Cancer-related immunosuppressant inhibitors

Abstract

The present invention relates to a compound having a glycosylated Fc fragment for use as an immunosuppressant inhibitor in the treatment of cancer-associated immunosuppression. The present invention further relates to a pharmaceutical composition comprising at least the compound having a glycosylated Fc fragment. [Selection diagram] None

Description

[Technical field]

[0001] The present invention relates to the treatment of an immunosuppressive state that may arise during cancer disease. [Background technology]

[0002] Cancer immune evasion is a major obstacle in designing effective anticancer therapeutic strategies. Although considerable progress has been made in understanding how cancers evade destructive immune systems, measures to block tumour evasion have lagged behind.

[0003] Long-term patient survival is considered the "gold standard" of success in cancer treatment, with disease-free survival being the ultimate goal from the patient's perspective. It is increasingly believed that long-term and disease-free survival largely depend on the enhancement of the patient's individual immune system to generate an effective anti-tumor response. Evidence of a strong immune response to treatment, even to the point of inducing autoimmune symptoms, may be a favorable indicator of long-term survival in cancer patients (Burkholder et al., 2014, Biochimica et Biophysica Acta, Vol. 1845:182-201).

[0004] Although various drugs have been screened for their antitumor effects and selected drugs have been approved for the treatment of cancer patients, chemotherapy, radiotherapy, and surgery remain the mainstay of standard cancer treatment strategies (Vinay et al., 2015, Seminars in cancer biology, Vol. 35: 5185-5198). The drawback of these therapies is their ability to cause transient immunosuppression, which may increase the risk of infection and also reduce the ability of the immune system to prevent further development of cancer.

[0005] Identifying an adapted overall cancer treatment strategy involves determining the extent to which immune-boosting therapies can enhance standard anti-cancer therapies. It is strongly suggested in the art that most, if not all, overall cancer treatment strategies should include relevant measures to boost anti-tumor immunity, regardless of the type of anti-tumor treatment used.

[0006] Immunotherapy has the potential to treat cancer because it acts through a different mechanism than chemotherapy or radiotherapy and represents a non-cross-resistant treatment with a completely different toxicity spectrum. Both T cells and B cells, through the genetic modification of their respective receptors, can have the ability to recognize a wide variety of potential tumor antigens, and more importantly, both T cells and B cells can distinguish subtle antigenic differences between normal and transformed cells, providing specificity while minimizing toxicity.

[0007] The importance of the immune response to cancer has been known for decades. However, recent advances in immuno-oncology have greatly improved our understanding of the interactions between the immune system and cancer. Immunoediting refers to the process by which the immune system can alter tumor progression. Immunoediting regulates both tumor quantity and quality. The process of immunoediting cancer has three distinct phases: elimination, equilibrium, and escape phases. The escape phase can occur at the tumor level or at the level of the tumor microenvironment. At the microenvironment level, the introduction of regulatory T cells (Treg) and myeloid derived suppressor cells (MDSC), or the expression of programmed death-1 (PD-1) / programmed death-ligand 1 (PD-L1) in the immune infiltrate, can result in an immunosuppressive tumor microenvironment.

[0008] Soluble factors produced by tumor-associated macrophages (TAMs), tumor-associated fibroblasts, Tregs, and suppressor cells all contribute to cancer-induced immunosuppression. TAMs can drive multiple tumor-promoting processes, including immunosuppression, angiogenesis, and direct secretion of tumor growth factors.

[0009] Thus, the immune system plays a key role in controlling and eradicating cancer. Nevertheless, in the setting of malignancies, multiple mechanisms of immunosuppression may exist that prevent effective antitumor immunity.

[0010] Antibody therapies directed against several negative immune regulators (checkpoints) have shown considerable success today and have the potential to become a mainstay of treatment for patients with a variety of malignancies.

[0011] The first such molecule, shown to inhibit both T-cell proliferation and IL-2 production, was cytotoxic T-lymphocyte associated protein 4 (CTLA-4). This discovery led to efforts directed at blocking this inhibitory pathway, with the aim of activating dormant T cells directed against cancer cells. Ipilimumab, the first antibody directed against CTLA-4, was rapidly introduced into clinical trials and approved by the US Food and Drug Administration (FDA) in 2011 for the treatment of metastatic melanoma. After the success with ipilimumab, other immune checkpoints were tested for inhibition as potential targets. One such interaction was that of the programmed cell death 1 (PD-1) T-cell receptor with its ligand, programmed death ligand 1 (PD-L1), found on many cancer cells.

[0012] However, such antibodies are effective in only a limited number of tumor types (mainly melanoma, lung cancer, and renal cancer), and even in sensitive tumors, a significant proportion of patients remain resistant to them.

[0013] Current knowledge related to anticancer therapeutic strategies dictates that in most situations, to achieve successful cancer therapy, a dual approach must be followed that seeks to (i) eliminate immune suppressive factors / mechanisms and (ii) enhance tumor-killing activity. Summary of the Invention [Problem to be solved by the invention]

[0014] Thus, there is a need in the art to provide additional therapeutic strategies for treating cancer. In particular, there is a need for new means to reduce or block the immune suppression that may occur in cancer patients in order to define potential new anti-cancer treatment strategies. [Means for solving the problem]

[0015] The present invention relates to glyco-engineered Fc fragment-bearing compounds for use as immunosuppressant inhibitors in the treatment of cancer-associated immunosuppression.

[0016] In particular, the present invention relates to compounds having a glycoengineered Fc fragment for use as T cell immunosuppressant inhibitors in the treatment of cancer-associated immunosuppression.

[0017] The present invention relates to CD8 inhibitors in the treatment of cancer-associated immunosuppression. + The present invention includes compounds having glycosylated Fc fragments for use as T cell immunosuppressant inhibitors.

[0018] In some embodiments, the compound having a glycoengineered Fc fragment is a hypofucosylated Fc fragment-bearing compound.

[0019] In some embodiments, the compound having the glycoengineered Fc fragment comprises two amino acid chains of SEQ ID NO. 70.

[0020] In some embodiments, the compound having a glycoengineered Fc fragment is a glycoengineered antibody, in particular a hypofucosylated antibody.

[0021] In some embodiments, the glycoengineered antibody is directed against a tumor associated antigen.

[0022] In some embodiments, the tumor-associated antigen is selected from the group consisting of HER2, HER3, HER4, and AMHRII.

[0023] In some embodiments, the antibody is selected from the group consisting of the antibodies designated 3C23K, 9F7F11, H4B121, and HE4B33, and variants thereof, as disclosed herein.

[0024] In some embodiments, the cancer treatment comprises administering to the individual an additional anti-cancer agent.

[0025] In some embodiments, the cancer treatment comprises administering to the individual an inhibitory immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1, PD-L2, BTLA, CTLA-4, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), IDO, KIR, LAG3, TIM-3, VISTA, CD137, OX40, OX40L, and B7S1.

[0026] In some embodiments, the inhibitor consists of an antibody, or an antigen-binding fragment thereof, directed against the inhibitory immune checkpoint.

[0027] The present invention also relates to a pharmaceutical composition comprising (i) a compound having a glycoengineered Fc fragment, and (ii) an inhibitory immune checkpoint inhibitor. [Brief description of the drawings]

[0028] [Figure 1A] Figure 1 shows that glycoengineered 3C23K mAb (GM102) reduces macrophage-induced T cell inhibition. Figure 1A: Measures of PBT proliferation co-cultured with MDM2 macrophages targeting COV434-AMHRII tumor cells. MDM2 were challenged with anti-CD3 / CD28 pre-activated peripheral blood T cells (also called "PBT") for 4 days prior to co-culture with COV434-AMHRII cell lines opsonized with either irrelevant mAb R565 (isotype control), anti-AMHRII FcKO, or anti-AMHRII 3C23K for an additional 4 days. Data represent Division Index (i.e., the average number of cell divisions undergone by cells in the initial population) ± standard deviation of pre-activated CD8+ T cells (data are representative of 3 independent experiments; p-value *<0.05). Abscissa, left to right: (i) naT cell control, (ii) aT cell control, (iii) isotype control, (iv) FcKO, and (v) 3C23K. [Figure 1B]Figure 1 shows that glycoengineered 3C23K mAb (GM102) reduces macrophage-induced T cell inhibition. Figure 1B: Measure of PBT proliferation co-cultured with MDM2 macrophages targeting mAb-treated polystyrene beads. MDM2 were challenged for an additional 4 days with cell trace violet-loaded PBT that had been pre-activated 24 hours prior to co-culture with uncoated polystyrene beads as a control or anti-AMHRII FcKO or anti-AMHRII 3C23K-coated polystyrene beads. Data represent division index values ​​(i.e., the average number of cell divisions undergone by cells in the initial population) ± standard deviation of pre-activated CD8+ T cells (data are representative of three independent experiments. p-value **<0.01). na refers to non-activated T cells, a. refers to activated T cells. Abscissa, left to right: (i) naT cell control, (ii) aT cell control, (iii) untreated beads, (iv) FcKO, and (v) 3C23K. [Figure 2A] Figure 2 shows that glycoengineered 3C23K (GM102) antibody does not affect proliferation of human T lymphocytes. CellTrace Violet-loaded T cells were activated with CD3 / CD28-coated beads in the presence or absence of 10 μg / ml of anti-AMHRII3C23K mAb. After 3 days, dilutions of CellTrace Violet were assessed by flow cytometry and presented as raw data (Figure 2A). [Figure 2B]Figure 2 shows that glycoengineered 3C23K (GM102) antibody does not affect proliferation of human T lymphocytes. CellTrace Violet-loaded T cells were activated with CD3 / CD28-coated beads in the presence or absence of 10 μg / ml of anti-AMHRII3C23K mAb. After 3 days, dilutions of CellTrace Violet were assessed by flow cytometry and expressed as % of T cells that had undergone 1, 2, 3, or 4 divisions (Figure 2B). In Figure 2B, the ordinate represents the percentage (%) at each peak; from bottom to top: (i) 0 divisions, (ii) 1 division, (iii) 2 divisions, (iv) 3 divisions, and (v) 4 divisions. [Figure 3A-1] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3A illustrates the decreased expression of markers associated with M2 phenotype macrophages, such as Sepp1 (mRNA - Figure 3A-1). The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3A-2] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3A illustrates the decreased expression of markers associated with M2 phenotype macrophages, such as Stab1 (mRNA - Figure 3A-2). The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3A-3]Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3A illustrates the decreased expression of markers associated with M2 phenotype macrophages, such as FOLFR2 (m-RNA - Figure 3A-3). The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3A-4] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3A illustrates the reduced expression of markers associated with M2 phenotype macrophages, such as CD163 (protein - Figure 3A-4). The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3B-1] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3B-1 (mRNA expression) illustrates increased expression of CD16. The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3B-2] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3B-2 (protein expression) illustrates increased expression of CD16. The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3C-1] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3C-1 (mRNA expression) illustrates increased expression of CD64. The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3C-2] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3C-2 (mRNA expression) illustrates increased expression of CD64. The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3C-3] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3C-3 (protein expression) illustrates increased expression of CD64. The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3D-1] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3D illustrates the increase in pro-inflammatory factors normally expressed by M1 macrophages, such as TNFα (Figure 3D-1). The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3D-2]Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3D illustrates the increase in pro-inflammatory factors normally expressed by M1 macrophages, such as IL1β (Figure 3D-2). The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3E] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3E illustrates the reduction of mRNA levels of the gene encoding the immunosuppressive factor TGFβ. The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3F] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3F illustrates the reduction of mRNA levels of the gene encoding the immunosuppressive factor IDO1. The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3G-1] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3G-1 (mRNA expression) illustrates the reduction of the immunosuppressive factor IL10. The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3G-2]Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3G-2 (protein expression) illustrates the reduction of the immunosuppressive factor IL10. The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3H] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3H (mRNA expression) illustrates the reduction of the pro-angiogenic factor PDGFα. The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3I] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3I (mRNA expression) illustrates the reduction of the pro-angiogenic factor VEGFβ. The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3J] Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3J (mRNA expression) illustrates the reduction of the angiogenic factor HGF. The left box illustrates M2 macrophages grown in wells without antibody. The middle box illustrates M2 macrophages cultured in wells with FcKO antibody. The right box illustrates M2 macrophages cultured in wells with hypofucosylated R18H2 antibody. [Figure 3K]Figure 3 shows activation of TAM-like macrophages by Fc-bearing glycoengineered compounds. Figure 3K shows PDL2 expression on the surface of M2 macrophages. The white box illustrates an M2-type macrophage cultured in a well with FcKO antibody. The black box illustrates an M2-type macrophage cultured in a well with hypofucosylated R18H2 antibody. [Figure 4A] Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immune suppression, which leads to activation of the immune system. Figure 4A: ADCC generated by TAM-like macrophages on SKOV3-AMHRII cells incubated with anti-AMHRII antibody for 4 hours at 37°C. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data from macrophages from three donors tested in triplicate are presented. [Figure 4B] Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immunosuppression, which leads to activation of the immune system. Figure 4B shows cytolysis of SKOV3-AMHRII cancer cells by TAM-like macrophages incubated with anti-AMHRII antibody for 4 days. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data from macrophages from three donors tested in triplicate are presented. [Figure 4C] Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immune suppression, which leads to activation of the immune system. Figure 4C: Percentage (%) of CD8 memory (CD8+CD25+) macrophages after co-incubation of TAM-like macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody for 4 days. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data are presented for macrophages from three donors tested in triplicate. [Figure 4D]Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immune suppression, which leads to activation of the immune system. Figure 4D: Percentage (%) of Th1 (CD4+CD183+) macrophages after co-incubation of TAM-like macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody for 4 days. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data for macrophages from three donors tested in triplicate are presented. [Figure 4E] Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immune suppression, which leads to activation of the immune system. Figure 4E: Percentage (%) of Th2 (CD4+CD183-) macrophages after co-incubation of TAM-like macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody for 4 days. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data for macrophages from three donors tested in triplicate are presented. [Figure 4F] Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immune suppression, which leads to activation of the immune system. Figure 4F: Detection of CXCL9 in the culture medium after 4 days of co-incubation of TAM-like macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data from macrophages from three donors tested in triplicate are presented. [Figure 4G]Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immune suppression, which leads to activation of the immune system. Figure 4G: Detection of CXCL10 in the culture medium after 4 days of co-incubation of TAM-like macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data are presented for macrophages from three donors tested in triplicate. [Figure 4H] Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immune suppression, which leads to activation of the immune system. Figure 4H: Detection of CCL2 in the culture medium after co-incubation of TAM-like macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody for 4 days. Data from three donors (triplicates) were treated separately due to different baselines for each donor. Donors from left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). [Figure 4I] Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immune suppression, which leads to activation of the immune system. Figure 4I: Detection of IL1β in the culture medium after 4 days of co-incubation of TAM-like macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data from macrophages from three donors tested in triplicate are presented. [Figure 4J]Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immune suppression, which leads to activation of the immune system. Figure 4J: Detection of IL6 in the culture medium after 4 days of co-incubation of TAM-like macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody. Left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20. Data are presented for macrophages from three donors tested in triplicate. [Figure 4K] Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immune suppression, which leads to activation of the immune system. Figure 4K: Detection of CCL5 in culture medium after 4 days of co-incubation of TAM-like macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data from macrophages from three donors tested in triplicate are presented. [Figure 4L] Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immunosuppression, which leads to activation of the immune system. Figure 4L: Detection of IL12 in the culture medium after 4 days of co-incubation of undifferentiated macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data are presented for macrophages from three donors tested in triplicate. [Figure 4M]Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immunosuppression, which leads to activation of the immune system. Figure 4M: Detection of IL6 in the culture medium after 4 days of co-incubation of undifferentiated macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data are presented for macrophages from three donors tested in triplicate. [Figure 4N] Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immune suppression, which leads to activation of the immune system. Figure 4N: Detection of IL1β in the culture medium after 4 days of co-incubation of undifferentiated macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data from macrophages from three donors tested in triplicate are presented. [Figure 4O] Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immune suppression, which leads to activation of the immune system. Figure 4O: Detection of IL23 in culture medium after 4 days of co-incubation of undifferentiated macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data from macrophages from three donors tested in triplicate are presented. [Figure 4P]Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immune suppression, which leads to activation of the immune system. Figure 4P: Detection of CXCL9 in culture medium after 4 days of co-incubation of undifferentiated macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data from macrophages from three donors tested in triplicate are presented. [Figure 4Q] Figure 4 shows that glycoengineered 3C23K (GM102) antibody blocks immune suppression, which leads to activation of the immune system. Figure 4Q: Detection of CXCL10 in culture medium after 4 days of co-incubation of undifferentiated macrophages with SKOV3-AMHRII cancer cells and anti-AMHRII antibody. From left to right: 3C23K-FcKO, 3C23K CHO, and 3C23K YB20 (meaning 3C23K YB2 / 0). Data are presented for macrophages from three donors tested in triplicate. [Figure 5A] Figure 5 shows activation of macrophages present in tumor tissues of cancer patients administered glycoengineered antibodies. Figure 5A (cd16): Results of immunofluorescence assay of cell markers performed on tumor tissues of cancer patients administered 3C23K antibody. Figure 5A: Percentage (%) of CD16 positive tumor tissues in patients 04-01 and 01-01 before (baseline) and during treatment. Black boxes illustrate patient 04-01 and grey boxes illustrate patient 01-01. [Figure 5B]Figure 5 shows activation of macrophages present in tumor tissue of cancer patients administered glycoengineered antibodies. Figure 5B (Granzyme): shows the results of immunofluorescence assay of cell markers performed on tumor tissue of cancer patients administered 3C23K antibody. Figure 5B: shows the percentage (%) of cells in patient 01-01 before (baseline) and during treatment. Black boxes illustrate the percentage (%) of cells CD8+GZB+ / mm2; grey boxes illustrate the percentage (%) of cells CD16+GZB+ / mm2; and white boxes represent the percentage (%) of NKp46+GZB+ / mm2. [Figure 6A] Figure 6 shows activation of NK cells, monocytes and ICOS+ T cells in cancer patients receiving glycoengineered antibodies. Figure 6A: Flow cytometric quantification (mean flow intensity) of CD4+ICOS+ cells present in blood samples of patients treated with GM102(3C23K). Samples were taken before (C1J0) and during (C1J0+4H) cycle 1 injection of GM102(3C23K), then before cycles 2 and 3 (C2J1 and C3J1, respectively). [Figure 6B] Figure 6 shows activation of NK cells, monocytes and ICOS+ T cells in cancer patients receiving glycoengineered antibodies. Figure 6B: Flow cytometric quantification (mean flow intensity) of CD8+ICOS+ cells present in blood samples of patients treated with GM102 3C23K at Gustave Roussy. Samples were taken before (C1J0) and during the injection period (C1J0+4H) of GM102 (3C23K) in cycle 1, then before cycles 2 and 3 (C2J1 and C3J1, respectively). [Figure 7] FIG. 7 shows the percentage (%) of typical, intermediate, and atypical monocyte subsets present within CD14+ cells in patients post-treatment and during treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Using an in vitro model of cancer tissue containing cancer cells and immune system cells, such as T cells and macrophages, the inventors found that inhibition of T cell activation could be serendipitously induced by M2 tumor-associated macrophages (TAMs).

