Anti-CSF-1R antibody

Abstract

The present invention relates to a monoclonal antibody or a fragment thereof that binds to CSF-1R (colony-stimulating factor 1 receptor), in particular human CSF-1R. The present invention further relates to the in vitro use of the monoclonal antibody or a fragment thereof of the present invention for the detection of CSF-1R in a sample. The present invention also encompasses a complex comprising the monoclonal antibody or a fragment thereof of the present invention and CSF-1R, such as a human CSF-1R polypeptide.

Description

Technical Field

[0001] The present invention relates to monoclonal antibodies or fragments thereof that bind to CSF-1R ( C olony s timulating f actor 1 r eceptor: colony stimulating factor 1 receptor), particularly human CSF-1R. The present invention further relates to the in vitro use of the monoclonal antibodies or fragments thereof of the present invention for the detection of CSF-1R in a sample. Complexes comprising the monoclonal antibodies or fragments thereof of the present invention and CSF-1R such as human CSF-1R polypeptide are further encompassed by the present invention.

Background Art

[0002] The human CSF-1 receptor (CSF-1R; colony stimulating factor 1 receptor) is a growth factor and is encoded by the c-fms proto-oncogene (reviewed, for example, in Roth, P., and Stanley, E.R., Curr. Top. Microbiol. Immunol. 181 (1992) 141-167).

[0003] Currently, two CSF-1R ligands that bind to the extracellular domain of CSF-1R are known. The first is CSF-1 (colony stimulating factor 1), which is found extracellularly as a disulfide-bonded homodimer (Stanley et al., Stem Cells 12 Suppl. 1 (1995) 15-24). The second is IL-34 (Hume et al., Blood 119 (2012) 1810-1820). The main biological effects of CSF-1R signaling are the differentiation, proliferation, migration, and survival of hematopoietic progenitor cells into macrophage-lineage strains. Activation of CSF-1R is mediated by its CSF-1R ligands, CSF-1 and IL-34. Binding of CSF-1 to CSF-1R induces homodimer formation and activation of the kinase by tyrosine phosphorylation (Stanley et al., Mol. Reprod. Dev. 46 (1997) 4-10).

[0004] CSF-1R is associated with many diseases and disorders (see, e.g., Cannarile, M.A., et al. (2017), J Immunother Cancer 5(1):53, Ries, C.H., et al. (2014), Cancer Cell 25(6):846-859). For example, CSF-1R plays an important role in the initiation of inflammatory, cancer, and bone disorders. Accordingly, many CSF-1R inhibitors (such as small molecule inhibitors and monoclonal antibodies) have been described and are being analyzed in clinical trials (reviewed by El-Gamal et al., J Med Chem. 2018, 61(13):5450-5466).

[0005] Anti-CSF-1R antibodies are disclosed, for example, in International Publication No. WO 2011 / 123381, International Publication No. WO 2011 / 140249, and International Publication No. WO 2012 / 110360.

[0006] The extracellular domain of the CSF-1 receptor (CSF-1R-ECD) contains five subdomains (D1-D5). Biologically active homodimeric CSF-1 binds to CSF-1R within subdomains D1-D3. Subdomains D4-D5 of the extracellular domain are not involved in CSF-1 binding (Wang et al., Molecular and Cellular Biology 13(1993)5348-5359). Subdomain D4 is involved in dimerization (Pixley et al., Trends Cell Biol. 14(2004)628-638).

[0007] Many therapeutic candidate anti-CSF-1R antibodies bind to the extracellular domain of the CSF-1 receptor (CSF-1R-ECD). For example, International Publication No. WO 2009 / 026303 and International Publication No. WO 2009 / 112245 disclose anti-CSF-1R antibodies that bind to CSF-1R within the first three subdomains (D1-D3) of the extracellular domain. International Publication No. WO 2011 / 070024 discloses anti-CSF-1R antibodies that bind to CSF-1R within the dimerization domain (D4-D5).

[0008] In addition to antibodies that bind to the extracellular domain of the CSF-1 receptor, antibodies that bind to other domains such as antibodies that bind to the CSF-1R kinase domain are described. For example, polyclonal antibodies that bind to phosphorylation sites of CSF-1R are commercially available from Thermo Fisher, such as phospho-CSF1R(Tyr561), phospho-CSF1R(Tyr699), phospho-CSF1R(Tyr708), phospho-CSF1R(Tyr723), phospho-CSF1R(Tyr809), phospho-CSF1R(Tyr921), etc. The polyclonal antibodies were generated by using a phosphopeptide chemically synthesized as an immunogen.

