method to increase m1 macrophage clustering in a patient

专利摘要:
METHOD FOR INCREASING M1 MACROPHAGE GROUPING IN A PATIENTThe invention in general concerns the promotion of an M1-type immune response (polarization of the M1 macrophage) by administering a compound that modulates macrophage activation. The invention relates to the use of an antibody capable of binding to CSF-1R to modulate macrophage activation. The invention also relates to methods for assessing the dose effectiveness of an antibody capable of binding CSF-1R in a patient, by evaluating the in vivo or in vitro activation of macrophages. The invention additionally relates to complementary testing and post-treatment testing to assess the effect of an antibody capable of binding CSF-1R on a subject being treated.
公开号:BR112014008540A2
申请号:R112014008540-4
申请日:2012-10-19
公开日:2020-12-01
发明作者:Hélène Haegel；Rémy Hallet
申请人:Transgène S.A.；
IPC主号:

专利说明:

[0001] [0001] The invention in general concerns the promotion of an immune response type M1 (polarization of the M1 macrophage) by administering a compound that modulates macrophage activation, also called macrophage polarization. The invention relates to the use of an antibody capable of binding to CSF-IR to modulate macrophage activation / polarization. The invention also relates to methods for assessing the dose effectiveness of an antibody capable of binding CSF-IR in a patient by assessing the in vivo or in vitro polarization of macrophages. The invention further relates to complementary testing and post-treatment testing to assess the effect of an antibody capable of binding CSF-IR on a subject being treated.
[0002] [0002] During inflammation, circulating monocytes are recruited to the site of inflammation where they adopt a macrophage phenotype dictated by the presence of specific cytokines and growth factors. Mature macrophages are divided into two populations, polarized Ml, or "classically activated", and polarized M2, or "alternatively activated." Macrophages are important tumor infiltrating cells and play essential roles in tumor growth and metastasis. In more solid tumors, the existence of macrophages is advantageous for tumor growth and metastasis. Recent studies indicate that tumor-associated macrophages (TAMs) show an M2 phenotype. These tumor-associated macrophages (TAM) produce interleukin IL-10 and transforming growth factor 3 (TGF) to suppress general anti-tumor immune responses. However, TAMs promote tumor neoangiogenesis by secreting proangiogenic factors and define the invasive microenvironment to facilitate metastasis and tumor spread. For these reasons, selective depletion of TAMs M2 was considered an unprecedented approach to anticancer therapy (Sica et al., 2006, European Journal of Cancer, 42,717-727).
[0003] [0003] Macrophages participate in immune responses to tumors in a polarized manner. Differentiation of M1 is triggered by GM-CSF and further stimulated with interferon-y (IFN-y), bacterial lipopolysaccharide (LPS), or tumor necrosis factor a (TNFa), and is mediated by several signal transduction pathways involving transcriptional signal transducer and activator (STAT), enhancer of the activated B cell kappa light chain factor (NFKB), and mitogen-activated protein kinase (MAPK). These events increase the production of agents such as reactive oxide and nitric (NO) oxygen species and promote subsequent inflammatory immune responses, increasing the ability of antigen presentation and inducing Th1 immunity through the production of cytokines, such as IL12 . In contrast, M2 macrophage activation is used to describe activated macrophages in ways other than MI activation including IL4 / IL13-stimulated macrophages, IL10-induced macrophages and triggered immune complex macrophages. Among many molecular differences between activation of M1 versus M2, the IL12 and ILIO production ratio is critical to distinguish M1 and M2 macrophages. Notably, TAMs share many properties of M2 macrophages.
[0004] [0004] TAMs exhibit an M2 profile characterized not only by an IL-12 "* IL-10" 8 "phenotype but also a high FcR-mediated phagocytic capacity associated with regulatory functions (Schmieder et al. 2012, Semin Cancer Biol., 22 , 289-297) .The hemoglobin-removing receptor (CD163) has been identified as a marker of M2 polarized macrophages that is expressed by TAMs (Ambarus et al. 2012, 375,196-206). TAMs may represent the most abundant immunosuppressive cell population in the tumor microenvironment, recruited by CSF-1 and CCL-2 (MCP-1) (Sica et al. 2006, Eur J Cancer., 42, 717-727).
[0005] [0005] Similarly, the alternatively activated M2 macrophages have been implicated in several pathologies, the most prominent of which are allergy and asthma (Duffield, 2003, Clin. Sci. 104, 27; Gordon, 2003, Nat. Rev. Immunol., 3 , 23; Dagupta and Keegan, J. Innate Immun., 2012, 4, 478).
[0006] [0006] The inventors have recently shown that certain monoclonal antibodies are able to shift M2 macrophages towards MI macrophages (i.e., to induce differentiation of M1, rather than M2 macrophages). They showed that said monoclonal antibodies are capable of infrarregular surface FoeyRI (CD64) and FeyRII (CDI16) of infrarregular MCP-1 (Macrophage Chemistry Protein 1, also called CCL-2), IL-6, production of MMP9 and / or IL-10, and promote production of IL-12, IL-11, TNF-o. They further showed that said monoclonal antibodies exhibit the differentiation of type CDI163 + M2 macrophages from human monocytes and increase ratios of M1 / M2 macrophages.
[0007] [0007] The invention relates in general to methods for immunomodulation by modulating macrophage activation.
[0008] [0008] As used throughout the application, the terms “one” and “one” are used in the sense that they mean “at least one”, “at least one first”, “one or more” or “a plurality ”Of the referenced components or steps, unless the context clearly dictates otherwise. For example, the terms "a cell" include a plurality of cells, including their mixtures.
[0009] [0009] The term "and / or" used here anywhere includes the meaning of "e", "or" and "any or all other combination of elements connected by said term".
[00010] [00010] The terms "about" or "approximately" in the manner used herein mean 20%, preferably 10%, and more preferably 5% of a given value or range. The terms "about x" additionally include the value of x.
[00011] [00011] As used herein, "comprising" and "comprising" are intended to indicate that the kits of parts, products, compositions and methods include the referenced components or steps, but not excluding others. For example, "a composition comprising x and y" encompasses any composition that contains x and y, no matter how many components may be present in the composition. Likewise, “a method comprising the step of x” encompasses any method in which x is performed, whether x is just the step in the method or just one of the steps, no matter how many other steps there may be and no matter how simple or complex x can be compared to them.
[00012] [00012] "Consisting essentially of" when used to define products, compositions and methods must mean excluding other components or steps of any essential significance. Thus, a composition consisting essentially of the aforementioned components would not exclude trace contaminants and pharmaceutically acceptable vehicles. “Consisting of” should mean excluding more than trace elements from other components or stages.
[00013] [00013] According to a first embodiment, the present invention relates to an immunomodulation method modulating activation of M2 macrophage in a patient suffering from undesirable conditions associated with polarization of the M2 macrophage, wherein said method comprises the step of administering in said patient an effective amount of an antibody capable of binding CSF-IR. The invention is more specifically directed to a method for immunomodulation like this, wherein said patient is additionally suffering from conditions associated with CSF-IR activity.
[00014] [00014] In accordance with a special embodiment, the present invention relates to the use of an antibody capable of binding to human CSF-IR to modulate the macrophage polarization M2, especially in a patient suffering from undesirable conditions associated with macrophage polarization M2. According to a special modality, said patient is additionally suffering from conditions associated with CSF-IR activity.
[00015] [00015] The present application relates to “macrophage polarization / activation modulation”. These terms mean that the modulatory antibodies of the invention cause a decrease in M2 macrophage cluster activation and / or an increase in MI macrophage cluster, preferably a decrease in M2 macrophage cluster activation and an increase in M1 macrophage cluster. Thus, the MI / M2 ratio increases. This can be indicated, as revealed here, by changes in the levels of factors that are associated with MI and M2 macrophages. Examples of such factors are membrane markers such as CD64 or CDI63, cytokines such as IL6, ILIO or IL12, interferons, MCP-I, MMPS, etc. This "modulation of macrophage activation" in patients can be perceived, for example, by measuring the increase in the IL12 / IL10 ratio, MCP-1 or IL-6 level, or the CD163- / CD163 + macrophage ratio after administration of an antibody capable of binding the CSF-IR of the invention in a patient.
[00016] [00016] According to another embodiment, the present invention relates to a method for increasing clustering of M1 macrophages in a patient suffering from conditions associated with undesirable M2 polarization, wherein said method comprises the step of administering to said patient an effective amount of an antibody capable of binding CSF-IR. The invention more specifically concerns such a method for immunomodulation, wherein said patient is additionally suffering from conditions associated with CSF-IR activity.
[00017] [00017] According to a special modality, said method for increasing clustering of M1 macrophages in a patient suffering from conditions associated with undesirable M2 polarization, additionally decreases clustering of M2 macrophages.
[00018] [00018] "Patient" means a vertebrate, such as a mammal, such as a human. Mammals include, but are not limited to, humans, dogs, cats, horses, cows and pigs. In accordance with the present invention, the "patient" is suffering from conditions associated with undesirable M2 activation and, according to a particular modality, is additionally suffering from conditions associated with CSF-IR activity.
