Methods and pharmaceutical composition for treatment of cancers resistant to immune checkpoint therapy

Abstract

To provide methods and pharmaceutical compositions for the treatment of cancers resistant to immune checkpoint therapy.SOLUTION: The present invention provides a method of increasing an amount of tumor infiltrating CD8+ T cells in a patient suffering from cancer, comprising administering to the patient a therapeutically effective amount of an agent capable of depleting a population of CD163+ tumor associated macrophages. The present invention also provides a method of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective combination of at least one immune checkpoint inhibitor and an agent capable of depleting a population of CD163+ tumor associated macrophages.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to methods and pharmaceutical compositions for the treatment of cancers resistant to immune checkpoint therapy. [Background technology]

[0002] Tumor-associated macrophages (TAMs) are the most abundant immune cells in solid tumors and have been widely reported to play an important role in tumor progression. 1 In addition to their trophic functions, supporting angiogenesis, invasion, and metastasis, TAMs also upregulate the immunosuppressive cytokine IL-10. 2 T cell proliferation and activation via release of arginine, a stimulant for which T cells are highly dependent 3 and tryptophan 4 It has also been suggested that macrophages inhibit the localized depletion of TAMs. The important function of macrophages with respect to tumor progression has already attracted great interest in the development of new therapeutic strategies targeting TAMs. Current strategies in clinical trials include blocking the chemokine CCL2 or its receptor CCR2, which inhibits TAM recruitment by neutralizing the migration of bone marrow-derived monocytes, and blocking the macrophage growth factor receptor CSF1R (M-CSFR; c-FMS; CD115). 5 Blockade of CCL2 / CCR2 only targets the recruitment of monocyte-derived macrophages, whereas CSF1 plays an essential role in both the survival and differentiation of tissue-resident macrophages as well as in the maturation of monocyte-derived macrophages. 6 Indeed, although clinical data are still limited, we reported that TAM numbers were reduced after treatment with a monoclonal antibody (mAB) against CSF1R, which may be an interesting therapeutic option for tenosynovial giant cell tumors. 7,8 .

[0003] Despite the strong association between TAM accumulation and poor clinical outcomes in large-scale clinical trials, several reports have linked the number or accumulation of TAMs in specific TAM subsets with a favorable prognosis. 9.10 HLA-DR is associated with beneficial outcomes in + The frequency of TAMs, which is a protective immune response 11 In this regard, recent paired single-cell studies using mass cytometry and RNA sequencing revealed unprecedented levels of diversity in tumor-infiltrating myeloid cells (TIMs) from patients with lung adenocarcinoma and renal cell carcinoma (RCC). 12,13 In RCC, 17 different TAM phenotypes have been reported. 12 Although a deep understanding of the functions of different TAM subsets and their respective contributions to tumor progression is lacking, it is tempting to consider that selectively targeting TAM subsets that halt tumor-promoting mechanisms while retaining innate immune functions could promote antitumor immunity and provide meaningful clinical benefit.

[0004] Expression of CD163 by TAMs has been shown to be a particularly strong indicator of poor prognosis in several different cancers. CD163 is a macrophage- and monocyte-specific transmembrane protein that functions as a scavenger receptor for haptoglobin-hemoglobin complexes, which are formed during intravascular hemolysis. 14 Expression of CD163 is induced by tumor-promoting cytokines such as IL-6 and IL-10, whereas inflammatory stimuli including lipopolysaccharide (LPS), TNFα and IFNγ promote rapid downregulation and clearance of membrane-bound CD163 expression via proteolytic shedding. 15,16 This, together with the generation of anti-inflammatory heme metabolites from hemoglobin scavenging, promotes CD163 + Linking macrophages to anti-inflammatory functions 15 However, CD163 +The association between TAM accumulation and tumor progression is based solely on clinical advances, and experimental evidence for their specific tumor-promoting functions is still lacking.

[0005] The recent development of immune checkpoint inhibitors (ICIs), such as anti-PD-1, has had a profound impact on cancer treatment, particularly melanoma. 17,18 Increased expression of PD-1 ligand (PD-L1) on cancer cells promotes the uptake of PD-1-expressing CD8 + Suppresses activation of cytotoxic T cells (CTL) 19 Thus, blocking PD-1 / PD-L1 signaling using anti-PD-1 or PD-L1 mAbs increases CTL activation and ultimately leads to unexpected rates of tumor regression. 20 Unfortunately, only a small minority of patients respond to ICI treatment, the reasons for which are currently under investigation. Furthermore, the main reason for the limitation of ICI treatment is the indiscriminate activation of T cells, which leads to severe immune-related side effects and makes continuous treatment impossible. 21,22 Therefore, there is a great need for novel therapeutic strategies to promote antitumor immunity that can overcome ICI resistance or ameliorate severe side effects. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention relates to methods and pharmaceutical compositions for the treatment of cancers resistant to immune checkpoint therapy. In particular, the invention is defined in the claims. [Means for solving the problem]

[0007] Detailed Description of the Invention Recent work in understanding macrophage biology has revealed that tumor-associated macrophages are highly heterogeneous, with several different subsets coexisting in the tumor microenvironment. These subsets differ not only in their expression profile and origin, but also in whether they display tumor-promoting or antitumor functions. Herein, we describe macrophage subsets in a mouse model of metastatic melanoma that express CD163 and immunomodulatory cytokines such as IL10, Ido1, and Lgals1. Specific depletion of CD163-expressing cells in an anti-PD-1 checkpoint inhibitor-resistant melanoma model using cytotoxic lipid nanoparticles conjugated with αCD163 mAb resulted in the depletion of CD4 + and activated CD8 + Resulting in a large infiltration of T cells. CD163 + Specific depletion of TAMs only is CD163 neg This leads to the accumulation of inflammatory macrophages, which, together with activated T-cells, induces an antitumor immune response and tumor regression. Taken together, these data suggest that CD163 + Macrophages have a strong immunosuppressive function and express CD163 + It appears that loss of macrophages leads to a reeducation of the tumor immune microenvironment, indicating that CD163+ macrophages are crucial for maintaining a pro-tumor tumor immune microenvironment and that targeting this population represents an attractive therapeutic target for immune checkpoint inhibitor-resistant tumors.

[0008] As used herein, "CD163" (Cluster of Differentiation 163), also known as M130 MM130 or SCARI1, has its general meaning in the art and refers to the protein encoded in humans by the CD163 gene [Gene ID: 9332]. CD163 is expressed exclusively in monocytes and macrophages. CD163 functions as an acute phase regulatory receptor involved in the export and endocytosis of hemoglobin / haptoglobin complexes by macrophages, and may therefore protect tissues from oxidative damage mediated by free hemoglobin. The protein may also function as an innate immune sensor for bacteria and local inflammatory inducers. The molecular size is 130 kDa. The receptor belongs to the scavenger receptor cysteine-rich family type B and consists of an extracellular domain of 1048 amino acid residues, a single transmembrane segment and a cytoplasmic tail with several splice variants. An example of the human amino acid sequence is shown in SEQ ID NO:1. The extracellular domain of CD163 is from amino acid residue 42 to amino acid residue 1050 of SEQ ID NO:1. [Table 1]

[0009] As used herein, the term "tumor associated macrophage" or "TAM" has its general meaning in the art and refers to a type of cell belonging to the macrophage lineage that is found in the immediate vicinity of or within tumor masses. TAMs are derived from circulating monocytes or resident tissue macrophages and form the major leukocyte infiltrate found in the stroma of many tumor types. Thus, the term "CD163+ tumor associated macrophages" refers to a subset of TAMs that are characterized by expression of CD163. In certain embodiments, the CD163+ tumor associated macrophage population of the present invention is further characterized by expression of immune-modulating cytokines such as IL10, Ido1, and Lgals1.

[0010] Thus, a first object of the present invention relates to a method for increasing the number of tumor-infiltrating CD8+ T cells in a patient suffering from cancer, comprising administering to the patient a therapeutically effective amount of an agent capable of depleting the population of CD163+ tumor-associated macrophages.

[0011] As used herein, the term "CD8+ T cells" has its general meaning in the art and refers to a subset of T cells that express CD8 on their surface. They are MHC class I restricted and function as cytotoxic T cells. "CD8+ T cells" are also called cytotoxic T lymphocytes (CTLs), T killer cells, cytolytic T cells, or killer T cells. The CD8 antigen is a member of the immunoglobulin supergene family and is an associative recognition element in major histocompatibility complex class I restricted interactions. As used herein, the term "tumor-infiltrating CD8+ T cells" refers to the pool of CD8+ T cells in a patient that have left the bloodstream and migrated to the tumor.

[0012] As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood-borne tumors. The term "cancer" includes diseases of the skin, tissues, organs, bone, cartilage, blood, and blood vessels. Furthermore, the term "cancer" includes primary and metastatic cancers. Examples of cancers that may be treated with the methods and compositions of the present invention include, but are not limited to, cancer cells originating from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testes, tongue, or uterus. Furthermore, the cancer may be of the following histological types, among others, but not limited to: malignant neoplasm; carcinoma; undifferentiated carcinoma; giant cell spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; stromal carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; mixed cholangiocarcinoma and hepatocellular carcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyps; familial polyposis coli; solid tumors; malignant cysts; Rutinoid tumor; bronchiole-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granulocytic carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; Cancer; apocrine adenocarcinoma; sebaceous adenocarcinoma; cerumen adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the mammary gland; Adenoid cell carcinoma;Adenosquamous carcinoma;Adenocarcinoma with squamous metaplasia;Malignant thymoma;Malignant ovarian stromal carcinoma;Malignant theca cell tumor;Malignant granulosa cell tumor;Malignant neuroblastoma;Sertoli cell carcinoma;Malignant Leydig cell carcinoma;Malignant lipid cell carcinoma;Malignant paraganglioma;Malignant extra-adrenal paraganglioma;Pheochromocytoma;Glomus sarcoma;Malignant melanoma;Amelanotic melanoma;Superficial spreading melanoma;Malignant melanoma in giant pigmented nevus;Epithelioid cell melanoma;Malignant blue nevus;Sarcoma;Fibrosarcoma;Fibrous histiocytoma; Myxosarcoma;Liposarcoma;Leiomyosarcoma;Rhabdomyosarcoma;Embryonic rhabdomyosarcoma;Ovarioid rhabdomyosarcoma;Basal sarcoma;Malignant mixed tumor;Müllerian mixed tumor;Nephroblastoma;Hepatoblastoma;Carcinosarcoma;Malignant mesenchymoma;Malignant Brenner tumor;Malignant phyllodes tumor;Synovial sarcoma;Malignant mesothelioma;Dysgerminoma;Embryonal carcinoma;Malignant teratoma;Malignant ovarian thyroid carcinoma;Choriocarcinoma;Malignant mesonephric carcinoma;Angiosarcoma;Malignant hemangioepithelioma;Kaposi's sarcoma;Malignant hemangiopericytoma;Lymphangiosarcoma;Osteosarcoma;Periosteal osteosarcoma;Chondrosarcoma;Malignant chondroblastoma;Mesenchymal chondrosarcoma;Giant cell tumor of tendon sheath;Ewing's sarcoma;Malignant odontogenic tumor;Myeloblastic odontoma;Malignant ameloblastoma;Ameloblastic fibrosarcoma;Malignant pineal tumor;Chordoma;Malignant glioma;Ependymoma;Astrocytoma;Protoplasmic astrocytoma;Fibrillary astrocytoma;Astroblastoma;Glioblastoma;Oligodendroglioma;Oligodendroglioma;Primitive neuroectodermal tumor;Cerebellar sarcoma;Ganglioblastoma;Neuroblastoma;Retinoblastoma;Olfactory neurogenic tumor;Malignant meningioma;Neurofibrosarcoma;Malignant neurilemmoma;Malignant granular cell tumor;Malignant phosphorus lymphoma;Hodgkin's disease;Hodgkin's lymphoma;paragranuloma;small lymphocytic lymphoma;diffuse large cell lymphoma;malignant follicular lymphoma;mycosis fungoides;other specified non-Hodgkin's lymphoma;malignant histiocytosis;multiple myeloma;mast cell sarcoma;immunoproliferative small intestinal disease;leukemia;lymphocytic leukemia;plasma cell leukemia;erythroleukemia;lymphoblastic cell leukemia;myeloid leukemia;basophilic leukemia;eosinophilic leukemia;monocytic leukemia;mast cell leukemia;megakaryoblastic leukemia;myeloid sarcoma;and hairy cell leukemia.;

[0013] In some embodiments, the subject suffers from melanoma. As used herein, "melanoma" refers to a condition characterized by the growth of tumors derived from the melanocyte system in the skin and other organs. Most melanocytes arise in the skin, but also in the meninges, gastrointestinal tract, lymph nodes, and eye. When melanoma arises in the skin, it is called subcutaneous melanoma. Melanomas also arise in the eye, and are called ocular or intraocular melanoma. Rarely, melanomas arise in the meninges, gastrointestinal tract, lymph nodes, or other areas where melanocytes are found. 40-60% of melanomas have activating mutations in the gene that encodes the serine-threonine protein kinase B-RAF (BRAF). Of the BRAF mutations found in melanoma, over 90% are at codon 600, of which over 90% are single nucleotide mutations that result in the substitution of glutamic acid for valine (BRAFV600E).

[0014] A further object of the present invention relates to a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective combination of at least one immune checkpoint inhibitor and an agent capable of depleting the population of CD163+ tumor-associated macrophages.

[0015] As used herein, the term "treatment" or "treating" refers to both preventative or preventative measures, as well as therapeutic or disease-modulating measures, including treatment of patients at risk of or suspected of having a disease, and patients who have been diagnosed as having a disease or suffering from a disease or medical condition, and also includes suppression of clinical recurrence. Treatment is administered to patients with a medical condition or patients who have ultimately acquired a disease, to prevent, cure, delay the onset, reduce the severity, reduce one or more symptoms of the disease or disease recurrence, or prolong the survival of the patient beyond that expected in the absence of such treatment. A "treatment regimen" refers to a pattern of disease treatment, such as a dosing pattern used during treatment. A treatment regimen can include an initial regimen and a maintenance regimen. The term "initial regimen" or "initial" refers to a treatment regimen (or a portion of a treatment regimen) used in the initial treatment of a disease. The general goal of an initial regimen is to provide high levels of drug to the patient early in the treatment regimen. The initial plan may use (partially or entirely) an "escalation plan", which may include administering a higher dose than the doctor uses in the maintenance plan, administering more frequently than the doctor uses in the maintenance plan, or both. The term "maintenance plan" or "maintenance phase" refers to a treatment plan (or part of a treatment plan) that is used to maintain a patient in the treatment of a disease, for example, to keep the patient in remission for an extended period of time (months or years). A maintenance plan may use continuous treatment (e.g., administering a drug periodically, such as weekly, monthly, or yearly), or may use intermittent treatment (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or when certain predefined criteria are reached (e.g., pain, disease spread, etc.)).