[0009] Furthermore, monoclonal antibodies were prepared by immunizing rabbits with a synthetic phosphopeptide corresponding to the residues surrounding Tyr708 of the human CSF-1R receptor protein. An antibody called "Phospho-M-CSF Receptor (Tyr708)(D5F4Y) Rabbit mAb #14591" is commercially available from Bioke (Leiden, Netherlands). The antibody recognizes the endogenous level of CSF-1R only when phosphorylated at Tyr708, but can cross-react with other activated protein tyrosine kinases such as phospho-Src. Therefore, the antibody may not be suitable for immunohistochemistry (IHC).

[0010] WO 2012 / 150320 describes a scaffold technology for the production of antibodies. This document describes a fusion polypeptide comprising one or more fragments of a peptidyl-prolyl cis / trans isomerase or FKBP domain family member, and its use in methods for antibody screening / selection, its use for epitope mapping, and its use as an immunogen for the production of antibodies that specifically bind to an immunogenic peptide or secondary structure presented by the fusion polypeptide. WO 2015 / 044083 discloses that the Thermus thermophilus SlyD FKBP domain is suitable for a fusion polypeptide for generating antibodies.

[0011] The inventors transplanted a fragment of the kinase domain of human CSF-1R onto the Thermus thermophilus SlyD FKBP scaffold. The resulting fusion polypeptide was used as an immunogen for the production of monoclonal antibodies in rabbits. The antibodies were shown to exhibit high antibody / receptor affinity and specificity for CSF-1R, particularly for an antibody designated "1H11". Since the antibodies exhibited excellent immunohistochemical performance, they could be applied to clinical trials using small molecules or antibodies targeting CSF-1R. Surprisingly, the antibodies were able to detect both native and denatured CSF-1R.

Summary of the Invention

[0012] The present invention relates to a monoclonal antibody or a fragment thereof that binds to CSF-1R ( C olony s timulating f actor 1 r eceptor: colony stimulating factor 1 receptor), particularly human CSF-1R.

[0013] In one embodiment of the present invention, the monoclonal antibody or a fragment thereof comprises a light chain variable domain that is at least 85% identical to the light chain variable domain having the sequence shown in SEQ ID NO: 2, and / or the monoclonal antibody or a fragment thereof comprises a heavy chain variable domain that is at least 85% identical to the heavy chain variable domain having the sequence shown in SEQ ID NO: 3.

[0014] In one embodiment of the present invention, the monoclonal antibody or a fragment thereof (a) comprises a light chain variable domain including: (a1) a light chain CDR1 that differs from the light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS) by a total of 3 or fewer amino acid additions, substitutions, and / or deletions, (a2) A light chain CDR2 having an amino acid sequence shown in SEQ ID NO: 5 (EASKVAS) and differing from it by addition, substitution and / or deletion of a total of 3 or fewer amino acids, and / or (a3) A light chain CDR3 having an amino acid sequence shown in SEQ ID NO: 6 (AGGYDVSDDA) and differing from it by addition, substitution and / or deletion of a total of 3 or fewer amino acids, (b) A heavy chain variable domain comprising: (b1) A heavy chain CDR1 having an amino acid sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT) and differing from it by addition, substitution and / or deletion of a total of 3 or fewer amino acids, (b2) A heavy chain CDR2 having an amino acid sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG) and differing from it by addition, substitution and / or deletion of a total of 3 or fewer amino acids, and / or (b3) A heavy chain CDR3 having an amino acid sequence shown in SEQ ID NO: 9 (GGQNNGYDL) and differing from it by addition, substitution and / or deletion of a total of 3 or fewer amino acids, Or (c) Both the light chain variable domain defined in (a) and the heavy chain variable domain defined in (b).

[0015] In one embodiment of the monoclonal antibody or fragment thereof of the present invention, the epitope of the monoclonal antibody or fragment thereof comprises the sequence shown in SEQ ID NO: 1 (YKNIHLEKKY). Preferably, at least one, particularly both, of the two tyrosine residues of the epitope are phosphorylated.

