Generation and in vivo characterization of a novel high-affinity human antibody targeting carcinoembryonic antigen

Figures & data

Figure 1. In vitro characterization of new anti-CEA antibodies. (a) SPR sensorgram of G9, F7, and F4 in a scFv format. K_D values were calculated to be 640 nM, 50 nM, and 7.7 nM, respectively. (b) Flow cytometry analysis with the new antibodies in IgG format on CEA-expressing CT26 cells and on CEA-negative CT26 wild-type cells. (c) Immunofluorescence staining with IgG formats on the human colon adenocarcinoma xenograft LS174T. Anti-CEA antibodies were detected in green. Blood vessels were detected by CD31 staining (red). 20× magnification, scale bars = 100 μm.

A: Three SPR line graphs plotting antibody binding over time. All clones show a fast association. G9 has fast dissociation, F7 has intermediate dissociation, and F4 has slow dissociation. B: Three titration flow cytometry graphs plotting mean fluorescence intensity over increasing concentration. G9 values on CT26-CEA cells are moderately increasing in high concentrations. F7 and F4 display an increasing sigmoidal curve on CT26-CEA cells. No clone binds to CT26-wildtype cellsC: Immunofluorescence staining with the new antibodies. Intermediate green staining is present with G9, and strong green staining is present with F7 and F4.

Figure 2. Immunofluorescence analysis of human cancer sections. Human tissue microarrays with cancer sections and corresponding healthy controls were analyzed by immunofluorescence analysis with F4, the positive control Sm3E, and an irrelevant isotype control (KSF). Antibody binding was detected in green. Blood vessels were detected with CD31 staining (red) and nuclei with DAPI (blue). 20× magnification, scale bars = 100 μm.

Immunofluorescence staining of a colon tumor, healthy colon, lung tumor, healthy lung, pancreas tumor, and healthy pancreas with the antibodies F4, Sm3E, and KSF. Strong green staining is present in the colon tumor and lung tumor with antibodies F4 and Sm3E and in slightly lower intensity in the pancreas tumor. No green staining is present with KSF and in all healthy organs.

Figure 3. Ex vivo immunofluorescence-based biodistribution analysis. Immunofluorescence analysis assessed tumor targeting of new anti-CEA antibodies in IgG format. Two hundred micrograms of IgG-FITC were injected intravenously into LS174T-bearing mice. Tumors were excised 24 hours after injection. IgG-FITC was detected in green; blood vessels were detected through CD31 staining (red). 20× magnification, scale bars = 100 μm.

Immunofluorescence staining of the tumor, liver, lung, spleen, heart, kidney, and intestine from the groups F4, Sm3E, G9, F7, and KSF. Strong green staining is present in the tumor of the F4 group and at a slightly lower intensity in the Sm3E group. Minimal green staining is present in the tumor of the F7 group. No green staining is present in the tumors of the G9 and KSF groups and in all healthy organs.

Figure 4. Quantitative biodistribution with radiolabeled anti-CEA antibodies in diabody format. Quantitative biodistribution analysis of radio iodinated anti-CEA diabodies in BALB/c nude mice bearing subcutaneous LS174T colon adenocarcinomas. Organs were harvested 24 hours after intravenous injection, and radioactivity was quantified. Results are shown as the percentage of injected dose per gram (ID/g (%)). Error bars = SEM; n = 4.

Four panels of bar charts plotting the percentage of injected dose per gram for the tumor, liver, lung, spleen, heart, kidney, intestine, and blood. The four panels represent the antibodies F4, F7, G9, and KSF. Significantly higher accumulation in the tumor compared to the organs is observed for antibodies F4 and F7.

Figure 5. Preclinical characterization of F4-IL12. (a) Schematic representation of F4-IL12. P35 and p40 of murine IL12 were genetically linked to F4 in single-chain diabody format. (b) Surface plasmon analysis of F4-IL12 on recombinant CEA. (c) IFNγ release assay with F4-IL12 on murine splenocytes. (D and G) Flow cytometry analysis on CEA⁺ CT26 (d) and C51 (g) cells. As a negative control, cells were treated with the detecting secondary antibody only. (E and H) Tumor growth of (e) CT26-CEA bearing BALB/c mice treated with 3 × 12 μg F4-IL12, KSF-IL12, or saline (n = 3) and (h) C51-CEA bearing BALB/c mice treated with the same compounds (n = 8). Mice were randomized when tumors reached an average volume of 100 mm³. Arrows indicate days of injection. †=mouse excluded due to ulceration. Bodyweight change of treated mice bearing (f) CT26-CEA tumors and (i) C51-CEA tumors. Error bars = SEM.