[0016] As used herein, the term "immune checkpoint inhibitor" has its general meaning in the art and refers to any compound that inhibits the function of an immune inhibitory checkpoint protein. As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule expressed in T cells that either up-regulates (stimulatory checkpoint molecules) or down-regulates (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art as constituting immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see, e.g., Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480- 489). Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. Inhibition includes reduced function and complete inhibition. Preferred immune checkpoint inhibitors are antibodies that specifically recognize immune checkpoint proteins. Numerous immune checkpoint inhibitors are known, and as well as these known immune checkpoint inhibitors, alternative immune checkpoint inhibitors will be developed in the (near) future. Immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. Examples of immune checkpoint inhibitors include PD-1 antagonists, PD-L1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-H3 antagonists, B7-H4 antagonists, and BTLA antagonists.

[0017] In some embodiments, the PD-1 (Programmed Death-1) axis antagonist includes a PD-1 antagonist (e.g., an anti-PD-1 antibody), a PD-L1 (Programmed Death Ligand-1) antagonist (e.g., an anti-PD-L1 antibody), and a PD-L2 (Programmed Death Ligand-2) antagonist (e.g., an anti-PD-L2 antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (also known as nivolumab, MDX-1106-04, ONO-4538, BMS-936558, and Opdivo®), Merck 3475 (also known as pembrolizumab, MK-3475, lambrolizumab, Keytruda®, and SCH-900475), and CT-011 (also known as pidilizumab, hBAT, and hBAT-1). In some embodiments, the PD-1 binding antagonist is AMP-224 (also known as B7-DCIg). In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of YW243.55.S70, MPDL3280A, MDX-1105, and MEDI4736. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007 / 005874. Antibody YW243.55.S70 is an anti-PD-L1 antibody described in WO 2010 / 077634 A1. MEDI4736 is an anti-PD-L1 antibody described in WO2011 / 066389 and US2013 / 034559. MDX-1106, also known as MDX-1106-04, ONO-4538 or BMS-936558, is an anti-PD-1 antibody described in US Pat. No. 8,008,449 and WO2006 / 121168. Merck 3745, also known as MK-3475 or SCH-900475, is an anti-PD-1 antibody described in US Pat. No. 8,345,509 and WO2009 / 114335.CT-011 (pidilizumab), also known as hBAT or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Atezolimumab is an anti-PD-L1 antibody described in US Pat. No. 8,217,149. Avelumab is an anti-PD-L1 antibody described in US 20140341917. CA-170 is a PD-1 antagonist described in WO2015033301 and WO2015033299. Other anti-PD-1 antibodies are described in US Pat. No. 8,609,089, US 2010028330, and / or US20120114649. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody selected from nivolumab, pembrolizumab, or pidilizumab. In some embodiments, the PD-L1 antagonist is selected from the group consisting of avelumab, BMS-936559, CA-170, durvalumab, MCLA-145, SP142, STI-A1011, STIA1012, STI-A1010, STI-A1014, A110,KY1003, and atezolimumab, with avelumab, durvalumab, or atezolimumab being preferred.

[0018] In some embodiments, the CTLA-4 (cytotoxic T-lymphocyte antigen-4) antagonist is an anti-CTLA-4 antibody, a human anti-CTLA-4 antibody, a murine anti-CTLA-4 antibody, a mammalian anti-CTLA-4 antibody, a humanized anti-CTLA-4 antibody, a monoclonal anti-CTLA-4 antibody, a polyclonal anti-CTLA-4 antibody, a chimeric anti-CTLA-4 antibody, MDX-010 (ipilimumab), tremelimumab, an anti-CD28 antibody, an anti-CTLA-4 adnectin, an anti-CTLA-4 domain antibody, a single chain anti-CTLA-4 fragment, a heavy chain anti-CTLA-4 fragment, a light chain anti-CTLA-4 fragment, an inhibitor of CTLA-4 that activates the costimulatory pathway, an antibody disclosed in PCT Publication WO 2001 / 014424, an antibody disclosed in PCT Publication WO The CTLA-4 antibody is selected from the group consisting of the antibodies disclosed in US Pat. Nos. 5,811,097; 5,855,887; 6,051,227; and 6,984,720; PCT Publications WO 01 / 14424 and WO 00 / 37504; and US Publication Nos. 2002 / 0039581 and 2002 / 086014. Other anti-CTLA-4 antibodies that can be used in the methods of the invention include, for example, those disclosed in WO 98 / 42752; US Pat. Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17): 10067-10071 (1998); Camacho et al., J. Clin: Oncology, 22(145): Abstract No. 2505 (2004) (antibody CP-675206); Mokyret et al., Cancer Res., 58:5301-5304 (1998), and US Pat. Nos. 5,977,318, 6,682,736, 7,109,003, and 7,132,281.A preferred clinical CTLA-4 antibody is the human monoclonal antibody (also called MDX-010 or ipilimumab having CAS No. 477202-00-9, available from Medarex, Inc., Bloomsbury, NJ) disclosed in WO 01 / 14424. CTLA-4 antagonists (antibodies) are known and include tremelimumab (CP-675,206) and ipilimumab.

[0019] In one embodiment, the immunotherapy comprises administering to the patient a combination of a CTLA-4 antagonist and a PD-1 antagonist.

[0020] Other immune checkpoint inhibitors include lymphocyte activation gene-3 (LAG-3) inhibitors such as IMP321, a soluble Ig fusion protein (Brignone et al., 2007, J. Immunol. 179:4202-4211). Other immune checkpoint inhibitors include B7 inhibitors such as B7-H3 and B7-H4 inhibitors, particularly the anti-B7-H3 antibody MGA271 (Loo et al., 2012, Clin. Cancer Res. July 15 (18) 3834). Additionally, TIM-3 (T-cell immunoglobulin domain and mucin domain 3) inhibitors (Fourcade et al., 2010, J. Exp. Med. 207:2175-86 and Sakuishi et al.,2010, J. Exp. Med. 207:2187-94). As used herein, the term "TIM-3" has its general meaning in the art and refers to T-cell immunoglobulin and mucin domain-containing molecule 3. The natural ligand of TIM-3 is galactin 9 (Gal9). Thus, as used herein, the term "TIM-3 inhibitor" refers to a compound, substance, or composition that can inhibit the function of TIM-3. For example, the inhibitor can inhibit the expression or activity of TIM-3, modulate or block the TIM-3 signaling pathway, and / or block the binding of TIM-3 to galactin-9. Antibodies with specificity for TIM-3 are known in the art and are typically described in WO2011155607, WO2013006490, and WO2010117057.

[0021] In some embodiments, the immune checkpoint inhibitor is an IDO inhibitor. Examples of IDO inhibitors are described in WO 2014150677. Non-limiting examples of IDO inhibitors include 1-methyl-tryptophan (IMT), β- (3-benzofuranyl)-alanine, β- (3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6-fluoro-tryptophan, 4-methyl-tryptophan, 5-methyl-tryptophan, 6-methyl-tryptophan, 5-methoxy-tryptophan, 5-hydroxy-tryptophan, indole 3-carbinol, 3,3'-diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9-vinylcarbazole, acemetacin, 5-bromo-tryptophan, 5-bromoindoxyl diacetate, 3- The IDO inhibitor is selected from 1-methyl-tryptophan, β-(3-benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3-amino-naphthoic acid and β-[3-benzo(b)thienyl]-alanine or a derivative or prodrug thereof.

[0022] As used herein, the term "co-administration" refers to the procedure of administering a combination of an agent capable of depleting a population of CD163+ tumor-associated macrophages and an immune checkpoint inhibitor to the same patient. The agent capable of depleting a population of CD163+ tumor-associated macrophages and the immune checkpoint inhibitor can be administered simultaneously or sequentially. If the administration is sequential, the agent capable of depleting a population of CD163+ tumor-associated macrophages is administered before the immune checkpoint inhibitor. The agent capable of depleting a population of CD163+ tumor-associated macrophages and the immune checkpoint inhibitor do not have to be administered in the same vehicle. The agent capable of depleting a population of CD163+ tumor-associated macrophages and the immune checkpoint inhibitor are administered one or more times, and the number of administrations of each component of the combination can be the same or different. Additionally, the agent capable of depleting a population of CD163+ tumor-associated macrophages and the immune checkpoint inhibitor do not have to be administered at the same site.

[0023] As used herein, the terms "combination" and "combination treatment" are interchangeable and refer to treatments that include simultaneous, separate, or sequential administration of at least two compounds. As used herein, the term "co-administration" refers to a procedure in which a combination of at least two compounds is administered to the same patient. The at least two compounds can be co-administered essentially simultaneously or sequentially. The at least two compounds can be administered separately in different vehicles or compositions. The at least two compounds can also be administered in the same vehicle or composition (such as a pharmaceutical composition). The at least two compounds are administered one or more times, and the number of administrations of each component of the combination can be the same or different.

[0024] In particular, the method of the present invention is particularly suitable for the treatment of cancers characterized by low tumor infiltration of CD8+ T cells.Accordingly, a further object of the present invention relates to a method for treating cancer in a patient in need thereof, comprising:i) quantifying the density of CD8+ T cells in a tumor tissue sample obtained from the patient, ii) comparing the density quantified in step i) with a predefined reference value, and iii) administering to the patient a combined therapeutically effective amount of an agent capable of depleting a population of CD163+ tumor-associated macrophages and an immune checkpoint inhibitor when the density of CD8+ T cells quantified in step i) is lower than the corresponding predefined reference value.

[0025] In one embodiment, the method of the invention is particularly suitable for the treatment of cancers characterized by low tumor infiltration of CD8+ T cells and high tumor infiltration of CD163+ tumor associated macrophages.Accordingly, a further object of the invention relates to a method for treating cancer in a patient in need thereof, comprising:i) quantifying the density of CD8+ T cells and the density of CD163+ tumor associated macrophages in a tumor tissue sample obtained from the patient,ii) comparing the densities quantified in step i) with predefined reference values, andiii) administering to the patient a combined therapeutically effective amount of an agent capable of depleting the population of CD163+ tumor associated macrophages and an immune checkpoint inhibitor when the density of CD163+ tumor associated macrophages quantified in step i) is higher than the corresponding predefined reference value and the density of CD8+ T cells quantified in step i) is lower than the corresponding predefined reference value.

[0026] As used herein, the term "tumor tissue sample" refers to any tissue tumor sample from a patient. The tissue sample is obtained for the purpose of in vitro evaluation. In some embodiments, the tumor sample may be obtained from a tumor excised from a patient. In some embodiments, the tumor sample may be obtained from a biopsy performed on the patient's primary tumor or from a metastatic sample distant from the patient's primary tumor. For example, an endoscopic biopsy performed on the intestine of a patient with colorectal cancer. In some embodiments, the tumor tissue sample includes (i) the entire primary tumor (whole), (ii) a tissue sample from the center of the tumor, (iii) a tissue sample obtained from tissue immediately surrounding the tumor, specifically referred to as the "invasive periphery" of the tumor, (iv) lymph islands close to the tumor, (v) lymph nodes closest to the tumor, (vi) tumor tissue samples collected at a previous surgery (e.g., for patient follow-up after treatment), and (vii) a distant metastasis. As used herein, "invasive periphery" has its general meaning in the art and refers to the cellular environment surrounding the tumor. In one embodiment, the tumor tissue sample includes a piece or slice of tissue removed from the tumor center or from the invasive periphery of the tumor, whether it is from the tumor center, the invasive periphery of the tumor, or from the nearest lymph node, after surgical tumor resection or after collection of a tissue sample for biopsy, and after quantification of one or several biological markers, in particular by histological or immunohistochemical methods, by flow cytometric methods, gene or protein expression analysis, including genomic and proteomic analysis. Of course, the tumor tissue sample may be subjected to various well-known post-collection preparation and preservation methods (fixation, preservation, freezing, etc.). The sample may be fresh, frozen, fixed (e.g., formalin-fixed), or embedded (e.g., paraffin-embedded).