[00019] [00019] The invention also more specifically concerns a method of reducing macrophage pro-tumor functions (i.e. tumorigenicity) and / or decreasing macrophage tumor suppression activity in a patient, especially in a patient suffering from conditions associated with undesirable M2 polarization and / or conditions associated with CSF-IR activity, wherein said method comprises the step of administering to said patient an effective amount of an antibody capable of binding to CSF-IR.
[00020] [00020] According to a special modality, the method of the invention suppresses at least one macrophage pro-tumor function selected from the group consisting of tumor invasion, metastasis, tumor cell proliferation, tumor growth, tumor survival, neoangiogenesis, suppression of innate or adaptive immunity and remodeling of the extracellular matrix.
[00021] [00021] Thus, with respect to the invention, "modulation of macrophage activation / polarization" may additionally mean that the modulatory antibodies of the invention reduce at least one macrophage pro-tumor function (i.e. tumorigenicity) selected from the group consisting of invasion tumor, metastasis, tumor cell proliferation, tumor growth, tumor survival, neoangiogenesis, suppression of adaptive or innate immunity and remodeling of the extracellular matrix.
[00022] [00022] According to a special embodiment, the method of the invention inhibits production of macrophage MCP-1, MMP-9 and IL-6 by macrophages, especially human macrophages.
[00023] [00023] According to a special modality, the method of the invention infrarulates expression of FeyRI (CD64) and FeyRIII (CDI16) on the surface in macrophages, especially human macrophages.
[00024] [00024] According to a special modality, the method of the invention promotes production of IL-12 (more particularly, the P70 form of IL-12) by macrophages, especially human macrophages and / or superstructure IL-12 / I1- ratios 10.
[00025] [00025] According to a special modality, the method of the invention modulates the activation state of macrophages by means of secreted factors.
[00026] [00026] According to a special modality, the method of the invention reduces at least one of the following: and recruitment of TAM into the tumor; and at least a macrophage pro-tumor function; * tumor angiogenesis; and tumor invasion and metastasis; and tumor growth; and tumor cell proliferation in patients, especially in patients suffering from undesirable conditions associated with M2 macrophage polarization. According to a special modality, said patient is additionally suffering from conditions associated with CSF-IR activity.
[00027] [00027] The invention also relates to methods for conducting macrophages in favor of an MI-like immune response! (M1 macrophage polarization) and / or against an M2 type immune response (M2 macrophage polarization) in patients, especially in patients suffering from undesirable conditions associated with M2 macrophage polarization or in a patient suffering from conditions associated with CSF activity -IR, wherein said method comprises the step of administering to said patient an effective amount of an antibody capable of binding CSF-IR.
[00028] [00028] The invention further relates to methods for conducting macrophages in favor of a Th1 immune response or against a Th2 immune response in patients, especially in patients suffering from undesirable conditions associated with M2 macrophage polarization or in a patient suffering of conditions associated with CSF-IR activity, wherein said method comprises the step of administering to said patient an effective amount of an antibody capable of binding CSF-IR.
[00029] [00029] The invention also relates to the use of an antibody capable of binding to CSF-IR to modulate macrophage polarization. The invention also relates to the use of an antibody capable of binding to CSF-IR to drive macrophages in favor of an M1-type immune response (polarization of the M1 macrophage). The invention also relates to the use of an antibody capable of binding to CSF-IR to induce macrophages to stimulate a Th1l-like immune response. The invention also concerns the use of compositions, such as pharmaceutical compositions, comprising an antibody capable of binding CSF-IR to modulate macrophage activation, to drive macrophages in favor of an MI-type immune response (polarization of the M1 macrophage) and / or by inducing macrophages to stimulate a Th1-type immune response.
[00030] [00030] As used herein, the terms "able to bind to" refer to a binding reaction that is determinative of the presence of a target protein in the presence of a heterogeneous population of proteins and other biological substances. Thus, under designated test conditions the antibody, according to the invention, binds preferentially to at least part of the CSF-IR and preferably does not bind in a significant amount to other components present in a test sample. Specific binding between the antibody according to the invention and the target CSF-IR means that the binding affinity is at least 10º M "', and preferably 105 M", 10º M ", 107 ML, 10º M1, 10th M 'or 10th M ".
[00031] [00031] As used herein, the term "CSF-IR" refers to the human CSF1 receptor.
[00032] [00032] As used herein, "antibody" or "Ab" is used in the broadest sense. Therefore, an "antibody" or "Ab" can be naturally occurring or produced by man, such as monoclonal antibodies (mAbs) produced by conventional hybridoma technology, recombinant technology and / or a functional fragment thereof. Antibodies of the present invention are preferably monoclonal antibodies (mAb).
[00033] [00033] As used herein, the terms "variable region" refer to the variable region or domain, of the light chain (VL) or heavy chain (VH) that contains the determinants for specificity of link recognition. The variable domains are involved in antigen recognition and form the antigen binding site. The variable region of both the heavy and light chain is divided into segments comprising four sub-regions of the structure (FR1, FR2, FR3, and FR4), interrupted by three sections of hypervariable sequences, or the complementarity determining regions (CDR's), defined in Kabat database, with CDRI1 positioned between FR1I and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying particular subregions such as FR1, FR2, FR3 or FR4, a region of the structure referred to by others, it represents the FR's combined in the variable region of a single naturally occurring immunoglobulin chain. As used here, an FR represents one of the four subregions, and FR's represent two or more of the four subregions that make up a region of the structure. The region of the structure of an antibody is the region of the combined structure of the constituent light and heavy chains and serves to position and align the CDR's. CDR's are basically responsible for the formation of an antibody binding site, giving specificity and binding affinity for an antigen epitope. Within the variable regions of the H or L chains that provide antigen binding, regions are smaller than sequences called “hypervariables” due to their extreme variability between antibodies of different specificity. Such hypervariable regions are also referred to as “complementarity determining regions” or “CDR” regions. These CDR regions take into account the basic specificity of the antibody for a particular antigenic determining structure. The variable heavy and light chains of all antibodies each have 3 CDR regions, each not contiguous with the other (called L1, L2, L3, H1, H2, H3) for the respective light (L) and heavy (H ).
[00034] [00034] "Co-administer" means to administer one together with another, among themselves, in a coordinated manner, including simultaneous or sequential administration of two or more agents.
[00035] [00035] "Effective amount" in general means an amount that provides the local or desired systemic effect, for example, effective to mitigate undesirable effects of inflammation, including modulation of macrophage activation, etc. For example, an effective amount is an amount sufficient to produce a beneficial or desired clinical outcome. The effective quantities can be supplied all at once, in a single administration or in fractional quantities that provide the effective quantity in several administrations. The precise determination of what would be considered an effective amount can be based on individual factors for each subject, including their size, age, injury and / or illness or injury being treated, and the amount of time since the injury occurred or the disease started. Versed in the technique, they will be able to determine the effective quantity for a given subject based on those considerations that are routine in the technique. As used herein, "effective dose" means the same as "effective amount".
[00036] [00036] According to another embodiment, the present invention relates to the use of an antibody capable of binding to human CSF-IR to reduce at least one macrophage pro-tumor function selected from the group consisting of tumor invasion, metastasis , tumor growth, tumor survival, neoangiogenesis, suppression of innate or adaptive immunity and matrix remodeling (i.e., tumorigenicity) and / or increased macrophage tumor suppression activity in patients, especially patients suffering from conditions associated with activation of undesirable M2 and, according to a particular modality, which additionally suffers from conditions associated with CSF-IR activity.
[00037] [00037] According to another embodiment, the present invention relates to the use of an antibody capable of binding to human CSF-1R to inhibit MCP-1, MMP-9 and production of IL-6 by macrophages, especially human macrophages .
[00038] [00038] According to another embodiment, the present invention relates to the use of an antibody capable of binding to human CSF-IR to infraregulate expression of FceyRI (CD64) and / or FeyRIII (CD16) from the surface in macrophages, especially macrophages of human.
[00039] [00039] According to another embodiment, the present invention relates to the use of an antibody capable of binding to human CSF-1R to promote production of IL-12 (more particularly P70 form of IL-12) by macrophages, especially macrophages of human and / or to suppress the IL-12 / I1-10 ratio.
[00040] [00040] According to another embodiment, the present invention relates to the use of an antibody capable of binding to human CSF-IR to modulate the activation state of macrophages by means of secreted factors.
[00041] [00041] According to another embodiment, the present invention relates to the use of an antibody capable of binding to human CSF-1R to reduce recruitment of TAM and / or tumor angiogenesis in patients, especially a patient suffering from undesirable conditions associated with polarization of the M2 macrophage, and according to a particular modality, which additionally suffers from conditions associated with CSF-IR activity.
[00042] [00042] The invention also more specifically concerns the use of an antibody capable of binding to human CSF-IR to reduce TAM recruitment and / or tumor invasion and metastasis in patients, especially a patient suffering from undesirable conditions associated with polarization of the M2 macrophage, and according to a particular modality, which additionally suffers from conditions associated with CSF-IR activity.
[00043] [00043] The invention also more specifically concerns the use of an antibody capable of binding to human CSF-IR to reduce TAM recruitment and / or tumor growth in patients, especially a patient suffering from undesirable conditions associated with macrophage polarization M2, and according to a particular modality, which additionally suffers from conditions associated with CSF-IR activity.