[0030] Furthermore, the inventors have shown that such TAM-induced inhibition of T cell activation can be reduced or blocked by adding glycoengineered antibodies to this in vitro cancer tissue model. Glycoengineered antibodies, in particular hypofucosylated antibodies, are known in the art to bind with high affinity to Fc receptors, particularly those present on macrophage membranes, in particular FcγRIIIa (also referred to in the art as "CD16a").

[0031] Without wishing to be bound by any particular theory, the inventors believe that the binding of glycoengineered antibodies to Fc receptors present on the macrophage membrane induces the release of soluble factors, e.g., cytokines, which have an inhibitory blocking effect on T cells present in the tumor tissue environment or cause activation of T cells present in the tumor tissue environment. Thus, the inventors believe that glycoengineered antibodies can reduce or block the inhibition of immune responses against cancer cells in certain individuals affected by cancer, by blocking T cell inhibition or activating T cells.

[0032] The inventors further show herein that tumor cells themselves are not necessarily required for the immune stimulatory effect obtained in the presence of glycoengineered antibodies.

[0033] Furthermore, the inventors have shown that the glycoengineered antibodies described above, which have been shown to bind with high affinity to Fcγ receptors on TAM-like macrophages, induce TAM-like macrophages of the immunosuppressive M2 phenotype towards a non-immunosuppressive M1 phenotype with a concomitant reduction in immunosuppressive cytokines such as IL-10. The inventors have also shown that in the presence of the glycoengineered antibodies described herein, the TAM-like macrophages express more pro-inflammatory cytokines such as IL-1 beta, without significant changes in the expression of tumor-promoting genes such as VEGF alpha, VEGF beta, PDGF beta, and hepatocyte growth factor.

[0034] It has also been shown herein that administration of the glycoengineered antibodies described herein induces increased levels of CD8+ T cells in cancer patients. Without wishing to be bound by any particular theory, the inventors believe that the glycoengineered antibodies induce macrophages to release T cell activating cytokines, thus allowing for the removal of T cell inhibition that occurs in cancer patients experiencing an immunosuppressive state, thus causing activation of CD8+ T cells.

[0035] Furthermore, it is shown herein that the glycoengineered antibodies described herein induce an increase in CD4+ T cells of Th1 phenotype and a decrease in CD4+ T cells of Th2 phenotype. Such a shift in the balance between Th1 and Th2 T cells is expected to aid in increasing the immune response against tumor cells.

[0036] The inventors have also shown that the glycoengineered antibodies described herein modulate the expression of cytokines such as IL1beta, IL6, IL10, IL12, and IL23.

[0037] The glycoengineered antibodies described herein are also shown to induce naive macrophages to reduce their production of immunosuppressive cytokines, such as IL-10.

[0038] The inventors have further shown that administration of the glycoengineered antibodies described herein to cancer patients induces an increase in the number of CD16+ (Fc gamma RIII+) cells in tumor tissue. Thus, administration of the glycoengineered antibodies described herein to cancer patients causes an increase in anti-tumor activated macrophages in tumor tissue. It has further been shown that administration of the glycoengineered antibodies described herein to cancer patients increases the levels of granzyme B-producing activated macrophages in tumor tissue, the inhibition of which immunosuppressive state should contribute to tumor cell cytolysis. Still further, administration of the glycoengineered antibodies described herein to cancer patients also increases the number of NK cells in tumor tissue, the inhibition of which immunosuppression should likewise contribute to tumor cell killing.

[0039] The inventors have also shown that administration of the glycoengineered antibodies described herein to cancer patients (i) increases the expression of CD16 (Fc gamma RIII) by NK cells, (ii) increases the expression of CD69 on monocytes, and (iii) increases the expression of ICOS (inducible T cell costimulatory molecule) on T cells, which are other parameters that result in the inhibition of the immunosuppressive state experienced by cancer patients.

[0040] Overall, our findings indicate that glycoengineered antibodies can reduce or block macrophage-induced suppression of T cell anti-tumor activity.

[0041] Our results allow them to be considered as a therapeutic tool based on the administration of glycoengineered antibodies, aiming at reducing or blocking the immunosuppressive state that may arise in individuals affected by cancer.

[0042] Without wishing to be bound by any particular theory, the inventors believe that the immunostimulatory effect elicited by glycoengineered antibodies is due to the high affinity that said glycoengineered antibodies have for Fc receptors present on cell membranes, in particular for Fc receptors present on macrophage membranes, whether said antibodies have an associated antigen-binding domain or not.

[0043] As shown in the Examples herein, reducing or blocking the immunosuppressive state is obtained even in models in which tumor antigen expressing cells are not present, which may mean that the reduction in tumor cell mass induced by the binding of said glycoengineered antibodies to tumor antigens and phagocytosis itself may not be required to induce their immunostimulatory effect, and in particular may not be required to reduce or block macrophage-induced T cell inhibition. In other words, the inventors believe that the blocking of T cell inhibition by said glycoengineered antibodies is related to the behavior of these antibodies as compounds with glycoengineered Fc fragments.

[0044] The present invention relates to compounds having a glycoengineered Fc fragment for use as immunosuppressive inhibitors in the treatment of cancer in an individual.

[0045] The present invention relates to compounds having a glycoengineered Fc fragment for use in preventing or treating an immunosuppressive state in individuals affected by cancer.

[0046] The present invention relates to the use of a compound having a glycoengineered Fc fragment as an immunosuppressant inhibitor for the preparation of a medicament for the treatment of cancer.

[0047] The present invention relates to the use of a compound having a glycomodified Fc fragment for the preparation of a medicament for the prevention or treatment of an immunosuppressive state in an individual affected by cancer.

[0048] The present invention relates to a method for treating cancer, comprising the step of administering to an individual in need thereof a compound having a glyco-modified Fc fragment as an immunosuppressant inhibitor.

[0049] The present invention relates to a method for preventing or treating an immunosuppressive state in an individual affected by cancer, the method comprising the step of administering to an individual in need thereof a compound having a glycosylated Fc fragment.

[0050] The present invention relates to the use of a compound having a glycomodified Fc fragment for the preparation of a medicament for reducing or blocking the immunosuppressive state caused by macrophage-induced T cell inhibition occurring in individuals affected by cancer.

[0051] The present invention relates to the use of a compound having a glycoengineered Fc fragment for the preparation of a medicament for reducing or blocking the immunosuppressive state caused by macrophage-induced T cell inhibition occurring in individuals affected by cancer.

[0052] The present invention also relates to a method for reducing or blocking an immunosuppressive state caused by macrophage-induced T cell inhibition in an individual affected by cancer, comprising the step of administering to an individual in need thereof a compound having a glycomodified Fc fragment.

[0053] From the preceding embodiments it follows that the individuals with cancer concerned by the present invention are individuals who are also affected by immunosuppression.

[0054] In some embodiments, individuals with cancer concerned by the present invention are also individuals affected by immune suppression caused by anti-cancer treatment.

[0055] definition According to the present invention, the expression "comprising", e.g. "comprising the steps of", is also understood as "consisting of", e.g. "consisting of the steps of".

[0056] As used herein, the term "cancer-associated immunosuppression," "cancer-associated immunosuppression," or "immunosuppressed state" refers to a condition in which, when associated with an individual affected by cancer, the CD8 + It refers to a physiological state encompassing a situation in which a T cell has its ability to be activated and that ability is reduced or blocked, i.e., that the ability to be activated and that ability is partially or completely inhibited.

[0057] By way of illustration, in accordance with the present invention, a cancer-affected individual experiencing an immunosuppressive state may be screened for peripheral blood CD8+ in a sample previously collected from said individual. + This can be determined by an in vitro test method comprising a step of measuring the ability of peripheral blood T cells to proliferate, wherein the peripheral blood T cells are subjected to a pre-activation step before measuring their proliferative ability.

[0058] Thus, in some embodiments, an immunosuppressive state may be detected in the tested individual if the tested individual's ability to expand CD8+ T cells is lower than a reference CD8+ T cell proliferation capacity value indicative of the absence of an immunosuppressive state. In some embodiments, the reference CD8+ T cell proliferation capacity value may be the average CD8+ T cell proliferation capacity value found in healthy individuals who are not immunosuppressed. In some other embodiments, the reference value may be a threshold value that allows for the distinction between (i) a CD8+ T cell proliferation capacity value lower (or higher depending on the index unit used) than a threshold value indicative of an immunosuppressive state and (ii) a CD8+ T cell proliferation capacity value higher (or lower depending on the index unit used) than a threshold value indicative of the absence of an immunosuppressive state.

[0059] In some embodiments, the CD8 T cell proliferation capacity value is a CD8+ T cell proliferation index value, as shown in the Examples herein.

[0060] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. Although not excluded, it will be recognized that treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

[0061] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the probability of developing a disorder or condition in a subject who does not have the disorder or condition but is at risk of or susceptible to developing it.

[0062] As used herein, a compound having a glycoengineered Fc fragment encompasses any compound comprising an Fc fragment of an antibody with altered glycosylation that allows binding of said Fc fragment with high affinity to Fc receptors, in particular to Fc receptors present in macrophage membranes, which includes Fc receptors present in the membranes of tumor-associated macrophages.

[0063] In some embodiments, the Fc-containing protein comprises one or more polypeptides.

[0064] As used herein, an "Fc fragment-bearing protein" refers to a protein that comprises an Fc fragment fused to at least one other heterologous protein unit or polypeptide.

[0065] Compounds having a glycoengineered Fc fragment include (i) the glycoengineered Fc fragment itself, (ii) hybrid compounds comprising a glycoengineered Fc fragment covalently linked to a nonproteinaceous moiety, and (iii) proteinaceous compounds comprising a glycoengineered Fc fragment linked to a proteinaceous moiety.

[0066] Proteinaceous compounds having a glycoengineered Fc fragment include proteins, wherein said glycoengineered Fc fragment is covalently linked to an antigen-binding domain of an antibody, e.g., covalently linked to a variable region of an antibody.

[0067] Proteinaceous compounds having a glycoengineered Fc fragment include proteins, in which said glycoengineered Fc fragment is directly or indirectly covalently linked to one or more other Fc fragments, e.g., covalently linked to one or more other glycoengineered Fc fragments. Examples of compounds having such glycoengineered Fc fragments include compounds known in the art as "Fc multimers" as described, for example, by Thiruppathi et al. (2014, J Autoimmun, Vol. 52:64-73), by Jain et al. (2012, Arthritis Research and Therapy, Vol. 14:R192), or by Zhou et al. (2017, Blood advances, Vol. 1(n°6):DOI 10.1182 / biooadvances.2016001917).

[0068] In some preferred embodiments, compounds having a glycoengineered Fc fragment according to the present invention include glycoengineered antibodies.

[0069] In some preferred embodiments, glycoengineered antibodies include antibodies directed against tumor-associated antigens.

[0070] As used herein, the term "antibody" refers to an assembly (e.g., a natural antibody molecule, an antibody fragment, or a variant thereof) having significant and known specific immunoreactive activity against an antigen of interest, particularly against a tumor-associated antigen of interest. Antibodies and immunoglobulins comprise light and heavy chains with or without interchain covalent bonds between them.

[0071] Immunoglobulin light chains are classified as kappa or lambda (κ, λ). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, the light and heavy chains are covalently linked to each other, and when the immunoglobulin is produced by a hybridoma, a B cell, or a genetically engineered host cell, the "tail" portions of the two heavy chains are linked to each other by a covalent disulfide bond or a non-covalent bond.

[0072] Both the light and heavy chains are divided into regions of structural and functional homology. The term "region" refers to a portion or part of an immunoglobulin or antibody chain, including the constant or variable region, as well as more discrete parts or portions of said regions. For example, the light chain variable region contains "complementarily determining regions" or "CDRs" interspersed with "framework regions" or "FRs" as defined herein.

[0073] Regions of an immunoglobulin heavy or light chain may be defined as "constant" (C) regions or "variable" (V) regions, based on the relative lack of sequence variation within the region among different class members, in the case of "constant regions," or the significant variation within the region among different class members, in the case of "variable regions."

[0074] By convention, the numbering of variable constant region domains increases as they move away from the antigen-binding site or amino-terminus of an immunoglobulin or antibody. The N-terminus of each of the heavy and light immunoglobulin chains is the variable region, and the C-terminus is the constant region; the CH3 and CL domains comprise the carboxy-terminus of the heavy and light chains, respectively. Thus, the light chain immunoglobulin domains are arranged in a VL-CL orientation, while the heavy chain domains are arranged in a VH-CH1-hinge-CH2-CH3 orientation.

[0075] Amino acid positions within the heavy chain constant region, including those within the CH1, hinge, CH2, CH3, and CL domains, may be numbered according to the Kabat index numbering system (see Kabat et al., "Sequences of Proteins of Immunological Interest", USDept. Health and Human Services., 5th edition, 1991). Alternatively, antibody amino acid positions may be numbered according to the EU index numbering system (see Kabat et al., ibid.).

[0076] As used herein, the term "Fc region" is defined as the portion of the heavy chain constant region beginning in the hinge region immediately upstream of the papain cleavage site (i.e., residue 216 in IgG, given that the first residue of the heavy chain constant region is 114) and ending at the C-terminus of the antibody. Thus, a complete Fc region includes at least the hinge, CH2, and CH3 domains.

[0077] As used herein, the term "Fc fragment" refers to a molecule that contains the sequence of a non-antigen-binding fragment resulting from the digestion of an antibody or generated by other means, whether in monomeric or multimeric form, and may include a hinge region. The original immunoglobulin source of the Fc fragment can be of human origin and can be any immunoglobulin, such as IgG1 or IgG2. The Fc fragment consists of monomeric polypeptides that can be linked into a dimeric or multimeric form by covalent associations (i.e., disulfide bonds) and non-covalent associations. The number of intermolecular disulfide bonds between the monomeric subunits of the Fc fragment ranges from 1 to 4 depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of an Fc fragment is a disulfide-linked dimer resulting from papain digestion of IgG. As used herein, the term "Fc fragment" generally refers to monomeric, dimeric and multimeric forms.

[0078] As used herein, the term "Fc fragment-bearing protein" or "Fc fragment-containing protein" refers to a protein that comprises an Fc domain or an Fc receptor-binding fragment thereof (including N-glycans). In certain embodiments, the N-glycan is an N-linked biantennary glycan present in the CH2 domain of the immunoglobulin constant (Fc) region (e.g., at EU position 297). The "N-glycan" is linked to the amide nitrogen of an asparagine or arginine residue in the protein via an N-acetylglucosamine residue. These "N-linked glycosylation sites" occur, for example, in peptide primary structures that contain the amino acid sequence: asparagine-X-serine / threonine, where X is any amino acid residue except proline and aspartic acid. Such N-glycans are fully described, for example, in Drickamer K, Taylor ME (2006). Introduction to Glycobiology, 2nd ed., incorporated herein by reference in its entirety.