[0027] In one embodiment, cell density is quantified by immunohistochemistry (IHC). For example, cell density is quantified by contacting the tumor tissue sample with a binding partner (such as an antibody) specific for a cell surface marker of the cells. Typically, cell density is quantified by contacting the tumor tissue sample with a binding partner (such as an antibody) specific for CD8 for CD8+ cells and CD163 for CD163+ tumor-associated macrophages. Typically, cell density is measured as the number of cells counted per unit of surface area of ​​the tumor tissue sample, e.g., cm of surface area of ​​the tumor tissue sample. 2 or mm 2 In one embodiment, cell density is expressed as the number of cells counted per unit volume of sample, e.g., per cm of a tumor tissue sample. 3It may also be expressed as the number of cells per 1000. In some embodiments, cell density may consist of the percentage of a particular cell per total cells (which is taken as 100%). Immunohistochemistry typically involves the following steps: i) fixing the tumor tissue sample with formalin, ii) embedding the tumor tissue sample in paraffin, iii) sectioning the tumor tissue sample for staining, iv) incubating the sections with a binding partner specific for the marker, v) rinsing the sections, vi) incubating the sections with a secondary antibody, typically biotinylated, and vii) revealing the antigen-antibody complex, typically with an avidin-biotin-peroxidase complex. Thus, the tumor tissue sample is first incubated with the binding partner. After washing, the labeled antibody bound to the marker of interest is revealed by an appropriate technique, e.g., radioactive, fluorescent or enzymatic labeling, depending on the type of label generated by the labeled antibody. Multiple labels may be performed simultaneously. Alternatively, the method of the invention may use a secondary antibody and an enzymatic molecule coupled to an amplification system (for intensity staining signal). Such conjugated second antibodies are commercially available, e.g., Dako, EnVision systems. Counterstaining can be, e.g., H&E, DAPI, Hoechst. Other staining methods can be performed using any suitable method, e.g., automated, semi-automated, or manual systems, as will be apparent to those skilled in the art. For example, one or more labels can be attached to the antibody to allow detection of the target protein (i.e., marker). Examples of labels include radioisotopes, fluorescent dyes, ligands, chemiluminescent agents, enzymes, and combinations thereof. In some embodiments, the label is a quantum dot. Non-limiting examples of labels that can be conjugated to the first and / or second affinity ligand include fluorescent dyes or metals (fluorescein, rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g., rhodopsin), chemiluminescent compounds (e.g., luminal, imidazole, etc.) and bioluminescent proteins (e.g., luciferin, luciferase), heptanes (e.g., biotin).Many other useful fluorophores and chromophores are described in Stryer L (1968) Science 162:526-533 and Brand L and Gohlke JR (1972) Annu.Rev. Biochem. 41:843-868. Affinity ligands include enzymes (horseradish peroxidase, alkaline phosphatase, β-lactamase, etc.), radioisotopes (. 3 H, 14 C, 32 P, 35 S or 125I) and particles (e.g., gold). Different types of labels can be attached to the affinity ligand using various chemical reactions, e.g., amine or thiol reactions. However, reactive groups other than amines and thiols, e.g., aldehydes, carboxylic acids, glutamine, etc., can also be used. Many enzymatic staining methods are known in the art for the detection of a protein of interest. For example, enzymatic interactions can be visualized using peroxidase or alkaline phosphatase, or using different chromogens, such as DAB, AEC, or Fast Red. In other examples, antibodies can be conjugated to peptides or proteins and detected via a labeled binding partner or antibody. In indirect IHC assays, the second antibody or second binding partner is not labeled, and it is necessary to detect binding with the first binding partner. The resulting stained specimen is imaged using a system for visualizing the detectable signal and obtaining an image, such as a digital image of the staining, respectively. Methods for obtaining images are well known to those skilled in the art. For example, once a sample is stained, the stain or biomarker label can be detected using either optical or non-optical imaging devices, such as upright or inverted optical microscopes, confocal scanning microscopes, cameras, scanning or tunneling electron microscopes, scanning probe microscopes, and imaging infrared detectors. In some instances, the image is captured digitally. The resulting image is then used to quantitatively or semi-quantitatively determine the amount of marker in the sample. A variety of automated sample processing, scanning and analysis systems suitable for use in immunohistochemistry are available in the art. Such systems include automated staining and microscope scanning, computerized image analysis, serial section comparison (to control for variations in sample orientation and size), digital report generation, and archival and tracking of samples (such as slides bearing tissue sections). Cell imaging systems are commercially available that couple conventional light microscopes to digital image processing systems to perform quantitative analysis of cells and tissues, including immunostained samples. See, for example, the CAS-200 system (Becton, Dickinson & Co.). Notably, detection can be performed manually or by image processing methods involving computer processors and software.For example, such software can be used to configure, adjust, standardize and / or visualize images based on factors including staining quality and staining intensity using methods known to those of skill in the art (see, e.g., published US Patent Publication No. US20100136549). Images are quantitatively or semi-quantitatively analyzed and scored based on the staining intensity of the sample. Quantitative or semi-quantitative histochemistry refers to a method of scanning and scoring a sample where histochemistry is performed to identify and quantify the presence of a particular biomarker (i.e., marker). Quantitative or semi-quantitative methods can use image software to detect staining density or amount, or can also detect staining by human eye, where a trained operator evaluates the results numerically. For example, images can be quantitatively analyzed using pixel counting algorithms (such as Aperio Spectrum Software, Automated Quantitative Analysis platform (AQUA® platform)) or other standard methods that quantitatively or semi-quantitatively measure the intensity of staining; see, for example, US Pat. No. 8,023,714; US Pat. No. 7,257,268; US Pat. No.7,219,016; US Pat. No. 7,646,905; published US Patent Publication Nos. US20100136549 and 20110111435; Camp et al. (2002) Nature Medicine, 8:1323-1327; Bacuset al. (1997) Analyt Quant Cytol Histol, 19:316-328. The ratio of strongly positive staining (e.g., brown staining) to the sum of all stained areas is calculated and evaluated. The amount of detected biomarker (i.e., marker) is quantified as a percentage of positive pixels and / or score. For example, the amount can be quantified as a percentage of positive pixels. In one example, the amount can be quantified as a percentage of stained area, e.g., a percentage of positive pixels.For example, a sample may have at least, or approximately at least, or approximately 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more positive pixels relative to the total stained area. In some embodiments, a sample is given a score that is a numerical representation of the intensity or amount of histochemical staining of the sample, which represents the amount of target biomarker (e.g., marker) present in the sample. The optical density or area percentage values ​​can be scaled scores, e.g., integer scales. Thus, in one embodiment, the method of the present invention comprises the steps of: i) providing one or more immunostained slices of a tissue section obtained by an automated slide staining system using a binding partner capable of selectively interacting with a marker (such as an antibody as described above), ii) digitizing the slide of step i) by high-resolution scan capture, iii) detecting the slices of the tissue section on the digital photograph, iv) providing a size reference grid with uniformly distributed units having the same surface, said grid being adapted to the size of the tissue section to be analyzed, and v) detecting, quantifying and measuring the intensity of stained cells in each unit, thereby assessing the number or density of stained cells in each unit.

[0028] In some embodiments, the predefined value is the threshold or cut-off value. Typically, the "threshold" or "cut-off value" can be determined experimentally, empirically, or theoretically. The threshold value can also be arbitrarily selected based on existing experimental and / or clinical conditions, as will be appreciated by those skilled in the art. For example, past measurements of cell density in properly stored past patient samples can be used to establish the predefined reference value. The threshold value must be determined to obtain optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical outcomes of false positive and false negative). Typically, the optimal sensitivity and specificity (and thus the threshold value) are determined using a receiver operating characteristic (ROC) curve based on experimental data. For example, after quantifying the cell density in the reference group, an algorithmic analysis can be used for statistical processing of the measured densities in the test samples to obtain classification criteria that have significance for classifying the samples. The full name of the ROC curve is the receiver operator characteristic curve, which is also known as the receiver operating characteristic curve. It is mainly used in clinical biochemical diagnostic tests. The ROC curve is a comprehensive index that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It reveals the relationship between sensitivity and specificity in an image synthesis manner. A series of different cut-off values ​​(threshold or critical value, boundary value between normal and abnormal results of a diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Thus, to draw the curve, sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate. The more the area under the curve (AUC), the higher the diagnostic accuracy. On the ROC curve, the point closest to the top left of the coordinate diagram is the critical point with both high sensitivity and high specificity value. The AUC value of the ROC curve is between 1.0 and 0.5. If AUC>0.5, the closer the AUC is to 1, the better the diagnostic result is. If AUC is between 0.5 and 0.7, the accuracy is low. If AUC is between 0.7 and 0.9, the accuracy is moderate. If AUC is greater than 0.9, the accuracy is extremely high.The algorithmic method is preferably implemented on a computer. ROC curves can be drawn using software or systems existing in the art, such as MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VI0.0 (Dynamic Microsystems, Inc. Silver Spring, Md., USA). In some embodiments, the predetermined reference value is related to the survival time of the patient. Those skilled in the art will recognize that OS survival is expressed based on the percentage of people who survive a certain type of cancer for a certain period of time. Overall 5-year survival rates are often used in cancer statistics. In general, OS rates do not specify whether cancer survivors are still undergoing treatment at 5 years or have become cancer-free (recovered). DSF gives more specific information, which is the number of people with a particular cancer who have been cured. Additionally, progression-free survival (PFS) rates (the number of people who still have cancer but whose disease does not progress) include people who have had some success with treatment, but whose cancer has not completely disappeared. As used herein, the term "short survival" means that a patient has a shorter survival time than the median (or average) seen in the general population of patients with the same cancer. When a patient has a short survival time, it means that the patient has a "poor prognosis". Conversely, the term "long survival" means that a patient has a longer survival time than the median (or average) seen in the general population of patients with the same cancer. When a patient has a long survival time, it means that the patient has a "good prognosis".

[0029] A further object of the present invention relates to a method for treating cancer resistant to immune checkpoint therapy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent capable of depleting the population of CD163+ tumor-associated macrophages.

[0030] As used herein, the term "resistant to immune checkpoint therapy" is used in the broadest context to refer to the reduced effectiveness of at least one immune checkpoint inhibitor (such as a PD-1 antagonist) to inhibit cell growth, kill cells, or inhibit one or more cellular functions; and the ability of a cell to survive exposure to an agent designed to inhibit cell growth, kill the cell, or inhibit one or more cellular functions. The resistance exhibited by a cell may be acquired, for example, by previous exposure to the agent, or may be inherent or innate. The resistance exhibited by a cell may be complete, in the sense that the agent is completely ineffective against the cell, or partial, in the sense that the effectiveness of the agent is reduced. Thus, the term "resistance" refers to repeated outbreaks of cancer, or the progression of cancer, independent of whether the disease had been cured prior to the outbreak or progression.

[0031] A further object of the present invention relates to a method for increasing the efficacy / potency of an immune checkpoint inhibitor administered as part of a treatment regimen to a subject suffering from cancer, said method comprising administering to the subject a pharmacologic effective amount of an agent capable of depleting the population of CD163+ tumor-associated macrophages in combination with at least one immune checkpoint inhibitor.

[0032] As used herein, the phrase "increases the effectiveness of immune checkpoints" refers to the ability of an agent capable of depleting a population of CD163+ tumor-associated macrophages to increase the ability of an immune checkpoint inhibitor to enhance the proliferation, migration, persistence and / or cytotoxic activity of CD8+ T cells. The ability of an immune checkpoint inhibitor to increase T CD8 cell killing activity can be determined using any assay known in the art. Typically, the assay is an in vitro assay in which CD8+ T cells are contacted with target cells (e.g., target cells that are recognized and / or lysed by CD8+ T cells). For example, immune checkpoint inhibitors of the present invention can be selected for their ability to increase specific lysis by CD8+ T cells by about 20% or more, preferably at least about 30%, at least about 40%, at least about 50%, or more than the specific lysis obtained at the same effector:target cell ratio as the CD8+ T cells or CD8 T cell lines contacted with the immune checkpoint inhibitor of the present invention. Examples of classical cytotoxicity assays are routine.

[0033] As used herein, in the context of cancer, the phrase "increased therapeutic efficacy" refers to a delay or reduction in the growth of cancer cells or solid tumors, or a reduction in the total number of cancer cells or overall tumor burden. Thus, "improved therapeutic outcome" or "increased therapeutic efficacy" refers to an improvement in a patient's condition by any clinically acceptable criterion, including, for example, a reduction in tumor size, an increase in time to cancer progression, an increase in progression-free survival, an increase in overall survival time, an increase in life expectancy, or an improvement in quality of life. In particular, "improvement" or "increase" refers to an improvement or increase of any clinically acceptable indicator of therapeutic outcome or efficacy by 1%, 5%, 10%, 25%, 50%, 75%, 100%, or more than 100%. As used herein, when used in the context of comparing the activity and / or efficacy of a combination composition comprising an immune checkpoint inhibitor and an agent capable of depleting a population of CD163+ tumor-associated macrophages, the phrase "compared to" refers to a comparison using amounts known to be comparable to those skilled in the art.

[0034] A further object of the present invention relates to a method for preventing resistance to an administered immune checkpoint inhibitor in a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of an agent capable of depleting the population of CD163+ tumor-associated macrophages.

[0035] As used herein, the term "agent capable of depleting a population of CD163+ tumor-associated macrophages" refers to any compound capable of depleting said population. As used herein, the term "deplete" with respect to CD163+ tumor-associated macrophages refers to a measurable reduction in the number of CD163+ TAMs in the tumor of a subject. The reduction may be at least about 10%, e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In one embodiment, the term refers to a reduction in the number of CD163+ TAMs in a subject's tumor below the limit of detection.

[0036] In one embodiment, the agent is an antibody that has binding affinity for CD163 and leads to depletion of CD163+ TAMs in the subject's tumor. In particular, the antibody binds to the extracellular domain of CD163 as defined above.

[0037] Thus, as used herein, the term "antibody" refers to any antibody-like molecule that has an antigen-binding region, and thus includes antibody fragments that contain an antigen-binding domain, such as: Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimers, Fv, scFv (single-chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibodies, tribodies (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabodies; kappa(lamda) bodies (scFv-CL fusions); BiTE (bispecific T-cell Engager, scFv-scFv tandems for attracting T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immune protein, a type of minibody); SMIP ("small molecule immunopharmaceutical agent" scFv-Fc dimers; DART (ds-stabilized diabodies "Dual Affinity Antibody fragments include, but are not limited to, antibody fragments that are not specifically retargeted; small antibody mimetics that contain one or more CDRs; and the like. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art (Kabat et al., 1991, specifically incorporated herein by reference). Further, inter alia, diabodies are described in EP 404,097 and WO 93 / 1 1 161; and linear antibodies are described in Zapata et al. (1995). Antibodies can be fragmented by conventional techniques. For example, F(ab')2 fragments can be produced by treating an antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bonds to produce Fab' fragments. Fab fragments can be formed by papain digestion. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, Bispecific antibody fragments, and other fragments, can also be synthesized recombinantly or chemically. Techniques for producing antibody fragments are well known in the art and described in the literature.See, for example, Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and Young et al., 1995, which describe and enable the generation of effective antibody fragments. In one embodiment, the antibody of the invention is a single chain antibody. As used herein, the term "single domain antibody" has its general meaning in the art and refers to a single heavy chain variable domain of an antibody of the type found in camelid mammals that do not naturally have light chains. Such single domain antibodies are also "nanobodies®". A general description of (single) domain antibodies can be found in the above mentioned prior art and in EP 0 368 684, Ward et al. (Natur. e 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and WO 06 / 030220, WO 06 / 003388.

[0038] As used herein, the term "binding" indicates that an antibody has affinity with a surface molecule. As used herein, the term "affinity" refers to the strength with which an antibody binds to an epitope. The affinity of an antibody is indicated by the dissociation constant, Kd, ​​which is defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant, Ka, is defined as 1 / Kd. Preferred methods for determining the affinity of mAbs can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), all of which are incorporated herein by reference. A preferred standard method known in the art for determining the affinity of mAbs uses a Biacore instrument.

