NZ621911B2 - Modulation of macrophage activation - Google Patents

Abstract

Disclosed is a use of an effective amount of an antibody able to bind to CSF-1R in the manufacture of a medicament for treating a condition associated with undesirable M2 activation in a patient, wherein administration of the medicament increases the M1 macrophage pool in the patient and wherein the antibody binds to CSF-1R at least one epitope located between amino acid positions 20-41 of SEQ ID NO: 23, wherein the sequence is as disclosed in the specification. antibody binds to CSF-1R at least one epitope located between amino acid positions 20-41 of SEQ ID NO: 23, wherein the sequence is as disclosed in the specification.

Description

/070805 MODULATION OF MACROPHAGE TION SUMMARY OF THE INVENTION The invention is generally directed to promoting Ml~type (macrophage Ml polarization) immune response by administering a compound that modulates macrophage activation, also called macrophage polarization. The invention is directed to the use of an antibody able to bind to CSF—lR for modulating macrophage activation/ polarization. The ion is also directed to Hethods for evaluating the dose efficacy' of an dy able to bind to CSF—lR in a patient by assessing the in vivo or in vitro polarization of macrophages. The invention is further directed to post—treatment companion test and assays to assess the effect of an antibody able to bind to CSF—lR on a subject being treated.

During inflammation, circulating monocytes are recruited to the site of inflammation where they adopt a inacrophage ype dictated by the presence of specific cytokines and growth factors. Mature macrophages are divided into two populations, Ml—polarized or “classically activated” and M2~ polarized or “alternatively activated.” hages are important tumoreinfiltrating cells and play pivotal roles in tumor growth and metastasis. In most solid tumors, the existence of macrophages is ageous for tumor growth and metastasis. Recent studies indicate that associated macrophages (TAMs) show a M2 phenotype. These tumor~associated macrophages (TAM) e interleukin IL—lO and transforming growth factor (TGF) B to suppress general antitumor immune responses. Meanwhile, TAMs e tumor neo—angiogenesis by the secretion of pro—angiogenic factors and define the invasive microenvironment to facilitate tumor metastasis and dissemination. For these reasons, selective depletion (sf M2 TAMS has been considered as a novel approach to anti—cancer therapy (Sica et al., 2006, European Journal of Calicer, 42,717—727).

Macrophages participate in immune responses to tumors in a polarized manner. The M1 differentiation is triggered by GMv CSF and further stimulated by with interferon—y (IETJ~Y), bacterial lipopolysaccharide (LPS), or tumor necrosis factor a (TNFd), and is mediated by several signal transduction pathways involving signal ucer and activator of ription (STAT), nuclear factor light—chain— enhancer of activated B cells (NFKB), and mitogen~activated protein kinases (MAPK). These events e the production of agents such as the ve oxygen s and nitric oxide (NO) and promote the subsequent inflammatory immune responses by increasing antigen presentation ty and inducing~ the Th1 immunity through the production of cytokines such as IL12.

In contrast, M2 macrophage activation is used to describe macrophages activated in ways other than the M1 activation including IL4/IL13~stimulated macrophages, ILlOeinduced macrophages and immune complex—triggered. macrophages. .Among many molecular differences between Ml versus M2 activation, the ratio of ILl2 and ILlO production is critical to distinguish M1 and M2 hages. Noticeably, TAMs share many ties of M2 macrophages.

TAMs exhibit a M2 profile characterized not only by a IL~ lZl‘WIL—lohigh phenotype but also high EbR~mediated phagocytic capacity associated with regulatory functions (Schmieder et al. 2012, Semin Cancer Biol., 22, 289—297). The haemoglobin ger receptor (C0163) has been identified as a marker of M2—polarized hages which is expressed by TAMs (Ambarus et al. 2012, 375,196~206). TAMs can represent the most abundant immunosuppressive cell population in the tumor microenvironment, recruited by CSF—l and COL—2 (MCP—l) (Sica et al. 2006, Eur J Cancer., 42, 717-727).

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. l., 3, 23; Dagupta and , J. Innate Immun., 2012, 4, 478).

The Inventors have now shown that certain. monoclonal dies are able to switch M2 macrophages towards M1 macrophages (i.e. to induce the differentiation of M1 rather than M2 macrophages). They have shown that the said monoclonal antibodies are able to down regulate surface chRI (CD64) and FcyRIII (CD16) to down regulate MCP—l phage Chemotactic Protein 1, also called CCL—2), IL—6, MMP9 and/or IL—10 production, and to e IL-12, IL—lB, TNF—a production.

They have further shown that the said monoclonal antibodies inhibit the differentiation of CD163+ M2-type macrophages from human monocytes and increases M1/M2 hage ratios.

Disclosure of Invention The invention is y directed to methods for immunomodulation by modulating macrophage activation.

In particular, the present invention provides the use of an effective amount of an antibody able to bind to CSF~1R in the manufacture of a medicament for treating a condition associated with undesirable M2 activation in a patient, n administration of the medicament increases the M1 macrophage pool in said patient and wherein said antibody able to bind to CSF—lR is an antibody that binds to at least one epitope d between position amino acids 20 to 41 of SEQ ID NO:23.

As used throughout the entire application, the terms ”a" and "an" are used in the sense that they mean "at least one", "at least a first", "one or more" or "a plurality" of the [FOLLOWED BY PAGE 3a] referenced components or steps, unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

The term "and/or" er used herein includes the meaning of "and", "or" and "all or any other combination of the elements ted by said term".

The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within % of a given. value or range. The term “about x" further includes x value.

[FOLLOWED BY PAGE 4] 2012/070805 As used herein, ”comprising" and \\ comprise" 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 asses any composition that ns x and y, no matter what other components may be present in the composition. Likewise, “a method comprising the step of x” encompasses any method in which x is carried out, whether x is the only step in the method or it is only one of the steps, no matter how many other steps there may be and no matter how simple or complex X is in comparison to them.

"Consisting essentially of" when used to define products, compositions and methods, shall mean excluding <3ther components or steps of any essential significance. Thus, a composition consisting essentially of the recited components would not exclude trace contaminants and pharmaceutically acceptable carriers. ”Consisting of" shall mean excluding more than trace elements of other components or steps.

According to a first embodiment, the present invention concerns an modulation method by modulating M2 hage activation in a patient suffering from conditions associated with undesirable M2 macrophage polarization, wherein said method comprises the step of administering to the said patient an effective amount of an antibody able to bind to CSF~1R. The invention is more ically directed to such a method for immunomodulation wherein said patient is further suffering from conditions associated with CSF—lR activity.

According to one l embodiment, the present invention concerns the use of an antibody able to bind to human CSF—lR for modulating M2 macrophage zation, especially in a patient suffering from conditions associated with undesirable M2 hage polarization. According to one special embodiment, said patient is further suffering from conditions associated with R activity.

The present application refers to “modulating macrophage polarization/activation”. This term means that the modulatory antibodies of the Invention cause a decrease in the M2 macrophage activation pool and/or increase in Ml macrophages pool, preferably a decrease in the M2 macrophage activation pool and an increase in M1 macrophages pool. Thus, the Ml/MZ ratio increases. This can be indicated, as disclosed herein, by changes in the levels of factors that are associated with Ml and M2 macrophages. Examples of such s are membrane markers such as CD64 or CD163, cytokines such as 1L6, ILlO or IL12, interferons, MCP—l, MMP9, etc... This “modulating macrophage activation” in patients can be appreciated for example by measuring se of the ILlZ/ILlO ratio, MCP~1 or IL—6 level, or the /CD163+ macrophage ratio following stration to a t of an antibody able to bind to CSF—lR of the Invention.

According to another embodiment, the present invention concerns a method for increasing Ml macrophages pool in a patient suffering from conditions associated with undesirable M2 polarization, wherein said method comprises the step of administering to the said patient an effective amount of an dy able to bind to CSF~lR. The invention is more specifically directed to such a method for immunomodulation wherein said patient is further suffering from conditions associated with CSF-lR activity.

According to special embodiment, said method for increasing M1 hages pool in a t suffering from conditions associated with undesirable M2 polarization, further decreases the macrophage M2 pool.

“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. According to the pruasent invention, the “patient” is suffering from conditions associated with undesirable M2 activation, and accordirmg to particular embodiment is r suffering from condifi:ions associated with CSF—lR activity.

The invention is also more specifically directed tC) one method for reducing macrophage pro—tumoral functions (i.e. tumorigenicity) and/or increasing macrophage tumor suppression activity fill a patient, especially in patient suffering from ions associated with undesirable M2 polarization annd/or from ions associated with CSF~1R activity, wherein. said method ses the step of administering to the said patient an effective amount of an dy able to bind to CSE—lR.

According to special embodiment, the method of the invention ses at least one hage pro—tumoral functions selected in the group consisting of tumor invasion, metastasis, tumor cell proliferation, tumor growth, tumor survival, neo—angiogenesis, ssion of innate or adaptive immunity and extracellular matrix ling.

Thus, with respect to the invention, “modulating macrophage tion/ polarization” can further mean that the modulatory antibodies of the Invention reduce at least one macrophage pro~tumoral functions (i.e. tumorigenicity) selected in the group consisting of tumor invasion, metastasis, tumor cell proliferation, tumor , tumor survival, neo—angiogenesis, suppression of adaptive or innate immunity and extracellular matrix remodeling.