[0079] In one embodiment, "N-glycan" refers to the Asn-297N-linked biantennary glycans present in the CH2 domain of the immunoglobulin constant (Fc) region. These oligosaccharides may contain terminal mannose, N-acetyl-glucosamine, galactose, or sialic acid.

[0080] As used herein, the term "glycoengineering" refers to any art-recognized method of altering the glycoform profile of a binding protein composition. Such methods include expression of a binding protein composition in genetically engineered host cells (e.g., CHO cells) that have been genetically engineered to express a heterologous glycosyltransferase or glycosidase. In other embodiments, the glycoengineering method includes culturing the host cells under conditions that bias a particular glycoform profile.

[0081] As used herein, a "glyco-modified Fc fragment" includes (i) a hyper-galactosylated Fc fragment, (ii) a hypomannosylated Fc fragment, including an amannosylated Fc fragment, and (iii) a hypofucosylated Fc fragment, including an afucosylated Fc fragment. As used herein, a glyco-modified fragment includes an Fc fragment having an altered glycosylation selected from the group consisting of one or more of the following altered glycosylation: (i) hypergalactosylated, (ii) hypomannosylated, and (iii) hypofucosylated. Thus, glyco-modified Fc fragments for use in accordance with the present invention include examples of hypergalactosylated, hypomannosylated, and hypofucosylated Fc fragments.

[0082] Those skilled in the art may refer to well-known techniques for obtaining hypergalactosylated, hypomannosylated, and hypofucosylated Fc fragments, which are known to bind to Fc receptors with higher affinity than unmodified Fc fragments.

[0083] As used herein, the term "hypergalactosylated population" refers to a population of Fc domain-containing binding proteins that have an increased galactose content of the N-glycans compared to a reference population of Fc domain-containing binding proteins having the same amino acid sequence. A hypergalactosylated population can be expressed as a population that has an increased number of G1 and G2 glycoforms compared to a reference population of Fc domain-containing binding proteins.

[0084] As used herein, the term "hypomannosylated population" refers to a population of Fc domain-containing binding proteins having a reduced mannose content of N-glycans compared to a reference population of Fc domain-containing binding proteins having the same amino acid sequence. A hypomannosylated population can be described as a population having a reduced number of oligomannose glycoforms (e.g., M3-M9 glycoforms) compared to a reference population of Fc domain-containing binding proteins. In some embodiments, the mannose content is determined by measuring the content of one or more oligomannose glycoforms selected from the group consisting of Man3, Man4, Man5, Man6, Man7, Man8, and Man9. In other embodiments, the oligomannose content is determined by measuring at least Man5, Man6, and Man7. In some embodiments, the oligomannose content is determined by measuring all glycoforms M3 through M9. As used herein, the terms "G0 glycoform," "G1 glycoform," and "G2 glycoform" refer to N-glycan glycoforms having zero, one, or two terminal galactose residues, respectively. These terms include G0, G1, and G2 glycoforms that are fucosylated or contain bisecting N-acetylglucosamine residues. In some embodiments, the G1 and G2 glycoforms further contain a sialic acid residue attached to one or both of the terminal galactose residues to form the G1S1, G2S1, and G2S2 glycoforms. As used herein, the terms "G1S1 glycoform," "G2S1 glycoform," and "G2S2 glycoform" refer to N-glycan glycoforms having a sialic acid residue attached to only one terminal galactose residue in the G1 glycoform, one terminal galactose residue in the G2 glycoform, or both terminal galactose residues in the G2 glycoform, respectively. These terms include G1S1, G2S1, and G2S2 glycoforms that are fucosylated or contain bisecting N-acetylglucosamine residues.In certain embodiments, the sialic acid residues of the G1S1, G2S1, and G2S2 glycoforms are linked by alpha-2,6-sialic acid linkages to terminal galactose residues of each glycoform to enhance the anti-inflammatory activity of the binding molecule (see, e.g., Anthony et al., PNAS 105: 19571-19578, 2008).

[0085] The definitions of "hypofucosylation" or "afucosylation" below relate generally to the glycoengineered Fc-bearing compound of interest, as applied to specific embodiments of the glycoengineered Fc-bearing compound comprising an antibody.

[0086] A "hypofucosylated" antibody preparation refers to an antibody preparation in which less than 50% of the N-linked oligosaccharide chains contain α1,6-fucose linked to the CH2 domain. Typically, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than 5%, or less than 1% of the N-linked oligosaccharide chains contain α1,6-fucose linked to the CH2 domain in a "hypofucosylated" antibody preparation. As used herein, an antibody preparation in which less than 50% of the N-linked oligosaccharide chains contain α1,6-fucose linked to the CH2 domain refers to an antibody preparation in which less than 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 41%, 42%, 43 ... These include preparations in which less than 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% contain α1,6-fucose linked to the CH2 domain.

[0087] Thus, the terms "afucosylated" and "nonfucosylated" are used interchangeably herein to refer to antibodies lacking α1,6-fucose in the carbohydrate linked to the CH2 domain of the IgG heavy chain. Umana et al., Nat. Biotechnol 17:176-180, 1999, describe a bisected GlcNac that results in 10-fold increased ADCC. Umana points out that such bisected molecules result in less fucosylation. Davies et al., Biotechnol. Bioeng. 74:288-294, 2001, describe CHO cells inserted with the enzyme β1-4-N-acetylglucosaminyltransferase III (GnTIII) (leading to a bisected GlcNac structure) that results in increased ADCC of anti-CD20 antibodies. By way of illustration, US Pat. No. 6,602,684 describes cells engineered to produce a bisecting GlcNac glycoprotein.

[0088] Further examples of methods for reducing fucosylation of antibody preparations are presented in Shields et al., J Biol Chem 277:26733-26740, 2002, which describes a fucosylation-deficient CHO cell (Lec13) for producing IgG1 and further describes that binding of fucose-deficient IgG1 to human FcγRIIIA was improved by up to 50-fold, enhancing ADCC. Furthermore, Shinkawa et al., J Biol Chem 278:3466-3473, 2003, compares IgG produced in YB2 / 0 and CHO cells. YB2 / 0 cells have reduced fucosylation and increased bisecting GlcNac content. Niwa et al., Clinc. Cancer Res. 1-:6248-6255, 2004, compared anti-CD20 antibodies with antibodies made in YB2 / 0 cells (hypofucosylated) and observed enhanced ADCC for the latter. Examples of techniques for producing afucosylated antibodies are provided, for example, in Kanda et al., Glycobiology 17:104-118, 2006. U.S. Patent No. 6,946,292 (Kanda) describes fucosyltransferase knockout cells for producing afucosylated antibodies. U.S. Patent No. 7,214,775 and PCT application WO 00 / 61739 describe antibody preparations in which 100% of the antibody is afucosylated.

[0089] Still further techniques for modifying glycosylation are also known and are described, for example, in US Patent Application No. 2007 / 248600; US Patent No. 2007 / 178551 (the GlycoFi technique, which utilizes engineered lower eukaryotic cells (yeast) to produce "human" glycosylation structures); US Patent No. 2008 / 060092 (the Biolex technique, which utilizes engineered plants to produce "human" glycosylation structures), and US Patent No. 2006 / 253928 (which also describes the engineering of plants to produce "human" antibodies).

[0090] Additional technologies for reducing fucose include the ProBioGen technology (von Horsten et al., Glycobiology, (advance access publication Jul. 23, 2010); the Potelligent™ technology (Biowa, Inc., Princeton, NJ); and the GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland).

[0091] The N-linked oligosaccharide content of an antibody can be analyzed by methods known in the art. The following is an example of such a method: the antibody is subjected to digestion with the enzyme N-glycosidase F (Roche; TaKaRa). The released carbohydrates are analyzed by matrix assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) using the positive ion mode (Papac et al., Glycobiol. 8:445-454, 1998). The monosaccharide composition is then characterized by modified high performance anion exchange chromatography (HPAEC) (Shinkawa et al., J. Biol. Chem. 278:3466-3473, 2003).

[0092] In some embodiments, the compounds having glycoengineered Fc fragments of the invention are produced in a mammalian cultured host cell line (e.g., a CHO cell line). In some embodiments, the host cell line is engineered by glycoengineering to produce the hypergalactosylated and / or hypomannosylated binding proteins of the invention. In some exemplary embodiments, the binding proteins of the invention are obtained from glycoengineered CHO cells. In one exemplary embodiment, the glycoengineered CHO cells contain a heterologous galactosyltransferase gene (e.g., mouse galactosyltransferase beta-1,4). In another exemplary embodiment, the glycoengineered CHO cells contain a knockdown of one allele of the beta-galactosidase gene.

[0093] In accordance with the present disclosure, the term "3C23K" refers to the anti-AMHRII humanized monoclonal antibody 3C23K. AMHRII may also be referred to as MSRII.

[0094] In accordance with the disclosure of the present specification, the term "GM102" refers to an anti-AMHRII humanized antibody having light and heavy chains with the same amino acid sequences as the 3C23K antibody, but engineered by glycosylation, more particularly hypofucosylated. "GM102" may also be referred to as "R18H2" herein.

[0095] According to the present disclosure, "YB2 / 0 cells" (EMABling®) or "YB20" refers to a cell line for producing recombinant monoclonal hypofucosylated antibodies.

[0096] In accordance with the disclosure herein, 3C23K-CHO consists of a 3C23K antibody with normal glycosylation, which encompasses the 3C23K antibody produced by a CHO cell line.

[0097] In accordance with the disclosure herein, 3C23K-FcKO consists of the 3C23K antibody lacking the Fc fragment.

[0098] Compounds having glycosylated Fc fragments The terms "compound having a glycoengineered Fc fragment," "molecule having a glycoengineered Fc fragment," "compound containing a glycoengineered Fc fragment," and "molecule containing a glycoengineered Fc fragment" may be used interchangeably herein to mean a compound that comprises an Fc fragment of an antibody with altered glycosylation that provides the Fc fragment with a higher affinity for an Fc receptor compared to the same Fc fragment with unaltered glycosylation.

[0099] In some preferred embodiments, a compound having a glycoengineered Fc fragment has a higher affinity for FcγRIIIa (also referred to as “CD16a”) than the same Fc fragment that has not undergone glycoengineering.

[0100] This is exemplified in the present example by a hypofucosylated Fc fragment-bearing compound called "3C23K," which has high affinity for human FcγRIIIa (CD16a) with a constant Kd value of less than 50 nM as measured using the well-known Biacore® method.

[0101] In some preferred embodiments, the compound having a glycoengineered Fc fragment consists of the glycoengineered Fc fragment itself, and thus the compound does not contain an antigen-binding region.

[0102] In some preferred embodiments, the compound having a glycoengineered Fc fragment consists of a protein having a glycoengineered Fc fragment, wherein said glycoengineered Fc fragment is covalently linked to another protein moiety, which is either (i) a protein that comprises an antigen-binding region, or (ii) a protein that does not comprise an antigen-binding region.

[0103] In some of these preferred embodiments, the compound having a glycoengineered Fc fragment comprises only one glycoengineered Fc fragment.

[0104] Thus, the invention encompasses the use of a compound having a glycoengineered Fc fragment comprising (i) a polypeptide monomer unit comprising a glycoengineered Fc fragment, and (ii) another polypeptide covalently linked to said polypeptide monomer unit. The other polypeptide may be an antigen-binding region of an antibody, such as the VH and VL chains of an antibody. The other polypeptide may be a ligand-binding protein portion of a receptor protein, such as a VEGF receptor or a VEGF binding domain of a VEGF receptor, or a TNF alpha receptor or a TNF binding domain of a TNF alpha receptor.

[0105] In some of these preferred embodiments, the other protein moiety may comprise another Fc fragment, in particular another glycoengineered Fc fragment. In some embodiments, the two glycoengineered Fc fragments have the same amino acid sequence. In some other embodiments, the two glycoengineered Fc fragments have different amino acid sequences. In some embodiments, the two Fc fragments have the same amino acid sequence but different altered glycosylation patterns. In some other embodiments, the two Fc fragments have the same amino acid sequence and have the same altered glycosylation pattern.

[0106] Thus, a compound having a glycosylated Fc fragment includes a protein compound that comprises more than one Fc fragment, provided that at least one of the Fc fragments contained therein is glycosylated, e.g., at least one of the Fc fragments contained therein is hypomannosylated, hypergalactosylated, or hypofucosylated.

[0107] As already described elsewhere herein, Fc fragment-bearing compounds comprising more than one Fc fragment, for example comprising 2, 3, 4, 5 or 6 Fc fragments, are well known in the art and may be referred to as "Fc multimers". Such Fc multimer constructs have been disclosed, inter alia, by Thiruppathi et al. (2014, J Autoimmun, Vol.five2:64-73), by Jain et al. (2012, Arthritis Research and Therapy, Vol. 1four:R192) or by Zhou et al. (2017, Blood advances, Vol.1(n°6):DOI 10.1182 / biooadvances.2016001917).

[0108] Thus, compounds having a glycomodified Fc that may be used in accordance with the present invention include multimeric proteins comprising two or more polypeptide monomer units, (i) where each polypeptide monomer unit comprises an Fc fragment, and (ii) where at least one polypeptide monomer unit comprises a glycomodified Fc fragment, e.g., a hypomannosylated Fc fragment, a hypergalactosylated Fc fragment, or a hypofucosylated Fc fragment.

[0109] Such Fc multimers are also disclosed in US Patent Application No. 2017 / 088063.

[0110] In some embodiments of the Fc multimeric compound, the compound also includes an antigen-binding domain therein, such as the antigen-binding domain disclosed by Zhang et al. (2016, J Immunol, Vol. 196:1165-1176).

[0111] In some other preferred embodiments, as exemplified in the Examples herein, the compound having a glycoengineered Fc fragment consists of a glycoengineered antibody.

[0112] In some preferred embodiments, as illustrated in the Examples herein, the compound having a glycoengineered Fc fragment consists of a hypofucosylated Fc fragment-bearing compound, such as a hypofucosylated antibody.

[0113] In some other embodiments, the compound with a glycoengineered Fc fragment, more precisely the compound bearing a hypofucosylated Fc fragment, consists of an afucosylated Fc fragment bearing compound, such as an afucosylated antibody.

[0114] In another embodiment, the compound having a glycoengineered Fc fragment consists of a hypergalactosylated Fc fragment-bearing compound, such as a hypergalactosylated antibody.

[0115] In yet other embodiments, the compound having a glycoengineered Fc fragment consists of a hypomannosylated Fc fragment-bearing compound, such as a hypomannosylated antibody.

[0116] As previously described elsewhere herein, reducing or blocking immune suppression, e.g. macrophage-induced immune suppression, occurring in cancer diseases by compounds having a glycoengineered Fc fragment, e.g. glycoengineered antibodies, does not require the presence of tumor cells and therefore does not require binding of the antibody to target tumor cells.

[0117] This explains why the inventors believe that reducing or blocking immune suppression, in particular the inhibition of T-cell activation, should be obtained by compounds with glycoengineered Fc fragments that do not contain an antigen-binding region, e.g. that do not contain a tumor-associated antigen-binding region.

[0118] However, it is also illustrated in the Examples herein that reducing or blocking immunosuppression is achieved when using a glycoengineered antibody as the compound having a glycoengineered Fc fragment.

[0119] Furthermore, the inventors believe that the beneficial effect of a compound having a glycoengineered Fc fragment as defined herein may be further enhanced when using a compound having a glycoengineered Fc fragment consisting of a glycoengineered antibody directed against a relevant tumor-associated antigen (meaning a glycoengineered antibody directed against a tumor-associated antigen expressed by tumor cells present in the tumor tissue or in the body fluids of the cancer individual to be treated).

[0120] Thus, in some preferred embodiments, the compound having a glycoengineered Fc fragment consists of a glycoengineered antibody directed against a tumor-associated antigen expressed by tumor cells of the cancer individual to be treated.

[0121] In some preferred embodiments, as illustrated in the Examples herein, said glycoengineered antibody comprises a hypofucosylated antibody.

[0122] Without wishing to be bound by any particular theory, the inventors believe that the use of glycoengineered antibodies directed against a tumor-associated antigen expressed by tumor cells of the cancer individual to be treated makes it possible (i) to reduce or block immune suppression, e.g. inhibition of T cell activity, in particular inhibition of CD8+ T cell activity, e.g. macrophage-induced immune suppression, and (ii) to destroy, e.g. by ADCC or ADC activity, the tumor cells expressing the tumor-associated antigen against which said glycoengineered antibodies are directed.

[0123] As used herein, the term "tumor-associated antigen" refers to an antigen that can be present or presented on the surface located on tumor cells or within tumor cells. These antigens can be presented on the cell surface with the extracellular portion often combined with the transmembrane and cytoplasmic portions of the molecule. These antigens can, in some embodiments, be presented only by tumor cells and not by normal cells (i.e., non-tumor cells). Tumor antigens can be expressed exclusively on tumor cells or can display tumor-specific mutations compared to non-tumor cells. In such embodiments, individual antigens can be referred to as tumor-specific antigens or tumor-associated antigens (also referred to as "TAA"). Some antigens, which can also be referred to as tumor-associated antigens, are presented by both tumor cells and non-tumor cells. These tumor-associated antigens can be overexpressed on tumor cells when compared to non-tumor cells, or are more likely to participate in antibody binding within tumor cells because the structure of tumor tissue is less compact compared to non-tumor tissue. In some embodiments, the tumor-associated surface antigen is located in the vasculature of the tumor.