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A: Cartoon showing the p40 domain linked to the p35 domain, which is linked to VH-VL-VH-VL. B: SPR line graph plotting binding over time, displaying fast association and slow dissociation. C: Line graph plotting the percentage of IFN gamma release over increasing concentration, showing an increasing sigmoidal curve. D: Flow cytometry histogram plotting relative count over fluorescence intensity on CT26-CEA cells. F4-IL12 displays increased fluorescence intensity compared to the negative control. E: Line graph plotting tumor volume over time for saline, KSF-IL12, or F4-IL12 treated mice. The curve for the saline group increases rapidly until day 18. The curve for the KSF-IL12 group initially increases and then remains stable. The curve for F4-IL12 decreases and finally remains stable. F: Line graph plotting the percentage of bodyweight change over days after tumor implantation. All groups show a constant bodyweight over time. G: Flow cytometry histogram plotting relative count over fluorescence intensity on C51-CEA cells. F4-IL12 displays increased fluorescence intensity compared to the negative control. H: Line graph plotting tumor volume over time for saline, KSF-IL12, and F4-IL12 treated mice. The curve for the saline group increases rapidly until day 15. The curve for the KSF-IL12 group increases slightly slower than for saline and stops at day 19. The curve for F4-IL12 increases slowly until day 24. I: Line graph plotting the percentage of bodyweight change over days after tumor implantation. All groups show a constant bodyweight over time.

Figure 6. Ex vivo analysis of F4-IL12 treated tumor-bearing mice. (a) Analysis of tumor-infiltrating lymphocytes in CT26-CEA (left) and C51-CEA (right). Tumors were removed 48 hours after the third injection and analyzed by immunofluorescence staining. Markers specific for CD4⁺ T cells (CD4), CD8⁺ T cells (CD8), Natural killer cells (NKp46), and regulatory T cells (Foxp3) were used (green). Vasculature was visualized through CD31 staining (red) and nuclei with DAPI (blue). 20× magnification; scale bar = 100 μm. (b) Phenotype analysis of CD4⁺ and CD8⁺ T cells in the tumor of treated C51-CEA bearing mice. Tumors were removed 48 hours after the third injection. Bar plots show expression levels of IFNγ, PD-1 (both in CD4⁺ and CD8⁺ T cells), the ratio of CD4⁺ and CD8⁺ T cells, and expression of Perforin and Granzyme B in CD8⁺ T cells. Statistical differences were assessed between mice receiving saline, KSF-IL12, and F4-IL12. *p < 0.05; **p < 0.01 (regular one-way ANOVA test with Tuckey posttest). Error bars = SEM; n = 3.

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A: Immunofluorescence staining of the tumors of the three different treatment groups in the CT26-CEA and C51-CEA models. In CT26-CEA tumors, the saline group and the KSF-IL12 group display a green signal for the CD8 marker and a minimal green signal for the CD4 marker but no signal for NKp46 and Foxp3. The F4 IL12 group displays a green signal for the markers CD4, CD8, and NKp46 but not for Foxp3. In C51-CEA tumors, the F4-IL12 group displays a moderate green signal for the markers CD4, CD8, and NKp46 but no signal for Foxp3. The saline and KSF-IL12 groups are negative for all markers. B: Bar charts plotting the expression of different markers on tumor infiltrating T cells. IFN gamma expression in the F4-IL12 group is significantly overexpressed compared to the saline and the KSF-IL12 groups in CD4-positive T cells and compared to the saline group in the CD8-positive T cells. The expression of PD-1 in CD4-positive and CD8-positive T cells and the expression of granzyme B and perforin in CD8-positive T cells is slightly higher in the F4-IL12 group, compared to the saline and KSF-IL12 groups, although not significant.