[00044] [00044] According to another embodiment, the present invention relates to the use of an antibody capable of binding to human CSF-IR to drive macrophages in favor of an M1-type immune response (polarization of the M1 macrophage) and / or against a Type M2 immune response (M2 macrophage polarization) in patients, especially patients suffering from undesirable conditions associated with M2 macrophage polarization, and according to a particular modality, who additionally suffer from conditions associated with CSF-IR activity.
[00045] [00045] According to another embodiment, the present invention relates to the use of an antibody capable of binding to human CSF-1R to drive macrophages in favor of a Th1 immune response and / or against a Th2 immune response in patients , especially patient who suffers from undesirable conditions associated with polarization of the M2 macrophage, and according to a particular modality, who additionally suffers from conditions associated with CSF-IR activity.
[00046] [00046] According to preferred embodiments, said antibody capable of binding to human CSF-IR is an antibody that binds to at least one epitope located between amino acid position 20 to 41 of SEQ ID NO: 23 (i.e., part of the N termination of the human DI domain). In a preferred embodiment, the antibody, according to the invention, binds to an epitope located between amino acid position 20 to 39 of SEQ ID NO: 23 (i.e., part of the N-terminus of the human DI domain), in amino acids Asn72, Ser94-Ala95-Ala96, Lys102, Aspl131-Pro132-Val133 and Trp159 of SEQ ID NO: 23.
[00047] [00047] In another embodiment, the antibody, according to the invention, binds to an epitope located between amino acid position 20 to 41 of SEQ ID NO: 23 (i.e., part of the N-terminus of the human DI domain) and does not bind in any epitope located between amino acid position 42 to 90, and / or between amino acid position 91 to 104, and / or between amino acid position 105 to 199, and / or between amino acid position 200 to 298 SEQ ID NO0: 23. According to the preferred embodiment, the antibody of the present invention is able to recognize the minimal epitope located between the amino acid position 20 to 41 of SEQ ID NO: 23 (i.e., part of the N-terminus of the human DI domain), preferably in the epitope between amino acid position 20 to 39 of SEQ ID NO: 23.
[00048] [00048] In a preferred embodiment, said antibody capable of binding to human CSF-IR is an antibody that does not compete with IL-34 ligand for binding to the CSF-IR receptor. The terms "does not compete with IL-34 ligand" in the manner used herein refer to any inhibition of the IL34 ligand in its CSF-IR binding of the receptor.
[00049] [00049] In a preferred embodiment, said antibody capable of binding to human CSF-IR is an antibody that partially competes with CSF-1 ligand for binding at the CSF-IR receptor. The terms "partially competes with CSF-1 ligand" in the manner used herein refer to an inhibition of the CSF-1 ligand in its CSF-IR receptor binding which is less than 100%, preferably less than 50%, and even more preferably less than 20%, and advantageously less than 10%. This partial inhibitor only reduces, but does not completely exclude ligand binding, the inhibition is called partial inhibition. In a preferred embodiment, said antibody capable of binding to CSF-IR is an antibody which is capable of partially preventing the binding of CSF1 to its CSF-IR of the receptor, and is not capable of fully displaying said binding. More particularly, the antibodies according to the invention are able to decrease the binding of CSF-1 to CSF-1R by approximately 5 to 10%.
[00050] [00050] According to one embodiment, said antibody capable of binding to human CSF-1R is an antibody comprising: (1) at least one CDR in which said CDR is comprising at least five consecutive amino acids of the sequence starting at position 45 and ending at position 54 of SEQ ID NO: 1, the sequence starting at position 66 and ending at position 87 of SEQ ID NO: 1 or the sequence starting at position 117 and ending at position 126 of SEQ ID NO: 1; or, (1) at least one CDR in which said CDR is comprising at least five consecutive amino acids of the sequence starting at position 44 and ending at position 56 of SEQ ID NO: 2, of the sequence starting at position 66 and ending at position 76 SEQ ID NO: 2 or the sequence starting at position 109 and ending at position 117 of SEQ ID NO: 2.
[00051] [00051] According to a preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1R, and comprises the following CDRs comprising at least five consecutive amino acids:
[00052] [00052] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, and comprises at least one CDR selected, independently of one another, in the CDR group as shown: - in the sequence starting at position 45 and ending at position 54 of SEQ ID NO: 1, - in the sequence starting at position 66 and ending at position 87 of SEQ ID NO: 1, - —in the sequence starting at position 117 and ending at position 126 of SEQ ID NO: 1, - in the sequence starting at position 44 and ending at position 56 of SEQ ID NO: 2, - in the sequence starting at position 66 and ending at position 76 of SEQ ID NO: 2 and - at sequence starting at position 109 and ending at position 117 of SEQ ID NO: 2.
[00053] [00053] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, and comprises CDR as shown: - in the sequence starting at position 45 and ending at position 54 of SEQ ID NO: 1, - in the sequence starting at position 66 and ending in position 87 of SEQ ID NO: 1, - in the sequence starting at position 117 and ending at position 126 of SEQ ID NO: 1, - in the sequence starting at position 44 and ending at position 56 of SEQ ID NO: 2, - in the sequence starting at position 66 and ending at position 76 of SEQ ID NO: 2 and - in the sequence starting at position 109 and ending at position 117 of SEQ ID NO :two.
[00054] [00054] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, and comprises at least one CDR comprising an amino acid sequence shown in any of the SEQ IDs NOs: 5, 6,7,8,9 or 10.
[00055] [00055] According to a preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, and comprises CDRs comprising amino acid sequences shown in SEQ ID NOs: 5, 6 , 7, 8, 9 or 10.
[00056] [00056] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, and comprises (1) at least one CDR comprising an amino acid sequence displayed in any one SEQ ID NOs: 11, 12 or 13; or (ii) at least one CDR comprising an amino acid sequence shown in any of SEQ ID NOs: 14, 15 or 16.
[00057] [00057] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, and comprises the CDR comprising amino acid sequences shown in SEQ ID NOs: 11.12, 13, 14, 15 or 16.
[00058] [00058] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1IR, and comprises (1) at least one CDR comprising an amino acid sequence displayed in any one SEQ ID NOs: 17, 18 or 19; or (ii) at least one CDR comprising an amino acid sequence shown in any of SEQ ID NOs: 20, 21 or 22.
[00059] [00059] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1IR, and comprises at least one CDR comprising an amino acid sequence shown in any of the SEQ IDs NOs: 17, 18, 19, 20,21 or 22.
[00060] [00060] According to a preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, and comprises the CDR comprising amino acid sequences shown in SEQ ID NOs: 17, 18 , 19, 20, 21 or 22.
[00061] [00061] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1IR, and comprises a variable region, wherein said variable region comprises the three CDRs shown in SEQ ID NOs: 5, 6, and 7.
[00062] [00062] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, and comprises a variable region, wherein said variable region comprises the three CDRs presented in SEQ ID NOs: 8, 9, and 10.
[00063] [00063] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1IR, and comprises a variable region, wherein said variable region comprises the three CDRs shown in SEQ ID NOs: 11, 12, and 13.
[00064] [00064] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1IR, and comprises a variable region, wherein said variable region comprises the three CDRs shown in SEQ ID NOs: 14, 15, and 16.
[00065] [00065] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1IR, and comprises a variable region, wherein said variable region comprises the three CDRs shown in SEQ ID NOs: 17, 18, and 19.
[00066] [00066] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1IR, and comprises a variable region, wherein said variable region comprises the three CDRs shown in SEQ ID NOs: 20, 21, and 22.
[00067] [00067] According to a preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1R, and comprises a variable region, wherein the variable region comprises a displayed amino acid sequence in SEQ ID NO: 3.
[00068] [00068] In a more preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, and comprises a variable region, where the variable region is shown in SEQ ID NO: 3.
[00069] [00069] In another preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1R, wherein the variable region comprises an amino acid sequence shown in SEQ ID NO: 4.
[00070] [00070] In another more preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, and comprises a variable region, where the variable region is shown in SEQ ID NO: 4.
[00071] [00071] According to a preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1R, and comprises: - a variable region comprising the three CDRs shown in SEQ ID NOs: 5, 6, e7, e - a variable region comprising the three CDRs presented in SEQ ID NOs: 8, 9, and 10.
[00072] [00072] According to a preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1R, and comprises: - a variable region comprising the three CDRs shown in SEQ ID NOs: 11, 12, and 13, and - a variable region comprising the three CDRs presented in SEQ ID NOs: 14, 15, and 16.
[00073] [00073] According to a preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1R, and comprises: - a variable region comprising the three CDRs shown in SEQ ID NOs: 17, 18, and 19, and - a variable region comprising the three CDRs presented in SEQ ID NOs: 20, 21, and 22.
[00074] [00074] According to a preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1R, and comprises: -. — A variable region shown in SEQ ID NO: 3 and - —A variable region shown in SEQ ID NO: 4.
[00075] [00075] According to a preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to CSF-
[00076] [00076] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, and comprises (i) a heavy chain variable region comprising the three CDRs shown in SEQ ID NOs: 5, 6, and 7, and (1) a light chain variable region comprising the three CDRs presented in SEQ ID NOs: 8.9, and 10.