[0124] A list of tumor-associated antigens is disclosed, inter alia, by Liu et al. (2016, European Journal of Cancer Care, doi:10 / 1111 / ecc.12446), to which the skilled person may refer.

[0125] A list of tumor antigens recognized by T cells is disclosed by Renkvist et al. (2001, Cancer immunology and immunotherapy, Vol. 50(n°1) 3-15), to which the skilled artisan may refer.

[0126] Examples of tumor-associated surface antigens include CD10, CD19, CD20, CD22, CD33, Fms-like tyrosine kinase 3 (FLT-3), CD135, chondroitin sulfate proteoglycan 4 (CSPG4, melanoma-associated chondroitin sulfate proteoglycan), epidermal growth factor receptor (EGFR), Her2neu, Her3, IGFR, CD133, IL3R, and fibroblast activating protein (FAP). These include CDCP1, delrin 1, tenascin, frizzled 1-10, vascular antigens VEGFR2 (KDR / FLK1), VEGFR3 (FLT4, CD309), PDGFR-a (CD140a), PDGFR-β (CD140b), endoglin, CLEC14, Tem1-8, and Tie2. Further examples are A33, CAM PATH-1 (CDw52), carcinoembryonic antigen (CEA), carboanhydrase IX (MN / CA IX), CD21, CD25, CD30, CD34, CD37, CD44v6, CD45, CD133, de2-7EGFR, EGFRvIII, EpCAM, Ep-CAM, folate binding protein, G250, Fms-like tyrosine kinase 3 (FLT-3, CD135), c-Kit (CD117), CSF1R (CD115), HLA-DR, IGFR, IL-2 receptor, IL3R, MCSP (melanoma-associated cell surface chondroitin sulfate proteoglycan), Muc-1, prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), prostate specific antigen (PSA), Examples of antigens expressed on the extracellular matrix of tumors are tenascin and fibroblast activating protein (FAP).

[0127] Preferred tumor associated antigens (TAA) are CD45, IL-3Ra (also known as CD123), CD33, CD20, CD22, CD19, EpCAM (also known as "Epithelial Cell Adhesion Molecule"), HER2, TROP-2 (also known as "Trophoblast cell surface antigen 2"), GNMB ("Glycoprotein non-metastatic B"). B), MMP9, EGFR, PD-L1 (CD274), CTLA4, GM3, mesothelin, folate receptor 1, fibronectin extra domain B, endoglin, CD22, IL-1 alpha, HER3, cMet, phosphatidylserine, MUC5AC, NeuGc ganglioside, CD2, CD38, EGFR, HGF / SF, PD1, GD2, ST4, and folate receptor alpha.

[0128] The most preferred tumor-associated antigens according to the invention are antigens selected from the group comprising HER2, HER3, HER4 and AMHRII.

[0129] Preferred embodiments of glycoengineered antibodies that may be used according to the present invention are selected from the group comprising the glycoengineered antibodies referred to herein as 3C23K, 9F7F11, H4B121, and HE4B33.

[0130] Exemplary embodiments of compounds having glycoengineered Fc fragments Exemplary embodiments of Fc fragment-bearing compounds include compounds that comprise a glycoengineered Fc fragment comprising two amino acid chains of SEQ ID NO:70 as described herein.

[0131] The amino acid chain of SEQ ID NO: 70 consists of the heavy chain constant region of a human IgG1 antibody, including the CH1 domain, the hinge region, the CH2 domain, and the CH3 domain.

[0132] As disclosed in the Examples, compounds with glycoengineered Fc fragments, in particular compounds carrying hypofucosylated Fc fragments, can be obtained by a method comprising expressing a nucleic acid sequence encoding said Fc fragment in YB2 / 0 cells. Such a method can be the well-known method called EMABling®, which is described in the Examples.

[0133] In some embodiments, a compound having a glycomodified Fc fragment, in particular a hypofucosylated Fc fragment-bearing compound, can be obtained by a method comprising expressing the nucleic acid sequence of SEQ ID NO: 69 in YB2 / 0 cells.

[0134] In some embodiments, the compound having the above-mentioned glycomodified Fc fragment comprises a glycomodified antibody, in particular a hypofucosylated antibody; the glycomodified Fc fragment comprises two amino acid chains of SEQ ID NO:70.

[0135] Embodiments of glycoengineered antibodies comprising glycoengineered Fc fragments are described herein below.

[0136] Antibodies as compounds having glycoengineered Fc fragments Thus, an embodiment of the compound having a glycoengineered Fc fragment consists of an antibody, in particular a glycoengineered antibody directed against a tumor-associated antigen.

[0137] In some embodiments, compounds having a glycoengineered Fc fragment include glycoengineered multispecific antibodies, particularly glycoengineered bispecific antibodies. Illustratively, these glycoengineered antibodies include antibodies comprising a glycoengineered Fc fragment as described herein and (i) a first antigen-binding region that binds to a tumor antigen, and (ii) a second antigen-binding region that binds to a T cell antigen, e.g., CD3, or an inhibitory immune checkpoint protein, e.g., with a view to simultaneously (i) being directed against a tumor antigen-expressing cell and (ii) activating T cells.

[0138] Examples of such glycoengineered antibodies include antibodies directed against tumor-associated antigens, such as AMHRII, HER2, HER3, and HER4.

[0139] Such antibodies may be described with reference to their antigen-binding regions, in particular their heavy chain variable region (VH) and light chain variable region (VL).

[0140] Exemplary embodiments of anti-AMHRII antibodies PCT Application No. PCT / FR2011 / 050745 (International Publication No. WO / 2011 / 141653) and US Pat. No. 9,012,607 (each of which is incorporated herein by reference) disclose novel humanized antibodies derived from the murine 12G4 antibody. The humanized antibodies may be used as AMHRII binding agents for the purposes of the present invention. In particular embodiments disclosed in PCT Application No. WO / 2011 / 141653, the antibodies are those identified as 3C23 and 3C23K. The nucleic acid and polypeptide sequences of these antibodies are provided herein as SEQ ID NOs: 1-16. In some aspects of the present invention, the anti-AMHRII antibodies of interest may be referred to as "comprising a light chain comprising SEQ ID NO: and a heavy chain comprising SEQ ID NO:." Thus, in various embodiments, particularly preferred antibodies include: a) a light chain comprising SEQ ID NO:2 and a heavy chain comprising SEQ ID NO:4 (leaderless 3C23 VL and VH sequences); b) a light chain comprising SEQ ID NO:6 and a heavy chain comprising SEQ ID NO:8 (leaderless 3C23K VL and VH sequences); c) a light chain comprising SEQ ID NO:10 and a heavy chain comprising SEQ ID NO:12 (leaderless 3C23 light and heavy chains); d) A light chain comprising SEQ ID NO: 14 and a heavy chain comprising SEQ ID NO: 16 (leaderless 3C23K light and heavy chains).

[0141] Other antibodies (eg, humanized or chimeric antibodies) can be based on the heavy and light chain sequences described herein.

[0142] Exemplary embodiments of anti-AMHRII antibodies comprising / containing CDRs comprising (or consisting of) the following sequences are as follows: CDRL-1: RASX1X2VX3X4X5A (SEQ ID NO: 71), where X1 and X2 are independently S or P, X3 is R or W or G, X4 is T or D, and X5 is I or T; CDRL-2 is PTSSLX6S (SEQ ID NO:72), where X6 is K or E; and CDRL-3 is LQWSSYPWT (SEQ ID NO:73); CDRH-1 is KASGYX7FTX8X9HIH (SEQ ID NO:74), where X7 is S or T, X8 is S or G, and X9 is Y or N; CDRH-2 is WIYPX10DDSTKYSQKFQG (SEQ ID NO:75), where X10 is G or E; and CDRH-3 is GDRFAY (sequence number 76).

[0143] Antibodies (e.g., chimeric or humanized) within the scope of this application include those disclosed in the table below: 3C23K antibody: SEQ ID NO: 17 for the VH amino acid sequence SEQ ID NO: 34 for the VL amino acid sequence is defined as follows:

[0144] Table 1 below lists anti-AMHRII humanized antibodies that may be used in accordance with the present invention.

[0145] [Table 1] JPEG0007678493000002.jpg255139JPEG0007678493000003.jpg235170

[0146] Exemplary embodiments of anti-HER3 antibodies Exemplary embodiments of glycoengineered anti-HER3 antibodies are those referred to herein as 9F7F11 and H4B121.

[0147] The 9F7F11 antibody comprises (i) a heavy chain variable region of SEQ ID NO:63, and (ii) a light chain variable region of SEQ ID NO:64.

[0148] The H4B121 antibody comprises (i) a heavy chain variable region of SEQ ID NO:65, and (ii) a light chain variable region of SEQ ID NO:66.

[0149] Exemplary embodiments of anti-HER4 antibodies An exemplary embodiment of an anti-HER4 antibody is the antibody designated herein as HE4B33.

[0150] The HE4B33 antibody comprises (i) a heavy chain variable region of SEQ ID NO:67, and (ii) a light chain variable region of SEQ ID NO:68.

[0151] For clarity, all of the above antibodies include glycoengineered Fc fragments as described herein, and in particular include hypofucosylated Fc fragments as described herein.

[0152] In some preferred embodiments, these antibodies comprise a glycoengineered Fc fragment having two glycoengineered amino acid chains of SEQ ID NO:70, particularly a hypofucosylated Fc fragment having two hypofucosylated amino acid chains of SEQ ID NO:70.

[0153] Combination of a compound having a glycoengineered Fc fragment with one or more other active agents The compounds having a glycoengineered Fc fragment as defined herein make it possible to reduce or block the immunosuppressive state in cancer patients and are therefore useful for enhancing the anti-cancer activity of known anti-cancer treatments, including surgical, radiotherapy and chemotherapy treatments.

[0154] Furthermore, compounds with glycoengineered Fc fragments as defined herein make it possible to reduce or block the immunosuppressive state in cancer patients and therefore possibly act as active agents that should enhance the beneficial effects of other compounds aimed at blocking immunosuppression or at inducing immune stimulation or immune activation in immunosuppressed cancer patients. Moreover, compounds with glycoengineered Fc fragments may also contribute to affect the resistance of cancer cells to immunosuppressive inhibitors (checkpoint inhibitors) or immune stimulatory drugs.

[0155] Thus, in a further aspect, the compounds having a glycoengineered Fc fragment as defined herein may be used in combination with another anti-cancer treatment, in particular in combination with one or more different compounds consisting of anti-cancer agents.

[0156] In a further aspect, the present invention relates to compounds having a glycoengineered Fc fragment and their use as immunosuppressive inhibitors in the treatment of cancer in an individual, in combination with one or more different anti-cancer agents.

[0157] The present invention further relates to the use of a compound with a glycoengineered Fc fragment in combination with one or more different anti-cancer agents for the preparation of a medicament for treating cancer.

[0158] The present invention also relates to a method for treating cancer comprising administering to an individual in need thereof a compound having a glycomodified Fc fragment in combination with one or more different anti-cancer agents.

[0159] Anti-cancer agents include compounds that have anti-cancer activity, such as anti-proliferative active agents, many of which are well known to those skilled in the art. Anti-cancer agents also include inhibitors of inhibitory immune checkpoint proteins, as detailed elsewhere herein.

[0160] "Anti-cancer agent" is used according to its ordinary meaning and refers to a composition (e.g., a compound, drug, antagonist, inhibitor, modulator) that has anti-neoplastic properties or the ability to inhibit cell growth or proliferation. In some embodiments, the anti-cancer agent is a chemotherapeutic agent. In some embodiments, the anti-cancer agent is an agent identified herein that has utility in methods for treating cancer. In some embodiments, the anti-cancer agent is an agent approved by the FDA or a similar regulatory agency in a country other than the United States for treating cancer.

[0161] In some embodiments, the cancer medicament does not consist of an antibody-derived compound, such as the antibody itself or an antigen-binding fragment or antigen-binding format thereof.

[0162] ;Talimustine;Tamoxifen methiodide;Tauromustine;Tazarotene;Tecogalan sodium;Tegafur;Terlapyrylium;Telomerase inhibitors;Temoporfin;Temozolomide;Teniposide;Tetrachlorodecaoxide;Tetrazomine;Taliblastine;Thiocoraline;Thrombopoietin;Thrombopoietin mimetics;Thymalfasin;Thymopoietin receptor agonists;Thymotrin;Thyroid-stimulating hormone;Tin ethyl etiopurpurin;Tirapazamine;Titanocene dichloride;Topsentin;Toremifene;Totipotent stem cell factor;Translation inhibitors;Tretinoin;Triacetyluridy ;Triciribine;Trimetrexate;Triptorelin;Tropisetron;Turosteride;Tyrosine kinase inhibitors;Tyrphostin;UBC inhibitors;Ubenimex;Urogenital sinus-derived growth inhibitory factor;Urokinase receptor antagonists;Vapreotide;Variolin B;Vector systems, red blood cell gene therapy;Veraresol;Veramine;Verdine;Verteporfin;Vinorelbine;Vinxartin;Vitaxin;Vorozole;Zanoteron;Zeniplatin;Zilascorub;Zinostatin stimalamer, Adriamycin, Dactinomycin, Bleomycin, Vinblastine, Cisplatin,Acivicin;Aclarubicin;Acodazole hydrochloride;Acronine;Adzelesin;Aldesleukin;Altretamine;Ambomycin;Amethanthrone acetate;Aminoglutethimide;Amsacrine;Anastrozole;Anthramycin;Asparaginase;Asperlin;Azacitidine;Azetepa;Azotomycin;Batimastat;Benzodepa;Bicalutamide;Bisantrene hydrochloride;Bisnafide dimesylate;Bizeresin;Bre sulfate Omycin;Brequinar sodium;Bropirimine;Busulfan;Cactinomycin;Calsterone;Caracemide;Carbetimer;Carboplatin;Carmustine;Carubicin hydrochloride;Carzercin;Cedefingol;Chlorambucil;Ciloremycin;Cladribine;Crisnatol mesylate;Cyclophosphamide;Cytarabine;Dacarbazine;Daunorubicin hydrochloride;Decitabine;Dexormaplatin;Desaguanine;Desaguanine mesylate Anin;Diaziquone;Doxorubicin;Doxorubicin hydrochloride;Droloxifene;Droloxifene citrate;Dromostanolone propionate;Duazomycin;Edatrexate;Eflornithine hydrochloride;Elsamitrucin;Enloplatin;Enpromate;Epipropizine;Epirubicin hydrochloride;Elburozole;Esorubicin hydrochloride;Estramustine;Estramustine sodium phosphate;Etanidazole;Etoposide;Phosphate Etoposide; etopurine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; fluorocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; irmofosine; interleukin II (including recombinant interleukin II, or rIL.sub.2),Interferon alpha-2a;Interferon alpha-2b;Interferon alpha-n1;Interferon alpha-n3;Interferon beta-1a;Interferon gamma-1b;Iproplatin;Irinotecan hydrochloride;Lanreotide acetate;Letrozole;Leuprolide acetate;Liarozole hydrochloride;Lometrexol sodium;Lomustine;Losoxantrone hydrochloride;Masoprocol;Maytansine;Mechlorethamine hydrochloride;Megestrol acetate;Melengestrol acetate;Melphalan;Menogaril;Mercaptopurine;Methotrexate;Methotrexate sodium thorium; metoprine; meturedepa; mitindomide; mitocalcin; mitochromin; mitogillin; mitomarcine; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; pegaspargase; periomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; promestane; porfimer sodium; porfiromycin; prednisone ;Procarbazine hydrochloride;Puromycin;Puromycin hydrochloride;Pyrazofurin;Riboprin;Rogletimide;Safingol;Safingol hydrochloride;Semustine;Simtrazene;Sparphosate sodium;Sparsomycin;Spirogermanium hydrochloride;Spiromustine;Spiroplatin;Streptonigrin;Streptozocin;Sulofenur;Tallysomycin;Tecogalan sodium;Tegafur;Teroxantrone hydrochloride;Temoporfin;Teniposide;Teroxylon;Testolactone;Thiamiprine;Thioguanine;Thiotepa;Thiazo Furin;Tirapazamine;Toremifene citrate;Trestron acetate;Triciribine phosphate;Trimetrexate;Trimetrexate glucuronate;Triptorelin;Tubrozole hydrochloride;Uracil mustard;Uredep;Vapreotide;Verteporfin;Vinblastine sulfate;Vincristine sulfate;Vindesine;Vindesine sulfate;Binepidine sulfate;Vingrisinate sulfate;Vinleurosine sulfate;Vinorelbine tartrate;Vinrocidine sulfate;Vinzolidine sulfate;Vorozole;Zeniplatin;Zinostatin;Zorubicin hydrochloride, arrests cells within the G2-M phase,and / or agents that modulate the formation or stability of microtubules (e.g., Taxol™ (i.e., paclitaxel), Taxotere™), compounds containing a taxane skeleton, elbrozole (i.e., R-55104), dolastatin 10 (i.e., DLS-10 and NSC-376128), mibobulin isethionate (i.e., as CI-980), vincristine, NSC-639829, discodermolide (i.e., as NVP-XX-A-296), ABT-751 (Abbott, i.e., E-7010), altrlutin (e.g., altrlutin A and altrlutin B), rurtin C), spongistatins (e.g., spongistatin 1, spongistatin 2, spongistatin 3, spongistatin 4, spongistatin 5, spongistatin 6, spongistatin 7, spongistatin 8, and spongistatin 9), cemadotin hydrochloride (i.e., LU-103793 and NSC-D-669356), epothilones (e.g., epothilone A, epothilone B, epothilone C (i.e., desoxyepothilone A or dEpoA), epothilone D (i.e., KOS-862, dEpoB, and desoxyepothilone B), epothilone E, epothilone F, epothilone B N-oxide, epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B (i.e., BMS-310705), 21-hydroxyepothilone D (i.e., desoxyepothilone F and dEpoF), 26-fluoroepothilone, auristatin PE (i.e., NSC-654663), sobridotin (i.e., TZT-1027), LS-4559-P (Pharmacia, i.e., LS-4577), LS-4578 (Phase I, Pharmacia, Inc. ... armacia, i.e. LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, i.e. WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian Academy of Sciences), BSF-223651 (BASF, i.e. ILX-651 and LU-223651),SAH-49960 (Lilly / Novartis), SDZ-268970 (Lilly / Novartis), AM-97 (Armad / Kyowa Hakko), AM-132 (Armad), AM-138 (Armad / Kyowa Hakko), IDN-5005 (Indena), cryptophycin 52 (i.e., LY-355703), AC-7739 (Ajinomoto, i.e., AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto, i.e., AVE-8062, AVE-8062A, CS-39-L-Ser.HCl, and RPR-258062A), bitilebamide, tubulysin A, canadensol, centaureydin (i.e., NSC-106969), T-138067 (Tularik, i.e., T-67, TL-138067, and TI-138067), COBRA-1 (Parker Hughes), Institute, i.e. DDE-261 and WHI-261), H10 (Kansas State University), H16 (Kansas State University), oncocidin A1 (i.e. BTO-956 and DIME), DDE-313 (Parker Hughes Institute), physianolide B, laulimalide, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute, i.e. SPIKET-P), 3-IAABU (Cytoskeleton / Mt. Sinai School of Medicine, i.e. MF-569), narcosine (also known as NSC-5366), nascapine, D-24851 (Asta Medica), A-105972 (Abbott), hemiasterlin, 3-BAABU (Cytoskeleton / Mt. Sinai School of Medicine, i.e. MF-569), Medicine, i.e. MF-191), TMPN (Arizona State University), vanadocene acetylacetonate, T-138026 (Tularik), monsatrol, inanocin (i.e. NSC-698666), 3-IAABE (Cytoskeleton / Mt. Sinai School of Medicine), A-204197 (Abbott), T-607 (Tuiarik,i.e., T-900607), RPR-115781 (Aventis), eluterobin (e.g., desmethyleluterobin, desethyleluterobin, isoeluterobin A, and Z-eluterobin), carbaeoside, carbaeolin, halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica), diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott), diozostatin, (-)-phenylahistine (i.e., NSCL-96F037), D-68838 (Asta Medica), D-68836 (Asta Medica), myoseverin B, D-43411 (Zentaris, i.e., D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286 (i.e., SPA-110, trifluoroacetate) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCI), resbelostatin sodium phosphate, BPR-OY-007 (National Health Research Institute) Institutes), and SSR-250411 (Sanofi), steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, gonadotropin releasing hormone agonists (GnRH) (e.g., goserelin or leuprolide), corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol), antiestrogens (e.g., tamoxifen), androgens (e.g., propionic acid, testosterone, fluoxymesterone), antiandrogens (e.g. flutamide), immunostimulants (e.g. Bacillus Calmette-Guerin Calmette-Guerin (BCG), levamisole, interleukin-2, alpha-interferon, etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin, epirubicin, topotecan, itraconazole, vindesine, cerivastatin, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFR inhibitors, epidermal growth factor receptor (EGFR)-targeted therapies or therapeutic agents (e.g., gefitinib (Iressa™), erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™), panitumumab (Vectibix™), vandetanib (Caprelsa™), afatinib / BIBW2992, CI-1033 / canertinib, neratinib / HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib / PF299804, These include, but are not limited to, OSI-420 / desmethylerlotinib, AZD8931, AEE788, pelitinib / EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib, dasatinib, hormonal therapy, etc. Details of the administration route, dosage, and treatment regimen of anticancer drugs are known in the art, for example, as described in "Cancer Clinical Pharmacology" (2005) ed. By Jan HM Schellens, Howard L. McLeod and David R. Newell, Oxford University Press.