[0016] In one embodiment, the monoclonal antibody or fragment thereof (a) A light chain variable domain comprising: (a1) A light chain CDR1 having the sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS), (a2) A light chain CDR2 having the sequence shown in SEQ ID NO: 5 (EASKVAS), and (a3) A light chain CDR3 having the sequence shown in SEQ ID NO: 6 (AGGYDVSDDA), and (b) a heavy chain variable domain comprising: (b1) a heavy chain CDR1 having the sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT), (b2) a heavy chain CDR2 having the sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG), and (b3) a heavy chain CDR3 having the sequence shown in SEQ ID NO: 9 (GGQNNGYDL).

[0017] The present invention further relates to the in vitro use of any one of the monoclonal antibodies or fragments thereof of the present invention for the detection of CSF-1R in a sample.

[0018] The present invention further relates to a method for detecting CSF-1R in a sample, comprising: (a) contacting a sample containing CSF-1R with a monoclonal antibody or fragment thereof of the present invention to thereby form a complex comprising CSF-1R and the monoclonal antibody or fragment thereof, and (b) detecting the complex formed in step (a) to thereby detect CSF-1R in the sample.

[0019] The present invention further contemplates a complex comprising a monoclonal antibody or fragment thereof of the present invention and CSF-1R such as a human CSF-1R polypeptide. The drawings show the following: BRIEF DESCRIPTION OF THE DRAWINGS

[0020]

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[0021] As described above, the present invention relates to a monoclonal antibody or a fragment thereof that binds to CSF-1R ( C olony s timulating f actor 1 r eceptor: colony stimulating factor 1 receptor), particularly the human CSF-1R.

[0022] CSF-1R is a polypeptide belonging to class III subfamily of receptor tyrosine kinases and is encoded by the c-fms proto-oncogene. Synonyms of CSF-1R are macrophage colony-stimulating factor 1 receptor, which is the M-CSF receptor, Fms proto-oncogene and c-fms. Binding of CSF-1 or IL-34 induces receptor dimerization, followed by autophosphorylation and activation of downstream signaling cascades. Activation of CSF-IR regulates survival, proliferation and differentiation of monocytes and macrophages (Xiong, Y. et al., J. Biol. Chem. 286 (2011) 952-960). Preferably, the monoclonal antibody (or antigen-binding fragment thereof) of the present invention binds to the human CSF-1R polypeptide. Human CSF-IR has been known since 1986 (Coussens, L., et al, Nature 320 (1986) 277-280). Cloning of the receptor was first described in Roussel, M.F., et al., Nature 325 (1987) 549-552. CSF-1R is a single-pass transmembrane receptor tyrosine kinase (RTK) and is a member of the family of immunoglobulin (Ig) motifs containing an RTK characterized by five repeated Ig-like subdomains D1-D5 within the extracellular domain (ECD) of the receptor (Wang, Z., et al Molecular and Cellular Biology 13 (1993) 5348-5359). The intracellular domain of CSF-1R includes, among others, the kinase domain of CSF-1R.

[0023] The amino acid sequence of full-length human CSF-1R is as follows (SEQ ID NO: 10): MGPGVLLLLLVATAWHGQG IPVIEPSVPELVVKPGATVTLRCVGNGSVEWDGPPSPHWTLYSDGSSSILSTNNATFQNTGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVVVFEDQDALLPCLLTDPVLEAGVSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQSQDYQCSALMGGRKVMSISIRLKVQKVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDFHNNRYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFFRVVESAYLNLSSEQNLIQEVTVGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTYRHTFTLSLPRLKPSEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWLQCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTVQSLLTVETLEHNQTYECRAHNSVGSGSWAFIPISAGAHTHPPDEFLFTPVVVACMSIMALLLLLLLLLLYKYKQKPKYQVRWKIIESYEGNSYTFIDPTQLPYNEKWEFPRNNLQFGKTLGAGAFGKVVEATAFGLGKEDAVLKVAVKMLKSTAHADEKEALMSELKIMSHLGQHENIVNLLGACTHGGPVLVITEYCCYGDLLNFLRRKAEAMLGPSLSPGQDPEGGVDYKNIHLEKKYVRRDSGFSSQGVDTYVEMRPVSTSSNDSFSEQDLDKEDGRPLELRDLLHFSSQVAQGMAFLASKNCIHRDVAARNVLLTNGHVAKIGDFGLARDIMNDSNYIVKGNARLPVKWMAPESIFDCVYTVQSDVWSYGILLWEIFSLGLNPYPGILVNSKFYKLVKDGYQMAQPAFAPKNIYSIMQACWALEPTHRPTFQQICSFLQEQAQEDRRERDYTNLPSSSRSGGSGSSSSELEEESSSHLTCCEQGDIAQPLLQPNNYQFC