[00077] [00077] According to another embodiment, the antibody of the invention specifically binds to CSF-IR and comprises (1) a heavy chain variable region comprising the three CDRs shown in SEQ ID NOs: 11, 12, and 13, and (11 ) a light chain variable region comprising the three CDRs presented in SEQ ID NOs: 14, 15, and 16.
[00078] [00078] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1IR, and comprises (1) a heavy chain variable region comprising the three CDRs shown in SEQ ID NOs: 17, 18, and 19, and (11) a light chain variable region comprising the three CDRs presented in SEQ ID NOs: 20, 21, and 22.
[00079] [00079] According to a preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, and comprises (i) a heavy chain variable region shown in SEQ ID NO: 3 and (ii) a light chain variable region shown in SEQ ID NO: 4.
[00080] [00080] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, comprising: (a) a first variable region being defined by the following formula FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 where: FR1, FR2, FR3 and FR4 are each regions of the structure; CDRI1I, CDR2 and CDR3 are each determining regions of complementarity; where: CDRI has at least five consecutive amino acids in the sequence starting at position 45 and ending at position 54 of SEQ ID NO: 1; CDR2 has at least five consecutive amino acids in the sequence starting at position 66 and ending at position 87 of SEQ ID NO: 1; and CDR3 has at least five consecutive amino acids in the sequence starting at position 117 and ending at position 126 of SEQ ID NO: 1; and (b) a second variable region being defined by the following formula FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 where:
[00081] [00081] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1IR, comprising: (a) a first variable region being defined by the following formula FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 where: FR1, FR2, FR3 and FR4 are each regions of the structure; CDR1I, CDR2 and CDR3 are each determining regions of complementarity; where: CDRI has an amino acid sequence selected from the group consisting of: SEQ ID NO: 5, 11 and 17; CDR2 has an amino acid sequence selected from the group consisting of: SEQ ID NO: 6, 12 and 18: and CDR3 has an amino acid sequence selected from the group consisting of: SEQ ID NO: 7, 13 and 19; and (b) a second variable region being defined by the following formula FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 where: FR1, FR2, FR3 and FR4 are each regions of the structure; CDR1I, CDR2 and CDR3 are each determining regions of complementarity; where: CDRI1 has an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, 14 and 20; CDR2 has an amino acid sequence selected from the group consisting of: SEQ ID NO: 9, 15 and 21; and CDR3 has an amino acid sequence selected from the group consisting of: SEQ ID NO: 10, 16 and 22.
[00082] [00082] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1IR, comprising any of the following (i), (11) or (1ii):
[00083] [00083] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, comprising: -. a first variable region comprising the amino acid sequence of SEQ ID NO: 3; and
[00084] [00084] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1IR, comprising: - a first variable region comprising the amino acid sequence of SEQ ID NO: 1 ; and - —a second variable region comprising the amino acid sequence of SEQ ID NO: 2.
[00085] [00085] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1IR, comprising: - - a heavy chain selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 25, and - - a light chain selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.
[00086] [00086] According to another embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, comprising: - a first variable region selected from the group consisting of SEQ ID NO: 29 and SEQ ID NO: 30; and - a second variable region selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33.
[00087] [00087] According to a preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1R, comprising (a) a heavy chain consisting of SEQ ID NO: 24, and (b) a light chain consisting of SEQ ID NO: 26.
[00088] [00088] According to another preferred embodiment, said antibody capable of binding CSF-IR is an antibody that specifically binds CSF-
[00089] [00089] According to an advantageous embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1R, comprising (a) a heavy chain consisting of SEQ ID NO: 24, and (b) a light chain consisting of SEQ ID NO: 28. Example of said monoclonal antibody is monoclonal antibody H27K15.
[00090] [00090] According to a preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, comprising (a) the first variable region consisting of SEQ ID NO: 29, and (b) a second variable region consisting of SEQ ID NO: 31.
[00091] [00091] According to another preferred embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-1R, comprising (a) first variable region consisting of SEQ ID NO: 30, and (b) a second variable region consisting of SEQ ID NO: 32.
[00092] [00092] According to an advantageous embodiment, said antibody capable of binding to CSF-IR is an antibody that specifically binds to human CSF-IR, comprising (a) the first variable region consisting of SEQ ID NO: 29, and (b) a second variable region consisting of SEQ ID NO: 33. Example of said monoclonal antibody is monoclonal antibody H27K15.
[00093] [00093] The antibody, more specifically the human antibody, according to the invention, can be of different isotypes, such as IgG, IgA, IgM or IgE. In a preferred embodiment the antibody, more specifically the human antibody, according to the invention, is an IgG.
[00094] [00094] The antibody according to the invention can be glycosylated or non-glycosylated.
[00095] [00095] As used herein, the term "glycosylation" refers to the presence of carbohydrate units that are covalently attached to the antibody.
[00096] [00096] The methods of the invention are used in treating conditions associated with undesirable M2 activation and associated with CSF-IR. The methods of the invention are used in the treatment of disease involving inflammation and associated with CSF-IR.
[00097] [00097] "Patients suffering from undesirable conditions associated with polarization of the M2 macrophage", according to the invention, designate cancer, especially metastatic cancer, progressive fibrotic diseases such as, for example, idiopathic pulmonary fibrosis (IPF), liver fibrosis or systemic sclerosis (Wynn and Barron, 2010, Semin. Liver Dis., 30, 245), allergy and asthma, atherosclerosis and Alzheimer's disease.
[00098] [00098] According to another embodiment, the present invention relates to methods for conducting macrophages in favor of an immune response triggered by type M1 (polarization of macrophage M1) and against an immune response triggered by type M2 (polarization of macrophage M2) in cancer patients.
[00099] [00099] According to another embodiment, the present invention relates to methods for conducting macrophages in favor of an MI type immune response (polarization of the M1 macrophage) and against an immune response triggered by type M2 (polarization of the M2 macrophage) in patients suffering from progressive fibrotic diseases.
[000100] [000100] According to another embodiment, the present invention relates to methods for conducting macrophages in favor of an immune response triggered by type M1 (polarization of macrophage M1) and against an immune response triggered by type M2 (polarization of macrophage M2) in patients suffering from allergy.
[000101] [000101] According to another embodiment, the present invention relates to methods for conducting macrophages in favor of an M1 type immune response (polarization of the M1 macrophage) and against an M2 type immune response (polarization of the M2 macrophage) in patients suffering of asthma.
[000102] [000102] As used herein, the term "cancer" refers, but without limitation, to adenocarcinoma, acinic cell adenocarcinoma, adrenal cortical carcinoma, alveolar cell carcinoma, anaplastic carcinoma, basaloid carcinoma, basal cell carcinoma, bronchiolar carcinoma , bronchogenic carcinoma, renaladinol carcinoma, embryonic carcinoma, anometroid carcinoma, fibrolamolar liver cell carcinoma, follicular carcinomas, giant cell carcinomas, hepatocellular carcinoma, intraepidermal carcinoma, intraepithelial carcinoma, carcinoma leptomangioma - melcinoma carcinoma - melcinoma carcinoma , menequal carcinoma, mesometonefic carcinoma, small cell carcinoma, squamous cell carcinoma, sweat gland carcinoma, transitional cell carcinoma, tubular cell carcinoma, amelioblastic sarcoma, angiolytic sarcoma, botryoid sarcoma, endometrial stromama sarcoma, sarcoma fascicular sarcoma, giant cell sarcoma, granular sarcoma ectus, immunoblastic sarcoma, juxacordial osteogenic sarcoma, coppices sarcoma, leukocytic sarcoma (leukemia), lymphatic sarcoma (lympho sarcoma), medullary sarcoma, myeloid sarcoma (granulocytic sarcoma), austiogenci sarcoma, histiocytic sarcoma, retinal cell, sarcoma round cell sarcoma, spindle cell sarcoma, synovial sarcoma, audiogenic telangiectatic sarcoma, Burkitt's lymphoma, NPDL, NML, NH and diffuse lymphomas. According to a preferred embodiment, the method according to the invention relates to the treatment of metastatic cancer in the bone, in which the metastatic cancer is breast, lung, kidney, multiple myeloma, thyroid, prostate, adenocarcinoma, cell malignancies blood, including leukemia and lymphoma; head and neck cancers; gastrointestinal cancers, including esophageal cancer, stomach cancer, colon cancer, intestinal cancer, colorectal cancer, rectal cancer, pancreatic cancer, liver cancer, gallbladder or gallbladder cancer; malignancies of the female genital tract, including ovarian carcinoma, uterine endometrial cancers, vaginal cancer, and cervical cancer; bladder cancer; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; and skin cancer, including malignant melanoma or squamous cell cancer.
[000103] [000103] The present invention additionally relates to a method for improving the treatment of a cancer patient who is undergoing chemotherapy with a therapeutic cancer agent, which comprises co-treatment of said patient together with a super-revealed method.