[0163] In some other embodiments, the additional anti-cancer agent comprises an anti-cancer antibody that is different from one or more Fc-bearing compounds used to inhibit cancer-associated immunosuppression. The anti-cancer antibody can be a monoclonal antibody (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonal antibodies), an immunotoxin (e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-Pseudomonas exotoxin conjugate, etc.), a radioimmunotherapy (e.g., 111 In, 90 Y, or 131 In some embodiments, these anti-cancer antibodies may themselves be glycomodified, e.g., hypofucosylated.

[0164] Anti-cancer drugs also include agents known to activate or reactivate the anti-cancer activity of the immune system. Agents that activate or reactivate the anti-cancer activity of the immune system include agents that are preferably agents that inhibit inhibitory immune checkpoints. These agents may be referred to herein as "inhibitory immune checkpoint inhibitors" or "immune checkpoint inhibitors." As known in the art, immune checkpoint inhibitors consist of agents that inhibit the activity of inhibitory immune checkpoint proteins.

[0165] The term "immune checkpoint protein" is known in the art. Within the known meaning of this term, it is clear to the skilled artisan that at the level of "immune checkpoint protein" the immune system provides inhibitory signals to its components to balance the immune response. Known immune checkpoint proteins include CTLA-4, PDl and its ligands PD-Ll and PD-L2, as well as LAG-3, BTLA, B7H3, B7H4, TIM3, KIR. It is recognized in the art that the pathway involving LAG3, BTLA, B7H3, B7H4, TIM3, and KIR constitutes an immune checkpoint pathway similar to the CTLA-4 and PD-1 dependent pathway (see, e.g., Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480-489).

[0166] Within the scope of the present invention, an immune checkpoint protein inhibitor is any compound that inhibits the function of an immune checkpoint protein. Inhibition includes reduction and complete blocking of function. In particular, the immune checkpoint protein is a human immune checkpoint protein. Thus, the immune checkpoint protein inhibitor is preferably an inhibitor of a human immune checkpoint protein. Immune checkpoint proteins are described in the prior art (see, for example, Pardoll, 2012. Nature Rev. cancer 12:252-264). The term immune checkpoint protein encompasses experimental demonstrations that inhibition of immune checkpoint proteins in vitro or in vivo stimulates antigen-receptor-triggered T lymphocyte responses, e.g., mice lacking expression of immune checkpoint proteins exhibit enhanced antigen-specific T lymphocyte responses or signs of autoimmunity (e.g., as disclosed in Waterhouse et al., 1995. Science 270:985-988; Nishimura et al., 1999. Immunity 11:141-151). Also, deliberate stimulation of immune checkpoint proteins in vitro or in vivo has been shown to result in enhanced antigen-receptor-triggered CD4 T cell responses. + or CD8 +It may also include the demonstration of inhibition of T cell responses (e.g., Zhu et al., 2005. Nature Immunol. 6:1245-1252). Preferred immune checkpoint protein inhibitors are antibodies that specifically recognize immune checkpoint proteins. Several CTLA-4, PD1, PDL-1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, and KIR inhibitors are known, and as well as these known immune checkpoint protein inhibitors, alternative immune checkpoint inhibitors may be developed in the (near) future. For example, ipilimumab is a fully human CTLA-4 blocking antibody currently marketed under the name Yervoy (Bristol-Myers Squibb). A second CTLA-4 inhibitor is tremelimumab (reviewed in Ribas et al., 2013, J. Clin. Oncol. 31:616-22). Examples of PD-1 inhibitors include, but are not limited to, humanized antibodies that block human PD-1, such as lambrolizumab (disclosed, for example, in WO 2008 / 156712; Hamid et al., N. Engl. J. Med. 369:134-144 2013 as hPD109A and its humanized derivatives h409A11, h409A16, and h409A17), or pidilizumab (disclosed in Rosenblatt et al., 2011. J Immunother. 34:409-18), as well as fully human antibodies, such as nivolumab (previously known as MDX-1106 or BMS-936558, Topalian et al., 2012. N. Eng. J. Med. 366:2443-2454; disclosed in U.S. Pat. No. 8,008,449). Other PD-1 inhibitors may also include presentation of soluble PD-1 ligands, including, but not limited to, the PD-L2 Fc fusion protein known as B7-DC-Ig or AMP-244 (disclosed in Mkrtichyan M et al., J Immunol. 189:2338-47 2012), and other PD-1 inhibitors currently under investigation and / or development for use in therapy.Additionally, immune checkpoint inhibitors may include, but are not limited to, humanized or fully human antibodies that block PD-L1, such as MEDI-4736 (disclosed in WO2011066389), MPDL328OA (disclosed in US8217149), and MIH1 (available from Affymetrix via eBioscience (16.5983.82)), as well as other PD-L1 inhibitors currently under investigation. In accordance with the present invention, the immune checkpoint inhibitor is preferably selected from CTLA-4, PD-1, or PD-L1 inhibitors, such as the known CTLA-4, PD-1, or PD-L1 inhibitors listed above (ipilimumab, tremelimumab, lambrolizumab, nivolumab, pidilizumab, AMP-244, MEDI-4736, MPDL328OA, MIH1). Known inhibitors of these immune checkpoint proteins may be used as is, or analogs may be used, particularly chimeric antibodies, humanized antibodies, or human forms of antibodies.

[0167] As those skilled in the art are aware, alternative and / or equivalent names may be used for certain antibodies described above. Such alternative and / or equivalent names are interchangeable in the context of the present invention. For example, it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.

[0168] It is more preferred to select the immune checkpoint inhibitor from a PD1 inhibitor and a PD-L1 inhibitor, such as the known PD-1 inhibitor or PD-L1 inhibitor described above, and most preferred to select from a PD-1 inhibitor, such as the known PD1 inhibitor described above. In a preferred embodiment, the PD1 inhibitor is nivolumab or pembrolizumab, or another antagonist antibody against human PD1.

[0169] The present invention also includes the selection of other immune checkpoint inhibitors known in the art to stimulate immune responses. This includes inhibitors that directly or indirectly stimulate or enhance antigen-specific T lymphocytes. These other immune checkpoint inhibitors include, but are not limited to, agents directed against immune checkpoint proteins and pathways involving PD-L2, LAG3, BTLA, B7H4, and TIM3. For example, human PD-L2 inhibitors known in the art include MIH18 (disclosed in Pfistershammer et al., 2006. Eur J Immunol. 36:1104-13). In another example, LAG3 inhibitors known in the art include soluble LAG3 (IMP321, or LAG3-Ig as disclosed in WO2009044273 and Brignon et al., 2009. Clin. Cancer Res. 15:6225-6231), as well as murine or humanized antibodies that block human LAG3 (e.g., IMP701 as disclosed in and derived from WO2008132601), or fully human antibodies that block human LAG3 (e.g., as disclosed in EP2320940). Another example is provided by the use of blocking agents against BTLA, including, but not limited to, antibodies that block the interaction of human BTLA with its ligands (e.g., 4C7 as disclosed in WO2011014438). Yet another example is provided by the use of agents that neutralize B7H4, including but not limited to antibodies against human B7H4 (disclosed in WO2013025779A1 and WO2013067492), or soluble recombinant forms of B7H4 (e.g., those disclosed in US20120177645, or anti-human B7H4 clone H74: eBiocience #14-5948). Yet another example is provided by agents that neutralize B7-H3, including but not limited to antibodies that neutralize human B7-H3 (e.g., MGA271, disclosed as BRCA84D, and derivatives described in US20120294796).Yet another example is provided by agents directed against TIM3, including but not limited to antibodies directed against human TIM3 (e.g., those disclosed in WO 2013 / 006490, anti-human TIM3, or the blocking antibody F38-2E2 disclosed by Jones et al., J Exp Med. 2008 Nov 24;205(12):2763-79). Known inhibitors of immune checkpoint proteins may be used in their known form, or analogs may be used, particularly chimeric forms of multiple antibodies, most preferably humanized forms.

[0170] The invention also includes the selection of more than one immune checkpoint inhibitor selected from a CTLA-4, PD-1 or PDL1 inhibitor for combination with a compound having a glycoengineered Fc fragment within the various aspects of the invention. For example, co-therapy with ipilimumab (anti-CTLA4) and nivolumab (anti-PD1) has demonstrated clinical activity that appears to be distinct from the activity achieved with monotherapy (Wolchok et al., 2013, N. Eng. J. Med., 369:122-33). Combinations of drugs that have been shown to improve the efficacy of checkpoint inhibitors, such as lirilumab (also known as anti-KIR, BMS-986015 or IPH2102, disclosed in U.S. Pat. No. 8,119,775, and Benson et al., Blood 120:4324-4333 (2012)) in combination with ipilimumab (Rizvi et al., ASCO 2013, and clinicaltrials.gov NCT01750580) or in combination with nivolumab (Sanborn et al., ASCO 2013, and clinicaltrials.gov NCT01714739), anti-PD-1 (Woo et al., 2012 Cancer Res. 72:917-27) or anti-PD-Ll (Butler NS et al., Nat Immunol. 2011, 11:131-132 (2012)). Also included are an agent directed against LAG3 in combination with anti-CTLA-4 (Fu et al., Cancer Res. 2011 71:5445-54), an agent directed against ICOS in combination with anti-CTLA-4 (Curran et al., PLoS One. 2011 6(4) el 9499).

[0171] In accordance with the present invention, preferred inhibitory immune checkpoint proteins that are targeted include those selected from the group consisting of PD-1, PD-L1, PD-L2, BTLA, CTLA-4, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), IDO, KIR, LAG3, TIM-3, and VISTA.

[0172] A preferred inhibitor of an inhibitory immune checkpoint protein of interest disclosed herein consists of an antibody directed against said inhibitory immune checkpoint protein of interest and inhibits the activity of said inhibitory immune checkpoint protein of interest.

[0173] Thus, in some preferred embodiments, inhibitors of inhibitory immune checkpoint proteins that may be used in accordance with the present invention include those selected from the group consisting of antibodies directed against one of PD-1, PD-L1, PD-L2, BTLA, CTLA-4, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), IDO, KIR, LAG3, TIM-3, and VISTA.

[0174] Cancers within the scope of the present invention include leukemia, acute lymphocytic leukemia, acute myeloid leukemia, myeloblastic promyelocytic, myelomonocytic erythroleukemia, chronic leukemia, chronic myelogenous (granulocytic) leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, primary central nervous system lymphoma, Burkitt's lymphoma and marginal zone B-cell lymphoma, polycythemia vera lymphoma, Hodgkin's disease, non-Hodgkin's disease, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors, sarcomas, and carcinomas. tumor, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphosarcoma, lymphangioendotheliosarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon sarcoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystic Alveolar carcinoma, Medullary carcinoma, Bronchogenic carcinoma, Renal cell carcinoma, Hepatoma, Cholangiocarcinoma, Choriocarcinoma, Seminoma, Embryonal carcinoma, Wilms' tumor, Cervical cancer, Uterine cancer, Testicular tumor, Lung cancer, Small cell lung cancer, Non-small cell lung cancer, Bladder cancer, Epithelial carcinoma, Glioma, Astrocytoma, Medulloblastoma, Craniopharyngioma, Ependymoma, Pinealoma, Hemangioblastoma, Acoustic neuroma, Oligodendroglioma, Meningioma, Melanoma, Neuroblastoma, Retinoblastoma, Nasopharyngeal carcinoma, Esophageal carcinoma, Basal cell carcinoma, Biliary tract cancer, Bladder cancer, Bone cancer, Brain and central nervous system Cancers of the central nervous system (CNS), cervical cancer, choriocarcinoma, colorectal cancer, cancer of connective tissue, cancer of the digestive tract, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, intraepithelial neoplasia, renal cancer, laryngeal cancer, liver cancer, lung cancer (small cell, large cell types), melanoma, neuroblastoma; oral cancer (e.g., lip, tongue, oral cavity, and pharynx), ovarian cancer, pancreatic cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer; respiratory system cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, and urinary system cancer.

[0175] Pharmaceutical compositions and methods of treatment As already described elsewhere herein, the compounds having a glycoengineered Fc fragment as defined herein may be advantageously used in a course of combination treatment with one or more further anti-cancer agents, including in a course of combination treatment with one or more further anti-cancer therapies, in particular in a course of combination treatment with one or more inhibitory immune checkpoint protein inhibitors.