According to special embodiment, the method of the invention inhibits macrophage MCP—l, MMP~9 and IL—6 production by macrophages, especially human macrophages.

According to special embodiment, the method of the invention down regulates surface FcyRI (CD64) and FcYRIII (CD16) expression on macrophages, especially liuman macrophages.

According to special embodiment, the method of the invention promotes IL—lZ (more particularly IL-lZ P70 form) IS production by hages, especially human macrophages r lates IL~12/IL~lO ratios.

According to special embodiment, the method of the invention modulates the activation state of macrophages by means of secreted factors.

According to l embodiment, the method of the invention reduces at least one of the followings: o TAM recruitment into tumor; 0 at least one macrophage pro—tumoral functions; a tumor angiogenesis; o tumor invasion and metastasis; a tumor growth; 0 tumor cell proliferation in patients, especially in patients suffering from conditions associated with undesirable M2 macrophage zation. fl3According to special embodiment, the said patient is further suffering from conditions associated with CSF—lR activity.

The invention is also directed to methods for driving macrophages towards a Ml—type phage Ml polarization) immune response and/or away front a MZ—type (macrophage M2 zation) immune response in patients, especially in patients suffering from conditions associated with undesirable M2 macrophage zation or in t suffering from conditions associated with CSF—lR activity, wherein said method comprises the step of administering to the said patient an effective amount of an antibody able to bind to CSF—lR.

The invention is further directed to methods for driving macrophages towards a Th1 immune response or away from 51 Th2 immune response in patients, ally in patients suffering from conditions associated with undesirable M2 hage polarization or in patient suffering from condjfi:ions associated with CSF—lR activity, wherein said method comprises the step of administering to the said patient an effective amount of an antibody able to bind to CSF~1R.

The invention is also directed to the use of an antibody able to bind to CSF~1R for modulating macrophage polarization.

The invention is also ed to the use of an antibody able to bind to CSF—lR for driving hages towards a Ml—type (macrophage Ml zation) immune response. The invention is also directed to the use of an antibody able to bind to CSF—lR for for ng macrophages to stimulate a Thl-type immune response. The invention is also directed to the use of compositions, such as pharmaceutical compositions, comprising an antibody able to bind CSF—lR. for' modulating macrophage activation, for driving macrophages towards a Ml—type (macrophage M1 polarization) immune response and/or inducing hages to stimulate a Th1~type immune response.

As used herein, the term "able to bind to" refers to a binding reaction which is determinative of the presence of a target protein in the presence of a heterogeneous population of proteins and other ics. Thus, under designated assay conditions, the antibody according to the invention bind preferentially to at least part of the CSF—lR and preferably do not bind in a icant amount to other components t in a test sample. Specific binding between the antibody according to the invention and the CSF—lR target means that the binding affinity is of at least 103 M4, and preferably 105 M1, 106 M1, 107 M1, 108 M1, 109 M‘1 or 101° M'l.

As used herein, the ’ternl “CSF-lR” refers to the human PCT/EP20121070805 CSFl receptor.

As used herein, ody" or “Ab” is used in the broadest sense. Therefore, an ody" or “Ab” can be naturally occurring or man—made such as monoclonal antibodies (mAbs) produced by tional hybridoma technology, inant technology and/or a functional fragment thereof.

Antibodies of the present invention are preferably monoclonal antibodies (mAb).

As used herein, the term "variable region" refers to the variable region, or domain, of the light chain (VL) or heavy chain (VH) which ns the determinants for binding recognition specificity. 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 framework sub—regions (FRl, FRZ, FR3, and FR4), interrupted by three stretches of hypervariable sequences, or the complementary determining regions (CDR's), as defined in Kabat‘s database, with the CDRl positioned between FRl and FRZ, CDRZ between FRZ and FR3, and CDR3 n FR3 and FR4. Without specifying the particular sub— regions as FRl, FRZ, FR3 or FR4, a framework region as referred by others, represents the combined FR‘s within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four sub—regions, and FR's ents two or more of the four sub—regions constituting a ork . The framework region of an antibody is the combined framework regions of the constituent light and heavy chains and serves to position and align the CDR's. The CDR's are primarily responsible for forming the binding site of an antibody conferring binding specificity and affinity to an epitope of an antigen. Within the le regions of the H or L chains that provide for the antigen binding regions are smaller ces dubbed "hypervariable" because of their extreme WO 57281 variability between antibodies of differing specificity. Such hypervariable regions are also referred to as ementarity ining regions" or ”CDR" regions. These CDR regions t for the basic specificity of the antibody for a particular antigenic determinant structure. The variable heavy and light chains of all antibodies each have 3 CDR regions, each non~contiguous with the others (termed L1, L2, L3, H1, H2, H3) for the respective light (L) and heavy (H) chains.

“Co—administer” means to administer in conjunction, with one another, together, coordinately, including aneous or tial administration of two or more agents.

“Effective amount” generally means an amount which provides the desired local or systemic effect, e.g., effective to ameliorate undesirable effects of inflammation, including modulation of activation of hages, etc. For e, an effective amount is an amount ient to effectuate a beneficial or desired clinical result. The effective amounts can be provided all at once in a single administration or in fractional amounts that provide the effective amount in several administrations. The precise determination of what would be considered an effective amount may be based on factors individual to each subject, including their size, age, injury, and/or disease or injury being treated, and amount of time since the injury occurred or the disease began. One skilled in the art will be able to determine the effective amount for a given subject based on these considerations which are routine in the art. As used herein, “effective dose” means the same as “effective amount”.

According to another embodiment, the present invention relates to the use of an antibody able to bind to human CSF—lR for reducing at least one macrophage pro—tumoral functions ed in the .5 group consisting oi tumor on, metastasis, tumor growth, tumor survival, neo—angiogenesis, suppression of innate or adaptive ty and matrix ling (i.e. tumorigenicity)and/or increasing macrophage tumor .suppression activity in patients, especially patient suffering from conditions associated with rable M2 activation, and according to particular embodiment r ing from conditions associated with CSF—lR activity.

According to another embodiment, the present invention relates to the use of an antibody able to bind to human CSF—lR for inhibiting MCP~1, MMP—9 and IL—6 production by macrophages, especially human macrophages.

According to r embodiment, the present invention relates to the use of an antibody able to bind to human CSF—lR for down regulating surface FcyRI (CD64) and/or FcyRIII (CD16) expression on macrophages, especially human macrophages.

According to another embodiment, the present ion relates to the use of an dy able to bind to human CSF—lR for promoting IL~12 (more particularly IL—lZ 970 form) production by macrophages, especially human macrophages and/or up—regulating the IL~l2/IL—10 ratio.

According to another embodiment, the present invention relates to the use of an antibody able to bind to human CSF—lR for modulating the activation state of macrophages by means of secreted factors.

According to another embodiment, the present invention relates to the use of an antibody able to bind to human CSF—lR for reducing TAM recruitment and/or tumor angiogenesis in patients, especially patient ing from. conditions associated. with undesirable M2 macrophage polarization, and according to particular embodiment further suffering from conditions associated with CSF-lR activity.

The invention is also more specifically ed to the use of an antibody able to bind to human CSF—lR for reducing TAM recruitment and/or tumor invasion and metastasis in patients, especially patient suffering from :ions associated ‘with undesirable M2 macrophage polarization, and according to particular embodiment further suffering from conditions associated with CSF-lR ty.

The invention is also more specifically directed tt) the use of an antibody able to bind to human CSF—lR for reducing TAM recruitment and/or tumor growth in ts, especially patient suffering from ions associated with undesirable M2 macrophage polarization, and according to ular embodiment further suffering from conditions associated Edith CSF—lR activity. ing to another embodiment, the present invention relates to the use of an antibody able to bind to human CSF—lR for driving macrophages towards a Ml—type phage M1 polarization) immune response and/or away from a MZ—type (macrophage M2 polarization) immune response in patients, especially patient suffering from conditions associated with undesirable M2 macrophage zation, and according to particular embodiment further suffering from conditions associated with CSF—lR activity.

According to another embodiment, the present invention relates to the use of an antibody able to bind to human CSF~1R for driving macrophages s a Thl immune response and/or away from a Th2 immune response in patients, especially patient suffering from conditions associated with undesirable M2 macrophage polarization, and according to particular embodiment r suffering from conditions associated lfiith CSF-lR activity.

According to preferred embodiments, the said ody able to bind to human CSF-lR is an antibody that binds to at least one epitope located between position amino acids 20 to 41 of SEQ ID NO:23 (i.e. N-terminal part of the human domain PCT/EP20121070805 D1). In preferred embodiment, the dy according tc> the Invention binds to one epitope d between position andno acids 20 to 39 of SEQ ID N0:23 (i.e. N~terminal part of the human domain D1), to amino acids Asn72, Ser94-Ala95—Jxla96, LyleZ, Aspl3l—Pr0132—Va1133 and Trp159 of SEQ ID N0223.