[0039] In a natural antibody, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains different sequence domains. Light chains contain two domains, the variable domain (VL) and the constant domain (CL). Heavy chains contain four domains, the variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). Both the light chain variable domain (VL) and the heavy chain variable domain (VH) determine the binding recognition and specificity of the antigen. The light chain constant domain (CL) and the heavy chain constant domain (CH) confer important biological properties to the antibody chain, such as association, secretion, transplacental mobility, complement fixation, and binding to Fc receptors (FcR). Fv fragments are the N-terminal part of the Fab fragment of immunoglobulins and consist of one light chain and one heavy chain variable part. The specificity of an antibody resides in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site consists mainly of residues from the hypervariable or complementarity determining regions (CDRs). Sometimes, residues from non-hypervariable or framework regions (FRs) participate in the antibody binding site or influence the overall domain structure and therefore the binding site. Complementarity determining regions or CDRs refer to the amino acid sequences that together determine the affinity and specificity of the native Fv region of a natural immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, called L-CDR1, L-CDR2, L- CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen binding site typically contains six CDRs, including a set of CDRs from each of the heavy and light chain variable regions. Framework regions (FRs) are amino acid sequences between the CDRs. Residues in antibody variable domains are conventionally numbered according to the system devised by Kabat et al.This system is defined in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter referred to as "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not necessarily correspond directly to the linear numbering of amino acid residues in the SEQ ID NO:. The actual continuous amino acid sequence may contain fewer or more amino acids than the strict Kabat numbering, corresponding to truncations or insertions of structural components of the basic variable domain structure, whether framework or complementarity determining regions (CDRs). The exact Kabat numbering of residues is determined for each antibody by aligning the antibody sequence with the "standard" Kabat numbered sequence to check for homology. The CDRs of the heavy chain variable domain are located at residues 31-35B (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) in the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system.

[0040] In some embodiments, the antibody is a humanized antibody. As used herein, "humanized" refers to an antibody in which some, most, or all of the amino acids outside the CDR regions are replaced with corresponding amino acids from human immunoglobulin molecules. Methods for humanization include, but are not limited to, those described in US Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205, which are incorporated herein by reference.

[0041] In some embodiments, the antibody is a fully human antibody. Fully human monoclonal antibodies can also be produced by transgenic immunization of mice with large portions of human immunoglobulin heavy and light chain loci. See, for example, US Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584 and the references cited therein, the contents of which are incorporated herein by reference.

[0042] In one embodiment, antibodies suitable for depletion of CD163+ TAMs mediate antibody-dependent cellular cytotoxicity. As used herein, the term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. Without intending to be limited to a particular mechanism of action, cytotoxic cells that mediate ADCC generally express Fc receptors (FcRs).

[0043] As used herein, "Fc region" includes the polypeptides comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, Fc refers to the two end constant region immunoglobulins of IgA, IgD, and IgG, and the three end constant region immunoglobulins of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc includes the immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 at the carboxy terminus, numbering herein is according to the EU index by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). "EU index as described in Kabat" refers to the residue numbering of the human IgG1 EU antibody as described in Kabat et al., supra. Fc refers to this region in isolation or in the context of an antibody, antibody fragment, or Fc fusion protein. An Fc variant protein can be an antibody, an Fc fusion, or any protein or protein domain that comprises an Fc region. Particularly preferred are proteins that comprise variant Fc regions, which are non-naturally occurring variants of an Fc region. The amino acid sequence of a non-naturally occurring Fc region (also referred to herein as a "variant Fc region") comprises a substitution, insertion, and / or deletion of at least one amino acid residue compared to the wild-type amino acid sequence. The new amino acid residue found in the sequence of the variant Fc region as a result of the insertion or substitution may be referred to as a non-naturally occurring amino acid residue. Note: Polymorphisms are observed at many Fc positions, including but not limited to Kabat 270, 272, 312, 315, 356, and 358, and thus there may be slight differences between the sequences shown and those of the prior art.

[0044] The term "Fc receptor" or "FcR" is used to refer to a receptor that binds the Fc region of an antibody. The primary cells for mediating ADCC, NK cells, express FcγRIII, whereas monocytes express FcγRI, FcγRII, FcγRIII and / or FcγRIV. Expression of FcR on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991). To assess ADCC activity of a molecule, an in vitro ADCC assay, such as that described in US Pat. No. 5,500,362 or 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model as described in Clynes et al., Proc. Natl. Acad. Sci. (USA), 95:652-656 (1998). As used herein, the term "effector cell" refers to a leukocyte that expresses one or more FcRs and performs effector function. The cell expresses at least FcγRI, FcγRII, FcγRIII and / or FcγRIV and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils.

[0045] In some embodiments, the antibody suitable for depletion of CD163+ TAMs is a full-length antibody. In some embodiments, the full-length antibody is an IgG1 antibody. In some embodiments, the full-length antibody is an IgG3 antibody.

[0046] In certain embodiments, an antibody suitable for depletion of CD163+ TAMs comprises a variant Fc region with increased affinity for FcγRIA, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, and FcγRIV. In certain embodiments, an antibody of the invention comprises a variant Fc region comprising at least one amino acid substitution, insertion, or deletion, wherein the substitution, insertion, or deletion of the at least one amino acid residue results in increased affinity for FcγRIA, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, and FcγRIV. In certain embodiments, an antibody of the invention comprises a variant Fc region comprising at least one amino acid substitution, insertion, or deletion, wherein the at least one amino acid residue is selected from the group consisting of residues 239, 330, and 332, where the amino acid residues are numbered according to the EU index. In one embodiment, an antibody of the invention comprises a variant Fc region comprising at least one amino acid substitution, wherein said at least one amino acid substitution is selected from the group consisting of S239D, A330L, A330Y, and 1332E, where amino acid residues are numbered according to the EU index.

[0047] In certain embodiments, the glycosylation of an antibody suitable for depletion of CD163+ TAMs is altered. For example, an aglycosylated antibody (i.e., an antibody lacking glycosylation) can be generated. For example, glycosylation can be altered to increase the affinity of the antibody to the antigen. Such carbohydrate alterations can be accomplished by changing one or more glycosylation positions within the antibody sequence. For example, one or more amino acid substitutions can eliminate one or more variable region framework glycosylation positions, thereby eliminating glycosylation at this position. Such aglycosylation can increase the affinity of the antibody to the antigen. Such approaches are described in further detail in US Patent Nos. 5,714,350 and 6,350,861 by Co et al. Additionally or alternatively, the antibody can be modified to have a different type of glycosylation, e.g., a hypofucosylated or nonfucosylated antibody with a low amount of fucosyl residues or no fucosyl residues, or an antibody with increased bisecting GlcNac structures. Such modified glycosylation patterns have been shown to increase the ADCC ability of the antibody. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with modified glycosylation machinery. Cells with modified glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the invention to produce antibodies with modified glycosylation. For example, Hang et al., EP 1,176,195, describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in the cell line exhibit hypofucosylation or no fucosyl residues. Thus, in certain embodiments, the human monoclonal antibodies of the invention can be produced by recombinant expression in a cell line that exhibits a hypofucosylated or nonfucosylated pattern, e.g., a mammalian cell line that has low expression of the FUT8 gene, which encodes a fucosyltransferase. PCT Publication WO 03 / 035835 by Presta describes Lecl3 cells, a variant CHO cell line that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates, resulting in hypofucosylation of antibodies expressed in the host cells.(See also Shields, RL et al, 2002 J. Biol. Chem. 277:26733-26740.) PCT Publication WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyltransferases, such as β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), resulting in antibodies expressed in the engineered cell lines exhibiting increased bisecting GlcNac structures and increased ADCC activity of the antibodies. (See also Umana et al, 1999 Nat. Biotech. 17: 176-180.) Additionally, Eureka Therapeutics describes engineered CHO mammalian cells capable of producing antibodies with modified mammalian glycosylation patterns that result in no fucosyl residues (http: / / www.eurekainc.com / a&boutus / companyoverview.html). Alternatively, the human monoclonal antibodies of the invention can be engineered with a mammalian-like glycosylation pattern and produced in yeast or filamentous fungi capable of producing antibodies that lack fucose as a glycosylation pattern (see, eg, EP1297172B1).

[0048] In some embodiments, antibodies suitable for depletion of CD163+ TAMs mediate complement dependent cytotoxicity. "Complement dependent cytotoxicity" or "CDC" refers to the ability of a molecule to initiate complement activation and lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (such as an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santaro et al., J. Immunol. Methods, 202:163 (1996), may be performed.

[0049] In one embodiment, antibodies suitable for depletion of CD163+ TAMs mediate antibody-dependent phagocytosis. As used herein, the term "antibody-dependent phagocytosis" or "opsonization" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγRs recognize bound antibody on a target cell and trigger phagocytosis of the target cell.

[0050] In one embodiment, the antibody suitable for depletion of CD163+ TAMs is a multispecific antibody comprising a first antigen-binding site for CD163 and at least one second antigen-binding site for the effector cells described above. In this embodiment, the second antigen-binding site is used to recruit the killing mechanism, for example by binding to an antigen on human effector cells. In one embodiment, the effector cells can induce ADCC, such as natural killer cells. For example, monocytes, macrophages expressing FcRs, are involved in the specific killing of target cells and presenting antigens to other components of the immune system. In one embodiment, the effector cells can phagocytose the target antigen or target cells. The expression of certain FcRs on effector cells may be regulated by humoral factors such as cytokines. The effector cells can phagocytose the target antigen or phagocytose or lyse the target cells. Examples of suitable cytotoxic agents and second therapeutic agents are provided below, and include toxins (such as radiolabeled peptides), chemotherapeutic agents, and prodrugs. In certain embodiments, the second binding site binds to an Fc receptor as defined above. In certain embodiments, the second binding site binds to a surface molecule of an NK cell to activate the cell. In certain embodiments, the second binding site binds to NKp46. Examples of multispecific antibody molecules of the invention include, but are not limited to, the following: (i) two antibodies, one specific for a particular surface molecule of an ILC and the other specific for a second antigen, crosslinked by chemical heteroconjugation; (ii) an antibody comprising two different antigen-binding regions; (iii) a single chain antibody comprising two different antigen-binding regions, e.g., two scFvs linked in tandem with an additional peptide linker; (iv) a dual variable domain antibody (DVD-Ig) in which each of the light and heavy chains comprises two variable domains connected in tandem by a short peptide bond (Wu et al., Generation and Characterization of a DualVariable Domain Immunoglobulin (DVD-Ig)). TM) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010); (v) chemically linked bispecific (Fab')2 fragments; (vi) Tandab, which is a tetravalent bispecific antibody fused to two single chain diabodies with a binding site for each of the target antigens; (vii) Flexibodies, which are a combination of scFvs and diabodies in a multispecific molecule; (viii) so-called "dock and lock" molecules based on the "dimerization and docking domain" in protein kinase A, which when applied to Fabs, results in a trivalent bispecific binding protein consisting of two identical Fab fragments bound to different Fab fragments; (ix) so-called Scorpion molecules, which contain, for example, two scFvs fused to both ends of a human Fab-arm; and (x) diabodies. Other examples of bispecific antibodies are IgG-like molecules with complementary CH3 domains that undergo heterodimerization. Such molecules can be prepared using known techniques, such as Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and Electrostatic Match (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonic (Merus) and DuoBody (Genmab A / S) technologies.

[0051] In one embodiment, antibodies suitable for depletion of CD163+ TAMs are conjugated to a therapeutic moiety, i.e., a drug. The therapeutic moiety can be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as "antibody-drug conjugates" or "ADCs."

[0052] In one embodiment, the antibody suitable for depletion of CD163+ TAMs is conjugated to a cytotoxic moiety. The cytotoxic moiety may be selected from the group consisting of: taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxyanthracin dione; tubulin inhibitors such as maytansine or an analogue or derivative thereof; antimitotics such as monomethyl auristatin E or F or an analogue or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; rol; puromycin; calichemicin or an analogue or derivative thereof; antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine or cladribine; mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, alkylating agents such as mitomycin C, platinum derivatives such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065) or analogs or derivatives thereof; antibiotics such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC), etc.; pyrrolo[2,lc][l,4]-benzodiazepines (PDB);Diphtheria toxin and related molecules such as diphtheria A chain and active fragments and hybrid molecules thereof, ricin toxins such as ricin A or deglycosylated ricin A chain toxins, cholera toxin, SLT I, SLT II, ​​SLT IIV, LT toxin, C3 toxin, Shiga-like toxins such as Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas aeruginosa exotoxins, allorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins such as PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, soapwort inhibitor, gelonin, mitgelin, restrictocin, phenomycin and enomycin toxins; ribonucleases (RNases); DNase I, Staphylococcal enterotoxin A, pokeweed antiviral protein, diphtherin toxin, and Pseudomonas aeruginosa endotoxin.

[0053] In some embodiments, the antibody suitable for depletion of CD163+ TAMs binds to an auristatin or a peptide analog, derivative, or prodrug thereof. Auristatins interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), and have anticancer (US5663149) and antifungal activity (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42:2961-2965). For example, auristatin E can react with paraacetylbenzoic acid or benzoylvaleric acid to generate AEB and AEVB, respectively. Other exemplary auristatin derivatives include AFP, MMAF (monomethylauristatin F), and MMAE (monomethylauristatin E). Suitable auristatins and auristatin analogs, derivatives and prodrugs, as well as suitable linkers for coupling auristatins to antibodies (Abs), are described, for example, in U.S. Patent Nos. 5,635,483, 5,780,588 and 6,214,345 and in International Patent Publications WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968 and WO205082023.

[0054] In one embodiment, an antibody suitable for depletion of CD163+ TAMs binds pyrrolo[2,lc][l,4]-benzodiazepine (PDB) or an analog, derivative, or prodrug thereof. Suitable PDB and PDB derivatives, and related techniques, are described, for example, in Hartley JA et al., Cancer Res 2010; 70(17): 6849-6858; Antonow D. et al., Cancer J 2008; 14(3): 154-169; Howard PW et al., Bioorg Med ChemLett 2009; 19: 6463-6466, and Sagnou et al., Bioorg Med Chem Lett 2000; 10(18): 2083-2086. In one embodiment, the antibody binds to a pyrrolobenzodiazepine (PBD), typically as described in WO2017059289.

[0055] In one embodiment, an antibody suitable for depletion of CD163+ TAMs binds to an antibody selected from the group consisting of anthracyclines, maytansine, calicheamicin, duocarmycin, rachelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, PDB, or any analog, derivative, or prodrug thereof.

