Antibodies targeting sialyl Lewis A mediate tumor clearance through distinct effector pathways

Abstract

Sialyl Lewis A (sLeA, also known as CA19-9), a tetrasaccharide selectively and highly expressed on advanced adenocarcinomas including colon, stomach, and pancreatic cancers, has long been considered as an attractive target for active and passive vaccination. While progress in antibodies targeting tumor-associated protein antigens resulted in an impressive array of therapeutics for cancer treatment, similar progress in exploiting tumor-associated carbohydrate antigens, such as sLeA, has been hampered by the lack of a detailed understanding of the singular characteristics of these antigens. We have addressed this issue by analyzing antibodies derived from patients immunized with an sLeA/KLH vaccine. These antibodies were engineered to mediate tumor clearance in vivo in preclinical models through Fc-FcγR interactions. However, in contrast to protein antigens in which hFcγRIIIA engagement was both necessary and sufficient to mediate tumor clearance in both preclinical and clinical settings, a similar selective dependence was not seen for anti-sLeA antibodies. Thus, re-engineering the Fc portion of sLeA-targeting antibodies to broadly enhance their affinity for activating FcγRs led to an enhanced therapeutic effect. These findings will facilitate the development of more efficient anticancer therapies and further advance this promising class of therapeutic antibodies into clinical use.

Keywords: Cancer immunotherapy; Immunoglobulins; Immunology.

Figure 1. Modeling sLeA-expressing murine tumor cell lines.

B16 melanoma cells and EL4 lymphoma cells were transduced to stably express the human enzyme fucosyltransferase III (FUT3), which synthesizes sLeA. (A) Surface expression of sLeA. B16 and EL4 tumor cells were labeled with an anti-sLeA primary Ab (5B1-hIgG1) followed by Alexa Fluor 488–conjugated goat anti–human IgG antibody. The panel shows a representative experiment (n > 3), all showing similar results. (B) Secretion of sLeA. Supernatants were collected from tumor cells 72 hours after seeding, filtered, and analyzed by sandwich ELISA for detection of extracellular sLeA. Data were pooled from n = 3 experiments and presented as mean ± SEM. (C) Lung colonization of sLeA⁺ B16 cells. WT C57BL/6 mice were inoculated i.v. with 5 × 10⁵ B16 or B16-FUT3 tumor cells. Fourteen days after inoculation, mice were euthanized, lungs were excised and fixed, and metastatic foci were counted. Data were pooled from n = 3 experiments, n ≥ 20/group. ***P < 0.005 (unpaired 2-tailed t test). The box extends from the 25th to 75th percentile, the line within the box represents the median value, and the whiskers correspond to the 5th to 95th percentile. (D) Tumor growth of B16 cells. WT C57BL/6 mice were inoculated subcutaneously with 5 × 10⁵ B16 or B16-FUT3 tumor cells. Average sizes of primary tumors ± SEM are presented in mm³, measured biweekly by caliper. Data were pooled from n = 2 experiments, n > 18/group. (E) Survival of mice inoculated with EL4 cells. WT C57BL/6 mice were inoculated i.v. with 5 × 10⁵ EL4 or EL4-FUT3 tumor cells. Survival was followed daily. Data were pooled from n = 3 experiments, n = 28/group.

Figure 2. sLeA-targeting Abs protect mice from sLeA⁺ tumor challenge.

(A) Anti-sLeA Abs inhibit lung colonization of sLeA⁺ B16 cells. WT C57BL/6 mice were inoculated i.v. with 5 × 10⁵ B16 or B16-FUT3 tumor cells. One hundred micrograms of anti-sLeA Abs (5B1-mIgG2a or 7E3-mIgG2a) or isotype-matched control Abs was administered i.p. on days 1, 4, 7, and 11. Fourteen days after inoculation, mice were euthanized, lungs were excised and fixed, and metastatic foci were counted. The panel summarizes data pooled from n = 3 experiments, and shows representative images of 3 excised lungs from mice inoculated with B16-FUT3 cells. n > 20 for all groups, except 7E3-mIgG2a (n = 6–8/group). **P < 0.01, ****P < 0.0001 (1-way ANOVA with Bonferroni’s post hoc test). The box extends from the 25th to 75th percentile, the line within the box represents the median value, and the whiskers correspond to the 5th to 95th percentile. (B) Anti-sLeA Abs rescue mice inoculated with sLeA⁺ EL4 cells. WT C57BL/6 mice were inoculated i.v. with 5 × 10⁵ EL4 or EL4-FUT3 tumor cells. One hundred micrograms of anti-sLeA Abs (B1: 5B1-mIgG2a; B2: 7E3-mIgG2a) or isotype-matched control Abs was administered i.p. on days 1, 4, 7, and 11. Survival was assessed daily. For 5B1, data were pooled from n = 2 experiments, n > 20/group. For 7E3, n = 10–20/group. ****P < 0.0001 (log-rank test). NS, not significant.