In another embodiment, the antibody ing to ‘the Invention binds to one epitope located between position amino acids 20 to 41 of SEQ ID NO:23 (i.e. N—terminal part of the human domain D1) and does not bind to any epitOpe located between position amino acids 42 to 90, and/or between position amino acids 91 to 104, and/or between position amino acids 105 to 199, and/or between position amino acids 200 to 298 of SEQ ID N0:23. According to preferred embodiment, the antibody of the present Invention is able to recognize the minimal epitope located between on amino acids 20 to 41 of SEQ ID N0:23 (i.e. N— terminal part of the human domain 01), preferably to epitope between position amino acids 20 to 39 of SEQ ID N0:23.

In red, embodiment, the said antibody able to bind to human CSF—lR is an antibody that does not compete with IL~34 ligand for binding to the CSF~1R receptor. The term “does not compete with IL—34 ” as used herein refers to no inhibition of the IL34 ligand to its receptor CSF—lR binding.

In preferred, embodiment, the said antibody able to bind to human CSF—lR is an antibody that competes partially with CSF—l ligand for binding to the CSF-lR or. The term “competes partially with CSF—l ligand” as used herein refers to an inhibition of the CSF~1 ligand to its receptor CSF—lR 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 totally exclude ligand binding, the ‘ 2012/070805 inhibition is called partial inhibition. In red embodiment, the said antibody able to bind to CSF-lR is an antibody that is able to partially prevent binding of CSFl to its receptor CSF—lR, and is not able to totally inhibit said binding. More particularly, the antibodies according to the Invention are able to se the CSF—l binding to CSF~ 1R by approximately 5 to 10%. ing to one embodiment, the said antibody able to bind to human CSF—lR is an antibody that ses: (i) at least one CDR wherein said CDR is comprising at least five consecutive amino acids of the sequence starting in position 45 and finishing in position 54 of SEQ ID NO:1, of the sequence starting in position 66 and finishing in position 87 of SEQ ID N021 or of the sequence starting in position 117 and finishing in position 126 of SEQ ID NO:1; (ii) at least one CDR wherein said CDR is comprising at least five utive amino acids of the sequence starting in position 44 and finishing in position 56 of SEQ ID NO:2, of the ce starting in position 66 and finishing in position 76 of SEQ ID NO:2 or of the sequence starting in position 109 and finishing in position 117 of SEQ ID NO:2. ing to a preferred embodiment, the said antibody able to bind to CSF—lR is an antibody‘ that binds specifically to human CSFelR, and comprises the following CDRs comprising at least five consecutive amino acids : - of the sequence starting in position 45 and finishing in position 54 of SEQ ID N011, - of the sequence starting in position 66 and finishing in position 87 of SEQ ID NO:l, — of the sequence starting in position 117 and finishing in on 126 of SEQ ID N021, ~ of the sequence starting in position 44 and finishing in position 56 of SEQ ID N022, - of the sequence starting in position 66 and finishing in position 76 of SEQ ID NO:2 - or of the sequence ng in position 109 and finishing in position 117 of SEQ ID N022.

According to another embodiment, the said antibody able to bind to CSF~1R is an antibody that binds specifically to human , and comprises at least one CDR selected, independently from one another, in the group) of the CDR as set forth in : ~ the sequence starting in position 45 and ing in position 54 of SEQ ID NO:1, - the sequence starting in position 66 and finishing in position 87 of SEQ ID NO:1, - the sequence starting in position 117 and ing in position 126 of SEQ ID NO:1, - the sequence starting in position 44 and finishing in position 56 of SEQ ID N012, - the sequence ng in position 66 and finishing in position 76 of SEQ ID NO:2 and - the sequence starting in position 109 and finishing in position 117 of SEQ ID N022.

According to r embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human , and ses the CDR as set forth in — the sequence starting in position 45 and finishing in position 54 of SEQ ID NO:1, — the sequence starting in position 66 and finishing in position 87 of SEQ ID N011, — the sequence starting in position 117 and finishing in position 126 of SEQ ID NO:1, - the sequence starting in position 44 and finishing in position 56 of SEQ ID NO:2, — the sequence starting in position 66 and finishing in position 76 of SEQ ID NO:2 and ~ the sequence starting in position 109 and finishing in on 117 of SEQ ID NO:2.

According to another embodiment, the said antibody able to bind to CSFelR is an antibody that binds specifically to human CSF~1R, and comprises at least one CDR comprising an amino acid sequence as set forth in any one of SEQ ID NOS: 5, 6, 7, 8, 9 or 10. ing to a preferred embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human CSF-lR, and comprises the CDRs sing amino acid sequences as set forth in SEQ ID N03: 5, 6, 7, 8, 9 or 10.

According to another ment, the said antibody able to bind to CSF—IR is an antibody that binds specifically to human CSF—IR, and comprises (i) at least one CDR comprising an amino acid sequence as set forth in any one of SEQ ID N03: 11, 12 or 13; or (ii) at least one CDR comprising an amino acid sequence as set forth in any one of SEQ ID N05: 14, 15 or 16.

According to r embodiment, the said antitxady able to bind to CSF—lR is an antibody that binds specifically to human , and comprises the (:DR comprising amino acid sequences as set forth in SEQ ID N08: 11, 12, 13, 14, 15 or 16.

According be another embodiment, the said antibody able to bind to CSF-lR is an antibody that binds specifically to human CSF~1R, and comprises (i) at least one CDR comprising an amino acid sequence as set forth in any one of SEQ ID N03: 17, 18 or 19; or (ii) at least one CDR comprising an amino acid sequence as set forth in any one of SEQ ID N08: 20, 21 or 22. ing" to another embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human CSF—lR, and ses at least one CDR comprising an amino acid sequence as set forth in any one of SEQ ID N03: 17, 18, 19, 20, 21 or 22.

According to one preferred ment, the said antibody able to bind to CSF—lR is an dy that binds specifically to human CSFle, and comprises the CDR comprising amino acid sequences as set forth in SEQ ID N08: 17, 18, 19, 20, 21 or 22.

According to another embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human CSF~1R, and comprises a variable region, wherein said variable region comprises the three CDRs as set forth in SEQ ID NOS: 5, 6, and 7.

According to another embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human CSF—lR, and comprises a variable region, wherein said variable region ses the three CDRs as set forth in SEQ ID NOs: 8, 9, and 10.

According to another embodiment, the said antibody able to bind to CSF~lR is an antibody that binds specifically to human CSF~1R, and comprises a le region, wherein said. variable region comprises the three CDRs set forth in SEQ ID NOs: 11, 12, and 13. ing to r embodiment, the said antikxady able to bind to CSF—lR is an antibody that binds specifically to human CSF—lR, and ses a variable region, wherein said variable region comprises the three CDRs as set forth in SEQ ID N08: 14, 15, and 16.

According to another embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human CSF—lR, and comprises a variable region, n said variable region comprises the three CDRs as set forth in SEQ ID N08: 17, 18, and 19.

According’ to another embodiment, the said. antibody able to bind to CSF~1R is an antibody that binds specifically to human CSF~1R, and comprises a variable region, wherein said, variable region comprises the three CDRs as set forth in SEQ ID N03: 20, 21, and 22.

According to one preferred embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human CSF—lR, and comprises a le region, wherein the variable region comprises an amino acid sequence as set forth in SEQ ID N023.

In a more preferred embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human CSF—lR, and comprises a variable region, wherein the le region is as set forth in SEQ ID NO:3.

In another preferred embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human CSF—lR, wherein the variable region ses an amino acid sequence as set forth in SEQ 1H) NOz4.

In another more preferred embodiment, the said antibody able to bind to CSF~1R is an antibody that binds specifically to human CSFelR, and comprises a variable region, n the le region is as set forth in :SEQ ID NO:4.

According to a preferred embodiment, the 'said antibody able to bind to CSF~1R is an antibody that binds specifically to human CSF~lR, and ses : — a variable region comprising the three CDRs as set forth in SEQ ID NOS: 5, 6, and 7, and - a variable region comprising the three CDRs as set forth in SEQ ID NOS: 8, 9, and 10.

According to a preferred embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human CSF—lR, and comprises : ~ a variable region comprising the three CDRs as set forth in SEQ ID N08: 11, 12, and 13, - a variable region comprising the three CDRs as set forth in SEQ ID N05: 14, 15, and 16.

According to a preferred embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human , and comprises - a variable region comprising the three CDRs as set forth in SEQ ID N03: 17, 18, and 19, and — a le region comprising the three CDRs as set forth in SEQ ID N03: 20, 21, and 22.

According to a preferred ment, the said antibody able to bind to CSF—lR is an antibody that binds ically to human CSF—lR, and comprises — a variable region as set forth in SEQ ID NO:3 ~ a variable region as set forth in SEQ ID NO:4.