[0056] In some embodiments, an antibody suitable for depletion of CD163+ TAMs binds to an anthracycline or an analog, derivative, or prodrug thereof. In some embodiments, the antibody binds to maytansine or an analog, derivative, or prodrug thereof. In some embodiments, the antibody binds to calicheamicin or an analog, derivative, or prodrug thereof. In some embodiments, the antibody binds to duocarmycin or an analog, derivative, or prodrug thereof. In some embodiments, the antibody binds to rachelmycin (CC-1065) or an analog, derivative, or prodrug thereof. In some embodiments, the antibody binds to dolastatin 10 or an analog, derivative, or prodrug thereof. In some embodiments, the antibody binds to dolastatin 15 or an analog, derivative, or prodrug thereof. In some embodiments, the antibody binds to monomethyl auristatin E or an analog, derivative, or prodrug thereof. In some embodiments, the antibody binds to monomethyl auristatin F or an analog, derivative, or prodrug thereof. In some embodiments, the antibody binds to pyrrolo[2,lc][l,4]-benzodiazepine or an analog, derivative, or prodrug thereof. In some embodiments, the antibody binds to irinotecan or an analog, derivative, or prodrug thereof.

[0057] In some embodiments, the antibody suitable for depletion of CD163+ TAMs binds to a nucleic acid or a nucleic acid-associated molecule. In one such embodiment, the nucleic acid bound is a cytotoxic ribonuclease (RNase) or deoxyribonuclease (e.g., DNase I), an antisense nucleic acid, an inhibitory RNA molecule (e.g., an siRNA molecule), or an immunostimulatory nucleic acid (e.g., an immunostimulatory CpG motif-containing DNA molecule). In some embodiments, the antibody binds to an aptamer or a ribozyme.

[0058] Techniques for conjugating molecules to antibodies are well known in the art (see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (Robinson et al. eds., Marcel Deiker,Inc., 2nd ed. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection and Thorpe et al., 1982, Immunol. Rev. 62:119-58. See also, e.g., PCT Publication WO 89 / 12624. Typically, the nucleic acid molecules are covalently coupled to lysines or cysteines on the antibody via N-hydroxysuccinimide esters or maleimides, respectively, depending on the functionality.Conjugation using engineered cysteines or the introduction of unnatural amino acids have been reported to improve conjugation homogeneity (Axup, JY, Bajjuri, KM, Ritland, M., Hutchins, BM, Kim, CH,Kazane, SA, Halder, R., Forsyth, JS, Santidrian, AF, Stafin, K., et al. (2012)). Site-specific synthesis of antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109, 16101-16106.; Junutula, JR,Flagella, KM, Graham, RA, Parsons, KL, Ha, E., Raab, H., Bhakta, S.,Nguyen, T., Dugger, DL, Li, G., et al. (2010). Engineered thio-trastuzumab-DM1 conjugates with improved therapeutic index targeting human epidermal growth factor receptor 2 receptor-positive breast cancer. Clin. Cancer Res.16, 4769-4778. Junutula et al. (2008) developed cysteine-based site-specific conjugates called "THIOMABs" (TDCs) that claim to show improved therapeutic index compared to traditional conjugation methods. Conjugation to non-natural amino acids incorporated into antibodies has also been explored for ADCs, although the generality of this approach has not yet been established (Axup et al., 2012). In particular, one skilled in the art can also consider engineered Fc-containing polypeptides with acyl donor glutamine-containing tags (e.g., Gin-containing peptide tags or Q-tags) or endogenous glutamines made reactive by polypeptide engineering (e.g., amino acid deletion, insertion, substitution, or mutation of the polypeptide).The transglutaminase can then be covalently cross-linked with an amine donating agent (e.g., a small molecule containing or coupled to a reactive amine) to form a stable and homogenous population of engineered Fc-containing polypeptide conjugates with the amine donating agent site-specifically coupled to the Fc-containing polypeptide via an acyl donor glutamine-containing tag or available / exposed / reactive endogenous glutamines (WO 2012059882).

[0059] Typically, the agent capable of depleting the population of CD163+ tumor-associated macrophages and the immune checkpoint inhibitor are administered to the patient in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic result at the required dosage and duration. The therapeutically effective amount of an active agent may vary depending on factors such as the individual's medical condition, age, sex, and weight, and the ability of the active agent to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects outweigh the toxic or deleterious effects of the antibody or antibody portion. An effective dosage or administration regimen of the active agent depends on the disease or condition to be treated and can be determined by one of ordinary skill in the art. A physician of ordinary skill in the art can easily determine and prescribe an effective amount of the pharmaceutical composition required. For example, a physician can start the dosage of the active agent used in the pharmaceutical composition at a level lower than that required to obtain the desired therapeutic effect, and gradually increase the dosage until the desired effect is obtained. In general, a suitable dosage of the composition of the present invention is the amount of the compound that is the minimum dosage required to provide a therapeutic effect according to a particular treatment regimen. Generally, such effective dosages will depend on the factors mentioned above. For example, a therapeutically effective amount for treatment can be measured by its ability to stabilize disease progression. Typically, the ability of a compound to inhibit cancer can be evaluated, for example, in an animal model system that predicts efficacy in human tumors. A therapeutically effective amount of a therapeutic compound can reduce tumor size or improve the patient's condition. One skilled in the art can determine the amount based on factors such as the size of the patient, the severity of the patient's condition, and the particular composition or route of administration selected. An example of a non-limiting range of a therapeutically effective amount of an inhibitor of the present invention is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, such as about 0.1-20 mg / kg, such as about 0.1-10 mg / kg, such as about 0.5, such as 0.3, about 1, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg. An example of a non-limiting range of a therapeutically effective amount of an inhibitor of the present invention is 0.02-100 mg / kg, such as about 0.02-30 mg / kg, such as about 0.05-10 mg / kg or 0.1-3 mg / kg, such as about 0.5-2 mg / kg.Administration may be, for example, intravenous, intramuscular, intraperitoneal or subcutaneous, e.g., near the target site. The dosing regimen in the above-mentioned methods and uses of treatment may be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, it may be administered as a single bolus, as several divided doses over time, or proportionally increased or decreased depending on the exigencies of the treatment situation. In some embodiments, the effectiveness of the treatment is monitored during the treatment, e.g., at predetermined time points. In some embodiments, the effectiveness of the treatment may be monitored by visualization of the diseased area, or by using a diagnostic method described herein, e.g., one or more PET-CT scans, e.g., a labeled inhibitor of the invention, a fragment derived from an inhibitor of the invention, or a mini-antibody. If desired, two, three, four, five, six or more separate doses of the effective daily dose of the pharmaceutical composition are administered separately at appropriate intervals throughout the day, optionally in unit dosage form. In some embodiments, the human monoclonal antibodies of the invention are administered by slow continuous infusion over an extended period of time, e.g., 24 hours or more, to minimize any undesirable side effects. An effective amount of an inhibitor of the invention may be administered using a dosing period of every one, two or three weeks. The dosing period may be limited, for example, to 8 weeks, 12 weeks or until clinical progression is established.Non-limiting examples of treatments according to the present invention include administering a daily dosage of an inhibitor of the present invention at about 0.1-100 mg / kg per day, e.g., 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg per day, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 10 The administration may be provided in a single or divided dose every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof, for at least one of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or for at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks, or any combination thereof.

[0060] Typically, the active agent is administered to the patient in the form of a pharmaceutical composition containing a pharma- ceutical acceptable carrier.The pharma-ceutical acceptable carriers that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffers such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, protamine sulfate, etc., disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylic acid, wax, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.For administration to a patient, the composition is formulated for administration to a patient.The composition of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally or by implanted reservoir. Here, administration is by subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion. Sterile injectable preparations of the compositions of the present invention may be aqueous or oily suspensions. Suspensions are formulated by techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may be conventionally employed as solvents or suspending agents. For this purpose, any brand of fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharma- ceutical acceptable oils such as olive oil and castor oil, particularly their polyoxyethylated versions. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersing agent, such as carboxymethyl cellulose or similar dispersing agents commonly used in the manufacture of pharma- ceutical acceptable dosage forms, including emulsions and suspensions.Other commonly used surfactants, such as Tween, Span, and other emulsifying agents, commonly used in the preparation of pharma- ceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes. The compositions of the present invention may be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions, or solutions. For tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. Diluents useful for oral administration in capsule form include, for example, lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. Certain sweetening, flavoring, and coloring agents may also be added, if desired. Alternatively, the compositions of the present invention may be administered in the form of suppositories for rectal administration. These may be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and therefore melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycols. The compositions of the invention may also be administered topically, particularly when the therapeutic target includes areas and organs readily accessible by topical administration, including the eye, the skin, or the lower intestinal tract. Suitable topical formulations can be readily prepared for each of these areas or organs. For topical administration, the compositions can be formulated in a suitable ointment comprising the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compositions of the invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the compositions can be formulated in a suitable lotion or cream comprising the active compound suspended or dissolved in one or more pharma- ceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Topical application to the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches can also be used. The compositions of the invention can also be administered by nasal aerosol or inhalation.Such compositions are prepared by techniques well known in the pharmaceutical manufacturing art and may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to increase bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents. For example, the antibody present in the pharmaceutical composition of the present invention may be provided at a concentration of 10 mg / mL in 100 mg (10 mL) or 500 mg (50 mL) single-use vials. For intravenous administration, the product is formulated in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and sterile water for injection. The pH is adjusted to 6.5. An example of a suitable dosage for the antibody in the pharmaceutical composition of the present invention is about 1 mg / m. 2 and 500 mg / m 2 It will be understood, however, that these schedules are exemplary and that optimal schedules and administration will be adopted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition, which must be determined in clinical trials. Pharmaceutical compositions of the invention for injection (e.g., intramuscular, intravenous) can be formulated to contain sterile buffered water (e.g., 1 ml for intramuscular use) and between about 1 ng and about 100 mg of an inhibitor of the invention, e.g., about 50 ng to about 30 mg, or more preferably about 5 mg to about 25 mg.

[0061] The present invention is further illustrated in the following figures and examples, which, however, should not be construed as limiting the scope of the invention in any way. [Brief description of the drawings]

[0062] [Figure 1-1]Figure 1: Depletion of CD163+ TAMs and targeted lipid nanoparticles promotes tumor regression. (Figure 1A) Schematic of γ-D163 mAb conjugated to lipid nanoparticles (LNPs). (Figure 1B) Therapeutic depletion of CD163+ TAMs in melanoma-bearing mice. Mice (n=6 per group) were injected subcutaneously in the right flank with 1x106 YUMM1.7 cells (mm x 5 mm) and treated with γ-D163-dxrLNPs ((γ-D163-dxr)) or ctrlIgG-dxrLNPs (ctrlIgG-dxr) (2 mg / kg dxr) every 2 days for 2 weeks. PBS (vehicle) or empty γ-D163-LNPs (γ-D163-ctl) were used as controls. [Figure 1-2] (Figure 1C) At the end of the study, the levels of TAM and CD163+ TAM were analyzed by flow cytometry and the frequency of viable cells was calculated. Results are representative of three independent experiments. (Figure 1D) Mice were inoculated with 1x106 YUMMER1.7 cells in the right flank and randomly divided into groups and treated with 2 mg / kg dxr or control every 2 days starting on day 10 for 2 weeks. Statistical significance was calculated using Mann-Whitney t-test; * p< 0.05, ** p< 0.01, *** p< 0.001 and **** p< 0.0001. [Diagram 2] Figure 2: Depletion of CD163+ TAMs promotes anti-PD-1 resistant CTL responses. (Figure 2A) Tumor-bearing mice were treated with ⅓D163-dxrLNP or vehicle in combination with aPD-1 mAb or isotype control IgG. (Figure 2B) Mice were treated with ⅓D163-dxrLNP or vehicle every 2 days for 10 days, then mice received aPD-1 mAb twice weekly. n=6 per group. **p<0.01. [Figure 3-1]Figure 3: Treatment study comparing the efficacy of CD163+ TAM depletion versus total macrophage depletion using SF1 blocking antibody (Figure 3A). Mice bearing palpable tumors were randomized into groups and treated with either IV injections of D163-dxr (n=6) or PBS (n=4) every 2 days for 2 weeks, or IP injections of SF1 (n=6) or control (CtrlIgG, n=6 or PBS, n=6) every 5 days. Statistical significance was calculated by 2-way ANOVA with Tukey post-hoc test; *** p< 0.001 and **** p< 0.0001. At the end of the study, the total numbers of mTAM, iTAM and Mn (Figure 3B) or CD4+ TILs and IFN-sup>+ CD8+ TILs (Figure 3C) were analyzed by flow cytometry and calculated from the frequency of viable cells. Data are presented as mean + / - SEM of n=6. [Figure 3-2] Figure 3: Treatment study comparing the efficacy of CD163+ TAM depletion versus total macrophage depletion using SF1 blocking antibody (Figure 3A). Mice bearing palpable tumors were randomized into groups and treated with either IV injections of D163-dxr (n=6) or PBS (n=4) every 2 days for 2 weeks, or IP injections of SF1 (n=6) or control (CtrlIgG, n=6 or PBS, n=6) every 5 days. Statistical significance was calculated by 2-way ANOVA with Tukey post-hoc test; *** p< 0.001 and **** p< 0.0001. At the end of the study, the total numbers of mTAM, iTAM and Mn (Figure 3B) or CD4+ TILs and IFN-sup>+ CD8+ TILs (Figure 3C) were analyzed by flow cytometry and calculated from the frequency of viable cells. Data are presented as mean + / - SEM of n=6. EXAMPLES

[0063] material and method Mice breeding and initiation of the murine melanoma model Spontaneous melanoma formation 23 To induce CA / + , PTENlox4-5 / lox4-5 and Tyr::CreER T2+ / - Mice bearing the IL-11 marker were shaved and 5-week-old mice were given 1 μL of 7.8 mg / mL 4-hydroxytamoxifen (4-HT) locally on the right flank. To induce syngeneic tumors, 8-week-old male or female mice were given 100 μl of Yale University Mouse Melanoma (YUMM1.7) in sterile PBS pH 7.4. 25 cells 1x10 6 1 were injected subcutaneously in the right hind flank. Tumor size was measured in x, y, and z with digital calipers and tumor volume was calculated using the equation for ellipse volume (volume = 0.5233xyz). All mice were housed in the animal facility of the Centre d'immunologie Marseille-Luminy, with water and food available ad libitum and a 12 h / 12 ​​h light / dark cycle. All animal experiments were approved and performed in accordance with the restriction principles for the use of animals in testing (the three Rs: replacement, reduction and refinement) and recognized by the French Ministry of Higher Education and Research.