[0176] According to these embodiments, the compound having a glycoengineered Fc fragment and the one or more additional anti-cancer agents are "co-administered."

[0177] As used herein, the term "co-administration" refers to the administration of at least two different substances sufficiently close in time to modulate an immune response. Preferably, co-administration refers to the administration of at least two different substances at the same time.

[0178] Thus, "co-administration" refers to the administration of two or more components in combination such that the therapeutic or prophylactic effect of the combination can exceed the therapeutic or prophylactic effect of either component administered alone. The two components can be co-administered contemporaneously or sequentially. Contemporaneously co-administered components can be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes the case where multiple components are administered such that each component can be present at the treatment site at the same time. Alternatively, sequential co-administration of two components can include the case where at least one component is removed from the treatment site, but at least one cellular effect (e.g., cytokine production, activation of a specific cell population, etc.) of the component administered persists at the treatment site until one or more additional components are administered to the treatment site. Thus, a co-administered combination can, in some circumstances, include components that are never present in a chemical mixture with each other.

[0179] In some embodiments, the selected compound having a glycoengineered Fc fragment and one or more additional anti-cancer agents are administered simultaneously to the cancer individual to be treated, and the two active agents may be contained in the same pharmaceutical composition or may be contained in separate pharmaceutical compositions. These two separate pharmaceutical compositions may be mixed together prior to use and then administered to the cancer individual to be treated. In other embodiments, these two separate pharmaceutical compositions may be administered to the cancer individual to be treated with a short time interval between them, for example, within 2-5 minutes.

[0180] The present invention further relates to a pharmaceutical composition comprising (i) a compound having a glycoengineered Fc fragment, and (ii) one or more different anti-cancer agents.

[0181] The present invention encompasses pharmaceutical compositions comprising (i) a compound having a glycoengineered Fc fragment, and (ii) one or more inhibitory immune checkpoint protein inhibitors.

[0182] In some preferred embodiments, the compound having a glycoengineered Fc fragment is a glycoengineered antibody directed against a tumor antigen.

[0183] In some embodiments, the tumor antigen is selected from the group consisting of HER2, HER3, HER4, and AMHRII.

[0184] In some embodiments, the glycoengineered antibody is selected from the group consisting of the glycoengineered antibodies designated 3C23K (or a variant thereof), 9F7F11, H4B121, and HE4B33, which are disclosed in detail elsewhere herein.

[0185] In some embodiments, the inhibitory immune checkpoint protein inhibitor is selected from the group consisting of inhibitors of PD-1, PD-L1, PD-L2, BTLA, CTLA-4, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), IDO, KIR, LAG3, TIM-3, and VISTA.

[0186] In some embodiments, the inhibitor consists of an antibody, or an antigen-binding fragment thereof, directed against the inhibitory immune checkpoint protein.

[0187] Methods for preparing and administering to a subject a compound having a glycoengineered Fc, or more generally, a polypeptide as disclosed herein, are well known to or readily determined by those of skill in the art. The route of administration of the polypeptides disclosed herein may be oral, parenteral, inhaled, or topical. As used herein, the term parenteral includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. Although all of these administration forms are expressly contemplated as being within the scope of the present disclosure, the administration form is a solution for injection, particularly for intravenous or intraarterial injection or infusion. Typically, a suitable pharmaceutical composition for injection may include a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer (e.g., human albumin), and the like. However, in other methods consistent with the teachings herein, a compound having a glycoengineered Fc can be delivered directly to the site of the harmful cell population, thereby increasing the exposure of the affected tissue to the therapeutic agent.

[0188] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. In the compositions and methods disclosed herein, pharma- ceutically acceptable carriers include, but are not limited to, 0.01-0.1M or 0.05M phosphate buffer, or 0.8% saline. Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose, and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present. More specifically, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the compositions must be sterile and fluid to the extent that easy syringability exists. The compositions should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0189] In any case, a sterile injectable solution can be prepared by incorporating the active compound (e.g., a compound having a glycosylated Fc fragment by itself or in combination with other active agents) in the required amount in a suitable solvent with one or a combination of the compounds listed herein, followed by filtered sterilization as necessary. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required compounds from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preparation method generally includes vacuum drying and lyophilization, which results in a powder containing the active ingredient plus any desired additional compounds from a previously sterile-filtered solution thereof. The preparations for injection are processed and filled into containers, such as ampoules, bags, bottles, syringes, or vials, and sealed under sterile conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit, such as those described in U.S. patent application Ser. Nos. 09 / 259,337 and 09 / 259,338, each of which is incorporated herein by reference.

[0190] The effective dose of the compositions disclosed herein for treating the above conditions will vary depending on many different factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or an animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Usually, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. Treatment doses can be titrated using routine methods known to those skilled in the art to optimize safety and efficacy.

[0191] The compounds having glycoengineered Fc fragments disclosed herein can be administered on multiple occasions. The interval between single doses can be one week, one month, or one year. The intervals can also be irregular as indicated by measuring the blood levels of the compounds having glycoengineered Fc fragments in the patient. In some methods, the dosage is adjusted to achieve a plasma concentration of the compounds having glycoengineered Fc fragments, particularly plasma concentrations of glycoengineered antibodies, of 1-1000 μg / ml, and in some methods 25-300 μg / ml. Alternatively, the compounds having glycoengineered Fc fragments can be administered as sustained release formulations, in which case less frequent administration is required. In the case of glycoengineered antibodies, the dosage and frequency vary depending on the antibody half-life in the patient. Generally, humanized antibodies exhibit the longest half-life, followed by chimeric and non-human antibodies.

[0192] Pharmaceutical compositions according to the disclosure herein typically include a pharma- ceutically acceptable, non-toxic, sterile carrier, such as physiological saline, non-toxic buffers, preservatives, etc. For purposes of this application, a pharma- ceutical sufficient amount of the compound having a glycoengineered Fc fragment must be maintained, meaning an amount sufficient to achieve a benefit, for example, to reduce or block an immunosuppressive state occurring in cancer patients. Of course, the pharmaceutical compositions disclosed herein may be administered in single or multiple doses to provide a pharma- ceutical effective amount of the compound having a glycoengineered Fc fragment. EXAMPLES

[0193] Example 1: Synthesis of compounds with glycoengineered Fc fragments A. Materials and Methods Cloning of chimeric 12G4, humanized 12G4 and 3C23K A pharmaceutical composition according to the chimeric 12G4 (ch12G4) of the present invention was constructed and expressed as previously described (27). L and V HThe DNA sequences were sequentially subcloned into the polycistronic CHK622-08 vector, which contains a promoter, a Kozak sequence, and a sequence for the human kappa / IgG1 constant region.

[0194] V of humanized 12G4 (h12G4) L and V H The DNA sequence encoding was synthesized using Genscript and then cloned into CHK622-08 by digestion and ligation as described above to obtain the HK622-18 vector. L Targeted mutagenesis of phage clone 3C23 was performed to introduce the E68K mutation to obtain the affinity matured form 3C23K V L and V H A DNA sequence encoding the 3C23K antibody was obtained. A signal peptide was added by PCR assembly using the humanized variable regions of h12G4 as a template and then cloned into HK622-18 as described above. The resulting vector expressing the humanized and affinity matured 3C23K antibody was called HK622-18MAO3C23K.

[0195] Generation and purification of ch12G4, h12G4, and 3C23K The different molecules were stably expressed as previously described (Siberil et al., 2006, Clin Immunol Orlando Fla, Vol. 118:170-179). CHO-S, HEK293, or YB2 / 0 cells were stably transfected with the appropriate linearized expression vector. Ch12G4, h12G4, and 3C23K antibodies were produced in YB2 / 0 cells using EMS (Invitrogen), 5% Ultra-low IgG fetal calf serum (FCS) (PAA), and 0.5 g / l G418 for 5-7 days. 3C23K-CHO-S was produced in CHO-S cells using ProCHO4 (Lonza), 4 mM glutamine, and 1 g / l G418 for 7 days.

[0196] MAbs were purified from culture supernatants by affinity chromatography using protein A Sepharose (GE-Healthcare). Aggregate and endotoxin levels were determined by gel filtration on Superdex HR / 200 (GE-Healthcare) and by LAL test, respectively. Antibody quality and purity were monitored by SDS-PAGE and Coomassie staining. In addition, the glycosylation pattern and core fucose percentage (%) of each purified antibody were determined by high performance capillary electrophoresis laser induced fluorescence (HPCE-Lif) (51).

[0197] SPR analysis SPR analysis was performed on a Bia3000 or T200 instrument at HBS-EP (GE Healthcare) at 25°C. For affinity measurements, MISRII was covalently immobilized (1000 RU) onto a CM5 sensor chip using EDC / NHS activation according to the manufacturer's instructions (GE Healthcare). Various concentrations (0.5-128 nM) of 12G4 or 3C23K were injected over the immobilized receptor for 180 s. After dissociation in running buffer for 400 s, the sensor chip was regenerated using Gly-HCl (pH 1.7). K DValues ​​were calculated using a Langmuir 1:1 fitting model (BiaEvaluation3.2, GE Healthcare) taking into account affinity and avidity. Antibody-FcγR measurements were performed at 100 μl / min with a single cycle titration on FcγR (Sigma) captured on covalently immobilized anti-His (R&D Systems) at 4000-5000 RU levels. Gamma receptor was injected at 20 nM for 60 s and five increasing concentrations of antibody were injected (injection time = 60 s). After a 600 s dissociation step in running buffer, the sensor surface was regenerated using 5 μl of glycine-HCl (pH 1.7). Kinetic parameters were evaluated from the sensorgrams using a heterogeneous ligand model or a steady-state fitting model on the T200 Evaluation Software 3.0 (GE Healthcare). All sensorgrams were corrected by subtracting low signals from a control reference surface (containing no immobilized protein) and a buffer blank injection prior to fitting evaluation.

[0198] antibody The mouse anti-MISRII MAb 12G4 was described by Salhi et al. and Kersual et al. (17, 22). The anti-idiotypic factor VIII chimeric IgG1 R565 EMABling® MAb and the anti-CEA MAb 35A7 (17) were used as irrelevant antibodies.

[0199] result Chimerization, humanization, and affinity maturation The 3C23K humanized antibody was originally derived from the variable regions of the murine 12G4 MAb (Sahli et al., 2004, Biochem J, Vol. 37:785-793). The humanization procedures included CDR grafting (MAb h12G4) and affinity maturation by random mutagenesis and phage display, resulting in the final molecule 3C23K.

[0200] In a first step, human template candidates for CDR grafting were identified by entering the sequences of the VL and VH domains separately in the IMGT / DomainGapAlign research program (28) and restricting the search to human sequences in IMGT / Gene-DB (29). The closest human VH gene, IGHV1-3, * 01 showed 67.34% identity with its murine counterpart. This identity increased to 92.85% after grafting the murine 12G4 CDR-IMGT to the human FR-IMGT. The closest human VL gene, IGK1-9 * IGK1-01 showed 62.76% identity with its mouse counterpart, although the IMGT / GeneFrequency tool (28) did not identify IGK1-9. * 01 suggested that it is not expressed very frequently, so IGKV1-5 * 01 was preferred. IGKV1-5 * 01 had 58.51% identity with the VL of 12G4, which increased to 88.29% after grafting.

[0201] To better define the binding properties, clone 3C23K was reformatted as an IgG1 antibody, produced in YB2 / 0 cells, and analyzed by surface plasmon resonance (SPR). The 3C23K antibody is a IgG1 antibody produced in YB2 / 0 cells and analyzed by surface plasmon resonance (SPR). D =7.9×10 -10 M) with a higher binding affinity (K D =5.5×10 -11 This latter value was very close to the value published in the original description of MAb12G4 (K D =8.6×10 -10 The increased binding affinity was also confirmed by flow cytometry using COV434-MISRII cells.

[0202] 3C23K production in YB2 / 0, CHO, or HEK293 cells, glycosylation analysis, and effect on binding to Fcγ receptors Oligosaccharide analysis of 3C23K expressed in YB2 / 0 cells (EMABling® version; 3C23K) (27), CHO-S cells (3C23K-CHO), or HEK293 (3C23K-HEK293) cells (used as comparators for functional assays) showed two distinct glycosylation patterns. The percentages (%) of fucosylated, galactosylated, and bisecting GlcNAc isoforms were 33.0%, 57.2%, and 1.8%, respectively, for 3C23K and 94.6%, 54.4%, and 2.0%, respectively, for 3C23K-CHO. The effect of these glycosylation differences on binding to FcγRs was analyzed by SPR. The binding affinity for hFcγRIIIa and hFcγRIIIb was clearly increased after fucose was reduced (1-12 nM and 86.0 nM for 3C23K, respectively, compared to 31-164 nM and 378 nM for 3C23K-HEK293), but this was not true for other FcγRs (hFcγRI, hFcγRIIa, hFcγRIIb) (see also Table 2 in Example 2 below).

[0203] Next, 3C23K was expressed in YB2 / 0 cells using the EMABling® technique to increase antibody interaction with the low / medium affinity Fc receptor CD16, which is mainly expressed on NK cells and macrophages (Siberil et al., 2006, Clin Immunol Orlando Fla, Vol. 118:170-179). This feature is related to the low expression of Fut8 gene in rat myeloma YB2 / 0 cells compared to other commonly used cell lines, such as CHO cells (Siberil et al., 2006, Clin Immunol Orlando Fla, Vol. 118:170-179).

[0204] As expected, 3C23K-YB2 / 0 showed higher binding affinity to CD16 than the fucose-rich 3C23K.

[0205] Example 2: Glycan analysis of the 3C23K (GM102) antibody background: GM102 is a humanized monoclonal antibody produced in YB2 / 0 cells (rat hybridoma YB2 / 3HL) using clone 18H2.

[0206] The carbohydrate moiety is the ASN of the heavy chain. 295 Located in. a) Glycan analysis results for GM102 :Table 2

[0207] [Table 2]

[0208] b) Characterization of the PB01 reference standard After N-deglycosylation with PNGase F and tagging of the released carbohydrate residues, analysis of these carbohydrate residues by UPLC-HILIC-FD revealed the presence of six major carbohydrate moieties: 1. Non-fucosylated (G0) 51.1% 2. Fucosylated (G0F) 12.8% 3. Monogalactosylated nonfucosylated (G1) 10.2% 4. Monogalactosylated fucosylated (G1F) 4.3% 5. Aglycosylated nonfucosylated (G0B) 9.9% 6. Fucosylated with bisecting GlcNAc (G0FB) 3.4% The total number of fucosylated residues was 23%.

[0209] [Table 3]

[0210] Example 3: High affinity of compounds with glycoengineered Fc fragments to Fc receptors A. Materials and Methods Surface plasmon resonance (SPR) analysis : Anti-histidine antibody (R&D Systems) was immobilized on a CM5 sensor chip using EDC / NHS activation according to the manufacturer's instructions (GE Healthcare) at a flow rate of 10 μl / min in HBS-EP on a T200 instrument at 25° C. The antibody was covalently immobilized on flow cell Fc2 at a level of 6900 RU, and a control reference surface (flow cell Fc1) was prepared using the same chemistry but without anti-His antibody.

[0211] All kinetic measurements for Fc1 and Fc2 were performed by single cycle titration on a T200 instrument at 25°C in HBS-EP at 100 μl / min. Each human gamma receptor (R&D Systems) was captured on an immobilized anti-His antibody at 20 nM for 60 s. Five increasing concentrations of antibody were injected (injection time = 120 s). After a 600 s dissociation step in running buffer, the sensor surface was regenerated using 5 μl of glycine-HCl (pH 1.7). All sensorgrams were corrected by subtracting low signals from control reference surface and buffer blank injections. Kinetic parameters were evaluated from the sensorgrams using a heterogeneous ligand model or a two-state model from the T200 evaluation software.

[0212] B. Results The results of determining the affinity constant (Kd) of the hypofucosylated anti-AMHRII3C23K antibody for human Fc receptors are shown in Table 4 below.

[0213] JPEG0007678493000006.jpg99170

[0214] The affinity constant is expressed as KD (units: nM). * KD was calculated using a heterogeneous ligand fitting model. ** When the fit did not fit the heterogeneous ligand model, KD values ​​were determined using a two-state reaction model.

[0215] Example 4: Reduced fucose antibodies block tumor-associated macrophage-induced immune suppression in cancer A. Materials and Methods In vitro immunological assays : T cell proliferation assays were performed as follows: Briefly, CMFDA stained COV434-AMHRII was treated with 10 μg / ml of irrelevant mAb R565, or anti-AMHRII FcKO, or anti-AMHRII 3C23K mAb for 1 h at 4° C. and incubated with unstained MDM2 for 4 days, followed by addition of CD3 / CD28 Dynabead preactivated CellTrace Violet (Molecular Probes®, Life Technologies™) stained T cells at a ratio of 1:8 as MDM2:T cells. After an additional incubation period of 4 days, cells were harvested and stained with anti-CD8 PerCP, CD11b PE-Cy7, and CD4 AF647 (BD Pharmingen®) before flow cytometry analysis. Dead cells were excluded by staining with Fixable Viability Dye eFluor® 506 (eBioscience®) prior to antibody staining. T cell proliferation was analyzed on CD8+ (CD11b−) T gated cells by measuring CellTrace Violet dilutions corresponding to cell division. A division index value, which is equal to the average number of cell divisions undergone by cells in the initial population, was calculated using FlowJo (TreeStar, version 7.6.5). A division index value, which is equal to the average number of cell divisions undergone by cells in the initial population, was calculated.