[0024] In the above sequence, it is drawn to the extracellular domain of human CSF-1R Underline and the intracellular domain is shown in italics. The first 19 amino acids (signal peptide) are cleaved after translation to form the mature form of the CSF-1R polypeptide. The epitope regions of the antibodies identified in the studies underlying the present invention are shown in bold. The sequence of the epitope is also shown in SEQ ID NO: 1 (YKNIHLEKKY). As described in more detail below herein, the tyrosine residue at position 699 and / or the tyrosine residue at position 708 is phosphorylated.

[0025] Advantageously, the antibodies of the present invention bind to both the native form, i.e., the non-denatured form, of CSF-1R and the denatured form of CSF-1R. Accordingly, the term "CSF-1R" includes native CSF-1R and denatured CSF-1.

[0026] The antibody or antigen-binding fragment of the present invention is assumed to have the following sequence: Preferably, the monoclonal antibody or fragment thereof that binds to CSF-1R (colony-stimulating factor 1 receptor) comprises a light chain variable domain having a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 2, and / or a heavy chain variable domain having a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 3, in that order. In particular, the monoclonal antibody or fragment thereof comprises a light chain variable domain having a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 2, and a heavy chain variable domain having a sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 3, in that order. For example, the monoclonal antibody or fragment thereof comprises a light chain variable domain having a sequence at least 85% identical to the sequence shown in SEQ ID NO: 2, and a heavy chain variable domain having a sequence at least 85% identical to the sequence shown in SEQ ID NO: 3, in that order.

[0027] In one aspect, the monoclonal antibody comprises a light chain variable domain having the sequence shown in SEQ ID NO: 2 and a heavy chain variable domain having the sequence shown in SEQ ID NO: 3. The sequence of the light chain variable region is as follows (SEQ ID NO: 2): AAVLTQTPSPVSAAVGGTVTISCQSSESVYSNNFLSWYQLKPGQRPRLLIYEASKVASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCAGGYDVSDDAFGGGTEVLVK The sequence of the heavy chain variable region is as follows (SEQ ID NO: 3): QSVEESGGRLVTPGTPLTLTCTASGFSLSRYWMTWVRQAPGKGLEYIGWIDRSGNTYFADWAKGRFTGSKTSTTRDLKITSPTTEDTATYFCGRGGQNNGYDLWGPGTLVTVSS

[0028] Alternatively or additionally, the monoclonal antibody or fragment thereof of the present invention (a) A light chain variable domain comprising: (a1) A light chain CDR1 that differs from the light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS) by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, (a2) A light chain CDR2 that differs from the light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5 (EASKVAS) by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, and / or (a3) A light chain CDR3 that differs from the light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6 (AGGYDVSDDA) by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, and / or (b) A heavy chain variable domain comprising: (b1) A heavy chain CDR1 that differs from the heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT) by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, (b2) A heavy chain CDR2 that differs from the heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG) by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, and / or (b3) A heavy chain CDR3 that differs from the heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 9 (GGQNNGYDL) by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, or (c) Both the light chain variable domain defined in (a) and the heavy chain variable domain defined in (b).

[0029] Alternatively or additionally, the monoclonal antibody or fragment thereof of the present invention (a) A light chain variable domain comprising: (a1) A light chain CDR1 having an amino acid sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS) and differing from it by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, (a2) A light chain CDR2 having an amino acid sequence shown in SEQ ID NO: 5 (EASKVAS) and differing from it by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, (a3) A light chain CDR3 having an amino acid sequence shown in SEQ ID NO: 6 (AGGYDVSDDA) and differing from it by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, and (b) A heavy chain variable domain comprising: (b1) A heavy chain CDR1 having an amino acid sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT) and differing from it by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, (b2) A heavy chain CDR2 having an amino acid sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG) and differing from it by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, (b3) A heavy chain CDR3 having an amino acid sequence shown in SEQ ID NO: 9 (GGQNNGYDL) and differing from it by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions.