According to a preferred embodiment, the said antibody able to bind to CSF~1R is an antibody that binds ically to human CSF—lR, and comprises (i) a heavy—chain variable region comprising - the CDR as set forth in the sequence starting in on 45 and finishing in position 54 of SEQ ID NO:1, — the CDR as set forth in the sequence starting in position 66 and finishing in position 87 of SEQ ID N021 and - the CDR as set forth in the sequence starting in position 117 and finishing in position 126 of SEQ ID NO:1; (ii) a light—chain variable region comprising ~ the CDR as set forth in the sequence starting in position 44 and finishing in position 56 of SEQ ID NO:2, ~ the CDR as set forth in the sequence starting in position 66 and finishing in position 76 of SEQ ID NO:2 and ~ the CDR as set forth in the sequence starting in position 109 and ing in position 117 of SEQ ID N022.

According to another embodiment, the said antikxady able to bind to CSF—lR is an antibody that binds specifically to human CSF~1R, and comprises (i) a heavy— chain variable region comprising the three CDRs as set forth in SEQ ID NOs: 5, 6, and 7, and (ii) a light—chain variable region comprising the three CDRs as set forth in SEQ ID NOS: 8, 9, and 10.

According to another embodiment, the antibody of the Invention binds specifically to CSF~1R and comprises (i) a chain variable region comprising the three CDRs as set forth in SEQ ID N03: ll, 12, and 13, and (ii) a light—chain variable region comprising the three CDRs as set forth. in SEQ ID N08: 14, 15, and 16.

According to another embodiment, the said. antibody able to bind to CSF—lR is an antibody that binds specifically to human , and comprises (i) a heavy— chain variable region comprising the three CDRs as set forth in. SEQ ID N05: 17, 18, and 19, and (ii) a light—chain variable region comprising the three CDRs as set forth in SEQ ID NOS: 20, 21, and 22. ing to a preferred embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human CSF—lR, and ses (i) a heavy- chain variable region as set forth in SEQ ID NO:3 and (ii) a light—chain variable region as set forth in SEQ ID N024.

According to another embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human , comprising: PCT/EP20121070805 (a) a first variable region being defined by the following formula FRl v CDRl - FRZ - CDRZ ~ FR3 ~ CDR3 — E‘R4 wherein: FRl, FRZ, FR3 and FR4 are each framework regions; CDRl, CDR2 and. CDR3 are each complementarity determining regions; CDRl has at least five consecutive amino acids of the sequence ng in position 45 and finishing in position 54 of SEQ ID NO:1; CDR2 has at least five consecutive amino acids of the sequence starting in position 66 and finishing in position 87 of SEQ ID NO:1; and CDR3 has at least five utive amino acids of the sequence starting in position 117 and finishing in position 126 of SEQ ID NO:1; (b) a second variable region being defined by the following formula FRl — CDRl - FRZ — CDRZ - FR3 — CDR3 ~ FR4 wherein: FRl, FR2, FR3 and FR4 are each framework regions; CDRl, CDR2 and CDR3 are each complementarity determining regions; wherein: CDRl has at least five consecutive amino acids of the sequence starting in position 44 and finishing in position 56 of SEQ ID NO:2; CDRZ has at least five consecutive amino acids of the sequence starting in position 66 and finishing in position 76 of SEQ ID N022; and CDR3 has at least five consecutive amino acids of the sequence starting in position 109 and finishing in position 117 of SEQ ID NO:2. ing to another embodiment, the said antibody able to bind to CSF—lR. is an antibody that Ioinds specifically to human CSF—lR, comprising: (a) 21 first variable region being d by 'the following formula FRl - CDRl ~ FRZ - CDRZ - FR3 — CDR3 * FR4 wherein: FRl, FR2, FR3 and FR4 are each framework regions; CDRl, CDR2 and CDR3 are each complementarity‘ determining regions; wherein: CDRl has an amino acid. sequence selected front the group consisting of: SEQ ID NO: 5, 11 and 17; 2O CDR2 has an amino acid sequence selected front the group consisting of: SEQ ID NO: 6, 12 and 18; and CDR3 has an amino acid sequence selected from. the group ting of: SEQ ID NO: 7, 13 and 19; (b) a second le region being d by the following formula FRl - CDRl — FRZ - CDRZ — FR3 — CDR3 — FR4 wherein: FRl, FRZ, FR3 and FR4 are each framework regions; CDRI, CDRZ and CDR3 are each complementarity determirxing regions; wherein: CDRI has an amino acid sequence selected from the group consisting of: SEQ ID NO: 8, I4 and 20; CDRZ 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.

According to another embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human , comprising any one of the following (i), (ii) or (iii) (a) a first variable region being defined by the following formula FRl * CDRl ~ FR2 — CDRZ M FR3 ~ CDR3 — FR4 wherein: FRI, FRZ, FRB and FR4 are each framework s; CDRl, CDRZ and. CDR3 are each complementarity determining wherein: CDRI is as set forth in SEQ ID NO: 5; CDRZ is as set forth in SEQ ID NO: 6; and CDR3 is as set forth in SEQ ID NO: 7; (b) a second variable region being defined by the following formula FRl ~ CDRl — FR2 - CDR2 - FR3 - CDR3 - FR4 2012/070805 wherein: FRI, FR2, FR3 and FR4 are each framework regions; CDRl, CDR2 and CDR3 are each complementarity determirring wherein: CDRl is as set forth in SEQ ID NO: 8; CDR2 is as set forth in SEQ ID NO: 9; and CDR3 is as set forth in SEQ ID NO: 10; (ii) (a) a first variable region being defined by the following formula FRl - CDRl _ FRZ - CDRZ - FR3 H CDR3 - FR4 wherein: FRI, 8R2, FR3 and FR4 are each framework regions; CDRl, CDR2 and. CDR3 are each complementarity' determining regions; wherein: CDRl is as set forth in SEQ ID NO: 11; CDRZ is as set forth in SEQ ID NO: 12; and CDR3 is as set forth in SEQ ID NO: 13; (b) a second variable region being defined by the following formula FRl - CDRl ~ FR2 — CDR2 — FR3 ~ CDR3 — FR4 n: FRI, FRZ, FRB and FR4 are each framework regions; CDRl, CDRZ and CDR3 are each complementarity determining regions; wherein: CDRl is as set forth in SEQ ID NO: 14; CDR2 is as set forth in SEQ ID NO: 15; and CDR3 is as set forth in SEQ ID NO: 16; (iii) (a) a first variable region being defined by the following formula FR]. - CDRl — FR2 — CDR2 — FR3 — CDR3 ~ FR4 FRl, FR2, FR3 and FR4 are each framework regions; CDRl, CDR2 and CDR3 are each. complementarity determining regions; wherein: CDRl is as set forth in SEQ ID NO: 17; CDR2 is as set forth in SEQ ID NO: 18; and CDR3 is as set forth in SEQ ID NO: 19; (b) a second variable region being defined by the following formula FRl - CDRl - FRZ — CDRZ ~ FR3 — CDR3 — FR4 wherein: FRl, FR2, FR3 and FR4 are each framework regions; CDRl, CDR2 and CDR3 are each mentarity deterndjiing regions; wherein: CDRl is as set forth in SEQ ID NO: 20; CDR2 is as set forth in SEQ ID NO: 21; and CDR3 is as set forth in SEQ ID NO: 22.

According to another embodiment, the said dy able to bind to CSF-lR is an dy that binds specifically to human CSF—lR, comprising — a first variable region comprising the amino acid sequence of SEQ ID NO: 3; and — a second variable region comprising the amino acid sequence of SEQ ID NO: 4.

According to another embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human CSF~1R, comprising — a first variable region comprising the amino acid sequence of SEQ ID NO: 1; and — a second variable region comprising the amino acid ce of SEQ ID NO: 2.

According to another embodiment, the said antibody able to bind to CSF—IR is an antibody that binds specifically to human CSF~1R, comprising — an heavy chain selected in the group consisting in SEQ ID NO :24 and SEQ ID NO :25, and — a light chain selected in the group ting in SEQ ID NO :26, SEQ ID NO :27 and SEQ ID NO :28.

According to another embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human CSF~1R, comprising — a first le region selected in the group consisting of SEQ ID NO :29 and SEQ ID NO :30; and — a second variable region selected in the group consisting of SEQ ID NO :31, SEQ ID NO :32 and SEQ ID N0 :33.

According to one preferred embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human CSF—lR,, comprising (a) an heavy chain consisting in SEQ ID NO :24, and (b) a light chain consisting in SEQ ID NO :26.

According to r preferred embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human CSF~lR, comprising (a) an heavy chain consisting in SEQ ID NO:25, and (b) a light chain consisting in SEQ ID NO :27. ing to one advantageous embodiment, the said antibody able to bind to CSF—lR is an antibody that binds ically to human CSF—lR, comprising (a) an heavy chain consisting in SEQ ID NO:24, and (b) a light chain consisting in SEQ ID NO:28. Example of the said monoclonal dy is monoclonal dy H27K15.

According to one preferred embodiment, the said antibody able to bind to CSFelR is an antibody that binds specifically to human CSleR,, comprising (a) first variable region consisting in SEQ ID NO :29, and (b) a second variable region consisting in SEQ ID NO :31.

According to another preferred embodiment, the said antibody able to bind to CSF—lR is an antibody that binds specifically to human ,, comprising (a) first variable region ting in SEQ ID NO :30, and (b) a second le region consisting in SEQ ID NO :32.