[000104] [000104] The present invention additionally relates to a method for improving the treatment of a cancer patient who is undergoing immunotherapy treatment with a therapeutic cancer vaccine, which comprises co-treatment of said patient together with a supra-revealed method. According to a preferred modality, said therapeutic vaccine for cancer is a viral-based therapeutic vaccine. More preferably, said viral-based therapeutic vaccine is an MVA-based therapeutic vaccine. Even more preferably, said MVA-based therapeutic vaccine carries and expresses human papilloma virus (HPV16) E6 and E7 oncoproteins and human interleukin-2 (e.g. TG4001 product) or expresses the Mucl! and human interleukin-2 (e.g., TG4010 product).
[000105] [000105] The invention additionally includes post-treatment monitoring assays, after administration to a patient of an antibody capable of binding CSF-IR to evaluate the effectiveness of said treatment, and / or to evaluate the clinical outcome of said treatment.
[000106] [000106] Monitoring assays include, but are not limited to, assays for circulating factors expressed and / or secreted by activated M1 macrophages or polarized M2 macrophages.
[000107] [000107] Factors expressed in the activation state of the M2 type macrophage include, but are not limited to, IL-10, IL-6 and MCP-1. Factors expressed in the M1 type macrophage activation state can also be assayed, for example, by measuring levels of IL-12, more particularly levels of the P70 form of IL-12. Activation of macrophages in favor of an immune response triggered by type M1 (polarization of macrophage M1) and against an immune response triggered by type M2 (polarization of macrophage M2) in patients can be perceived by measuring the increase in the ILI2 / IL10 ratio after administration to a patient of an antibody capable of binding CSF-1R of the invention.
[000108] [000108] These tests can be derived from serum, blood, tissue, etc. of the patient.
[000109] [000109] The invention additionally relates to post-treatment monitoring assays, after administration to a patient of an antibody capable of binding to CSF-IR to monitor macrophage activation and to establish and / or maintain an appropriate dosage regimen.
[000110] [000110] In this case, it is possible to obtain baseline levels by testing for the presence of M1 and / or M2 macrophages in tissues, either directly or by means of factors expressed and / or secreted by activated macrophages and then after administration of an antibody capable of binding to CSF-IR during treatment, monitoring one or more times for the presence of M1 versus M2 macrophages in tissues (tumor or normal tissues). It could then determine the optimized dose for treatment that would result in distortion of the response from type M2 macrophages to type MI macrophages.
[000111] [000111] The invention relates to materials and methods for evaluating the effectiveness of a treatment involving the administration of an antibody capable of binding CSF-IR to a patient using biological markers (biomarkers) that have been determined to have a substantially reliable signature that correlates with the desired immune response. Biomarkers are present in biological samples obtained from the patient. the ability to predict the clinical outcome of a treatment, long after it has started, will allow doctors and patients to identify ineffective therapy, make informed decisions regarding the course of treatment, including whether to abandon or allow the implementation of alternate therapy.
[000112] [000112] The invention relates to an ex vivo method for evaluating the effectiveness of a treatment involving an antibody capable of binding CSF-IR to a patient.
[000113] [000113] According to the invention, the term "evaluate" should be understood as "monitor, modify or adjust" a treatment involving the administration of an antibody capable of binding CSF-IR to a patient. [0001 14] In certain respects the method includes assessing the effectiveness of an antibody capable of binding to CSF-IR based on the levels of y interferon in the patient after immunotherapy treatment.
[000115] [000115] Monitoring assays include, but are not limited to, assays for circulating factors expressed and / or secreted by activated macrophages of M2 types M1 and / or.
[000116] [000116] Factors expressed in the activation state of the M2 type macrophage include, but are not limited to, IL-6, MMP9 and MCP-II. Factors expressed in the activation state of macrophage type M1 can also be assayed, for example, by measuring levels of IL-12, more particularly levels of the P70 form of IL-12, or ratios of IL-12 / I1-10.
[000117] [000117] In certain respects, the method includes measuring the patient's levels of interleukin-6, interleukin-12, MMP9 and / or MCP-1 after administration to the patient of an antibody capable of binding to CSF-IR; and to evaluate the effectiveness of the treatment based on the levels of interleukin-6, interleukin-12, MMP9 and / or MCP-I.
[000118] [000118] In certain respects, the method includes measuring the patient's levels of interleukin-6, interleukin-12, MMP9 and / or MCP-1 at least once for several weeks after administering to the patient an antibody capable of binding CSF -GO; and to assess the effectiveness of immunotherapy treatment based on levels of interleukin-6, interleukin-12, MMP9 and / or MCP-1.
[000119] [000119] In certain respects, the method may additionally include measuring the patient's levels of interleukin-6, interleukin-12, MMP9 and / or MCP-1 prior to administration of an antibody capable of binding to CSF-IR. According to the preferred embodiment, the values of the patient's levels of interleukin-6, interleukin-12, MMP9 and / or MCP-1l measured before said administration of an antibody capable of binding to CSF-IR are the “cut-off values” according to the present invention.
[000120] [000120] The time between administration of an antibody capable of binding in measurements of CSF-IR and interleukin-6, interleukin-12, MMP9 and / or MCP-1 can be 1 day to about 48 weeks or more (for example , about 1 day to about | week, about | week to about 2 weeks, about 2 weeks to about 4 weeks, about 4 weeks to about 8 weeks, about 8 weeks about 12 weeks, about 12 weeks to about 16 weeks, about 16 weeks to about 24 weeks, about 24 weeks to about 48 weeks, or more). In a preferred embodiment of the invention, the time interval is about 5 weeks. Similarly, additional measurements (ie, a third, fourth, fifth, etc. measurement) can be made at similar time intervals after the second measurement.
[000121] [000121] In related aspects the method includes determining the levels of interleukin-6, interleukin-12, MMP9 and / or MCP-1 in a patient after administering to the patient an antibody capable of binding to CSF-IR; comparing said levels with a cut-off value; and evaluate the effectiveness of immunotherapy treatment based on interleukin-6 levels,
[000122] [000122] In accordance with a special embodiment, the invention relates to a method for evaluating the effectiveness of a treatment involving the administration of an antibody capable of binding CSF-IR to a patient comprising: (1) administering one or more doses of said an antibody capable of binding CSF-IR in said subject; (11) measuring a level of interleukin-6, interleukin-12, MMP9 and / or MCP-I in the body of said subject after at least one of said administration.
[000123] [000123] According to the alternating embodiment of the invention, the method of the invention additionally comprises an initial step consisting of measuring the levels of interleukin-6, interleukin-12, MMP9 and / or MCP-1 in the patient's body prior to administration of the antibody capable of binding to CSF-IR.
[000124] [000124] According to the present invention, the levels of interleukin-6, interleukin-12, MMP9 and / or MCP-1I are measured in a biological sample obtained from the patient. Biological samples include, but are not limited to, blood, serum, tissue, and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen. In a preferred embodiment, the biological sample is blood, plasma or serum, in which case obtaining samples from a patient is a relatively simple and non-invasive procedure. Methods of obtaining blood or serum are well known in the art and are not part of the invention.
[000125] [000125] Furthermore, numerous methods for detecting and quantifying polypeptides, including the biomarkers in question, are known. Such methods include, but are not limited to, antibody-based methods, more specifically methods based on monoclonal antibodies. The particular methods of detecting and quantifying biomarkers are not important for the invention. For example, the materials and methods of the present invention can be used with Luminex technology (Luminex Corporation, Austin, Tex.) Or enzyme linked immunosorbent assays (ELISA, numerous ELISA kits are commercially available, for example, by CliniScience, Diaclone, Biosource ).
[000126] [000126] According to an embodiment of the invention, the levels of interleukin-6, interleukin-12, MMP9 and / or MCP-1 are determined using antibodies.
[000127] [000127] According to a specific embodiment of the invention, said antibody (s) is (are) specific to interleukin-6, interleukin-12, MMPS9 or MCP-I.
[000128] [000128] According to a specific embodiment of the invention, said antibodies are monoclonal antibodies.
[000129] [000129] According to a specific embodiment of the invention, said antibodies are labeled, for example, by fluorescence, radiolabeling, enzyme, biotin, or any other method designed to make cells labeled with said antibodies detectable. These techniques are widely used and known in technology.
[000130] [000130] The immunotherapy treatment of the invention will be considered efficient when the levels of interleukin-6, MMP9 and / or MCP-1 measured in a patient after administration of an antibody capable of binding to CSF-IR is below the levels of interleukin- 6 and / or MCP-1, respectively, measured in a patient before said administration (i.e., cut-off value).
[000131] [000131] Alternatively, the immunotherapy treatment of the invention will be considered efficient when the levels of interleukin-12 measured in a patient after administration of an antibody capable of binding to CSF-IR is above the levels of interleukin-12 measured in a patient before said administration (that is, cut-off value).
[000132] [000132] The invention additionally includes post-treatment monitoring assays, after administration to a patient of an antibody capable of binding to CSF-IR to monitor macrophage activation and establish and / or maintain an appropriate dosage regimen.
[000133] [000133] In this case, it is possible to obtain baseline levels by testing for the presence of Ml and / or M2 macrophages in the circulation, either directly or by means of factors expressed and / or secreted by activated macrophages and then after administration of an antibody able to bind to CSF-IR during treatment, monitor one or more times for the presence of macrophages in the circulation.