[0030] In one aspect, the monoclonal antibody or fragment thereof of the present invention is (a) A light chain variable domain comprising: (a1) A light chain CDR1 comprising at least 11, particularly at least 12 consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS), (a2) A light chain CDR2 comprising at least 5, particularly at least 6 consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 5 (EASKVAS), and / or (a3) A light chain CDR3 comprising at least 8, particularly at least 9, consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 6 (AGGYDVSDDA), and / or (b) A heavy chain variable domain comprising: (b1) A heavy chain CDR1 comprising at least 11, particularly at least 12, consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT), (b2) A heavy chain CDR2 comprising at least 11, particularly at least 12, consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG), and / or (b3) A heavy chain CDR3 comprising at least 7, particularly at least 8, consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 9 (GGQNNGYDL).

[0031] In one aspect, the monoclonal antibody or fragment thereof of the present invention (a) A light chain variable domain comprising: (a1) A light chain CDR1 having the sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS), (a2) A light chain CDR2 having the sequence shown in SEQ ID NO: 5 (EASKVAS), and / or (a3) A light chain CDR3 having the sequence shown in SEQ ID NO: 6 (AGGYDVSDDA), and / or (b) A heavy chain variable domain comprising: (b1) A heavy chain CDR1 having the sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT), (b2) A heavy chain CDR2 having the sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG), and / or (b3) A heavy chain CDR3 having the sequence shown in SEQ ID NO: 9 (GGQNNGYDL).

[0032] In one aspect, the monoclonal antibody or fragment thereof of the present invention (a) A light chain variable domain comprising: (a1) A light chain CDR1 having the sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS), and (a2) A light chain CDR2 having the sequence shown in SEQ ID NO: 5 (EASKVAS), (a3) A light chain CDR3 having the sequence shown in SEQ ID NO: 6 (AGGYDVSDDA), and (b) A heavy chain variable domain comprising: (b1) A heavy chain CDR1 having the sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT), (b2) A heavy chain CDR2 having the sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG), (b3) A heavy chain CDR3 having the sequence shown in SEQ ID NO: 9 (GGQNNGYDL).

[0033] Also, a monoclonal antibody or a fragment thereof that binds to CSF-1R (colony-stimulating factor 1 receptor) preferably comprises a light chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to the light chain variable domain having the sequence shown in SEQ ID NO: 2, and / or a heavy chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to the heavy chain variable domain having the sequence shown in SEQ ID NO: 3. The light chain variable domain (a1) A light chain CDR1 that differs from the light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS) by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, (a2) A light chain CDR2 that differs from the light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5 (EASKVAS) by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, (a3) A light chain CDR3 that differs from the light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6 (AGGYDVSDDA) by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, and the heavy chain variable domain (b1) a heavy chain CDR1 having an amino acid sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT), which differs from the heavy chain CDR1 by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, (b2) a heavy chain CDR2 having an amino acid sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG), which differs from the heavy chain CDR2 by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, (b3) a heavy chain CDR3 having an amino acid sequence shown in SEQ ID NO: 9 (GGQNNGYDL), which differs from the heavy chain CDR3 by a total of 3, 2, or particularly 1 or fewer amino acid additions, substitutions, and / or deletions, and

[0034] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after the sequences are aligned to achieve the maximum percent sequence identity and gaps are introduced as needed. Preferably, standard parameters are applied to determine the degree of sequence identity between two sequences. Preferably, the degree of identity should be determined by comparing two optimally aligned sequences over a comparison window, and the fragment of the amino acid sequence within the comparison window may include additions or deletions (e.g., gaps or overhangs) compared to the reference sequence (without additions or deletions) for optimal alignment. The percentage is calculated by determining the number of positions at which identical amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions within the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. The optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman Add.APL.Math.2:482 (1981), the homology alignment algorithm of Needleman and Wunsch J.Mol.Biol.48:443 (1970), the similarity search method of Pearson and Lipman Proc.Natl.Acad.Sci.(USA) 85:2444 (1988), computer implementations of these algorithms (the software packages of the Wisconsin Genetics Software Package (Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin), GAP, BESTFIT, BLAST, PASTA, and TFASTA), or by visual inspection. Considering that two sequences have been identified for comparison, it is preferred to use GAP and BESTFIT to determine their optimal alignment and thus the degree of identity. Preferably, default values of 5.00 for gap weight and 0.30 for gap weight length are used.In one embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm (Needleman 1970, J. Mol. Biol. (48):444-453), the BLOSUM62 scoring matrix, and a gap opening penalty of 10 and a gap extension penalty of 0.5, which are incorporated into the needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice, P., Longden, I., and Bleasby, A., Trends in Genetics 16(6), 276-277, 2000). Preferred non-limiting examples of parameters used to align two amino acid sequences using the needle program are the default parameters including the EBLOSUM 62 scoring matrix, a gap opening penalty of 10, and a gap extension penalty of 0.5.

