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. 2021 Apr 13;13(8):1845.
doi: 10.3390/cancers13081845.

Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation

Mélissa Prat  1 , Marie Salon  1   2 , Thibault Allain  1 , Olivier Dubreuil  3 , Grégory Noël  4 , Laurence Preisser  5 , Bérangère Jean  3 , Lydie Cassard  6 , Fanny Lemée  3 , Isabelle Tabah-Fish  3 , Bernard Pipy  1   2 , Pascale Jeannin  5 , Jean-François Prost  3 , Jean-Marc Barret  3 , Agnès Coste  1   2
Affiliations

Murlentamab, a Low Fucosylated Anti-Müllerian Hormone Type II Receptor (AMHRII) Antibody, Exhibits Anti-Tumor Activity through Tumor-Associated Macrophage Reprogrammation and T Cell Activation

Mélissa Prat et al. Cancers (Basel). .

Abstract

AMHRII, the anti-Müllerian hormone receptor, is selectively expressed in normal sexual organs but is also re-expressed in gynecologic cancers. Hence, we developed murlentamab, a humanized glyco-engineered anti-AMHRII monoclonal antibody currently in clinical trial. Low-fucosylated antibodies are known to increase the antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) potency of effector cells, but some preliminary results suggest a more global murlentamab-dependent activation of the immune system. In this context, we demonstrate here that the murlentamab opsonization of AMHRII-expressing ovarian tumor cells, in the presence of unstimulated- or tumor-associated macrophage (TAM)-like macrophages, significantly promotes macrophage-mediated ADCC and shifts the whole microenvironment towards a pro-inflammatory and anti-tumoral status, thus triggering anti-tumor activity. We also report that murlentamab orients both unstimulated- and TAM-like macrophages to an M1-like phenotype characterized by a strong expression of co-stimulation markers, pro-inflammatory cytokines and chemokines, favoring T cell recruitment and activation. Moreover, we show that murlentamab treatment shifts CD4+ Th1/Th2 balance towards a Th1 response and activates CD8+ T cells. Altogether, these results suggest that murlentamab, through naïve macrophage orientation and TAM reprogrammation, stimulates the anti-tumor adaptive immune response. Those mechanisms might contribute to the sustained clinical benefit observed in advanced cancer patients treated with murlentamab. Finally, the enhanced murlentamab activity in combination with pembrolizumab opens new therapeutic perspectives.