According to one advantageous embodiment, the said antibody able to bind to CSF-lR is an antibody that binds specifically to human CSF—lR, comprising (a) first variable region consisting in SEQ ID NO :29, and (b) a second variable region consisting in SEQ ID NO :33. Example of the said monoclonal antibody is monoclonal antibody H27K15.

PCT/EP20121070805 The antibody, more specifically the human antibody, according to the ion may be of different isotypes, such as IgG, IgA, IgM or IgE. In a preferred embodiment the antibody, more specifically the human antibody, ing to the invention is an IgG.

The antibody according to the invention may be glycosylated or non~glycosylated.

As used herein, the term "glycosylation" refers to the presence of carbohydrate units that are covalently attached to the antibody.

The methods of the ion are useful in treatment of conditions associated with undesirable M2 activation and associated with CSF-lR. The methods of the invention are useful in treatment of disease involving inflammation and associated with CSF-lR.

“Patients suffering from conditions associated with undesirable M2 hage polarization according to the Invention designate cancer, ally metastatic cancer, progressive ic diseases such as for example idiopathic pulmonary fibrosis (IPF), hepatic fibrosis or systemic sis (Wynn and Barron, 2010, Semin. Liver Dis., 30, 245), allergy and asthma, atherosclerosis and Altzheimer’s disease.

According to another embodiment, the present invention s to methods for driving macrophages s a Ml—type (macrophage M1 polarization)~driven immune response and away front a MZ—type (macrophage M2 polarization)~driven immune response in patients suffering from cancer. ing to another embodiment, the present invention relates to methods for driving macrophages towards a Ml—type (macrophage M1 polarization) immune response and away from a MZ—type (macrophage M2 polarization)—driven immune response in patients suffering from progressive fibrotic diseases.

PCT/EP20121070805 According to another ment, the present invention relates to s for driving macrophages towards a Ml—type (macrophage Ml polarization)—driven immune response and away from 23 M2~type phage M2 polarization)—driven inwmine response in patients suffering from allergy.

According to another embodiment, the present invention relates to methods for driving hages towards a Ml—type (macrophage M1 polarization) immune se and away from a e (macrophage M2 polarization) immune response in patients suffering from asthma.

As used herein, the term “cancer” refers but is not limited to adenocarcinoma, acinic cell arcinoma, adrenal cortical carcinomas, i cell carcinoma, anaplastic carcinoma, basaloid carcinoma, basal cell carcinoma, iolar carcinoma, bronchogenic carcinoma, renaladinol carcinoma, embryonal carcinoma, anometroid carcinoma, fibrolamolar liver cell carcinoma, follicular omas, giant cell carcinomas, hepatocellular carcinoma, intraepidermal carcinoma, intraepithelial carcinoma, leptomanigio carcinoma, medullary carcinoma, melanotic carcinoma, menigual carcinoma, mesometonephric carcinoma, oat cell carcinoma, squamal cell carcinoma, sweat gland carcinoma, transitional cell carcinoma, tubular cell carcinoma, amelioblastic sarcoma, angiolithic sarcoma, botryoid sarcoma, endometrial stroma sarcoma, swing sarcoma, fascicular sarcoma, giant cell a, granulositic sarcoma, immunoblastic sarcoma, ordial osteogenic sarcoma, coppices sarcoma, leukocytic sarcoma (leukemia), lymphatic sarcoma (lympho a), medullary sarcoma, myeloid sarcoma (granulocitic sarcoma), austiogenci sarcoma, periosteal a, reticulum cell sarcoma (histiocytic lymphoma), round cell sarcoma, spindle cell sarcoma, synovial sarcoma, telangiectatic audiogenic sarcoma, Burkitt‘s lymphoma, NPDL, NML, NH and diffuse lymphomas.

According to a preferred embodiment, the method according to the invention is directed to the treatment of metastatic cancer to bone, wherein the metastatic cancer is breast; lung, renal, multiple myeloma, thyroid, prostate, S adenocarcinoma, blood cell ancies, including leukemia and lymphoma; head and neck cancers; gastrointestinal cancers, including esophageal cancer, h cancer, colon cancer, intestinal cancer, ctal cancer, rectal cancer, pancreatic cancer, liver cancer, cancer of the bile duct or gall bladder; malignancies of the female genital tract, including ovarian carcinoma, uterine endometrial cancers, vaginal cancer, and al ; bladder cancer; brain cancer, including neuroblastoma; sarcoma, osteosarcoma; and skin cancer, including malignant melanoma or squamous cell cancer.

The t invention further concerns a method for improving the treatment of a cancer patient which is oing chemotherapeutic treatment with a cancer eutic agent, which comprises co—treatment of said t along with a method as above disclosed.

The present invention further concerns a method for improving the treatment of a cancer patient which is undergoing immunotherapy treatment with a cancer therapeutic vaccine, which comprises co~treatment of said patient along with aa method as above disclosed. According ix) preferred embodiment, said cancer therapeutic vaccine is a viral based eutic vaccine. More preferably said viral based therapeutic vaccine is a MVA based therapeutic vaccine. Even more ably, said MVA based therapeutic vaccine is carrying and expressing human papilloma Virus 16 (HPV16) E6 and E7 oncoproteins and human interleukin—2 (e.g. TG400l t) or is expressing the Mucl antigen and the human interleukin—2 (e.g. TG4010 product).

The invention further includes post—treatment monitoring assays, following administration to a t of an antibody able to bind to CSF~1R to assess efficacy of said treatment, and/or to evaluate the clinical e of the said treatment.

The monitoring assays include, but are not limited to, assays for circulating factors expressed and/or secreteci by activated macrophages M1- or M2spolarized macrophages.

Factors expressed in the macrophage MZ—type tion state include, but are not d to IL—10, IL~6 and MCP~1.

Factors expressed in the macrophage Ml~type activation state may also be assayed, for example by measuring IL~12 levels, more particularly IL—12 P70 form levels. g macrophages towards a Ml—type (macrophage Ml polarization~driven :hmmune response and away from a MZ—type (macrophage M2 polarization~ driven immune response in ts can be appreciated by measuring increase of the ILlZ/ILlO ratio following administration to a patient of an antibody able to bind to CSF-lR of the Invention.

These tests can be derived from the patient's serum“ blood, , etc.

The invention further includes post—treatment monitoring assays, ing administration to a patient of an antibody able to bind to CSFelR to monitor macrophage activation and establish and/or maintain a proper dosage regimen.

In this case, it is possible to obtain a baseline levels by assaying for the presence of macrophages M1 and/or M2 in tissues, either directly' or by’ means of factors sed and/or secreted by activated macrophages and, then, following administration of an antibody able to bind to CSF—lR during treatment, monitor one or more times for the presence of M1 versus M2 macrophages in tissues (tumoral or normal tissues).

One could then ine the optimized dose for treatment that will result in skewing fronx M2~type macrophages to Mix—type macrophages response.

The invention provides als and methods for assessing the efficacy of a treatment involving the administration of an antibody able to bind to CSF~1R ‘to a patient using biological markers (biomarkers) that have: been determined to be substantially reliable ure which correlates with the desired immune response. The biomarkers are present in biological samples ed from the patient.

The ability to t the clinical outcome of a treatment, soon after its initiation, will enable clinicians and ts to identify ineffective therapy, make informed decisions regarding the course of treatment, including r to abandon or to allow alternate therapy implementation.

The invention es an ex—vivo method for assessing the efficacy of a ent involving an antibody able to bind to CSF-lR to a patient.

According to the invention, the term “assessing” should be understood as “monitoring, modifying or adjusting” a ent involving the administration of an antibody able to bind to CSF-lR to a patient.

In certain aspects the method includes assessing the efficacy of an antibody able to bind to CSF~1R based on the levels of interferon y in the patient following immunotherapy treatment.

The monitoring assays include, but are not limited to, assays for circulating factors expressed and/or secreted by activated hages of M1 and/or M2 types.

Factors expressed in the macrophage M2—type activation state e, but are not limited to IL—6, MMP9 and MCP—l.

Factors expressed in the macrophage M1~type activation state may also be assayed, for example by nmasuring IL~12 levels, more particularly IL—lZ P70 form levels, or IL~12/IL—lO ratios.

In certain aspects, the method includes measuring a patient‘s levels of interleukin—6, eukin—12, MMP9 and/or MCP—l following administration into patient of an antibody able to bind to {BF-1R; and assessing the efficacy of the treatment based on the levels of the interleukin—6, interleukin~12, MMP9 and/or MCP—l.

In certain aspects, the method es measuring a patient's levels of interleukin—6, interleukin~12, MMP9 and/or MCP—l at least once several weeks following administration into patient of an antibody able to bind to ; and assessing the efficacy of the immunotherapy treatment based on the levels of the interleukin—6, interleukin~12, MMPQ and/or MCP-l.

In certain aspects, the method can further include ing a patient's levels of interleukin—6, eukin~12, MMP9 and/or MCP—l prior to stration of an antibody able to bind to CSF~lR. According to preferred embodiment, the values of patient‘s levels of eukin—6, interleukin—12, MMP9 and/or MCP—l measured before said administration <3f an antibody able to bind to CSF-lR are the “cut—off values” according to the present invention.