[0035] The term "antibody" refers to, for example, but not limited to, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, such as mammals including goats, rabbits, and mice, and immunoglobulins or immunoglobulin-like molecules including shark immunoglobulins. For example, an antibody can be a rabbit antibody. The term "antibody" includes intact immunoglobulins, as well as "antibody fragments" or "antigen-binding fragments" that specifically bind to a molecule of interest (or a group of molecules of interest that are very similar) while substantially excluding binding to other molecules. The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized mouse antibodies), heteroconjugate antibodies (e.g., bispecific antibodies), etc. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

[0036] In particular, the term "antibody" means a polypeptide ligand that specifically recognizes and binds to an epitope of an antigen and that includes at least light and heavy chain immunoglobulin variable regions. An antibody is composed of heavy and light chains, each of which has a variable region called the variable heavy (VH) region and the variable light (VL) region. Together, the VH region and the VL region are responsible for the binding of the antigen recognized by the antibody. Typically, the antibodies of the present invention have heavy (H) and light (L) chains interconnected by disulfide bonds. As used herein, the term "light chain" includes full-length light chains and fragments thereof having a variable region sequence sufficient to confer binding specificity. A full-length light chain includes the variable domain V L and the constant domain C L . The variable domain of the light chain is at the amino terminus of the polypeptide. Light chains include kappa and lambda chains. The term "heavy chain" includes full-length heavy chains and fragments thereof having a variable region sequence sufficient to confer binding specificity. A full-length heavy chain includes the variable domain V H , as well as three constant domain domains C H 1, C H 2, and C H 3, with the V H domain at the amino terminus of the polypeptide, the C H domain at the carboxyl terminus, and the C H 3 closest to the carboxyl terminus of the polypeptide.

[0037] There are five major heavy-chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains a constant region and a variable region (the regions are also known as "domains"). When combined, the heavy and light chain variable regions specifically bind an antigen. The light and heavy chain variable regions contain "framework" regions interrupted by three hypervariable regions, also called "complementary determining regions" or "CDRs". The CDRs are mainly responsible for binding to the epitope of the antigen. The CDRs of each chain are usually consecutively numbered, starting from the N-terminus, as CDR1, CDR2, and CDR3, and are also usually identified by the chain in which a particular CDR is located. Thus, VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while VL CDR1 is CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies that bind to CSF-1R have specific VH region and VL region sequences, and thus specific CDR sequences.

[0038] The antibody or fragment thereof of the present invention can be a single-chain antibody, IgD antibody, IgE antibody, IgM antibody, IgG antibody, and fragments thereof. For example, the antibody can be an IgG antibody, such as an IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody. In one embodiment, the antibody or fragment thereof is recombinantly produced.

[0039] Fragments of the monoclonal antibodies of the present invention are also encompassed by the present invention. The fragments shall be immunologically functional fragments, i.e., antigen-binding fragments. Therefore, fragments of the monoclonal antibodies of the present invention shall be capable of binding to CSF-1R such as human CSF-1R. Therefore, the term "immunologically functional fragment" of an antibody, as used herein, refers to a portion of an antibody that lacks at least a part of the amino acids present in the full-length chain but is capable of specifically binding to CSF-1R. Immunologically functional immunoglobulin fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments. Methods for producing antigen-binding fragments are well known in the art. For example, the fragments can be produced by enzymatic cleavage of the antibodies of the present invention. Further, the fragments can be generated by synthetic or recombinant techniques. Fab fragments are preferably produced by papain digestion of the antibody, Fab' fragments by pepsin digestion and partial reduction, and F(ab')2 fragments by pepsin digestion. Fv fragments are preferably produced by molecular biology techniques.

[0040] The antibody fragment may be a diabody, which is a small antibody fragment having two antigen-binding sites. A diabody preferably comprises a heavy-chain variable domain connected to a light-chain variable domain within the same polypeptide chain.

[0041] The antibodies of the present invention are preferably monoclonal antibodies. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of B lymphocytes or by cells transfected with the light-chain and heavy-chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by producing hybrid antibody-forming cells from the fusion of myeloma cells and immune spleen cells.