Keywords: adaptive immunity; glyco-engineered antibody; immunotherapy; murlentamab; ovarian cancer; tumor-associated macrophages.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Variation of pharmacodynamic markers measured in blood samples and biopsies of patients from C201 and C101 studies. (A) Detection of CD69 as a marker of activation of monocytes, regulatory and CD8+ T cells in blood samples of patients (n = 20) with colorectal cancer treated with murlentamab (single agent and in combination with trifluridine/tripiracil). Data shown (boxplots) are the results from 20 patients. * p < 0.05; ** p < 0.01. p values were determined using one-way ANOVA analysis followed by Tukey’s multiple comparisons test. (B) Detection of ICOS (inducible co-stimulatory molecule) as a marker of lymphocyte activation in blood samples of patients with ovarian cancer treated at Gustave Roussy (Paris, France) with murlentamab in combination with carboplatin + paclitaxel (n = 4). (C) Detection of CXCL9 (n = 16) and CXCL10 (n = 15) release in blood samples of all patients treated with murlentamab single agent in C201. (D) Detection of co-staining CD14/CD163 (n = 8) and of ICOS (n = 4) as markers of immune system regulation in FFPE (Formalin-Fixed Paraffin-Embedded) biopsies obtained from the C201 study. (E) Image and quantification of CD16 (n = 5), co-staining CD16/granzyme B (GrZB) (n = 4), and CD8 (n = 4) as markers of immune system activation in FFPE biopsies obtained from the C201 study. (F) Image and quantification of CD16/granzyme B (GrZB) co-staining in FFPE biopsies obtained from the C101 study (n = 2).
Figure 2
Figure 2
Murlentamab opsonization of SKOV3-R2+ increases macrophage anti-tumoral activity and antibody-dependent cell-mediated cytotoxicity (ADCC) killing. SKOV3-R2+ ovarian tumor cells were labeled with different 3C23K antibodies (3C23K-FcKO control, 3C23K-CHO normally fucosylated or murlentamab the low fucosylated form) and cultured in the presence of human monocyte-derived macrophages from healthy donors unstimulated (M0) or stimulated with M-CSF and IL-10 (tumor-associated macrophages (TAMs)). (A) Opsonized-SKOV3-R2+ cell number was determined by flow cytometry after one and two days of co-culture with M0 or TAMs. * p < 0.05; ** p < 0.01 compared 3C23K-FcKO vs. murlentamab at a given time (B) ADCC was performed after 4 h of co-culture between SKOV3-R2+ cells and M0 or TAMs. Data shown (mean ± SEM) are the results from three different experiments (performed with three different healthy donors). * p < 0.05; **** p < 0.0001. p values were determined using one-way ANOVA analysis followed by Tukey’s multiple comparisons test.
Figure 3
Figure 3
Murlentamab opsonization of SKOV3-R2+ orients naïve macrophages and reprograms TAMs towards an M1-like profile. SKOV3-R2+ ovarian tumor cells were labeled with different 3C23K antibodies (3C23K-FcKO control, 3C23K-CHO normally fucosylated or murlentamab the low fucosylated form) and cultured in the presence of human monocyte-derived macrophages from healthy donors unstimulated (M0) or stimulated with M-CSF and IL-10 (TAMs). (A) The proportion of macrophages expressing M1/M2 membrane markers (CD32, CD64, CD80, TLR2, CD163, CD36 and CD206) was determined by flow cytometry after three days of co-culture with SKOV3-R2+ cells. (B) The release of cytokines (IL1β, IL12, TNFα, IL6, IFNγ, IL10) and chemokines (CCL2, CCL4, CCL5, CXCL9 and CXCL10) in the culture medium was determined by AlphaLISA after three days of co-culture with SKOV3-R2+ cells. Data shown (boxplots) are the results from three different experiments (performed with three different healthy donors). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. p values were determined using one-way ANOVA analysis followed by Tukey’s multiple comparisons test.
Figure 4
Figure 4
Murlentamab opsonization of SKOV3-R2+ activates an effective anti-tumor T cell immune response. SKOV3-R2+ ovarian tumor cells were labeled with different 3C23K antibodies (3C23K-FcKO control, 3C23K-CHO normally fucosylated or murlentamab the low fucosylated form) and cultured in the presence of human monocyte-derived macrophages from healthy donors unstimulated (M0) or stimulated with M-CSF and IL-10 (TAMs). After 3 days of co-culture, activated T cells coming from the same healthy donor were added in the culture well for 4 more days. (A) The CD4+ Th1/Th2 polarization profile, (B) the proportion of CD3+ CD4+ CD25+ regulatory T cells and (C) the activation of T CD8+ cells were determined by flow cytometry after four days of co-culture. Data shown (boxplots) are the results from two different experiments (performed with two different healthy donors). * p < 0.05; ** p < 0.01; *** p < 0.001. p values were determined using one-way ANOVA analysis followed by Tukey’s multiple comparisons test.
Figure 5
Figure 5
Murlentamab/pembrolizumab combination accentuates the anti-tumoral effect of murlentamab monotherapy through the enhancement of T cell activation. (A–C) SKOV3-R2+ ovarian tumor cells were labeled with different 3C23K antibodies (3C23K-FcKO control or murlentamab the low fucosylated form) and cultured in the presence of human monocyte-derived macrophages from healthy donors stimulated with M-CSF and IL-10 (TAMs). After 3 days of co-culture, activated T cells coming from the same healthy donor were added in the culture well for 4 more days. Pembrolizumab was added into co-culture wells everyday from day 3 to day 10. (A) Opsonized-SKOV3-R2+ cell number was determined by flow cytometry after one and two days of co-culture with TAMs. Data shown (mean ± SEM) are the results from three different experiments (performed with one healthy donors). ** p < 0.01 compared 3C23K-FcKO vs. Murlentamab. # p < 0.05; ## p < 0.01 compared 3C23K-FcKO + anti-PD-1 vs. Murlentamab + anti-PD-1 as determined using one-way ANOVA analysis followed by Dunnett’s multiple comparisons test. (B,C) The CD4+ Th1/Th2 polarization profile and the activation of T CD8+ cells were determined by flow cytometry after four days of co-culture. Data shown (mean ± SEM) are the results from three different experiments (performed with one healthy donors). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. p values were determined using one-way ANOVA analysis followed by Tukey’s multiple comparisons test. (D,E) 10 × 106 COV434-R2+ ovarian tumor cells were transplanted subcutaneously into humanized GM-CSF/IL3/IL4 hu-NOG (NOD/Shi-scid/IL2Rγnull ) mice (Taconic). After 35 days, when tumors were big enough, mice were i.p treated or not with murlentamab (5 mg/kg) +/− pembrolizumab (25 mg/kg) twice a week for 4 weeks. (D) Quantification of circulating CD86+ and CD163+ cells by flow cytometry from blood of tumor-bearing mice before treatment and after 24 days of treatment with murlentamab (5 mg/kg) or pembrolizumab (25 mg/kg) as single agents or murlentamab/pembrolizumab combo-therapy. Data are represented as boxplots. *** p < 0.001, **** p < 0.0001 in comparison to baseline. (E) In vivo tumor growth. Data are represented as mean + SEM.
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