[0216] B. Results It is clearly established that intratumoral macrophages suppress the antitumor activity of T cells. We hypothesized that engagement of macrophages with 3C23K anti-AMHRII antibody alters their T cell suppressive function. To support this hypothesis, COV434-AMHRII target cells were treated with either irrelevant mAb R565, anti-AMHRII FcKO, or anti-AMHRII 3C23K mAb and co-cultured with MDM for 4 days, followed by the addition of CD3 / CD28 preactivated PBT. CD8 + T cell proliferation was analyzed by flow cytometry. As expected, MDMs strongly impaired T cell proliferation in the presence of control mAbs (irrelevant isotype control R565 and FcKO anti-AMHRII mAbs) or in the absence of treatment. Notably, MDM-mediated T cell immunosuppression was significantly reduced when co-cultured tumor cells were treated with 3C23K anti-AMHRII mAb, as indicated by a large increase in the proliferation index of CD8 T cells (Figure 1A).

[0217] The reduction in tumor cell numbers could partially explain this "immunostimulatory" effect, since tumor cells are known to directly exert T cell suppressive functions. To test whether the 3C23K anti-AMHRII mAb could also act on MDMs to make them less immunosuppressive, a tumor cell-free experiment was designed. Inert Sphero® polystyrene beads were used as a surrogate for tumor target cells. These beads were treated with mAb in the same conditions as the tumor cells, i.e., MDMs were first co-cultured with mAb-treated beads and then co-cultured with activated PBT. In this condition, CD8 + T cell proliferation was partially restored when MDMs were co-cultured with 3C23K-coated Sphero® polystyrene beads (Figure 1B). As a control, we checked that T cell proliferation observed in the absence of MDMs was not affected by 3C23K (Figure 2). These experiments strongly suggest that 3C23K directly alters the T cell suppressive capacity of MDMs.

[0218] Collectively, these results indicate that the glycoengineered humanized monoclonal anti-AMHRII antibody, 3C23K, can efficiently target tumor cells through the antigen-binding site and direct pro-tumor macrophages against tumor cells by recognizing the Fc domain. Thus, the mAb-activated macrophages triggered ADCC and ADCP against tumor cells and reduced their immunosuppressive behavior toward T cells.

[0219] Discussion of results ADCC / ADCP may not be the only mechanism induced by macrophages in response to treatment with mAbs. Tumor-associated macrophages have been described to suppress T-cell activation (Biswas et al., 2010, Nat. Immunol., Vol. 11 (n° 10): 889-896), and our data showing contact between lymphocytes and macrophages support the idea of ​​direct mutual interference between both cell types. By using in vitro assays, we found that FcR engagement by 3C23K reduces the immunosuppressive phenotype of macrophages. In such conditions, preactivated T cells regain their proliferation capacity that is blocked in the absence of 3C23K. The concept that therapeutic mAbs can engage not only innate but also adaptive immune cells is consistent with previous studies. In mouse tumor models, treatment with antitumor antigen Abs has been demonstrated to induce cellular immune responses, including T cells, required for long-term survival (Montalvao et al., 2013, J Clin Invest, Vol. 123:5098-5103; Gul et al., 2014, J Clin Invest, Vol. 124:812-823). However, the induction of adaptive immune responses in cancer patients treated with antitumor mAbs has not yet been extensively studied.

[0220] The mechanism by which 3C23K alters the phenotype of macrophages to relieve T cell suppression is currently unknown. However, various hypotheses can be envisaged. It has been shown that the interaction of antibodies with Fc receptors expressed by macrophages triggers several signal cascades that regulate the function of these cells (Biswas et al., 2010, Nat. Immunol., Vol. 11 (n° 10): 889-896). Our preliminary data show that macrophages activated via FcR with 3C23K produce several pro-inflammatory cytokines, including IL-1 beta and IL-6, which have been described to exert beneficial effects on T cells (Grugan et al., 2012, J Immunol., Vol. 189: 5457-5466). Indirect effects are also possible. In particular, tumor cell death can result in the release of several danger-associated molecular pattern molecules (DAMPs), such as calreticulin, which then activates innate and adaptive immune cells (Yatim et al., 2017, Nat Rev Immunol, Vol. 17(n°4): 262-275). The role of dendritic cells in mediating this immunogenic cell death has been well described. Evidence also suggests that calreticulin released during cell death activates macrophages that produce IL-6 and TNF-α, which are likely to exert beneficial effects on T cells (Duo et al., 2014, Int J Mol Sci, Vol. 15(n°2): 2916-2928).

[0221] Example 5 Activation of TAM-like macrophages by glycosylated compounds with Fc A. Materials and Methods Preparation of human monocyte-derived macrophages Peripheral blood mononuclear cells (PBMCs) were obtained from healthy blood donors.

[0222] PBMCs were classically isolated using positive magnetic selection of CD14+ cells. Monocytes were cultured in RPMI supplemented with 10% fetal bovine serum at 37°C and 5% CO2, then differentiated into M2-type macrophages by adding 50ng / mL M-CSF for 4 days. The phenotype of converted M2-type macrophages is CD14 high, CD163 high, IL10 high, IL12 low.

[0223] In vitro activation of macrophages by antibodies A 10 μg / mL solution of the antibody, i.e., hypofucosylated anti-AMHRII, named R18H2, or its FcKO counterpart that does not contain any binding to Fcγ receptors, was adsorbed onto a 24-well plate by incubating at 4°C for 24 h. This experimental condition mimicked the situation in which the antibody recognized its antigen. The uncoated antibody was discarded by washing with PBS solution. Next, M2-type macrophages (cells 10 6 Cells (cells / mL culture medium) were incubated in antibody-coated (or not, in the case of negative controls) wells at 37°C for 1-3 days.

[0224] Analysis of macrophages after antibody activation Macrophages incubated with antibodies were stimulated with 100 ng / mL LPS for 6 or 24 h and then analyzed by qRT-PCR or flow cytometry, respectively. Transcription of PDGFα, VEGFβ, HGF, TGFβ, IDO1, IL10, Sepp1, Stab1, FOLR2, CD64a, CD64b, and CD16a genes was quantified and normalized by using RPS18, B2M, and EF1a genes as references.

[0225] The changes in expression were confirmed at the protein level by flow cytometry for membrane proteins expressed by macrophages and by typical ELISA assays for soluble factors, such as IL10, IL1β, or TNFα, using samples from the culture medium.

[0226] B. Results Antibodies adsorbed on multiwell plates differentially stimulated M2 macrophages depending on their ability to bind to the Fcγ receptors of macrophages. Overall, no significant changes in markers were observed when M2 macrophages were cultured in wells without any antibodies. Only minor changes corresponding to non-specific binding of those proteins to macrophages were observed when FcKO antibodies were used. In contrast, the obvious decrease in certain typical markers of M2 macrophages, such as Sepp1, Stab1, FOLFR2, and CD163, which decreased after 3 days of incubation, was reduced when macrophages interacted with hypofucosylated R18H2 antibodies, as shown in Figure 3A. These changes strongly suggest that the macrophage type may change under stimulation with hypofucosylated antibodies. These changes were accompanied by an increase in Fcg receptors that bind antibodies and are involved in ADCC and phagocytosis, such as CD16 (Figure 3B) and CD64 (Figure 3C).

[0227] Interestingly, after 3 days of coculture with hypofucosylated R18H2 antibody, the profile of cytokines and soluble peptides detected in the culture medium of M2 macrophages revealed a clear increase in proinflammatory factors normally expressed by M1 macrophages, such as TNFα, IL1β (Figure 3D), or IL6 (data not shown). Moreover, a decrease in immunosuppressive factors such as TGFβ, IDO1, and IL10 (Figures 3E, 3F, and 3G), as well as a decrease in proangiogenic factors such as PDGFα, VEGFβ, and HGF (Figures 3H, 3I, and 3J) were also observed.

[0228] Moreover, the reduction in PDL2 expression on the surface of M2 macrophages upon stimulation with hypofucosylated R18H2 antibody (as shown in Fig. 3K ), together with the reduction in IL10, suggests a tendency towards reduced immunosuppressive activity of these phenotypically modified macrophages.

[0229] Taken together, these results indicated that binding of low-fucose antibodies to M2 macrophages results in a shift to an intermediate macrophage phenotype between M2 and M1, and induces changes in several factors that induce indirect antitumor effects through inhibition of angiogenesis and stimulation of the immune system.

[0230] Example 6 :3C23K antibody blocks immunosuppression, which leads to activation of the immune system A. Materials and Methods Preparation of human monocyte-derived macrophages (MDM) Peripheral blood mononuclear cells (PBMCs) were obtained from healthy blood donors (Etablissement Francais de Sang, EFS).

[0231] Human monocytes were isolated from PBMCs using the Negative Selection Monocyte Isolation Kit II (Macs Miltenyi) as recommended by the manufacturer's protocol. Monocytes were cultured at 37°C and 5% CO2 in Macrophage-SFM (Gibco) supplemented with L-glutamine (Invitrogen) and penicillin / streptomycin (PS, Invitrogen).

[0232] Isolated monocytes were either kept undifferentiated (NS, unstimulated) or differentiated into antitumor (M1-like) or tumor-promoting (TMA-like) macrophages by stimulation with IFN-γ (Macs Miltenyi, 100UI / ml) + LPS (100ng / ml, Sigma) or M-CSF (Macs Miltenyi, 200UI / ml) + IL-10 (Macs Miltenyi, 50UI / ml), respectively, for 3 days.

[0233] Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) assay SKOV-R2+ cells were pretreated with 10 μg / mL of anti-AMHRII antibodies: GM102 (also named 3C23K-YB20), 3C23K-CHO, or 3C23K-FcKO at 4° C. Target SKOV-R2+ cells were loaded with BATDA (bis-acetoxymethyl-2,2′:6′,2″-terpyridine-6,6″-dicarboxylate), resuspended in DMEM (Gibco) supplemented with L-glutamine, PS, and 10% heat-inactivated FCS, and added to effector cells (human macrophages) at a 1:1 ratio for 4 h at 37° C.

[0234] ADCC was measured by using a DELFIA EuTDA-based cytotoxicity assay (PerkinElmer). After 4 hours of incubation between target and effector cells, the supernatant was incubated with Eu3+ solution and fluorescence was measured (Envision, PerkinElmer). Data were normalized to the highest (Triton-treated target cells) and lowest (effector cells alone) cell lysis and fitted to a sigmoidal dose-response model.

[0235] Flow cytometric evaluation of the cytotoxic effects of macrophages plus antibodies against ovarian cancer tumor cell lines (SKOV-R2+ cells) SKOV-R2+ cells were stained with the CellTrace™ Violet Cell Proliferation Kit (Molecular Probes™, Life Technologies), resuspended in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with L-glutamine, PS, and 10% heat-inactivated fetal calf serum (FCS, Sigma), and added to each type of human macrophage at a 1:1 ratio in the presence of each of the three anti-AMHRII antibodies.

[0236] To assess the number and proliferation of SKOV-R2+ cells, pretreated or untreated human macrophages were challenged with tumor cells for 3, 4, and 5 days. The number of SKOV-R2+ cells was calculated by detecting fluorescently labeled cells, and their proliferation was assessed by CellTrace dilution.

[0237] A population of 10,000 cells was analyzed for each data point. All analyses were performed on a BD Fortessa flow cytometer using Diva software, except where CellTrace dilutions were analyzed using Modfit software.

[0238] Assessment of macrophage differentiation by detection of receptor expression on human macrophages Receptor expression (CD11b, CD163, CD36, CD206, CD14, CD16, CD32, CD64, CD80, CD282) was assessed by flow cytometry on the membrane of human macrophages (i) after differentiation and (ii) after 3 days of co-culture between differentiated human macrophages and SKOV-R2+ tumor cells (treated with different anti-AMHRII antibodies).

[0239] Receptors were detected using Cd11b-FITC, CD163-PE, CD36-PE, CD206-APC, CD16-VioBright515, CD64-PerCP-Vio700, CD80-PE, CD32-PE-Vio770, CD282(TLR2)-APC, CD14-APC-Vio770 (Miltenyi) and compared with appropriate isotype controls.

[0240] A population of 10,000 cells was analyzed for each data point. Dead cells (positive cells) were removed from the analysis after labeling with Viability Fixable Dye (Miltenyi). Analysis was limited to CD14 or Cd11b positive cells. All analyses were performed on a BD Fortessa flow cytometer using Diva software.

[0241] Th1 / Th2 T-CD4 polarization and T-CD8 activation Human T cells were isolated from PBMCs using a negative selection PanT cell isolation kit (Macs Miltenyi) as recommended by the manufacturer's protocol. After isolation, cells were stained with CellTrace™ Violet cell proliferation kit (Molecular Probes™, Life technologies), resuspended in RPMI1640 medium (Gibco) supplemented with L-glutamine, PS, and 10% heat-inactivated FCS, and added to co-cultures of human macrophages + SKOV-R2 + tumor cells (treated with different anti-AMHRII antibodies as above) at a ratio of 1:8 for 4 days.

[0242] To assess Th1 / Th2 T-CD4 bias, T cells were labeled with CD183 (CD183(CXCR3)-APC, Miltenyi) and analysis was restricted to CD4 positive cells (CD4-VioBright FITC, Miltenyi).

[0243] To assess T-CD8 activation, T cells were labeled with CD183 (CD183(CXCR3)-APC, Miltenyi) and CD25 (CD25-PE, Miltenyi), and analysis was limited to CD8 positive cells (CD8-PE-Vio770, Miltenyi).

[0244] Proliferation of T-CD4 and T-CD8 cells was assessed by CellTrace dilution and analysis was limited to CD4 positive or CD8 positive cells.

[0245] A population of 10,000 cells was analyzed for each data point. All analyses were performed on a BD Fortessa flow cytometer using Diva software, except where CellTrace dilutions were analyzed using Modfit software.

[0246] Cytokine and chemokine production Release of cytokines (IL-1β, IL-2, IL-6, IL-10, IL-12, IL-23, TNF-α, and TGF-β) and chemokines (CCL2, CCL4, CCL5, CXCL9, and CXCL10) was quantified in (i) supernatants of differentiated human macrophages, (ii) supernatants after 3 days of coculture with differentiated human macrophages and SKOV-R2+ tumor cells (treated with different anti-AMHRII antibodies), and (iii) supernatants after 4 days of addition of this coculture plus T cells.

[0247] Quantification of cytokine and chemokine release was measured by AlphaLisa immunoassay according to the manufacturer's instructions (AlphaLisa kit, PerkinElmer).

[0248] B. Results All experiments were performed with PBMCs from three different independent healthy donors. The ADCC measured in the presence of undifferentiated macrophages or macrophages differentiated into TAM-like (by addition of M-CSF and IL-10) was found to be clearly higher in 3C23K-YB20 low fucose antibody compared to 3C23K-CHO or 3C23K-FcKO, which were used as inactive controls. The data in TAM-like are presented in Figure 4A. This cytolytic activity may at least partially explain the reduction of tumor cells after 4 days of co-incubation with TAM-like macrophages. This reduction was also higher in 3C23K YB20 than in 3C23K-CHO, as shown in Figure 4B.

[0249] When T cells were added to the co-culture of TAM-like macrophages and tumor cells, an increase in the percentage (%) of memory CD8+ lymphocytes was observed (Figure 4C). A trend towards an increase in Th1 CD4 regulatory T cells was also seen under identical experimental conditions (Figure 4D), in parallel with a decrease in Th2 CD4+ T cells (Figure 4E). All these modulations were due to an increase in long-term T cell-mediated antitumor activity. All these changes were higher in 3C23K-YB20 than in 3C23K-CHO and 3C23K-FcKO.

[0250] Interestingly, the cytokine and chemokine profiles detected in the media of co-cultures of TAM-like+ tumor cells in the presence of anti-AMHRII 3C23K-YB20 antibody revealed a clear increase in CXCL9 (Figure 4F) and CXCL10 (Figure 4G), two factors involved in T cell recruitment, and CCL2, involved in macrophage recruitment, while the basal levels of CCL2 were different in each donor (Figure 4H). Moreover, when T cells were added to the co-cultures, an increase in two pro-inflammatory cytokines, IL6 (Figure 4I) and IL1b (Figure 4J), as well as an increase in CCL5 (Figure 4K), a factor associated with T cell infiltration, was detected in 3C23K-YB20, which was also higher than that in 3C23K-CHO and 3C23K-FcKO.

[0251] Taken together, these results demonstrated that the addition of 3C23K-YB20 to co-cultures of tumor cells + TAM-like macrophages, followed by the addition of T cells, conditions that mimic the pathological situation within the tumor, resulted in direct tumor cell lysis and activation of anti-tumor T cell responses. All these observations were higher with 3C23K-YB20 than with other anti-AMHRII antibodies tested.