[000134] [000134] It could then determine the optimized dose for treatment that would result in distortion of a response triggered by type M2 macrophage to a type MI macrophage.
[000135] [000135] The methods of the invention are used in the treatment of disease involving inflammation and associated with CSF-IR.
[000136] [000136] Figure 1: H27KI5 is not cytotoxic to differentiate macrophages, while other anti-CDI15 mAbs induce massive cell death;
[000137] [000137] Cells were counted after a day 6 monocyte culture with GM-CSF and CSF-1, in the presence or absence of anti-CDI115 or F (ab ”) 2 mAbs or with GW2580. Controls induced cultures treated with rituximab or F (ab ') 2 of rituximab, or without any compound added. Average cell counts in 5 microscope fields + standard deviation obtained for each culture condition are shown.
[000138] [000138] Figure 2: Inhibition of CD64 expression (FceyRI) in human macrophages differentiated in the presence of monoclonal antibody H27K15
[000139] [000139] Macrophages obtained after a day 6 culture of monocytes from 3 different blood donors with GM-CSF and CSF-1 were analyzed by IC / FC for CD64 surface expression. Upper panels: staining of CD64 in cultures from donor 1 treated with monoclonal antibody H27K15 (left, bold line) or with GW2580 (right, bold line) or its respective rituximab negative controls (left, thin line) or no treatment (right , thin line). Bottom panel: Median fluorescence intensities in macrophage cultures treated with test compounds at 10, 1 or 0.1 ng / mL * were compared with those in the corresponding negative controls: H27K15 vs rituximab, F (ab ”), derived from H27K15 vs F (ab ”), derived from rituximab, 2-4A5 or 9-4D2 mAbs vs rat IgG1, GW2580 vs no treatment. Percentages of reduction in CD64 expression were calculated as: 100 - [100 x median fluorescence intensity with test compound / median fluorescence intensity with control]. The average percentages of reduction in CD64 expression of the 3 blood donors are shown. * Except for F (ab ”), which was used at equimolar concentrations: 6.6; 0.6; and 0.06 µg / ml.
[000140] [000140] Figure 3: Induction of a CD86 * "SSCP * macrophage population x x nor H27K15
[000141] [000141] Panels - upper: - point graphs showing CD86 staining (x-axis) and lateral dispersion (SSC, y-axis) of donor 3 macrophages differentiated for 6 days in the presence of H27K15 (left) or negative control rituximab (right ). A control was established in the H27K15-induced CD86trilhante SS C low cell population. Bottom panel: Percentages of CD $ 6 cells containing key SSH with test compounds at 10, 1 or 0.1 µg / mL * were compared to those in the corresponding negative controls: H27K15 vs rituximab, derived from H27K15 F (ab '), vs derived from rituximab F (ab ”)., GW2580 vs no treatment. Percentages of increase in the CD86 cell population "SSCP shining ** º were calculated as: 100 x percentage of CD86 cells" SSC shining ”* i * º with test compound / CD86 cell count ES low with control. The average percentages of the increase in the CDg6trilhante SEChaixo cell population of the 3 blood donors are shown. * Except for F (ab ') ;: 6.6; 0.6; and 0.06 µg / ml.
[000142] [000142] Figure 4: H27K15 induces IL-12p70 secretion and increases macrophage IL-12 / IL-10 ratios
[000143] [000143] IL-12p70 and IL-10 were titrated in culture supernatants from day 6 macrophages differentiated in the presence of mAb H27K15 (1 µg / ml), GW2580 (1 µM) or their respective negative controls rituximab or no treatment. Left panel: IL-12p70 and IL-10 levels (pg / mL) in macrophage cultures from the 3 blood donors. Right: IL-12p70 ratios (pg / mLY / IL-10 (pg / mL) were calculated for each blood donor and each culture condition. In samples where IL-12p70 was undetectable (below the 11 pg detection limit / m) 1, its level was arbitrarily set at 1 pg / mL for the calculation of IL-12p70 / IL-10 ratios.
[000144] [000144] Figure 5: H27K15 inhibits secretion of MCP-1 / CCL2 and IL-6 by macrophages
[000145] [000145] MCP-I and IL-6 were titrated in culture supernatants from day 6 macrophages differentiated in the presence of mAb H27K15 (1 µg / mL), GW2580 (1 µM) or their respective rituximab negative controls or no treatment. Percentages of reduction in MCP-1 (upper panel) or in IL-6 production (lower panel) were calculated for the 3 blood donors as: 100 - [100 x cytokine concentration with test compound (pg / mL) / concentration of cytokine with control (pg / mL)].
[000146] [000146] Figure 6. Monocytes isolated from 3 different blood donors were differentiated for 6 days in the presence of GM-CSF and CSF-1, with or without mAb H27K15, Rituximab or GW2580. MAb H27K15 or Rituximab (0.1, 1 or 10 µg / mL) or equimolar concentrations of F (ab) ', derived from both mAbs were added to the cultures. MMP-9 was titrated by ELISA (R&D Systems) in supernatants from day 6 of the culture.
[000147] [000147] Figure 7. Cells obtained after day 6 culture with GM-CSF and CSF-1 were harvested and incubated for 20 minutes at 4 ° C with PBS containing human IZgG Fc fragments to saturate Fc receptors. Fluorochrome-conjugated mAbs (anti-CDI4-PerCP-Cy5.5, anti-CDI63-PE, anti-CD206-APC, anti-CD1a-FITC, BD Biosciences) were then incubated with each sample for 20 minutes at 4 ° C. FOM analysis was performed using a FACS LSR-I (BD biosciences) with the DIVA software. Data from 3 different blood donors are presented.
[000148] [000148] Figure 8. Monocytes from 2 different donors were cultured with GM-CSF and CSF-1 in the presence or absence of anti-CD115 mAb H27K5 or IgG rituximab, control at 1 µg / mL. Ratios between type M1 (CDI4'CDI63) and M2 (CDI4'CDI63 * ”) macrophages among the macrophage population were determined after 6 days of cell differentiation.
[000149] [000149] The following commercial monoclonal antibodies were used throughout the study: anti-human CD115 2-4A45-4 mAb (IgG; rat x, Santa Cruz), 9-4D2 (IgG, rat, Biolegend) and IZgG1 from isotype control rat (R&D Systems). 1.2 SM mAb is anti-CDI115 mAb of the 1.2 SM sequence published in patent application WO 2009/026303. Rituximab was obtained by Roche. F (ab ”) 2 were produced by Transgene by pepsin digestion of monoclonal antibodies, followed by purification by gel filtration.
[000150] [000150] Leather type coverings were provided by Etablissement Français du Sang (EFS, Strasbourg). Peripheral blood mononucleated cells (PBMC) were obtained by centrifugation on a ficoll gradient. Monocytes were purified by immunomagnetic cell classification, using microspheres coated with CDIl4 antibody (Miltenyii). Enriched monocyte suspensions were more than 95% pure. Monocytes were differentiated for 6 days in 48-well plates (3 x cells / well) in RPMI-GlutamaxTM medium supplemented with 10% thermally inactivated fetal calf serum and 1% tri-antibiotic mixture (penicillin, streptomycin, neomycin). GM-CSF (10 ng / mL) was added to the cell culture medium from day 0 to day 3. H27K15, other antibodies or internal control GW2580 (LC Labs) were added on day O. On day 3 post-isolation, monocytes were washed with PBS and additionally grown in medium supplemented with CSF-1 (10 ng / mL) and GM-CSF (2 ng / mL), in the presence or absence of antibodies or GW2580. On day 6, supernatants were collected and stored at -20 ºC. Cells were detached from the plastic and clusters of triplicates were analyzed by IC / FC (immunocytochemistry / flow cytometry) for cell sizes and FcyR and CD86 expression. Cytokines and chemokines were quantified in culture supernatants by multiplex (Bioplex, Bio-Rad) or by ELISA.
[000151] [000151] For IC / FC analysis of macrophage cultures, clusters of triplicates were centrifuged for 10 minutes at 1,500 rpm and incubated for minutes at 4 ºC with PBS containing 10% human AB serum to saturate Fc receptors. Anti-CD64-APC and anti-CD86-A1I700, anti-CD64, -CD86, -CD163 and / or -CD14 mAbs (BD fluorochrome-conjugated Biosciences) were then incubated with each sample for 20 minutes at 4 ° C. Cells were washed with PBS (5 minutes, 2,000 rpm at 4 ° C) and fixed with Cell-Fix (BD Biosciences, France). Flow cytometry analysis was performed using a FACS LSR-II (BD biosciences) with DIVA software for acquisition and Flow Jo software for analysis.