The time between the administration of an antibody able to bind to CSF~1R and interleukin—6, interleukin~12, MMP9 and/or MCP—l measurements may be 1 day to about 48 weeks or more (e.g., from about 1 day to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 4 weeks, from about 4 weeks to about 8 weeks, from about 8 weeks to about 12 weeks, from about 12 weeks to about 16 weeks, from about 16 weeks to about 24 weeks, from 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 {i.e., a third, fourth, fifth, etc. ement) may be taken at similar time als following the second measurement.

In related aspects the method includes determining’ the levels of interleukin~6, eukin—12, MMP9 and/or MCP~1 in a patient following administration into patient of an dy able to bind to CSF—lR; comparing said levels to a f value; and assessing the efficacy of immunotherapy treatment based. on the levels of interleukin—6, interleukin~12, MMP9 and/or MCP~l compared to the “cut—off value”.

According to special embodiment, the Invention concerns a method for assessing the efficacy of a treatment involving the administration. of an antibody’ able to bind to CSF—lR to a t comprising: (i) administering one or more doses of said an antibody able to bind to CSF—lR to said subject; (ii) measuring an interleukin—6, interleukin—12, MMP9 and/or MCP~l level in the body of said subject following at least one of the said administration. ing to alternate embodiment of the invention, the method of the invention further comprises an initial step consisting in measuring the interleukin—6, interleukin—12, MMP9 and/or MCP~l levels in the body of the patient before administration of the antibody able to bind to CSF—lR.

According to the present invention, the levels of interleukin—6, interleukin—12, MMP9 and/or MCP—l are measured in a biological sample obtained from the patient. Biological s include but are not limited to blood, serum, tissue, and other liquid samples of ical 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 the samples from a patient is relatively simple and non—invasive procedure. Methods of obtaining blood or serum are well—known in the art are not part of the invention.

In addition, numerous methods for detecting and quantifying polypeptides, ing the instant kers, are known. Such methods include but are not d to antibody—based methods, more specifically onal antibodies~based methods. The particular methods of detecting and quantifying the biomarkers are not important to the invention. For example the materials and methods of the t invention may be used with Luminex technology (Luminex Corporation, Austin, Tex.) or enzyme—linked immunosorbant assays (ELISA, numerous ELISA kits are commercially available e.g. by CliniScience, Diaclone, Biosource).

According to one embodiment of the Invention, the levels of eukin—6, interleukin—12, MMP9 and/or MCP-l are determined by using dies.

According to one ic embodiment of the Invention, said antibody(ies) is (are) specific of interleukin—6, interleukin—12, MMP9 or MCP—l.

According to one specific embodiment of the Invention, said antibodies are monoclonal antibodies.

According to one specific embodiment of the Invention, said antibodies are tagged for example by fluorescence, radiolabel, enzyme, biotin, or any other methods designed to render cells labelled with said antibodies detectable. These techniques are widely used and known in the art.

The immunotherapy ent of the Invention will be considered as efficient when the levels of interleukin—6, MMP9 and/or MCP~l measured in patient following administration of an antibody able to bind to CSF—lR is below the levels of 2012/070805 interleukin—6 and/or MCP—l, respectively, measured in patient before said administration (i.e. cut—off value).

Alternatively, the immunotherapy treatment of the Invention will be considered as ent when the levels of interleukin—12 measured in patient following administration of an antibody able to bind to CSF~1R is above the levels of interleukin—12 measured in patient before said administration (i.e. cut~off value).

The invention further includes reatment monitoring assays, following administration to a patient of an dy able to bind to CSF—lR to monitor macrophage activation and ish and/or maintain a proper dosage regimen.

In this case, it is possible to obtain a baseline levels by assaying for the presence of macrophages Ml and/or M2 in the circulation, either directly or by means of factors expressed and/or secreted by activated macrophages and, then, following administration of an antibody able to bind to CSF—lR during treatment, monitor one or more times for the presence of the macrophages in the circulation.

One could then determine the optimized dose for treatment that will result in skewing from an MZ-type hage to an e macrophage—driven response.

The methods of the invention are useful in ent of disease involving inflammation and associated with CSF—lR.

Figure legends Figure l : H27K15 is not cytotoxic to entiating macrophages while other anti-CD115 mAbs induce massive cell death Cells were counted following a 6—day culture of tes with GM—CSF and CSF—l, in the presence or absence of anti—CD115 mAbs or F(ab’)2 or with GW2580. Controls included cultures treated with rituximab or rituximab EWab’)2, or without any added compound. Shown are the means of cell counts in 5 microscope fields i standard deviation obtained in each culture condition.

Figure 2 : Inhibition of CD64 (FcyRI) sion in human macrophages differentiated in the ce of monoclonal antibody H27K15 Macrophages obtained following a 6—day culture of monocytes from 3 different blood donors with GM—CSF and CSF~1 were ed by IC/FC for surface expression of CD64. Upper panels: CD64 staining in cultures from donor 1 treated lfiith monoclonal antibody H27K15 (left, bold line) or with GW2580 (right, bold line) or their respective negative controls mab (left, thin line) or no treatment (right, thin line). Lower panel: Medians of fluorescence intensities in macrophage cultures treated with test nds at 10, l or 0.1 ug/ml* were compared with those in the corresponding negative controls : HZ7K15 vs rituximab, H27K15—derived F(ab’)2 vs rituximab—derived F(ab’)2, mAbs 2—4A5 or 9—4D2 vs rat IgGl, GW258O vs no treatment. Percentages of reduction_in CD64 expression were calculated as: 100 ~ [100 x Median fluorescence intensity with test nd / Median fluorescence intensity with control]. Shown are the mean percentages of reduction in CD64 expression from the 3 blood donors. *except for the F(ab’)2 which were used at equimolar concentrations : 6.6; 0.6; and 0.06 ug/ml.

Figure 3: Induction of a CD86bright SSClow macrophage population by H27K15 Upper panels : Dot plots showing CD86 staining (X~axis) and side scatter (SSC, y~axis) of macrophages from donor 3 differentiated for 6 days in the ce of H27K15 (left) or negative control rituximab (right). A gate was set on the CD86bright SSClm’cell population induced by H27K15. Lower panel Percentages of CD86b’ight sschm cells with the test compounds at 10, 1 or 0.1 ug/ml* were compared with those in the corresponding negative controls : H27K15 vs rituximab, 827K15— derived F(ab’)2 vs rituximab~derived F(ab’)2, GW2580 vs no treatment. Percentages of increase in the CD86bright SSCLow cell population were calculated as : 100 x percentage of CD86bright SSClow cells with test compound / percentage of CD86bright SSClow cells with control. Shown are the mean tages of increase in the the cpsobright sso10w cell population from the 3 blood donors. *except for F(ab’)2: 6.6; 0.6; and 0.06 ug/ml.

Figure 4: H27K15 induces 70 secretion and ses macrophage IL—12/1L~10 ratios IL—12p70 and IL~lO were ed in the culture supernatants from day—6 macrophages differentiated in the presence of mAb H27K15 (1 ug/ml), GWZSBO (1 pm) or their respective negative controls rituximab or no treatment. Left panel : IL—12p70 and IL—10‘ levels (pg/ml) in macrophage cultures from 3 blood donors. Right : 70 (pg/ml) / IL—lO (pg/ml) ratios were calculated for each blood donor anti each culture condition. In samples where IL—12p70 was undetectable (below the detection limit of 11 pq/m)l, its level was arbitrarily set at lpg/ml for the calculation of IL—lZp7O / IL—lO ratios.

Figure 5: H27K15 inhibits MCP~l / CCLZ and IL~6 ion by macrophages MCP—l and IL—6 were ed in the culture supernatants from day—6 macrophages differentiated in the presence of mAb H27K15 (1 ug/ml), GW2580 (1 uM) or their respective negative controls rituximab or no treatment. tages of reduction in MCP—l (upper panel) or in IL-6 production (lower panel) were ated for the 3 blood donors as : 100 — [100 x cytokine concentration with test compound (pg/ml) / cytokine concentration with control (pg/ml)}.

Figure 6. Monocytes isolated from 3 ent blood donors were differentiated for‘ 6 days ill the presence (of GM~CSF and CSF—l, with or t mAb H27K15, Rituximab or GW2580. MAb H27K15 or Rituximab (0.1, 1 or 10 #g/ml) or equimolar concentrations of F(ab)’2 derived from both mAbs were added to the cultures. MMP—9 was titrated by ELISA (R&D Systems) in day~6 culture supernatants.

Figure 7 Cells obtained after 6—day culture with GM—CSF and CSF—l were harvested and incubated for 20 min at 4°C with PBS containing human IgG Fc fragments to saturate Fc ors.

Fluorochrome—conjugated mAbs (anti—CD14—PerCP—Cy5.5, anti~CD163— PE, anti-CD206-APC, anti—CDla—FITC, BD Biosciences) were then incubated with each sample for 20 min at 4°C. FCM analysis was med ‘using a FACS LSR—II (BD biosciences) with, the DIVA software. Data from 3 different blood donors are presented.

Figure 8 e (CDl4+CDl63—) / M2—type (CD163+CD14+) macrophage ratios among the CD14+ cell population. at day 6.