[0042] In one embodiment, the antibody of the present invention is an isolated antibody. Therefore, the antibody is a purified antibody. Purification of the antibody can be achieved by methods well known in the art, such as size exclusion chromatography (SEC). Therefore, the antibody is assumed to be isolated from the cells in which the antibody was produced. In some embodiments, the isolated antibody is purified to more than 70% by weight of the antibody, in some embodiments 80%, 90%, 95%, 96%, 97%, 98% or 99% by weight, as measured by, for example, the Lowry method. In certain preferred embodiments, the isolated antibody according to the present invention is purified to a purity of more than 90% as determined by SDS-PAGE under reducing conditions using Coomassie blue staining for protein detection.

[0043] The monoclonal antibody or fragment thereof of the present invention is assumed to specifically bind to a CSF-1R polypeptide, such as a human CSF-1R polypeptide. The expressions "bind" and "specifically bind" are well understood and are used to indicate that the antibody (or fragment thereof) does not significantly bind to other biomolecules.

[0044] Preferably, the antibody (or fragment thereof) of the present invention is assumed to specifically bind to the intracellular domain of the CSF-1R polypeptide, particularly the kinase domain. The epitope of the monoclonal antibody or fragment thereof preferably includes the sequence shown in SEQ ID NO: 1 (YKNIHLEKKY). SEQ ID NO: 1 corresponds to amino acids 699-708 of human CSF-1R (see SEQ ID NO: 10). Therefore, the monoclonal antibody or fragment thereof of the present invention is assumed to specifically bind to this region. Preferably, at least one of the two tyrosine residues of the above epitope, i.e., the tyrosine residue at position 699 or the tyrosine residue at position 708, is phosphorylated. More preferably, both tyrosine residues of the epitope, i.e., the tyrosine residue at position 699 and the tyrosine residue at position 708, are phosphorylated. Therefore, it is assumed that the antibody or fragment thereof of the present invention binds to a human CSF-1R polypeptide in which the tyrosine residue at position 699 and / or the tyrosine residue at position 708 is phosphorylated.

[0045] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. Thus, this term preferably refers to a portion of a CSF-1R polypeptide (e.g., a human CSF-1R polypeptide) that can be specifically bound by an antibody (or a fragment thereof) of the present invention. An epitope usually consists of a chemically active surface population of molecules such as amino acids and usually has specific charge characteristics along with specific three-dimensional structural characteristics. Conformational epitopes and non-conformational epitopes are distinguished in that in the presence of a denaturing solvent, the binding to the former is lost but the binding to the latter is not.

[0046] In a preferred embodiment, the antibody or a fragment thereof of the present invention is linked to a detectable label. The detectable label described herein is preferably a label that is not naturally linked to the antibody or an antigen-binding fragment thereof. Thus, the detectable label is preferably heterologous with respect to the antibody. Suitable labels are any labels that are detectable by an appropriate detection method. In one embodiment, the detectable label can be an enzyme, biotin, a radioactive label, a fluorescent label, a chemiluminescent label, an electrochemiluminescent label, a gold label, or a magnetic label.

[0047] Examples of enzyme labels include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and luciferase. Substrates for these enzymes are well known in the art. Examples of substrates suitable for detection include di-amino-benzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate). Depending on the appropriate enzyme-substrate combination, a colored reaction product, fluorescence, or chemiluminescence may occur, which can be measured according to methods known in the art. Examples of fluorescent labels include 5-carboxyfluorescein, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, Cy2, Cy3, and Cy5, fluorescent proteins such as GFP (green fluorescent protein), Texas Red, and Alexa dyes. Examples of radioactive labels include radioactive isotopes of iodide, cobalt, selenium, tritium, carbon, sulfur, and phosphorus. Radioactive labels can be detected by any known and appropriate method, such as a photographic film or a phosphor imager. Examples of magnetic labels include paramagnetic labels and superparamagnetic labels. Examples of chemiluminescent labels that can be used include luminol, isoluminol, aromatic acridinium esters, imidazole, acridinium salts, or oxalate esters.

[0048] The above definitions and explanations in this specification preferably apply mutatis mutandis to the following.

[0049] The antibody or antigen-binding fragment thereof of the present invention is useful in methods for the localization and / or quantification of CSF-1R polypeptide. For example, the antibody or its fragment enables determination of the amount of CSF-1R polypeptide in a sample, use in diagnostic methods, or imaging of the CSF-1R polypeptide.