[0064] Tumor digestion, flow cytometry and cell sorting Tumors for flow cytometry and FACS sorting were minced and digested in RPMI1640 containing 1 mg / ml collagenase II (Sigma), 50 μg / ml DNAseI (Roche) and 0.1% (w / v) BSA for 30 min at 37 °C with gentle agitation. Single cell suspensions were then passed through a 70 μm cell strainer and collected by centrifugation. For RBC lysis, cell suspensions were incubated with 0.85% NH4Cl for 2 min at room temperature, collected by centrifugation and resuspended in FACS buffer (1xPBS pH 7.4, 1 mM EDTA pH 8.0, 3% FCS and 0.1% NaN3). For flow cytometry and FACS sorting, single cell suspensions from tumors were incubated with 2.4.G2 antibody for 10 min at 4 °C and then with specific antibodies (see Supplementary Table 1 for details) for 30 min at 4 °C. Prior to analysis, cells were incubated with SytoxBlue (Thermo Fischer Scientific) to distinguish dead cells. For intracellular staining of IFNγ, surface stained cells were incubated with Live / Dead Fixable Violet in PBS for 20 min to distinguish dead cells, then fixed, permeabilized, washed with BD Perm / Wash buffer (BD Biosciences), and incubated with IFNγ antibody diluted in Perm / Wash buffer for 30 min at 4°C. Analysis was performed on an LSR-2 or Fortessa X-20 (BD Biosciences) equipped with a 350 nm laser. Subsequent data analysis was performed with FlowJo software V10.4 for Mac (Tree Star). Immunophenotyping was performed (see Supplementary Table 2 for antibodies).

[0065] Preparation of liposomes Long-circulating liposomes encapsulating doxorubicin were prepared essentially as previously described. 26 Prepared as follows: 30, modified to target CD163. Briefly, liposome formulations were made from a mixture of HSPCs, mPEG2000-PE and cholesterol (55:40:5 molar ratio) (Lipoid GmBH, Ludwigshafen, Germany and Sigma Aldrich A / S, Glostrup, Denmark) using the ethanol injection method. Lipids were dissolved in EtOH for 15 min at 65°C and then hydrated (to 10% EtOH) for 1 h at 65°C in an aqueous buffer appropriate for further application. Liposomes were formed by extrusion 25 times through a 0.1 μm filter using an Avanti Mini Extruder Kit (Avanti Polar Lipids, AL, US) and dialyzed twice against 150 mM NaCl (0,9% NaCl), the second dialysis being performed overnight at 4°C. Encapsulation of calcein (calLNP) was performed by hydrating the lipids in 200 mM calcein solution (pH 7.4) and dialysis was repeated five times to remove excess calcein. For remote loading of doxorubicin, lipids were hydrated in 300 mM (NH4)1HPO3. After extrusion and dialysis, the (NH4)1HPO3 containing liposomes were mixed with doxorubicin hydrochloride in a doxorubicin:lipid ratio of 1:5 at 65 °C for 30 min. The lipid content, drug content and encapsulation efficiency were then evaluated by high-pressure size-exclusion chromatography (UV absorbance at 210 nm) using a Dionex Ultimate3000 HPLC system (Thermo Scientific, Hvidovre, Denmark) equipped with an Ascentis C18 column (Sigma Aldrich A / S). Liposome size was assessed using dynamic light scattering and a DynaPro NanoStar system (Wyatt Technology Europe GmbH, Dernbach, Germany). Liposome modification targeting CD163 was performed using the αCD163 antibody clone 3E10B10. 26,31 Alternatively, post-insertion of isotype control IgG (BioXcell) was performed as described above.

[0066] In vivo treatments When tumors reached a measurable size of approximately 5 mm x 5 mm, mice were injected retroorbitally with 2 mg / kg of doxorubicin encapsulated in lipid nanoparticles (dxrLNPs) conjugated to anti-CD163 IgG (αCD163-dxrLNPs). As controls, groups of mice received 2 mg / kg of doxorubicin in dxrLNPs conjugated to isotype control IgG (CtrlIgG-dxrLNPs), or the same amount of empty LNPs conjugated to αCD163 (αCD163-LNPs) or vehicle (sterile PBS pH 7.4). Mice were treated every 2 days for approximately 14 days. For in vivo experiments with calcein-loaded LNPs, mice were injected once with 100 μl of 0.67 mM lipid solution. CD4 + and CD8 + For T-cell depletion studies and anti-PD-1 mAb treatment, mice were injected intraperitoneally twice weekly with 250 μg of either aCD4 (clone GK1.5), aCD8b (clone 53-5.8), aPD-1 (clone RMP1-14), or isotype control IgG (IgG1 or IgG2a) (all BioXcell), with the first injection being 1 day before treatment with αCD163-dxrLNP.

[0067] Immunohistochemistry and immunofluorescence Whole tumors or 5 mm slices of back skin were fixed in 4% formalin and embedded in agarose for vibratome sectioning, OCT for cryostat sectioning, or paraffin for histology. For immunofluorescence on vibratome and cryostat sections, 200 μm or 10 μm thick sections were cut, respectively, and incubated with pAb rabbit anti-CD163-ATTO56532, CD146-Alexa647 (clone ME-9F1; BD Bioscience), CD3e-APC (clone 145-2C11; BD Bioscience) and CD8b-FITC (53-5.8; BD Bioscience) together with anti-FITC A488 (A11096; Life Technologies) in 0.1M Tris pH 7.2, 1% Triton X-100, 0.5% BSA for vibratome sections or in 1xPBS, 2%BSA for cryostat sections. Nuclei were visualized with Hoechst 33342 (Sigma Aldrich). Images were acquired on a Zeiss LSM780 confocal microscope using spectral resolution and a 20x objective. IHC sections were stained with H&E and pAb rabbit anti-CD163.

[0068] High-throughput gene expression analysis Total RNA was purified from the sorted populations using RNeasy Micro Kit (Qiagen) and the concentration was determined using the Quant-IT RiboGreen RNA Assay Kit (Thermo Fischer). First-strand cDNA synthesis was performed using the High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems) and 2.5 ng of total RNA was used to preamplify genes of interest using FluidigmPreAmp Master Mix (Fluidigm Corporation) according to the manufacturer's instructions. Exon-spanning primers designed to amplify genes of interest were calculated using Primer-Blast (see Supplementary Table 3 for details). Gene-specific assays were performed using a combination of forward and reverse primers of the genes of interest. For increased sensitivity, genes of interest were preamplified using pooled assays for 14 cycles of PCR and treated with Exonuclease 1 (New England Biolabs) to remove unincorporated primers. The final preamplified cDNA was diluted 1:5 in TE buffer. High-throughput gene expression analysis was performed using 96.96 Dynamic Arrays and the Biomark HD system from Fluidigm (Fluidigm Europe BV) according to the manufacturer's instructions and standard settings. The data obtained were analyzed with Real-Time PCR Analysis software (Fluidigm Europe BV) and the obtained C values ​​were normalized to Cph to obtain dC values. Heat maps, Z-scores and hierarchical clustering using One minus Pearson correlation were generated using Morpheus (https: / / software.broadinstitute.org / morpheus / ). PCA plots were generated using Qlucore Omics (Qlucore AB, Lund, Sweden).

[0069] statistical analysis Statistical analysis for treatment studies was performed by two-way ANOVA followed by Tukey post-hoc test. Statistical tests for comparison between groups were performed using non-parametric tests such as Mann-Whitney or Kruskal-Wallis where appropriate. Data were considered statistically significant when p<0.05. p-values ​​are indicated as * p< 0.05, ** p< 0.01, *** p< 0.001 and **** p< 0.0001. All statistical analyses were performed with Graphpad Prism 7 for Mac.

[0070] result CD163-expressing macrophages express naturally occurring Braf V600E Infiltrate induced melanoma Activating mutations in BRAF are the most common in human melanomas and are often associated with loss of tumor suppressor genes such as PTEN and CDKN2A. The increasing availability of genetically engineered mouse (GEM) models based on appropriate tumorigenic driver mutations has greatly improved the relevance of human disease to mouse tumor models. Tyr::CreER in metastatic melanoma; Braf CA ; Pten f / f The mouse model expresses tamoxifen-inducible Cre-recombinase (CreER) using the melanocyte-restricted tyrosinase (Tyr) promoter. T2 ), which in turn leads to the expression of constitutively active Braf V600E (Braf CA ) and the expression of the floxed Pten allele (Pten f / f ) 23 In such mice, subcutaneous (sc) administration of 4-hydroxytamoxifen (4-HT) initially produced small pigmented lesions around day 20 that progressed to amelanotic tumors around day 40 and then showed exponential growth (data not shown). In untreated mice, CD163 + Macrophages were uniformly distributed throughout the dermis and adipose tissue (data not shown). However, after 4-HT treatment, CD163 +Macrophages accumulate at the borders of pigmented premelanoma lesions in the dermis (data not shown). When pigmented lesions transform into rapidly growing amelanotic tumors, CD163 + Macrophages accumulate at the leading edge of the infiltration, whereas only a small amount of CD163 + Macrophages are present in the tumor (data not shown). V600E To further examine the tumor-infiltrating myeloid cell (TIM) compartment in tumors, flow cytometry of single-cell suspensions of tumor tissue was performed. CD45-negative tumor cells, lymphocytes and granulocytes (CD45.2 + , CD19 - , CD5 - , NK1.1 - ,SiglecF - , Ly6G - and CD11b + After gating out monocytes / macrophages, we found two major populations of monocytes / macrophages based on F4 / 80 and CD169 expression. 24 As mentioned above, F4 / 80 - CD169 - The population is mainly Ly6C + Monocytes (MN) and Ly6C + MHCII + The larger F4 / 80 consisted of immature TAMs. + CD169 + The population was negative for Ly6C and showed variable expression of CD163 and MHCII, suggesting a mature tumor-associated macrophage (TAM) phenotype (data not shown). Taken together, TAMs were by far the most abundant cell type and expressed CD11b + On average, TAMs comprised 20% of all tumor-infiltrating leukocytes, which were tumor-infiltrating CD8 + 100-fold higher than the number of T cells (data not shown). + Macrophages account for less than 25% of all TAMs and are more abundant in MHCII - population and less MHCII + The cells were then separated into a population of cells.

[0071] Symtopic Braf V600E Characterization of CD163-expressing TAMs in induced melanoma Apigmented stage of tumor growth 25 Braf, which imitates V600E Using cell lines derived from the induced mouse model, we tested whether transplanted orthotopic tumors with comparable driver mutations would give rise to a TAM population similar to that seen in spontaneous amelanotic tumors. The YUMM1.7 cell line (Yale University Mouse Melanoma) expresses the Braf V600E YUMM1.7 cells are derived from a spontaneous melanoma induced by activation of Braf and inactivation of Pten and Cdkn2a. V600E Flow cytometric analysis of YUMM1.7 tumors revealed that the spontaneous Braf tumor population was distinct from the monocyte / macrophage population based on F4 / 80 and CD169 expression. V600E However, F4 / 80 - CD169 - and F4 / 80 + CD169 + The populations appeared to be similar in the two models, with a prominent F4 / 80 population present in the orthotopic tumors. - CD169 + There was also a population (data not shown). This population contained some Ly6C + In addition to MN (data not shown), primary tumors consisted mainly of intTAMs (data not shown), as described in the spontaneous model. - CD169 - ) and intTAMs were present in a high proportion, whereas mature TAMs (F4 / 80 + CD169 + ) steadily increased and became most abundant in late-stage tumors (data not shown). Interestingly, orthotopic tumors were almost exclusively MHCII-negative, CD163 hi and CD163 lo Immunohistochemical (IHC) analysis revealed that CD163hi / lo TAMs differ in spatial distribution and express CD163 hi The cells were mainly localized in the tumor periphery, whereas CD163 lo The cells were located inside the tumor tissue (data not shown). - CD163 lo The proportion of TAMs increased with tumor progression, whereas MHCII - CD163 hi The proportion of cells remained constant (data not shown).

[0072] To further characterize the different monocyte / macrophage populations in this melanoma model, MNs, intTAMs and the four mature TAMs populations were isolated by flow cytometry and subjected to high-density quantitative PCR (qPCR) analysis of gene expression using the Fluidigm Biomark system (data not shown). hi / lo TAM (MHCII - CD163 lo ;MHCII - CD163 hi ) and CD163-negative TAM (MHCII - CD163 - ; MHCII + CD163 - We analyzed both MN and intTAM populations, along with both M2-macrophage-associated and M3-associated TAMs (data not shown). Gene expression data were analyzed by hierarchical clustering and principal component analysis, revealing that a cluster of major M2-macrophage-associated genes upregulated the CD163-expressing TAM subset (cluster III), including Il4ra, Mrc1, Stab1, and Slco2b1 (data not shown). Interestingly, CD163 hi A subset of CD8 includes Il10, Ido1 and Lgals1. + Genes known to be associated with effects on T cell function were also upregulated (data not shown).

[0073] We also observed that distinct clusters of inflammatory genes were upregulated in tumor-associated monocytes (MN) (cluster I; including Cxcl10, Il1b, Irf5, Ccr2, Il18) and intTAMs (cluster II; including Cxcl9, Ciita, Irf7), and that these genes were downregulated in mature TAMs, consistent with the acquisition of an M2-like phenotype (cluster III). Many of the genes represented in clusters I and II were IFN-responsive, reflecting an M1-like phenotype that is usually associated with immune stimulatory activity. This, together with principal component and network analysis (data not shown), suggests a progressive polarization of recruited MNs towards an M2-like TAM phenotype. This analysis also showed high expression of Nr4a1 in tumor-infiltrating MNs. The nuclear receptor Nr4a1 (Nur77) is generally regarded as a marker of so-called patrolling or non-classical monocytes, since high expression is normally only detected in circulating Ly6C monocytes (data not shown). To further study Nr4a1 expression in tumor-associated MN, we analyzed YUMM1.7 tumors derived from Tg(Nr4a1-GFP) mice and detected no Ly6C monocytes in the blood by flow cytometry analysis. + Tumor-associated MN and patrolling (Ly6C lo ) showed comparable levels of GFP expression in monocytes (data not shown), which gradually decreased in intTAMs and mature TAMs, respectively (data not shown), suggesting that tumor-associated MNs may originate from a circulating nonclassical monocyte population that downregulates Nr4a1 expression as it differentiates into mature TAMs.