[000152] [000152] anti-CD115 mAb H27K15 is not cytotoxic to macrophages but polarizes its differentiation in favor of type M1
[000153] [000153] To study whether H27K15 mAb would affect macrophage differentiation, purified CD14 * monocytes were cultured for 7 days in the presence of GM-CSF and CSF-1l, known to induce type M1 and M2 macrophages respectively (Akagawa KS, 2002; Verreck FA et al., 2004). Three different doses of H27K15 mAb or rituximab isotype control (0.1; 1 or 10 µg / ml) were added to the culture medium at the beginning of the culture and again 3 days later. F (ab ”) 2 generated from H27K15 or rituximab was tested in parallel to equivalent molar concentrations. F (ab ”) 2 generated from mAb 1.2 SM (WO 2009/026303) was tested for comparison with F (ab”) 2 derived from H27K15 or rituximab. The known blocking mAb for human CD115, 24A5, or the non-blocking mAb 9-4D2 (Sherr C.J. et al., 1989) was tested and compared with isotype control rat IgG1. As another control, the small molecule CD115 tyrosine kinase inhibitor GW2580 was added to some of the cultures at 1 µM, a concentration previously shown to exhibit CSF-1-dependent human monocyte proliferation and murine macrophage differentiation. in vitro (Conway JG et al., 2005; Paniagua RT et al., 2010).
[000154] [000154] Microscopic observation of cultures from day 6 showed no obvious difference between wells treated with H27K15, Rituximab, mAb 9-4D2, I8ZG1, H27K15 F (ab ') 2 or F (ab') 2 Rituximab, compared to non-Rituximab cells treated, regardless of the blood donor. For wells treated with mAb SM1.2 F (ab ”) 2, total cytotoxicity was observed for all donors for the 3 doses evaluated (0.066, 0.66 and 6.6 µg / mL). For wells treated with mAb 2-4A5, total cytotoxicity was observed for all donors at the 2 highest doses tested (1 and 10 µg / mL). For the lowest dose (0.1 µg / mL), cytotoxicity was only partial. Overall, those results did not reveal any toxicity from any antibodies except for mAb SM1.2 F (ab ”) 2 and mAb 2-4A5. For cells with the 1 µM control GW2580, depending on the microscope field, a relative cytotoxicity was visualized as a fragment and less cell density was observed in all blood donors. Cell viability was analyzed on day 6 by counting 5 microscope fields in each well of the plate (one in the center of the well and four fields the average distance between the center and the side of the well). Based on the previous observations, numbering was also done for wells treated with compounds exhibiting partial or total cytotoxicity (mnAb SM1.2 F (ab ') 2, mAb 2-4A5 and GW2580). The figure | shows the average of 5 fields counted per individual well + standard deviation. Some antibodies exhibited partial or total cytotoxicity. Thus, F (ab ”) 2 derived from mAb SM1.2 induced massive cell death at all concentrations and mAb 2-4A5 also dramatically reduced macrophage numbers compared to their respective controls. In the presence of GW2580, approximately 70% of the macrophage number remained on day 6 compared to untreated cultures.
[000155] [000155] Day 6 cultures were analyzed by IC / FC for surface expression of activation CD64 FcyR (FceyRI) and activation marker CD86. As shown in figure 2, CD64 surface expression has been drastically reduced by treatment with H27K15 or GW2580. H27K15 almost completely inhibited CD64 expression at all doses tested. Anti-human mouse CDI1I5 2-4A5 MAb also decreased CD64 surface expression, but in a dose-dependent manner. The non-CDI blocking mAb 15 9-4D2 did not change the expression level of CD64. Interestingly, F (ab *), derived from H27K15, also downregulated CD64 expression, but less potently than total mAb and only when tested at 1 or 10 µg / mL, suggesting that the effect of H27K15 was partially dependent on Fc.
[000156] [000156] When the expression of the co-stimulatory molecule / CD86 activation marker was analyzed in macrophages on day 6, we observed that a subpopulation of cells characterized by the CD8 & 6-rail SSCP * i * º phenotype appeared in cultures treated with both mAb H27K15 and GW2580 (Figure 3). Induction of this population of round-shaped cells that express high levels of CD86 was not observed with F (ab '*), derived from H27K15, suggesting a role for the Fc region of H27K15 in this phenomenon. The control in this population showed that CD86bright SSChaixo cells were mainly CD64 "** º 2 CD64 negative (data not shown).
[000157] [000157] IL-12p70 and IL-10 were titrated in supernatants from day 6 of the culture. Macrophages from the 3 donors tested did not produce any detectable IL-12p70 after culturing with IgG, human, or any reagent rituximab. Culture in the presence of H27K15 mAb induced secretion of IL-12p70 by macrophages from 2 of the 3 blood donors. In contrast, IL-12p70 was not detectable after treatment with GW2580 (Figure 4). IL-10, which was produced by resting macrophages from all donors, was suppressed by H27K15 in macrophages from donor 1, but not in donor 2 and was only slightly increased in donor 3. As a result, IL-12p70 / IL ratios -10 were overloaded in the 3 blood donors tested (Figure 4, right panel). Small molecule GW2580 also increased the ratios of IL-12p70 / IL-10, but more precisely because of the inhibition of IL-10 production.
[000158] [000158] These results show that targeting CDI15 with mAb H27K15 to differentiate macrophages not only dramatically downregulates CD64 / FeyRI expression, but also induces a population of SSCh * xº cells that express high levels of the CD86 activation marker. In addition, H27K15 can induce IL-12p70 production and suppress IL-12p70 / IL-10 ratios in all donors, indicative of macrophage polarization in favor of type M1.
[000159] [000159] Surprisingly, it was observed that the production of chemokine MCP-1 / CCL2 was almost completely suppressed when macrophages were differentiated in the presence of H27K15 or GW2580 mAb (Figure 5, top panel). Inhibition of MCP-1 secretion by H27K15 was effective in the 3 donors tested and ranged from 74% to 99%. Small molecule GW2580 was
[000160] [000160] anti-CD115 mAb H27K15 inhibits production of MMP-9 by monocytes cultured with GM-CSF and CSF-1
[000161] [000161] Tumor-associated macrophages are known to produce MMP-9 (matrix-metalloprotease 9), which promotes both tumor cell metastasis degrading the extracellular matrix and neoangiogenesis inducing VEGF release in the tumor microenvironment. MMP-9 produced by macrophages is a major regulator of angiogenic change in tumors.
[000162] [000162] CDI4 ”monocytes from 3 different donors were differentiated naturally in the presence of both GM-CSF and CSF-1, known to induce macrophage differentiation in favor of types M1 and M2 respectively. MAb H27K15 or Rituximab (used as a negative control) was added to the cultures at 0.1, 1 or 10 µg / ml. Equimolar concentrations of F (ab) ', derived from both mAbs were tested in parallel. The tyrosine kinase inhibitor GW2580 has previously been shown to exhibit CSF-II dependent proliferation of human monocytes and differentiation of murine macrophages in vitro was tested in the same assay. After 6 days of culture, concentrations of MMP-9 were measured in the supernatants by ELISA (Figure 6). In 2 of the 3 donors tested, there was less dose-dependent MMP-9 production when cultures were treated with mAb H27K15 or GW2580. With F (ab) '; derived from H27K15, there was an inhibitory effect on the same 2 donors. Thus, mAb H27K15 is able to decrease MMP-9 secretion by differentiating M2 macrophages.
[000163] [000163] These observations in macrophage cultures suggest that H27K15 administered to cancer patients may infregulate MMP-9 concentration in the tumor microenvironment.
[000164] [000164] MAb H27K15 inhibits the differentiation of CD163-positive type M2 macrophages.
[000165] [000165] The hemoglobin receptor remover (CDI163) has been identified as a marker of polarized M2 macrophages that is expressed by TAMs, notably in breast cancer. The surface expression of CDI63 was analyzed by flow cytometry in macrophages of day 6 derived from human monocytes cultured with GM-CSF and CSF-1. Figure 7 shows the percentages of CD163-positive cells in different cultures. Culture with H27K15 mAb inhibited the differentiation of the macrophage CD163 ”population in all 3 donors tested. In the presence of 1 µg / mL H27K15, percentages of CD163-positive cells decreased by 2.5 to 4 times compared to control cultures treated with rituximab. GW2580 had the same effect only in 2/3 donors. F (ab) ', derived from H27K15, had little or no effect on CD163 expression, indicating that the Fc region of the anti-CD115 mAb was involved in its mode of action.
[000166] [000166] As evidenced by these changes in surface CDI163 expression and in line with previous results, targeting CDII5 with mAb H27K15 inhibits the differentiation of type M2 macrophages.
[000167] [000167] MAb H27KI15 distorts monocyte differentiation from M2 to M1 macrophages
[000168] [000168] The ratios between Ml and M2 macrophages were analyzed in
[000169] [000169] Akagawa K.S. "Functional heterogeneity of colony-stimulating factor-induced human monocyte-derived macrophages." International journal of hematology. (2002) 76 (1): 27-34.
[000170] [000170] Conway JG, McDonald B., Parham J., Keith B., Rusnak DW, Shaw E., Jansen M., Lin P., Payne A., Crosby RM, Johnson JH, Frick L., Lin MH, Depee S., Tadepalli S., Votta B., James I., Fuller K., Chambers TJ, Kull FC, Chamberalain SD an Hutchins J.T. “Inhibition of colony-stimulating-factor-l signaling in vivo with the orally bioavailable cEMS kinase inhibitor GW2580.” Proceedings of the National Academy of Sciences of Unity States of America. (2005) 102 (44): 16078-16083.