Figure 3. Ab-induced antitumor effect is mediated by engagement of Fcγ receptors (FcγRs), and is dictated by the tumor.

(A) The anti-sLeA effect is fully mediated by FcγR engagement in an sLeA⁺ B16 tumor model. WT C57BL/6 or activating FcγR-null (aFcγR-null, γ chain–KO) mice (see Methods for detailed mouse strain information) were inoculated i.v. with 5 × 10⁵ B16-FUT3 tumor cells. One hundred micrograms of anti-sLeA Abs (5B1-mIgG2a or 5B1-mIgG1-D265A variants) or isotype-matched control Abs was administered i.p. on days 1, 4, 7, and 11. Fourteen days after inoculation, mice were euthanized, lungs were excised and fixed, and metastatic foci were counted. The panel summarizes the data pooled from n = 2 experiments, n ≥ 11/group, and shows representative images of 3 excised lungs. *P < 0.05, **P < 0.01 (1-way ANOVA with Bonferroni’s post hoc test). The box extends from the 25th to 75th percentile, the line within the box represents the median value, and the whiskers correspond to the 5th to 95th percentile. Data for groups WT mice – isotype, and WT – 5B1-mIgG2a also appear in Figure 2A. (B) The anti-sLeA effect is partially mediated by FcγR engagement in an sLeA⁺ EL4 tumor model. WT C57BL/6 or aFcγR–null mice (see Methods for detailed mouse strain information) were inoculated i.v. with 5 × 10⁵ EL4-FUT3 tumor cells. One hundred micrograms of anti-sLeA Abs (5B1-mIgG2a or 5B1-mIgG1-D265A) or isotype-matched control Abs was administered i.p. on days 1, 4, 7, and 11. Survival was assessed daily. n = 8/group. *P < 0.05, **P < 0.01 (log-rank test).

Figure 4. Engagement of either hFcγRIIA or hFcγRIIIA is necessary and sufficient for tumor clearance, mediated by sLeA-targeting Abs with an hIgG1 Fc.

Mice were inoculated i.v. with 5 × 10⁵ B16-FUT3 tumor cells. One hundred micrograms of anti-sLeA Abs or isotype-matched control Abs was administered i.p. on days 1, 4, 7, and 11. Fourteen days after inoculation, mice were euthanized, lungs were excised and fixed, and metastatic foci were counted. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (1-way ANOVA with Bonferroni’s post hoc test). For all panels, the box extends from the 25th to 75th percentile, the line within the box represents the median value, and the whiskers correspond to the 5th to 95th percentile. (A) Fc-engineered anti-sLeA Ab variants demonstrate superior antitumor efficacy. FcγR-humanized mice were treated with clones 5B1 or 7E3, hIgG1 or hIgG1-GAALIE with G236A/A330L/I332E mutations. Data were pooled from n = 2–3 experiments, n ≥ 13/group, except for 7E3-hIgG1-GAALIE (n = 7). (B) 5B1-hIgG1 Abs with enhanced binding affinity for hFcγRIIA, or hFcγRIIIA, or both demonstrate a superior antitumor effect. FcγR-humanized mice were treated with Fc variants 5B1-hIgG1, 5B1-hIgG1-GA with a G236A mutation, 5B1-hIgG1-ALIE with A330L/I332E mutations, or 5B1-hIgG1-GAALIE with G236A/A330L/I332E mutations. Data were pooled from n = 2–4 experiments. n ≥ 12/group. (C) hFcγRIIA or hFcγRIIIA engagement is essential for efficient tumor clearance of sLeA⁺ tumors. Activating FcγR-null (aFcγR-null, γ chain–KO), FcγR-humanized, hFcγRIIA/IIB–transgenic, and hFcγRIIIA/IIIB–transgenic mice were treated with the Ab 5B1-hIgG1-GAALIE with G236A/A330L/I332E mutations. Data were pooled from n ≥ 2 experiments for aFcγR-null and FcγR-humanized mice, n ≥ 12/group. For hFcγRIIA/IIB–transgenic and hFcγRIIIA/IIIB–transgenic, n = 5–8/group.