[0074] Specific depletion of CD163-expressing TAMs with targeted lipid nanoparticles promotes tumor regression CD163 +To deplete TAMs, we generated knock-in mice expressing iCre recombinase from the Cd163-IRES-iCre transcript (CD163-iCre) and crossed them with Csf1r-LSL-DTR mice expressing DTR under the control of the Csf1r promoter after cre-mediated depletion of LSL. A single injection of 4 ng / kg diphtheria toxin (DT) specifically depleted close to 50% of CD163+ TAMs after 24 h (data not shown). No effect was observed on the remaining myeloid compartment, except for a slight increase in recruited monocytes (data not shown). We next attempted to achieve sustained depletion of CD163+ TAMs by repeated DT injections. Compared to Csf1r-LSL-DTR wild type, CD163-iCre mice were significantly more depleted than Csf1r-LSL-DTR mice. + / - , Csf1r-LSL-DTR + / - Although tumor growth was significantly reduced in mice, sustained depletion of CD163+ macrophages caused severe side effects, indicating that the treatment is not suitable for continuous use (data not shown).

[0075] We previously reported the development of lipid nanoparticles (LNPs) conjugated with anti-CD163 mAb. 26We have developed a method to specifically target CD163-expressing cells using αCD163-LNPs (Figure 1A). These LNPs contain 5% polyethylene glycol (PEG; 2000 mw) to reduce nonspecific phagocytic uptake and increase the specificity of targeting to CD163+ cells. Anti-CD163 mAb is incorporated into the LNPs via a polyethylene (3400 mw) lipid anchor covalently attached to lysine side chains of the antibody (αCD163-LNPs). To demonstrate the specificity of αCD163-LNP, recombinant CHO K1 cells expressing mouse CD163 (data not shown) and mouse thioglycollate-elicited macrophages (data not shown) were used; αCD163-LNP were loaded with a self-quenching concentration of calcein (αCD163-cal-LNP; αCD163-cal) and cellular uptake was monitored, and controls included calcein-loaded LNPs alone (cal-LNP) and LNPs conjugated with an isotype control antibody (ctrl-IgG-cal-LNP; IgG-cal). Due to the encapsulation of a self-quenching concentration of calcein, fluorescence is only observed when calcein-loaded LNPs are endocytosed and the LNPs are degraded in the lysosomal compartment of the target cell. There was no increase in calcein fluorescence when non-targeted LNPs (cal-LNPs or ctrl-IgG-cal-LNPs) were incubated with CD163-expressing CHO K1 cells (data not shown), and only a slight increase was observed with ctrl-IgG-cal-LNPs in primary mouse macrophages (data not shown). These assays showed that high calcein fluorescence was specific to CD163-expressing cells upon incubation with αCD163-cal-LNPs. To evaluate the targeting of αCD163-LNPs to CD163-expressing TAMs in our melanoma model, tumor-bearing mice were intravenously (iv) injected with αCD163-LNPs and non-targeted control LNPs, followed by in vivo fluorescence imaging; fluorescence could be detected in the tumor area for both αCD163-targeted and non-targeted LNPs 4 hours after injection (data not shown). However, analysis of tumor tissue by flow cytometry showed that CD163 expression after administration of αCD163-LNPs was significantly higher than that after administration of non-targeted LNPs. +We demonstrated increased calcein uptake in TAMs (data not shown). Next, we generated cytotoxic LNPs to test the ability of αCD163-targeted LNPs to specifically deplete CD163+ macrophages in vivo. LNPs were loaded with the DNA damaging agent doxorubicin (dxr) and randomized groups of mice were intravenously injected with a single dose of either vehicle, empty αCD163-LNPs (αCD163-ctrl) or dxr-loaded targeted or non-targeted LNPs (αCD163-dxr or IgG-dxr, respectively) and the effect on CD163+ red pulp macrophages (RPM) in the spleen was measured 24 hours later by flow cytometry; a single injection of αCD163-dxr significantly reduced CD163+ macrophages compared to controls. + The number of RPMs was specifically reduced by approximately 50% (data not shown). Next, the effect of CD163 on tumor growth in melanoma-bearing mice was + The effect of TAM depletion was examined. + To achieve efficient and sustained depletion of TAMs, mice bearing palpable tumors were randomized and treated with αCD163-dxr or appropriate controls every 2 days for 2 weeks. Treatment with non-targeted cytotoxic LNP (IgG-dxr) was able to slow tumor growth, whereas mice treated with αCD163-dxr showed nearly complete tumor regression after 2 weeks (Figures 1B and 1D). Interestingly, subsequent flow cytometric analysis of tumors showed a reduction in total TAM numbers in mice treated with IgG-dxr (Figure 1C), suggesting indiscriminate total targeting of TAM subsets. However, CD163-targeted LNPs did not significantly increase the number of CD163 + Depletion of only a small fraction of TAMs had little effect on total TAM numbers (Figure 1C). Given the greater impact of CD163-targeted LNPs on tumor regression compared to non-targeted LNPs, this suggests that total targeting of TAM subsets is not sufficient to deplete CD163 TAMs. + These results suggest that a distinct TAM subset expresses CD163, which may effectively abolish the therapeutic benefit conferred by TAM depletion. + These results suggest that TAM depletion contributes to tumor regression.

[0076] CD163 + Targeted depletion of TAMs reeducates tumor-infiltrating myeloid cells CD163 + To further analyze the consequences of TAM depletion on the tumor immune microenvironment (TME), high-content immunophenotyping was performed by flow cytometry on tumors treated with αCD163-dxr compared to mice treated with empty CD163-targeted LNP (αCD163-ctrl) and vehicle. + Depletion of TAMs was associated with a highly significant overall expansion of the tumor-infiltrating leukocyte compartment, which increased from 5 to 30% of all cells (data not shown). Analysis of different immune cell types revealed that this was primarily CD4 + and CD8 + Both tumor-infiltrating T cells (TILs), and especially Ly6C + This was due to an increase in the number of monocytes (data not shown). + To further characterize the tumor-infiltrating myeloid (TIM) cell compartment after TAM depletion, we performed flow cytometry analysis using our previously established gating strategy (data not shown). In αCD163-dxr-treated mice, there was a dramatic increase in the recruitment of tumor-associated MN and intTAMs, totaling more than 300 million cells per gram of tissue (data not shown), resulting in a dramatic decrease in the proportion of mature TAMs (data not shown). Interestingly, CD163 + IntTAMs that infiltrated tumors after TAM depletion had a significant increase in CD11c expression (data not shown) and displayed a distinct gene expression profile compared to intTAMs from control tumors (data not shown), including increased expression of Ciita and Cxcl9 (data not shown), suggesting an immunostimulatory phenotype typical of activated monocyte-derived dendritic cells (moDCs). Importantly, intTAMs from αCD163-dxrLNP-treated mice showed significantly decreased expression of genes typically associated with patrolling or non-classical monocytes, such as Nr4a1 and Cx3cr1, while Cxcr4 expression was unchanged and expression of classical Ly6C was reduced. hiFcgr2b, which is associated with monocytes, was increased (data not shown). Furthermore, intTAMs from αCD163-dxrLNP-treated mice also showed increased expression of Pdl2 and CD209d, as well as the T cell chemokines Cxcl9 and Ccl17 (data not shown). + The decreased expression of genes associated with non-classical monocytes in intTAMs from TAM-depleted mice indicates that tumor-infiltrating myeloid cells may be reeducated by recruitment of classical inflammatory monocyte subsets.

[0077] Inflammatory monocytes (Ly6C + , Nr4a1 - , Cx3cr1 lo ) recruitment is highly dependent on the expression of the chemokine receptor CCR2, and in CCR2-deficient mice (Ccr2 - / - In contrast, patrolling monocytes (Ly6C lo , Nr4a1 + , Cx3cr1 hi ) distribution is Ccr2 - / - Only slightly affected in mice 27 CD163 + Ly6C after TAM depletion + To assess the contribution of CCR2-dependent monocyte recruitment to the accumulation of MN and intTAM, we performed a randomized controlled study of wild-type (WT) and melanoma-bearing Ccr2 - / - Mice were generated and treated with αCD163-dxr or vehicle as described above. Interestingly, tumor progression was not affected by CCR2-deletion in vehicle-treated mice (data not shown). Furthermore, accumulation of tumor-associated MN and intTAM was slightly reduced, whereas mature TAMs were significantly increased in vehicle-treated Ccr2-deleted mice. - / - However, the inhibition of tumor growth by αCD163-dxr treatment was not associated with Ccr2 - / - mice (data not shown), and CD163 + This was accompanied by a complete reversal of the intTAM recruitment caused by TAM depletion (data not shown). These data support the notion that CD163 TAM replenishment in melanoma-bearing mice is essential for the development of melanoma-bearing mice. +TAM depletion induces the depletion of fresh Ly6C + We demonstrated that this resulted in CCR2-dependent recruitment of monocytes and accumulation of immunostimulatory macrophages with an M1-like phenotype, which significantly contributed to the inhibition of tumor progression.

[0078] CD163 + TAM depletion promotes anti-PD-1 resistant CTL responses CD163 + Immunophenotyping of melanomas after TAM depletion showed a significant increase in tumor-infiltrating T cells (TILs) (data not shown). + To further analyze the effect of TAM depletion on TIL recruitment and activation, we performed additional flow cytometry analysis. + , CD19 - , NK1.1 - ,SiglecF - , Ly6G - and CD11b - , CD3e + , CD5 + ,) were gated to show that CD4+ cells were expressed in melanoma-bearing mice treated with αCD163-dxr compared to vehicle or empty αCD163-LNP (αCD163-ctrl). + and CD8 + In mice treated with the control, both CD8 TILs and CD8+ TILs were significantly increased (data not shown). + TILs showed heterogeneous expression of IFNγ and PD-1 (data not shown), whereas CD8 in mice treated with αCD163-dxr + The majority of TILs expressed high levels of IFNγ and did not express PD-1 (data not shown). We demonstrated that CD8 + We confirmed that TIL infiltration was increased (data not shown), which was due to CD163 + Consistent with the increased infiltration of activated CTL, CD163 +TAM depletion dramatically increased tissue-wide expression of IFNγ (Ifng) in tumors (data not shown), which was accompanied by increased expression of other proinflammatory cytokines, including TNFα, IL-1β (Il1b), and IL-18 (Il18) (data not shown), as well as the memory T cell-attracting chemokine CXCL9 (Cxcl9) (data not shown).

[0079] CD163 + The increased expression of Cxcl9 throughout the tissue upon TAM depletion correlated with a parallel increase in Cxcl9 expression in newly replenished intTAMs (data not shown). + It has been shown that CD163 is a potent chemoattractant for T cells, and induction of Cxcl9 expression in antigen-presenting cells (APCs) by T cell-derived IFNγ is essential for mounting CTL responses. + We decided to investigate whether CCR2-dependent intTAM recruitment induced by TAM depletion (data not shown) correlated with the changes observed in the TIL compartment. - / - TILs in melanomas from mice were analyzed by flow cytometry; WT and Ccr2 - / - CD163 in both mice + TAM depletion is CD4 + and CD8 + This resulted in an increase in TILs (data not shown), but + TILs and especially IFNγ-producing CD8 + The number of TILs was significantly increased in Ccr2 cells compared to WT mice. - / - In mice, CD163 expression was significantly decreased (data not shown). + CCR2-dependent recruitment of intTAMs after TAM depletion is conserved in CD4 + and CD8 + These results suggest that they contribute to TIL recruitment and activation, respectively.

[0080] CD163 + CD4 in tumor regression by TAM depletion +and CD8 + To establish the contribution of TILs, αCD4 and αCD8b mAbs were administered during treatment with αCD163-dxr to inhibit CD4 + and CD8 + TILs were depleted. CD4 + and CD8 + Depletion of both T cells completely reversed the control of tumor growth by αCD163-dxr-treatment in melanoma-bearing mice (data not shown). + This clearly indicates that the inhibition of tumor progression upon TAM depletion is determined by the activation of TILs. + Depletion of TILs alone resulted in CD163 + Infiltrating intTAMs and IFNγ-producing CD8s after TAM depletion + The number of TILs was significantly reduced (data not shown). Furthermore, gene expression analysis showed that CD4 + and CD8 + Depletion of both T cells reduced widespread Ifng expression, whereas widespread expression of Cxcl9 and Il1b was suppressed in CD4 + It was only reduced in T-cell depleted mice (data not shown).

[0081] YUMM1.7 cells after αCD163-dxr treatment 28 Given the expression of PD-L1 in TAMs and the increased expression of PD-L2 in intTAMs (data not shown), we sought to investigate whether the observed effects of treatment were influenced by inhibition of the PD-1 checkpoint. We administered αPD-1 mAb or control mAb to tumor-bearing mice alone, in combination with αCD163-dxr for 10 days (Figure 2A), or after αCD163-dxr treatment (Figure 2B). As shown in previous studies, αPD-1 mAb treatment alone had no effect on YUMM1.7 tumor growth (Figure 2A). Furthermore, αCD163-dxr efficiently controlled tumor growth with or without concomitant treatment with αPD-1 (Figure 2A), whereas tumors quickly recurred when αPD-1 alone was replaced by αCD163-dxr (Figure 2B).

[0082] Using αCSF1 blocking antibody, CD163 + Treatment study comparing efficacy of TAM depletion with total macrophage depletion Using αCSF1 blocking antibody, CD163 + To compare the efficacy of TAM depletion with total macrophages, mice bearing palpable tumors were treated with αCD163-dxr every 2 days for 2 weeks or with αCSF1 (n=6) intraperitoneally every 5 days. Total depletion of TAM subsets with anti-CSF1, compared with specific targeting of CD163+ TAMs, did not demonstrate any obvious inhibition of tumor growth (Figure 3A). Interestingly, anti-CSF1 treatment was associated with a strong reduction in all TAM subsets, including bone marrow-derived monocytes (MNs) (Figure 3B). Furthermore, the reduction in iTAMs after anti-CSF1 treatment was associated with a reduction in the number of tumor-infiltrating CD4+ and IFNγ-producing CD8+ T cells, compared with specific depletion of CD163+ TAMs (Figures 3B and 3C).

[0083] Discussion There is now ample experimental and clinical evidence highlighting many important disease-promoting functions of tumor-associated macrophages (TAMs), highlighting their crucial role in cancer progression. Thus, there is growing interest in developing novel therapeutic strategies targeting TAMs. To date, most strategies have focused on CSF-1 / CSF1R-signaling, which controls macrophage differentiation and survival, or the CCL2 / CCR2 axis, which controls monocyte recruitment and recruitment. However, these strategies have shown limited efficacy in experimental models and clinical trials. Recent studies have revealed that there is extensive heterogeneity in TAM subsets in human cancers, which may have important implications for clinical progression. Indeed, several clinical studies have shown that certain TAM subsets are associated with better patient prognosis. Thus, more data are needed to further understand the functions of specific TAM subsets to aid in the further development of more targeted therapies.

