation. (A) Black bars indicate numbers of patients evidencing CD4⁺ (upper lane) and/or CD8⁺ T-cell responses (lower lane) against 6 different pools of 5–6 peptides each at the given timepoints. (B) Exemplary IFN-γ ELISPOT results of patients GC-02 and GC-06 for CD4⁺ (two upper lanes) and CD8⁺ (two lower lanes) T-cell responses against single EpCAM peptides. Per well, 50 000 effector T cells were analyzed, background responses against irrelevant SSX2 peptide were usually < 10 spots/50 000 cells. (C) Frequencies of CD4⁺ and CD8⁺ T cells directed against individual EpCAM epitopes at the three timepoints (Pre, Post, EOT) are shown. Dots indicate spot numbers in ELISPOT assays with EpCAM peptide-pulsed target cells (T-APC) for 4 patients in whom the same individual EpCAM epitope was detectable at least at two of the three timepoints.

Figure 5. Application of catumaxomab results in an expansion of effector-type T cells, and not Tregs, in the peripheral blood and in the tumor environment. (A) Intracellular cytokine staining followed by flow cytometry was performed using CD4⁺ T cells of patient GC-02 after one round of in vitro sensitization with pooled EpCAM peptides. Representative results of intracellular co-staining of IFN-γ and FOXP3 are shown for EpCAM^–-specific CD4⁺ T cells obtained before initiation of catumaxomab therapy (Pre) and 4 weeks after completion of treatment (EOT). Dot plots show responses against the individual EpCAM peptide (upper row) and an SSX2 control peptide (lower row). (B) Bar graphs indicate mean numbers (+SEM) of CD4⁺ T cells specific for EpCAM amino acid region 151–210 (black bar) or control peptide SSX2 (white bar) within the malignant effusion of patient GC-02. Original ELISPOT data demonstrate spot numbers per well at 50 000 CD4⁺ T cells per well after presensitization with pooled EpCAM peptides.

Figure 6. Broad catumaxomab-induced modulation of humoral anti-tumor immunity. Lines indicate antibody responses against 6 different CT antigens in gastric cancer patients receiving catumaxomab in the adjuvant setting. Reciprocal IgG antibody titers against recombinant protein of the given CT antigen were measured by ELISA and are shown for two timepoints: before initiation of catumaxomab therapy (Pre) and 4 weeks after completion of treatment (EOT).