Monocytes from 2 different donors were cultured with GM-CSF and CSF—l in the presence or absence of D115 mAb H27K5 or control IgGl rituximab at 1 ug/ml. Ratios between M1 (CD14+CD163') and M2 (CD14+CD163+) —type macrophages among the macrophage population were determined after 6 days of cell differentiation.

Examples The following commercial monoclonal antibodies were used throughout the study: uman CD115 mAb 2—4A5—4 (rat IgGKK, Santa Cruz), 9—4D2 (rat IgGl, Biolegend) and isotype control rat IgGl (R&D Systems). mAb 1.2 SM is anti—CD115 mAb of sequence 1.2 SM published in patent application . mab was obtained from Roche. F(ab’)2 were produced at Transgene by pepsine digestion of the monoclonal antibodies, followed by cation by gel filtration.

Human macrophage differentiation assay: protocol Buffy coats were provided tn! the ssement Francais du Sang (EFS, Strasbourg). Peripheral blood mononucleated. cells (PBMC) were obtained by centrifugation on a ficoll gradient.

Monocytes were purified by immunomagnetic cell sorting, using CDl4—antibody—coated beads (Miltenyii). Enriched monocyte suspensions were more than 95% pure. Monocytes were differentiated for 6 days in 48-well plates (3 X 105 cells/well) in RPMI~GlutamaxTM medium mented with 10% heat inactivated fetal calf serum and 1% tri~antibiotic mixture (penicillin, streptomycin, neomycin). GM—CSF (10 ng/ml) was added in the cell culture medium from day 0 to day 3. H27K15, other antibodies or al control GW2580 (LC Labs) were added at day 0. On day 3 post—isolation, tes were washed with PBS and further cultivated in medium supplemented with CSF—l (10 ng/ml) and GM—CSF (2 , in the presence or absence of antibodies or GW2580. On day 6, supernatants were collected and stored at ~20°C. Cells were detached from the plastic and pools of triplicates were analyzed by IC/FC (immunocytochemistry / flow cytometry) for cell sizes and expression of FcyR and CD86. Cytokines and Chemokines were quantified in the culture supernatants by multiplex (Bioplex, Bio-Rad) or by ELISA.

For IC/FC analysis of hage cultures, pools of triplicates were centrifuged for 10 min at 1500 rpm and ted for 20 min at 4°C with PBS ning 10% human AB serum to saturate Fc receptors. Fluorochrome—conjugated nmbs anti~CD64nAPC and anti—CD86—AI700, anti—CD64, «CD86, —CD163 and/or —CDl4 (BD Biosciences) were then incubated with each sample for 20 min at 4°C. Cells were washed with PBS (5 min, 2000 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 the DIVA software for acquisition and the Flow Jo software for analysis.

Results Anti-CD115 mAb H27K15 is not cytotoxic to macrophages but polarizes their differentiation s the M1 type on3 To study whether H27K15 could affect macrophage entiation, purified CD14+ monocytes were cultured for 7 days in the of GMeCSF and CSF-l, known to induce presence respectively M1— and M2—type macrophages (Akaqawa K.S., 2002; doses of mab Verreck F.A. et al., 2004). Three ent H27K15 or isotype control rituximab (0.1 ; 1 or 10 ug/ml) were added to the e medium at the beginning of the culture H27K15 from and again 3 days later. F(ab’)2 generated from or rituximab were tested in parallel at equivalent molar mAb 1.2 SM trations. F(ab’)2 generated from (WO 2009/026303) comparison with the was tested for F(ab’)2 H27K15 to derived from or rituximab. The known blocking mAb human CD115, 2—4A5, or the ocking mAb 9—402 (Sherr C.J. isotype control at al., 1989) were assayed and compared with IgGl. As r, control, the small molecule CD115 rat tyrosine kinase inhibitor GW2580 was added to some of the cultures at l pm, a concentration previously shown to inhibit the CSF—l—dependent proliferation of human tes and the differentiation of murine macrophages in vitro (Conway J.G. et al., 2005; Paniagua R.T. et al., 2010).

Microscopical observation of day~6 cultures showed no obvious differences between wells treated with H27K15, Rituximab, mAb 9~4D2, IgGl, H27K15 F(ab’)2 or Rituximab F(ab’)2, compared with untreated cells, independently of the blood donor. For full the wells d with mAb SMl.2 F(ab’)2, cytotoxicity observed for all donors for the 3 doses evaluated (0.066, 0.66 and 6.6 ug/ml). For the wells treated with 2—4A5, full cytotoxicity was observed for all donors at the 2 highest doses tested (1 and 10 . For the lowest dose (0.1 ug/ml), cytotoxicity was only partial. Altogether, those results did not reveal any toxicity of any antibody except for mAb SMl.2 F(ab')2 and mAb 2—4A5. For the cells with the control GW2580 at 1 uM, depending on the microscope field, a relative cytotoxicity was visualized as debris and less density of cells was observed in all blood donors. Cell viability was analyzed at day 6 by counting 5 cope fields in each well of the plate (one in the center of well and four fields at mid~distance between the center and the side of the well). Based on the above observations, numeration was done also for wells treated with cmnpounds exhibiting partial or full cytotoxicity (mAb SMl.2 F(ab’)2, mAb 2—4A5 and GW2580). Figure 1 shows the means of 5 fields counted by individual well i standard deviation. Some antibodies exhibited partial or full cytotoxicity. Thus, the F(ab’)2 derived from mAb SMl.2 had induced massive cell death at all concentrations and mAb 2-4A5 also dramatically reduced macrophage numbers, in comparison with their respective controls. In the of GW2580, approximately 70% of the presence macrophage number ed at day 6 compared with untreated cultures.

Day 6 cultures were analyzed by IC/FC for surface sion of the ting FcyR CD64 (FcyRI) and. of the activation CD86. As shown on Figure 2, e expression of CD64 marker with was drastically d upon treatment with H27KlS or GWZSSO. H27K15 almost totally inhibited CD64 expression at all doses tested. Rat anti—human CD115 mAb 2e4A5 also decreased fashion. The surface CD64 expression, but in a dose—dependent 115 blocking mAb 9—4D2 did not modify CD64 expression level. Interestingly, F(ab’)g derived from H27K15 also down— regulated CD64 sion, but less potently than the full mAb and only when tested at l or 10 ug/ml, suggesting that the effect of H27K15 was partially endent.

When the expression of the activation marker / co—stimulatory molecule CD86 was analyzed in day—6 macrophages, we found that a sub~population of cells characterized by the ight SSClow phenotype had ed in the cultures treated with either mAb H27K15 or with GW2580 (Figure 3). Induction of this population of round—shaped cells expressing high levels of CD86 was not observed, with 827K15—derived F(ab’)2, suggesting a role for H27K15 Fc region in this phenomenon. Gating on this population showed that CD86bright SSClow cells were mainly CD64 to CD64— negative (data not shown).

IL~12p7O and IL—10 were ed in day—6 culture supernatants. Macrophages front the 23 donors tested. did not produce any detectable IL—12p70 following culture with human IgG1 rituximab or no reagent. Culture in the presence of mAb H27K15 induced IL—12p70 secretion by macrophages from 2 of the 3 blood donors. In contrast, lL-l2p70 was not detectable after treatment with GW258O (Figure 4). IL—lO, which was produced by resting hages from all , was up—regulated by H27K15 in macrophages from donor 1 but not in donor 2 and was only weakly increased in donor 3. As a result, IL-12p70 / IL— ratios were uperegulated, in the 3 blood donors tested (Figure 4, right panel). Small molecule GW2580 also increased IL—12p70 / IL—lO ratios, but rather due to the inhibition of IL-10 production.

These results show that targeting CD115 with mAb H27K15 on differentiating hages not only dramatically down— regulates the expression of CD64 / FcyRI, but also induces a tion of SSClow cells expressing high levels of the CD86 tion marker. In addition, H27K15 can induce IL—12p70 production and upregulates IL~12p7O / IL—lO ratios in all donors, indicative of macrophage polarization towards Ml—type.

Strikingly, production of the chemokine MCP—l / CCL2 was found to be almost totally suppressed when macrophages were differentiated in the presence of mAb H27K15 or GW2580 (Figure , upper panel). Inhibition of MCP—l secretion by H27Kl5 was effective in the 3 donors tested and ranged from 74 % to 99 %.

Small molecule GW2580 was as efficacious as H27K15, in suppressing MCP—l production. Levels of IL—6 in day—6 macrophage culture supernatants were also reduced by either H27K15 or GW2580 in all donors (Figure 5, lower panel).

Inhibition of IL—6 production was less drastic with H27K15 (from 27 % to 70 %) than with GW2580 (from 75 to 96%). H27K15— mediated inhibition of macrophage MCP~l and IL—6 production (Roca H. et al., 2009), two soluble factors implicated in M2 macrophage polarization, is another piece of evidence indicating that the anti~CD115 mAb repolarizes macrophages towards Ml.