[0252] Interestingly, the favorable effect of 3C23K-YB20 is not limited to conditions with TMA-like macrophages. Similar experiments with non-stimulated (NS) macrophages co-cultured with tumor cells and antibodies also allowed to show an increase in pro-inflammatory factors such as IL12 (Fig. 4L), IL6 (Fig. 4M), and IL1b (Fig. 4N), in parallel with a decrease in the pro-tumor and pro-angiogenic cytokine IL23 (Fig. 4O). Moreover, as described above for TAM-like macrophages, an increase in CXCL9 (Fig. 4P) and CXCL10 (Fig. 4Q), two anti-angiogenic chemokines involved in T cell recruitment, was also observed. All these changes were more significant with the 3C23K-YB20 low-fucose antibody than with the other antibodies. These results strongly suggest that 3C23K-YB20 may affect T cell anti-tumor activity via undifferentiated macrophages as well as via TAM-like macrophages.

[0253] Example 7: Activation of macrophages in tumor tissues of cancer patients administered glycoengineered antibodies A. Materials and Methods To identify multiple targets in the same tissue section, a multiplex immunofluorescence technique based on TSA is used in this study. Tyramide Signal Amplification (TSA) is based on a patented catalyzed reporter deposition (CARD) technology that uses derivatized tyramides. In the presence of small amounts of hydrogen peroxide, immobilized HRP converts a labeled substrate (tyramide) into a short-lived, highly reactive intermediate. The activated substrate molecule then reacts very rapidly with, and covalently binds to, electron-rich regions of neighboring proteins. This binding of activated tyramide molecules occurs only immediately adjacent to the site where the active HRP enzyme binds. Multiple depositions of labeled tyramides occur within a very short time (typically within 3-10 minutes). Subsequent detection of the label results in an efficient large-scale amplification of the signal. The advantage of this technique is that multiple primary antibodies generated in the same species can be detected on the same tissue slide. Each reaction is stopped when the TSA-fluorescent dye precipitates. This can be repeated until the five targets are reached. In our laboratory, a Ventana Discovery ULTRA automated slide stainer is available to automate the procedure. This instrument allows efficient and reproducible walk-away staining of FFPE tissue slides.

[0254] In multiplex applications, the following fluorophores disclosed in Table 5 below are used:

[0255] [Table 5]

[0256] These fluorophores, secondary antibody system, and primary antibodies represent assay-specific reagents. All other ancillary reagents used to perform the staining (pretreatment, washing, and denaturing buffers) are considered as generic reagents. The limitations of the assay are determined by the imaging platform available and used. All slide images are generated using a 3DHistech P250 Panoramic scanner equipped with appropriate filters to separate the fluorophores used (Rhodamin6G, RED610, DCC, FAM, Cy5). However, due to their spectral properties, the DAPI and DCC signals cannot be separated so far. Thus, the nuclear counterstain is excluded.

[0257] Development of multiplex immunofluorescence assays is needed for the following markers / objectives: For lymphocytes, cytokeratin (CK), CD3, CD4, CD8, FoxP3 CK, CD14, HLADR, CD206, and / or CD163 for macrophages CK, CD56, CD15, Granzyme, DC-Lamp for dendritic cells, polymorphonuclear cells, and natural killer cells CK, CD45, CD16, CD32, and CD64 for effector cells versus immune cells.

[0258] These four multiplex assays were validated, labeled as follows: 1. Anti-CD3 clone 2GV6, anti-CD4 clone SP35, anti-CD8 clone C8 / 144B, anti-FoxP3 clone D2W8E, and anti-CK clone cocktail AE1 / AE3 2. Anti-CD14 clone EPR3653, anti-CD68 clone KP-1, anti-CD163 clone MRQ-26, anti-MHC-II clone EPR11226, and anti-CK clone cocktail AE1 / AE3 3. Anti-CD16 clone SP175, polyclonal anti-granzyme B, anti-CD8 clone C8 / 144B, and anti-NKp46 4. Anti-CD15 clone MMA, anti-CD64 clone 3D3, polyclonal anti-CD206, and anti-LAMP3 clone 13A205.

[0259] B. Results During a Phase I trial of GM102, the presence of several cell types was investigated using multiplex fluorescent staining and FFPE paired and baseline ovarian cancer biopsy analysis. The multiplex staining was targeted to immune infiltrate and assessment of monocyte / macrophage differentiation and phagocytic activity. Baseline samples were biopsied 7-15 days prior to GM102, and a second biopsy was performed 1.5 months after treatment.

[0260] Because only two paired biopsies were examined, the effect of GM102 treatment was only evaluated descriptively, and no statistical analysis of the observed phenomena was performed. The first baseline samples were characterized by changes in the presence of immune infiltrates. The most striking observation of this study was the effect of GM102 on the monocyte-like CD16+ cell population, a cell type that was already abundant at baseline (Figure 5A). Under GM102 treatment, the CD16+ stained area was significantly increased in the examined patients, however, this was not reflected by an increase in CD16+ cell density as if the CD16+ cells were charged with CD16 upon GM102 treatment. This observation suggests that activation of CD16+ cells (effector cells, mainly macrophages) was involved in the antitumor activity of GM102.

[0261] Furthermore, an increase in Granzyme B expression was observed under GM102 treatment (Figure 5B). Granzyme B is a 29 kDa member of the granule serine protease family that is specifically stored in NK cells or cytotoxic T cells. Cytolytic T lymphocytes (CTL) and natural killer (NK) cells share the ability to recognize, bind to, and lyse specific target cells. They are believed to protect their host by lysing cells that bear "non-self" antigens, usually peptides or proteins, on their surface resulting from infection with intracellular pathogens. Granzyme B is important in cell-mediated immune responses for the rapid induction of target cell apoptosis by CTLs (Rousalova & Krepela, 2010, Int. J. Oncol. 37:1361-1378; Vaskoboinik et al., 2015, Nat. Rev. Immunol. 15:388-400). Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are key players in the elimination of neoplastic virus-infected cells. However, in these biopsies, natural killer cells were only sporadically identified CD8+ lymphocytes, as visualized by NKp46. This observation was confirmed in clinical samples, where treatment with GM102 induced the cytolytic activity of CD8+ T lymphocytes.

[0262] Example 8: Activation of NK cells, monocytes, and ICOS+ T cells in cancer patients treated with glycoengineered antibodies A. Materials and Methods In the Phase I study of GM102, 5 mL of blood was sampled for each patient in the escalation cohort at four time points: before the first GM102 infusion on day 1 (designated C1J1-SOI) and at the end of the first GM102 infusion (designated C1J1-EO1), before the second GM102 infusion on day 15 (C1J15-SOI), and at steady state, i.e., at the end of the second 28-day cycle on day 57 (C3J1-SOI).

[0263] For scientific discovery purposes, several different markers were monitored at each clinical site in the study.

[0264] At Gustave Roussy, 5 patients were included. The Laboratoire d'Immunomonitoring en Oncologie (LIO) received a total of 15 samples, as detailed in Table 6 below.

[0265] [Table 6]

[0266] SOI (Start Of Infusion): Start of infusion; EOI (End Of Infusion): End of infusion *Samples were collected at EOI on 9 February 2017 and received at LIO the following day. **Samples were collected at EOI on 29 May 2017 and received at LIO the following day.

[0267] All received samples were analyzed. PBMCs were isolated from all samples and stored in Gamamabs-GM102 test-specific boxes in liquid nitrogen tanks with access restricted to authorized personnel. All materials used are detailed below in Tables 7, 8, and 9:

[0268] [Table 7]

[0269] [Table 8]

[0270] [Table 9]

[0271] PBMCs were isolated according to the following procedure: 1. Disinfect the blood tubes by wiping them with Anios. 2. Take a 50 mL tube pre-filled with 15 mL of Ficoll and gently layer 35 mL of diluted blood on top of the Ficoll without mixing - the two layers should be clearly separated. (For other volumes, maintain a diluted blood:Ficoll ratio of approximately 2 / 3). 3. Seal the tube and centrifuge at 400g for 20 minutes at room temperature (RT), brake off. 4. Recover the mononuclear cell layer (ring) using a sterile, single-use 10 mL pipette and transfer it to a new 50 mL tube ( Optionally: The upper phase can be discarded before recovering the ring). 5. Add up to 50mL of PBS and centrifuge at 800RPM, 15°C for 15 minutes. 6. Using a pipette, quickly remove the supernatant, stopping at 3 mL before it reaches the bottom. 7. Shake to resuspend the pellet. 8. Add 50 ml of PBS onto the remaining cell pellet and mix to resuspend thoroughly. 9. Centrifuge at 300g, 15°C for 10 minutes. 10. Remove the tube, add 1 or 2 mL of PBS and count the cells.

[0272] PBMCs were stored according to the following procedure: 1. Place 90 μL of Blue Hayem in a 96 well plate and add 10 μL of pre-resuspended pellet to count PBMCs only. 2. Place 90 μL of Blue Trypan in a 96-well plate and add 10 μL of pre-resuspended pellet to count viable cells only. 3. Count the cells on Malassez slides and freeze 5-10 million viable cells in Hyclone (1 mL / cryotubes). 4. Place the cryotube in a "Mr Freeze" box and place it at -80°C for at least 24 hours, then transfer it into the nitrogen tank in the Gamamabs-GM102 box.

[0273] Blood immunophenotyping was performed by flow cytometry using the following procedures and markers listed in Tables 10-17 below.

[0274] [Table 10]

[0275] [Table 11]

[0276] [Table 12]

[0277] [Table 13]

[0278] [Table 14]

[0279] [Table 15]

[0280] [Table 16]

[0281] [Table 17]

[0282] B. Results Each of the above markers was measured and analyzed along the treatment. No significant changes were identified for the major immune populations of circulating cells (Ncells, monocytes, neutrophils, eosinophils, and T cells CD4+, CD8+, and Treg) in the five patients tested.

[0283] Several markers suggested activation of monocytes and NK cells. After decreasing during GM102 infusion, a significant increase in CD16 expression was observed on NK cells from C1J1-EOI to C1J15 (Figure 6A). A statistically non-significant trend towards an increase was also observed in CD69 expression on monocytes (Figure 6B). CD69 expression is known to increase after immune cell activation, although its immunoregulatory role remains unclear (Sancho et al., 2005, Trends in Immunology, Vol. 26(3):137-140). These increases could be interpreted as a sign of monocyte and NK cell activation.

[0284] Interestingly, the main and significant change was an increase in ICOS expression on T cells in C1J1-EOI to C1J15 (Figure 6C). ICOS is a receptor involved in T cell activation (Yao et al., 2013, Nature Reviews, Vol. 12: 130-146; Mahoney et al., 2015, Nature Reviews, Vol. 14: 561-584), and it is known as a pharmacodynamic marker of anti-CTLA4 antibody (ipilimumab), an inhibitor of immunological checkpoints (Tang et al., 2013, American association for cancer Research Journal, Vol. 1(4): 229-234). Therefore, this increase confirms that GM102 can reverse immune suppression in patients.

[0285] Example 9: Effect of GM102 on circulating monocytes A. Materials and Methods In the Phase I study of GM102, 5 mL of blood was sampled for each patient in the escalation cohort at four time points: before the first GM102 infusion on day 1 (designated C1J1-SOI) and at the end of the first GM102 infusion (designated C1J1-EO1), before the second GM102 infusion on day 15 (C1J15-SOI), and at steady state, i.e., at the end of the second 28-day cycle on day 57 (C3J1-SOI).

[0286] For scientific discovery purposes, several different markers were monitored at each clinical site in the study.

[0287] Human PBMCs were isolated from blood by density gradient centrifugation on Lymphoprep (Abcys). For infiltrating and activation of lymphocyte populations, PBMCs were labeled with the following antibodies: CD45-VioGreen, CD3-VioBlue, CD4-APCVio770, CD8-PerCP, CD25-PE, CD56-APC, CD19-PEVio770, and CD69-FITC (Myltenyi Biotec).

[0288] Typical, intermediate, and atypical populations of blood monocytes were assessed using the following antibodies: CD45-VioGreen, CD16-PE, and CD14-PerCPVio700 (Myltenyi Biotec). Appropriate fluorochrome-matched isotype antibodies were used to determine nonspecific background staining. All staining was performed in 100 μL of PBS- / -1% heat-inactivated fetal bovine serum. Populations of 10,000 cells were analyzed for each data point. All analyses were performed on a BD Fortessa flow cytometer using Diva software.

[0289] B. Results The percentages (%) of T cells, NK cells, and monocytes before the first infusion of 3C23K were found to vary among patients, indicating different immune capabilities among patients. No significant changes were observed in T cell and NK cell populations during and after treatment. In contrast, changes were observed in monocyte subsets.

[0290] Human blood monocytes are heterogeneous and are conventionally subdivided into three subsets based on CD14 and CD16 expression: «typical» monocytes (CD14 high CD16-) account for 90-95% of all monocytes in healthy donors, whereas «atypical» (CD14 low CD16+) and «intermediate» (CD14 high CD16+) populations account for smaller proportions (5-10%).

[0291] In three out of four patients with ovarian adenocarcinoma, the proportion of "typical monocytes" in the patients was significantly decreased before the first infusion compared to healthy patients (mean value=37.5%). This phenomenon was commonly observed in patients with ovarian cancer. As a result, the proportion of intermediate monocytes before the first infusion was increased in patients with ovarian adenocarcinoma compared to healthy donors (mean value=46.5%).

[0292] Interestingly, as illustrated in patients 04-06 in Figure 7, the percentage (%) measure during and after treatment with 3C23K revealed a significant increase in typical monocyte subsets with a decrease in the proportion of intermediate monocyte subsets in the patients (mean values ​​of 54.6% and 30.5%, respectively). Such changes in monocyte subsets were also observed in blood samples of four patients tested according to the same protocol at the Institut Bordet. These changes are indicative of a modification of the monocyte phenotype. It has recently been described that in response to immune checkpoint inhibitors, monocyte subpopulations change, leading to lymphocyte activation (due to blocking of inhibitory signals) (Krieg C., Nowicka M., Guglietta S., Schindler S., Hartmann FJ, Weber L..M et al., (2018). High-dimensional single-cell analysis predicts response to anti-PD-1 immunotherapy. Nat Med. 24, 144-153). Our data show that 3C23K can also modify the proportions of monocyte subsets and thus activate lymphocytes without binding to any immune checkpoints.

[0293] [Table 18] JPEG0007678493000021.jpg255164JPEG0007678493000022.jpg255164JPEG0007678493000023.jpg183169

Claims

1. An agent for use as an immunosuppressant inhibitor in treating cancer-associated immunosuppression in a subject, the agent being used to reduce macrophage-induced T cell inhibition and / or to activate TAM-like macrophages; The agent comprises a compound having a glycosylated Fc fragment, and the compound having a glycosylated Fc fragment consists of an anti-AMHRII antibody bearing a hypofucosylated Fc fragment.

2. The method of claim 1 , wherein the hypofucosylated Fc fragment-bearing anti-AMHRII antibody comprises a light chain variable region comprising SEQ ID NO:2 and a heavy chain variable region comprising SEQ ID NO:

4.

3. The agent described in claim 1, wherein the hypofucosylated Fc fragment-bearing anti-AMHRII antibody comprises a light chain variable region comprising SEQ ID NO:6 and a heavy chain variable region comprising SEQ ID NO:

8.

4. The agent described in claim 1, wherein the hypofucosylated Fc fragment-bearing anti-AMHRII antibody comprises a light chain variable region comprising SEQ ID NO: 10 and a heavy chain variable region comprising SEQ ID NO:

12.

5. The agent described in claim 1, wherein the hypofucosylated Fc fragment-bearing anti-AMHRII antibody comprises a light chain variable region comprising SEQ ID NO: 14 and a heavy chain variable region comprising SEQ ID NO:

16.

6. The method according to any one of claims 1 to 5, wherein the light and heavy chains of said hypofucosylated Fc fragment-bearing anti-AMHRII antibody are produced in YB2 / 0 cells.

7. The method of any one of claims 1 to 6, wherein the treatment of the cancer-associated immunosuppression further comprises administering to the subject an immune checkpoint protein inhibitor.

8. The agent according to claim 7, wherein the immune checkpoint protein inhibitor is selected from the group consisting of inhibitors of PD-1, PD-L1, PD-L2, BTLA, CTLA-4, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), IDO, KIR, LAG3, TIM-3 and VISTA.

9. The agent of claim 8 , wherein the immune checkpoint protein inhibitor comprises an antibody, or an antigen-binding fragment thereof, directed against the immune checkpoint protein inhibitor.

10. One or more further uses selected from the following: increasing a pro-inflammatory factor optionally selected from the group consisting of TNFα, IL1β, and IL6; Reducing an immunosuppressive factor optionally selected from the group consisting of TGFβ, IDO1, and IL10; reducing a pro-angiogenic factor optionally selected from the group consisting of PDGFα, VEGFβ, and HGF; and Activating ICOS+ T cells; The agent according to any one of claims 1 to 9 for the treatment of

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