[000171] [000171] Paniagua R.T., Chang A., Mariano M.M ,, Stein E.A., Wang Q., Lindstrom T.M., Sharpe O., Roscow C., Ho P.P., Lee D.M. and Robinson W.H. “c-Fms-mediated diferiation and priming of monocyte lineage cells play a central role in autoimmune arthritis.” Arthritis research & therapy. (2010) 12 (1): R32.
[000172] [000172] Roca H., Varsos Z.S., Sud S., Craig M.J., Ying C. and Pienta K.J. "CCL2 and interleukin-6 promote survival of human CD11b + peripheral blood mononuclear cell and induce M2-type macrophage polarization." The Journal of biological chemistry. (2009) 284 (49): 34342-34354.
[000173] [000173] Sherr C.J., Ashmun R.A., Downing J.R., Ohtsuka M., Quan
[000174] [000174] Verreck F.A., of Boer T., Langenberg D.M., Hoeve M.A ,, Kramer M,., Vaisberg E., Kastelein R., Kolk A., of Waal-Malefyt R. and Ottenhoff T.H. "Human IL-23-producing type 1 macrophages promote but IL-10-producing type 2 macrophages subvert immunity to (myco) bacteria." Proceedings of the National Academy of Sciences of the Unity and States of America. (2004) 101 (13): 4560-4565.

权利要求:
Claims (14)
[1]
1. Method for increasing clustering of M1 macrophages in a patient suffering from conditions associated with undesirable M2 activation, characterized by the fact that said method comprises the step of administering to that patient an effective amount of an antibody capable of binding CSF -GO.
[2]
2. Method, according to claim 1, characterized by the fact that the patient is additionally suffering from conditions associated with CSF-IR activity.
[3]
3. Method according to any one of the preceding claims, characterized by the fact that said method decreases the grouping of macrophage M2.
[4]
4, Method according to any one of the preceding claims, characterized by the fact that said method inhibits macrophage MCP-1, IL10 and production of IL-6.
[5]
5. Method, according to any of the preceding claims, characterized by the fact that said method infregulates expression of FcyRI (CD64) and FeyRII (CD16) surface in macrophages.
[6]
6. Method, according to any of the preceding claims, characterized by the fact that said method promotes production of IL-12 by macrophages.
[7]
7. Method according to any of the preceding claims, characterized by the fact that said method reduces at least one of the following: (1) recruitment of TAM in the tumor; (il) at least one macrophage pro-tumor function; (111) tumor angiogenesis; (iv) tumor invasion and metastasis; (v) tumor growth;
(vi) tumor cell proliferation.
[8]
8. Method according to any of the preceding claims, characterized by the fact that said conditions associated with undesirable M2 activation are selected from the group consisting of cancer, asthma, allergy and progressive fibrosis diseases.
[9]
9. Method according to any one of the preceding claims, characterized in that said antibody capable of binding to CSF-IR is an antibody that binds to at least one epitope located between the amino acid position 20 to 41 of SEQ ID NO: 23.
[10]
10. Method according to any one of the preceding claims, characterized in that said antibody capable of binding to CSF-IR is an antibody that binds to an epitope located between the amino acid position 20 to 39 of SEQ ID NO: 23, in amino acids Asn72, Ser94-Ala95-Ala96, Lys102, Asp131-Pro132-Val133 and Trp159 of SEQ ID NO0: 23.
[11]
11. Method according to any one of the preceding claims, characterized in that said antibody capable of binding to human CSF-IR is an antibody that does not compete with IL-34 ligand for binding to the CSF-IR receptor .
[12]
12. Method according to any one of the preceding claims, characterized in that said antibody capable of binding to human CSF-IR is an antibody that partially competes with CSF-1 ligand for binding to the CSF-IR receptor .
[13]
13. Method according to any one of the preceding claims, characterized in that said antibody capable of binding to human CSF-IR is an antibody that specifically binds to human CSF-IR, comprising: (a) a first variable region being defined by the following formula FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 where:
FR1, FR2, FR3 and FR4 are each regions of the structure;
CDRI1, CDR2 and CDR3 are each complementary determining regions;
on what:
CDRI has at least five consecutive amino acids in the sequence starting at position 45 and ending at position 54 of SEQ ID NO: 1;
CDR2 has at least five consecutive amino acids in the sequence starting at position 66 and ending at position 87 of SEQ ID NO: 1; and
CDR3 has at least five consecutive amino acids in the sequence starting at position 117 and ending at position 126 of SEQ ID NO: 1;
and
(b) a second variable region being defined by the following formula
FR1 - CDR1I - FR2 - CDR2 - FR3 - CDR3 - FR4 where:
FR1, FR2, FR3 and FRA4 are each regions of the structure;
CDRI1, CDR2 and CDR3 are each complementary determining regions;
on what:
CDRI1 has at least five consecutive amino acids in the sequence starting at position 44 and ending at position 56 of SEQ ID NO: 2;
CDR2 has at least five consecutive amino acids in the sequence starting at position 66 and ending at position 76 of SEQ ID NO: 2; and
CDR3 has at least five consecutive amino acids in the sequence starting at position 109 and ending at position 117 of SEQ ID NO: 2.
[14]
14. Method according to any one of the preceding claims, characterized in that said antibody capable of binding to CSF-IR is comprising (a) a heavy chain consisting of SEQ ID NO: 24, and (b) a light chain consisting of SEQ ID NO: 28.
: a 2 so ET - a 3 E S. - = a [= = D Fo 8 3 Í o; &: &
NOS:
CS SOS 1% oe "E e é É, 7 CU t Pas. 2 + Ea À“%., SEI ANAAHEIS “oo, Be, hi W Po mA: & ESSES E, o, Ss
A and
two. . 1 - ELES SAS OI =. << o Ta a a A NES, 8 8 888 "- 8 s o Tx o s 2 p or oduweo / ejnjão after o1lewWnnN
E E x É 3 345 2º a à 1 E E E E Ns. v E o =: o og, s% s Ss::% à "Or: mm Bmnjas sp wabequos
EEE
EEE <a CEEE DNS TERSSNERSTANS e. E o = E 1 2
RS T No and AAA O. re O Do O ESSSAANAA: rs ET & s' &
EDER o * ——— - i Fo | gRfESGRBS8 HS 3 = 7. OD emnoospwebeuos + 9Co I4IP9IW SP oBónpas ep%
And the o
E = Ss 3. E: Tiliageeo, [O = = IS GABEAS: q | | PRO : TAIS ST NSSWS OSS Sd Pi OA & Ar fo ISS E—— ws: EE & es E “|, and o | . IT o o, E a so | 9 IS ESA CNS & 1 PP S0SSonpOobdo. '2 les SSBRANRADOS SO 7 ISS E ——— OXILG-9SS UELIMA-GO S 9p ojuaume op%, 3 2: i: LL Jo
: $
O
And 3 s £:
PN 'Ss: 2 AND 3
AND IF SS 2288
EE UZ = eo S o = e Ss Ss S ". EN N" v oa = Es o E o "to 9 m and ov & Í - Naa = Bread 8 = S =), 8 Lu SostTNHOLENO: ATA A oocoo 'rt MIT MA 2 'oo Dx),; á R | Es a ES ES "7 2 = ES 2 = So =: 8ê EE Ss Oo h pr ee — n UTAMMTTTTTITTAETTATAAO
FE ERRARHAS RR AND HS ARO U / H EO
= 100 os & | =: “Es E = = In o Res SE o Í e Des So - E Da i = E EA Ss Í So É = = 8 40 EE SS E 8 2 o x. o Ro == E and 109 1 - S are | Are the EH27Ki5vsrituximab mm I ask == = RE * | ES = E AND GW2589 vs No treatment Sao | E and RS e) E ES es 8 | o = ds. And the o
RO RB o 1. CSS Co 'Donor 1 Donor2 —Donor3 Figure 5
: 6/8 ESSAS e.
E S 11%, Ps IS S, O [—— was | “Ge, ssssosp— 2 & o Casaca nemeaana) Fr A - o ——————“ “C&A - but SEVEN & 8 3 8 SS Ur o
S =: À x E (É hi 11% m be = =. 38 “oe, 8 -: ao & E—%, RR e“ &, PCA - 2 laughs and ss S É S o E 8 S 8 8 t = o
DOE Sa J i os rr: LE e ã% —— <e. S Ge, x ie) Yo and 6 E% Co, Ss “& a: PACO
O x Ss s bread 3 to 1U / BU E-IWIW
EzxÊ 25 , to if
PAIN —— o == = ——— o Space and ú and EL, o - UA a = - A 9% “mm 2%,
ECA ES E EF A 2 == mr With À) ds E m <=)% As, o - dp o = õ LM [S —— 2 o - “and A morena 4 o EE and SA -: Ge E O, “O, <AC sz 2, qo A is 2% to Es & fo E, o E— | | Po a Se. + £ 9,0 SOBejçI9EW ep%
8/8: Type M1 (CD14 + CD163 -) / Type M2 (CD163 + CD14 +) Macrophage ratios among the CD14 + cell population on day 6 [Rituximab
2.0 EM Ho7K1S q: = 15: No treatment =
And the 1.0 R e
If the
0.0 Donor 1 Donor 2 Figure 8

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法律状态:
2020-12-15| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2021-04-20| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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