[0084] Despite their intrinsic immune-stimulating potential, one of the major tumor-promoting functions of TAMs appears to be immunosuppression. It has been suggested that the tumor microenvironment (TME) polarizes macrophages to an alternative activation state associated with suppression of tumor-infiltrating T cell (TIL) function rather than activation. Immune-checkpoint inhibitors (ICIs), which trigger TIL activation, have had an unprecedented impact on the treatment of certain cancers, especially melanoma. However, the majority of patients do not respond to current ICI treatments, which often correlate with low TIL levels in the primary tumor.

[0085] We decided to study the role of TAMs in a clinically relevant mouse model of melanoma that is resistant to anti-PD-1, the current mainstay ICI treatment. First, we characterized the tumor-infiltrating myeloid (TIM) compartment in both autologous and orthotopic melanoma; TAMs express CD11b + They constitute up to 60% of white blood cells, and on average 20% of all tumor-infiltrating white blood cells are TAMs, which are CD8 + The number of TAMs was 100-fold higher than that of tumor-infiltrating T cells (TILs). Clinical data strongly correlated CD163 expression by TAMs with poor prognosis in a wide range of cancers, including melanoma. However, the functional relevance of these cells in tumor progression remains unclear. + TAMs represent only a small fraction (<25%) of total TAMs in mouse melanomas. + Gene expression analysis of TAMs revealed upregulation of a gene cluster associated with M2-like macrophages (including Il4ra, Mrc1, Stab1, Slco2b1). Interestingly, genes known to inhibit T cell activation, including Il10, Ido1, and Lgals1, were also specifically upregulated. This is in contrast to the signature cluster of inflammatory genes upregulated in tumor-infiltrating monocytes (MNs) (e.g., Cxcl10, Il1b, Irf5, Ccr2, Il18) and immature TAMs (intTAMs; Cxcl9, Ciita, Irf7). The gene is CD163+ down-regulated in MN and intTAMs, consistent with the acquisition of an M2-like phenotype. Many of the genes up-regulated in MN and intTAMs are IFN-responsive and typically associated with immune stimulatory activity and anti-tumor function, reflecting an M1-like phenotype, indicating that recruited MNs gradually polarize toward an M2-like TAM phenotype.

[0086] CD163 + To assess the specific contribution of TAMs to tumor progression, we developed CD163-targeted cytotoxic lipid nanoparticles (LNPs). + It depletes only a small fraction of TAMs and has little effect on total TAM numbers. However, CD163 + Selective depletion of TAMs significantly reduced tumor growth. Interestingly, we observed that non-targeted cytotoxic LNPs that significantly reduced total TAM numbers were not as effective as CD163-targeted LNPs in reducing tumor growth. This suggests that total targeting of TAM subsets is not sufficient to reduce tumor growth. + These results suggest that TAM depletion may in fact abrogate the therapeutic benefit conferred by specific TAM depletion, because other TAM subsets express CD163 + These results suggest that TAM depletion may result in tumor regression. To explore this hypothesis, we targeted CD163 to the TIM compartment in melanoma. + We assessed the impact of TAM depletion. + Tumor-infiltrating intTAMs after TAM depletion express CD11c hi We observed that IL-17 increased the expression of Cxcl9 and CD209d in monocyte-derived dendritic cells (moDCs). 29 This indicates that CD11c has a typical immune stimulatory phenotype. hi Recruitment of intTAMs is blocked in CCR2-deficient mice and CD163 + The reduction in tumor growth induced by TAM depletion was abolished, thus indicating that CCR2-dependent recruitment of inflammatory monocytes significantly contributed to tumor regression. + TAM depletion also inhibits CD4 T cells in melanoma, both of which are required for tumor growth control.+ and CD8 + Interestingly, CD4 + and CD8 + TIL recruitment is both CCR2-dependent and dependent on inflammatory monocyte recruitment and possibly CD11c hi We show that CD163 is required for the accumulation of intTAMs, possibly mediated by increased expression in these cells of CXCL9, a chemokine essential for memory T cell recruitment, and is coupled to enhanced antigen-presenting cell (APC) activity associated with increased MHC II expression via Ciita. As predicted, CD163 + CD4 after TAM depletion + Inhibition of TIL accumulation was observed in IFNγ-producing CD8 + The number of TILs was significantly reduced, and CD8 + CD4 helps activate TILs + Consistent with the role of T cells, however, CD8 + CD4, not TIL + Depletion of TILs reduces the expression of infiltrating CD11c hi The number of intTAMs was also significantly reduced. + TILs specifically contribute to the recruitment of inflammatory monocytes, which in turn recruit CD8 + These results suggest that it promotes the recruitment and accumulation of TILs.

[0087] Taken together, our study supports the role of CD163 in malignant melanoma. + In addition, CD163 + Specific depletion of TAMs results in the reprogramming of tumor-infiltrating monocytes towards immune stimulatory functions and tumor regression. These data not only suggest a novel therapeutic strategy based on specific targeting of TAM subsets, but also explain the low efficiency of therapeutic approaches that indiscriminately target monocyte-derived macrophages. Thus, the requirement of inflammatory monocytes to proliferate TIL recruitment and activation may limit the utility of therapies targeting all monocytes / macrophages.

[0088] literature Throughout this application, a number of references are described that describe the state of the art to which this invention pertains. The disclosures of these references are incorporated by reference into this disclosure. 1. Noy, R. &Pollard, JW Tumor-Associated Macrophages: From Mechanisms to Therapy.Immunity 41, 49-61 (2014). 2. Jiang, Y., Li,Y. & Zhu, B. T-cell exhaustion in the tumor microenvironment. Cell DeathDis 6, e1792-e1792 (2015). 3. Rodriguez, PCet al. Arginase I Production in the Tumor Microenvironment by Mature MyeloidCells Inhibits T-Cell Receptor Expression and Antigen-Specific T-CellResponses. Cancer Res 64, 5839-5849 (2004). 4. Munn, DH& Mellor, AL Indoleamine 2,3-dioxygenase and tumor-induced tolerance. J.Clin. Invest. 117, 1147-1154 (2007). 5. Li Yang, YZTumor-associated macrophages, potential targets for cancer treatment. BiomarkerResearch 5, 1423 (2017). 6. Wynn, TA, Chawla, A. & Pollard, JW Macrophage biology in development, homeostasis and disease. Nature 496, 445-455 (2013). 7. Cassier, P. A.et al. CSF1R inhibition with emactuzumab in locally advanced diffuse-typetenosynovial giant cell tumours of the soft tissue: a dose-escalation anddose-expansion phase 1 study. The Lancet Oncology 16, 949-956 (2015). 8. Ries, C. H. etal. Targeting Tumor-Associated Macrophages with Anti-CSF-1R Antibody Reveals aStrategy for Cancer Therapy. Cancer Cell 25, 846-859 (2014). 9. de Vos vanSteenwijk, P. J. et al. Tumor-infiltrating CD14-positive myeloid cells andCD8-positive T-cells prolong survival in patients with cervical carcinoma. Int.J. Cancer 133, 2884-2894 (2013). 10. Ino, Y. et al. Immunecell infiltration as an indicator of the immune microenvironment of pancreaticcancer. Br. J. Cancer 108, 914-923 (2013). 11. Mantovani, A. &Allavena, P. The interaction of anticancer therapies with tumor-associatedmacrophages. J. Exp. Med. 212, 435-445 (2015). 12. Chevrier, S. et al.An Immune Atlas of Clear Cell Renal Cell Carcinoma. Cell 169, 736-749.e18(2017). 13. Lavin, Y. et al.Innate Immune Landscape in Early Lung Adenocarcinoma by Paired Single-CellAnalyses. Cell 169, 750-765.e17 (2017). 14. Kristiansen, M. etal. Identification of the haemoglobin scavenger receptor. Nature 409, 198-201(2001). 15. Etzerodt, A. &Moestrup, S. K. CD163 and inflammation: biological, diagnostic, and therapeuticaspects. Antioxid. Redox Signal. 18, 2352-2363 (2013). 16. Etzerodt, A.,Maniecki, M. B., Moeller, K., Moeller, H. J. & Moestrup, S. K. Tumornecrosis factor α-converting enzyme (TACE / ADAM17) mediates ectodomain sheddingof the scavenger receptor CD163. J Leukoc Biol 88, 1201-1205 (2010). 17. Ugurel, S. et al.Survival of patients with advanced metastatic melanoma: the impact of noveltherapies-update 2017. Eur J Cancer 83, 247-257 (2017). 18. Robert, C. et al.Pembrolizumab versus Ipilimumab in Advanced Melanoma.http: / / dx.doi.org / 10.1056 / NEJMoa1503093 372, 2521-2532 (2015). 19. Pardoll, D. M. Theblockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer 12,252-264 (2012). 20. Tumeh, P. C. et al.PD-1 blockade induces responses by inhibiting adaptive immune resistance.Nature 515, 568-571 (2014). 21. Larkin, J. et al.Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N.Engl. J. Med. 373, 23-34 (2015). 22. Michot, J. M. et al.Immune-related adverse events with immune checkpoint blockade: a comprehensivereview. Eur J Cancer 54, 139-148 (2016). 23. Dankort, D. et al.BrafV600E cooperates with Pten loss to induce metastatic melanoma. Naturegenetics 41, 544-552 (2009). 24. Movahedi, K. et al.Different Tumor Microenvironments Contain Functionally Distinct Subsets ofMacrophages Derived from Ly6C(high) Monocytes. Cancer Res 70, 5728-5739 (2010). 25. Meeth, K., Wang, J.X., Micevic, G., Damsky, W. & Bosenberg, M. W. The YUMM lines: a series ofcongenic mouse melanoma cell lines with defined genetic alterations. PigmentCell Melanoma Res. 29, 590-597 (2016). 26. Etzerodt, A. et al.Efficient intracellular drug-targeting of macrophages using stealth liposomesdirected to the hemoglobin scavenger receptor CD163. J Control Release 160,72-80 (2012). 27. Tsou, C.-L. et al.Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow andrecruitment to inflammatory sites. J. Clin. Invest. 117, 902-909 (2007). 28. Natale, C. A. et al.Activation of G protein-coupled estrogen receptor signaling inhibits melanomaand improves response to immune checkpoint blockade. eLife 7, 116 (2018). 29. Menezes, S. et al.The Heterogeneity of Ly6Chi Monocytes Controls Their Differentiation into iNOS+Macrophages or Monocyte-Derived Dendritic Cells. Immunity 45, 1205-1218 (2016). 30. Fritze, A., Hens, F.,Kimpfler, A., Schubert, R. & Peschka-Suss, R. Remote loading of doxorubicininto liposomes driven by a transmembrane phosphate gradient. Biochim BiophysActa 1758, 1633-1640 (2006). 31. Torchilin, V. P. etal. p-Nitrophenylcarbonyl-PEG-PE-liposomes: fast and simple attachment ofspecific ligands, including monoclonal antibodies, to distal ends of PEG chainsvia p-nitrophenylcarbonyl groups. Biochim Biophys Acta 1511, 397-411 (2001). 32. Etzerodt, A. et al.Plasma Clearance of Hemoglobin and Haptoglobin in Mice and Effect of CD163 GeneTargeting Disruption. Antioxid Redox Signal 18, 2254-2263 (2013).

Claims

1. A method for increasing the amount of tumor-infiltrating CD8+ T cells in a patient suffering from cancer, comprising administering to the patient a therapeutically effective amount of an agent capable of depleting a population of CD163+ tumor-associated macrophages.

2. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective combination of at least one immune checkpoint inhibitor and an agent capable of depleting a population of CD163+ tumor-associated macrophages.

3. 3. The method of claim 1 or 2, wherein the cancer is melanoma.

4. 3. The method of claim 2, wherein the immune checkpoint inhibitor is selected from the group consisting of a PD-1 antagonist, a PD-L1 antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a VISTA antagonist, a TIM-3 antagonist, a LAG-3 antagonist, an IDO antagonist, a KIR2D antagonist, an A2AR antagonist, a B7-H3 antagonist, a B7-H4 antagonist, and a BTLA antagonist.

5. 3. The method of claim 2, comprising: i) quantifying a density of CD8+ T cells in a tumor tissue sample obtained from the patient; ii) comparing the density quantified in step i) with a predetermined reference value; and iii) administering to the patient a combined therapeutically effective amount of an agent capable of depleting a population of CD163+ tumor-associated macrophages and an immune checkpoint inhibitor when the density of CD8+ T cells quantified in step i) is lower than the corresponding predetermined reference value.

6. 3. The method of claim 2, comprising: i) quantifying the density of CD8+ T cells and the density of CD163+ tumor-associated macrophages in a tumor tissue sample obtained from the patient; ii) comparing the densities quantified in step i) with predetermined reference values; and iii) administering to the patient a combined therapeutically effective amount of an agent capable of depleting a population of CD163+ tumor-associated macrophages and an immune checkpoint inhibitor when the density of CD163+ tumor-associated macrophages quantified in step i) is higher than the corresponding predetermined reference values ​​and the density of CD8+ T cells quantified in step i) is lower than the corresponding predetermined reference values.

7. A method of treating cancer that is resistant to immune checkpoint therapy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent capable of depleting a population of CD163+ tumor-associated macrophages.

8. A method for increasing the effectiveness / potency of an immune checkpoint inhibitor administered to a subject suffering from cancer as part of a treatment regimen, the method comprising administering to the subject a pharmacologic effective amount of an agent capable of depleting a population of CD163+ tumor-associated macrophages in combination with at least one immune checkpoint inhibitor.

9. A method for preventing resistance to an administered immune checkpoint inhibitor in a subject suffering from cancer, comprising administering to the subject a therapeutically effective amount of an agent capable of depleting a population of CD163+ tumor-associated macrophages.

10. The method of any of the preceding claims, wherein the agent is an antibody that has binding affinity for CD163 and leads to depletion of CD163+ TAMs in the subject's tumor.

11. The method of claim 10, wherein the antibody binds to the extracellular domain of CD163.

12. The method of claim 10, wherein the antibody mediates antibody-dependent cell-mediated cytotoxicity.

13. The method of claim 10, wherein the antibody is a multispecific antibody comprising a first antigen-binding site directed against CD163.

14. The method of claim 10, wherein the antibody is an antibody-drug conjugate.

15. The method of claim 14, wherein the antibody is conjugated to a cytotoxic moiety.

Images