Anti—CD115 mAb H27K15 inhibits MMP—9 production by monocytes cultured with GM~CSF and CSF~l Tumor—associated hages are known to e MMP~9 2O (matrix—metalloprotease 9), which promotes both tumor cell asis by ing the extracellular matrix and iogenesis by inducing VEGF release in the tumor microenvironment. MMPe9 produced by macrophages is a major regulator of the angiogenic switch in tumors.

CD14+ monocytes from 3 different donors were allowed to differentiate in the presence of both GM—CSF and CSF—l, known to induce macrophage differentiation s respectively the M1— and MZ—types. MAb H27K15 or Rituximab (used as a negative control) were added to the es at 0.1, l or 10 ug/ml.

Equimolar concentrations of F(ab)'2 derived from both mAbs were assayed in parallel. The tyrosine kinase tor GW2580, previously shown to t the CSF—ledependent proliferation of human monocytes and the differentiation of murine macrophages in Vitro was tested iii the same assay; After 6 days of culture, MMP—9 concentrations were measured in the supernatants by ELISA (Figure 6). In 2 out of 3 donors tested, there was a dose—dependent decrease in MMP—9 production when es were treated with mAb H27K15 or with GW2580. With F(ab)'2 derived from H27K15, there was an inhibitory effect in the same 2 donors. Thus, mAb H27K15 is capable of sing MMP~9 secretion by differentiating M2 macrophages.

These observations in hage cultures suggest that H27K15 administered to cancer patients may down—regulate MMP~9 concentration in the tumor microenvironment.

MAb H27K15 inhibits the differentiation of positive M2— type macrophages The hemoglobin scavenger receptor (CD163) has been identified as a marker of arized macrophages which is expressed by TAMs, notably‘ in breast cancer. The surface expression of CD163 was analyzed by flow cytometry’ in day—6 macrophages derived from human monocytes cultured with GM—CSF and CSF—l.

Figure 7 shows the percentages of CD163¢positive cells in differentiated cultures. Culture with mAb H27K15 inhibited the differentiation of the CD163+ hage population 311 all 3 donors tested. In the presence of l ug/ml H27K15, percentages of CD163—positive cells decreased from 2.5 to 4 folds compared with l cultures treated with rituximab. GW2580 had the same effect in only 2/3 donors. F(ab)’2 derived frmn H27K15 had weak or no effect on CD163 expression, indicating that the Fc region of the anti~CDllS mAb was involved in its mode of action.

As evidenced by these changes in surface CD163 expression and in ent with the previous s, targeting CD115 with mAb H27K15 inhibits the differentiation of MZ—type macrophages.

MAb H27K15 skews monocyte differentiation from M2 to M1 macrophages The ratios between M1 and M2 macrophages were analyzeci in cells derived from monocytes cultured with GM—CSF and CSF—l, in the presence or absence of mAb H27K15 or rituximab. After a 6~days culture of cells fronx 2 different blood donors, Ml (CDl4+CDl63“) versus M2 (CDl4+CDl63+) macrophages were quantified by flow cytometry. Figure 8 shows the Ml/MZ macrophage ratios calculated among the CD14+ hage population for each e ion. MAb H27K15 at l ug/ml increased Ml/M2 ratios in macrophages from the 2 donors tested, compared to control IgG1 rituximab at the same concentration or to untreated cultures.

Akagawa K.S. "Functional geneity of colony—stimulating —induced human monocyte-derived macrophages.” International journal of hematology. (2002) 76(1): 27—34.

Conway J.G., McDonald 8., Parham J., Keith 8., Rusnak D.W., Shaw E., Jansen M., Lin P., Payne A., Crosby R.M., Johnson J.H., Erick L., Lin M.H., Depee 8., Tadepalli S., Votta 8., James 1., Fuller K., Chambers T.J., Kull F.C., Chamberlain S.D. and Hutchins J.T. "Inhibition of colony~ stimulating—factor~l signaling i11 vivo with time orally bioavailable CFMS kinase inhibitor GW2580." Proceedings of the National Academy of Sciences of the United States of America. (2005) ): 16078—16083.

Paniagua R.T., Chang A., o M.M., Stein E.A., Wang Q., Lindstrom T.M., Sharpe 0., Roscow 0., Ho P.P., Lee D.M. and Robinson W.H. "oeFms~mediated differentiation and priming of monocyte lineage cells play a central role in autoimmune arthritis " Arthritis research & therapy. (2010) 12(1): R32.

Roca H., Varsos 2.8., Sud 8., Craig M.J., Ying C. and Pienta K.J. "CCLZ and interleukin—6 promote survival of human CDllb+ peripheral blood mononuclear cells and induce M2~ type macrophage polarization." The deurnal of biological 40 try. (2009) 284(49): 34342~34354.

Sherr C.J., Ashmun R.A., Downing J.R., Ohtsuka M., Quan 8.6., Golde D.W. and Roussel M.F. ”Inhibition of colony— stimulating factor—1 activity by monoclonal antibodies to the human_ CSF—l receptor." Blood. (1989) 73(7): l786~ 1793.

Verreck F.A., de Boer T., Langenberg D.M., Hoeve M.A., Kramer M., Vaisberg E., Kastelein R., Kolk A., de Waal—Malefyt R. and Ottenhoff T.H. "Human producing type 1 macrophages promote but IL—lO—producinq type 2 macrophages subvert ty to (myco)bacteria." Proceedings of the National Academy of Sciences of the United States of America. (2004) ): 4560—4565.

Claims (14)

WHAT IS CLAIMED IS:

1. Use of an effective amount of an antibody able to bind to CSF—lR in the manufacture of a medicament for treating a condition associated with undesirable M2 activation in a patient, wherein administration. of the medicament increases the M1 macrophage pool in said. patient and wherein said antibody able to bind to CSF—lR is an antibody that binds to at least one epitope located n on amino acids 20 to 41 of SEQ ID NO:23. 10 2.

The use of claim 1, wherein patient is r suffering from conditions ated with CSF—lR activity.

The use of claim 1 or 2, wherein said medicament ses the macrophage M2 pool.

The use of any one of the preceding claims, wherein said 15 medicament ts macrophage MCP—l, ILlO and IL—6 production.

The use of any one of the ing claims, wherein said medicament down regulates surface chRI (CD64) and FcyRIII (CD16) expression on macrophages. 20 6.

The use of any one of the preceding claims, wherein said medicament promotes IL—12 production by macrophages.

The use of any one of the preceding claims, wherein said medicament reduces at least one of the following: (i) TAM recruitment into tumor; 25 (ii) at least one macrophage pro—tumoral function; (iii) tumor angiogenesis; (iv) tumor invasion and metastasis; (V) tumor growth; (vi) tumor cell proliferation

8. The use of any one of the preceding claims, wherein said conditions ated with undesirable M2 activation are selected. from the group consisting of cancer, asthma, allergy and progressive fibrosis diseases. 5

9. The use of any one of the preceding claims, wherein said antibody able to bind to CSF—lR is an antibody that binds to one e located between position amino acids 20 to 39 of SEQ ID NO:23, to amino acids Asn72, Ser94—Ala95— Ala96, LyleZ, AsplBl~Prol32~Vall33 and Trp159 of SEQ ID 10 NO:23.

10. The use of any one of the preceding , wherein said antibody able to bind to human CSF—lR is an antibody that does not compete with IL-34 ligand for binding to the CSF—lR receptor. 15

11. The use of any one of the preceding claims, wherein said dy able to bind to human CSF-lR is an dy that competes partially with CSF-l ligand for binding to the CSF—lR receptor.

12. The use of any one of the preceding claims, wherein said 20 antibody able to bind to human CSF—lR is an antibody that binds specifically to human CSF—lR, comprising: (a) a first variable region. being defined by the following formula FRl — CDRl - FR2 — CDR2 — FR3 — CDR3 — FR4 25 n: FRl, FR2, FR3 and FR4 are each framework regions; CDRl, CDR2 and CDR3 are each complementarity determining regions; wherein: 30 CDRl has at least five consecutive amino acids of the sequence starting in position 45 and finishing in position 54 of SEQ ID NO:1; CDR2 has at least five consecutive amino acids of the sequence starting in position 66 and finishing in position 87 of SEQ ID NO:1; CDR3 has at least five consecutive amino acids of the sequence starting in. position 117 and finishing in position 126 of SEQ ID NO:1; 10 and (b) a second variable region being defined by the following formula FRl — CDRl — FR2 — CDR2 — FR3 — CDR3 — FR4 wherein: 15 FRl, FR2, FR3 and FR4 are each ork regions; CDRl, CDR2 and CDR3 are each complementarity determining s; wherein: CDRl has at least five consecutive amino 20 acids of the sequence starting in position 44 and finishing in position 56 of SEQ ID NO:2; CDR2 has at least five utive amino acids of the sequence starting in position 66 and finishing in position 76 of SEQ ID NO:2; 25 and CDR3 has at least five consecutive amino acids of the sequence ng in position 109 and finishing in position 117 of SEQ ID NO:2.

13. The use of any one of the preceding claims, wherein said 30 antibody able to bind to CSF-lR comprises (a) an heavy chain consisting in SEQ ID NO:24, and (b) a light chain consisting in SEQ ID NO:28.

14. The use of claim 1, ntially' as herein described with reference to any one of the Examples and/or