[0050] Accordingly, the present invention provides a method for detecting CSF-1R in a sample, comprising (a) Contacting a sample containing CSF-1R with any monoclonal antibody or fragment thereof of the present invention, thereby forming a complex comprising CSF-1R and the monoclonal antibody or fragment thereof, and (b) Detecting CSF-1R in the sample by detecting the complex formed in step (a).

[0051] As used herein, a "subject" is preferably a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, the subject is a mammalian subject such as a human subject. In some embodiments of the above method, the sample is obtained from a subject diagnosed with cancer, suspected of having cancer, or at risk of having cancer.

[0052] As used herein, the term "sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject. Biological samples of the present disclosure include, but are not limited to, for example, whole blood, plasma, semen, saliva, tears, urine, fecal matter, sweat, buccal, skin, cerebrospinal fluid, and hair. Biological samples can also be obtained from biopsies of internal organs or cancers.

[0053] In one embodiment, the sample contains cancer cells. Cancer cells are cells that have undergone malignant transformation that makes them pathological for the patient. As used herein, the term "cancer cells" includes not only primary cancer cells but also any cells derived from the ancestors of cancer cells. This includes metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells.

[0054] Preferably, the sample containing cancer cells is a biopsy tissue containing cancer cells. Thus, the sample is obtained from a subject suffering from cancer.

[0055] According to the present invention, the sample is obtained from a subject who has been contacted with a candidate compound for the treatment of cancer. Therefore, the candidate compound has been administered to a subject, particularly a subject suffering from cancer, before obtaining the sample to be tested. Alternatively, the sample is contacted with a candidate compound for the treatment of cancer.

[0056] In one embodiment, the candidate compound for treating cancer is an inhibitor of CSF-1R, such as a small molecule inhibitor, or a monoclonal antibody that binds to CSF-1R, such as a monoclonal antibody that binds to the extracellular domain of CSF-1R.

[0057] In one embodiment, the inhibitor of CSF-1R is selected from cediranib, PLX7486, ARRY-382, JNJ-40346527, BLZ945, emactuzumab, AMG820, and IMC-CS4 (see Cannarile et al., 2017 Journal for Immunotherapy of Cancer. 5(1):53).

[0058] In one embodiment of the above method, the detection of CSF-1R is a quantitative detection of CSF-1R. Therefore, the amount of CSF-1R in the sample is determined. The determined amount of CSF-1R can be compared with the amount of CSF-1R in a control biological sample. Therefore, for example, it can be evaluated whether the amount of CSF-1R in the test sample is increased or decreased compared to the control sample.

[0059] In one embodiment of the above method, the detection of CSF-1R is an immunohistochemical detection of CSF-1R. Therefore, CSF-1R is identified in the context of intact cells by labeling the test sample with an antibody of the present invention (its antigen-binding fragment) in a manner that can be visualized by microscopy. By identifying CSF-1R in the tissue context or cell context, the spatial relationship between the biomarker and other morphological or molecular features of the cell or tissue sample can be elucidated, which can reveal information that is not apparent from other molecular or cell techniques.

[0060] To detect CSF-1R, the antibody (or fragment thereof) of the present invention may include a detectable label described elsewhere herein.

[0061] The present invention further relates to the in vitro use of the monoclonal antibody or fragment thereof of the present invention for the detection of CSF-1R, such as human CSF-1R, in a sample.

[0062] The terms "sample" and "subject" are defined above. The sample is a cancer cell or cancer tissue. The above definitions are applied as appropriate. As described above, the sample may be in contact with a candidate compound for the treatment of cancer. Alternatively, a sample is obtained from a subject that has been contacted with the candidate compound.

[0063] The present invention also relates to a complex comprising the monoclonal antibody or fragment thereof of the present invention and CSF-1R. Typically, the monoclonal antibody or fragment thereof is bound to CSF-1R (such as human CSF-1R) in the complex.

[0064] In one embodiment of the methods and uses of the present invention, the native form of CSF-1R (such as human CSF-1R) is detected. Thus, undenatured CSF-1R is detected. In an alternative embodiment, denatured CSF-1R is detected. Thus, the sample may be subjected to at least one pretreatment step that results in the denaturation of CSF-1R. For example, the sample may be heated or at least one denaturing agent may be added to the sample.

[0065] The present invention also relates to host cells that produce the antibody or antigen-binding fragment thereof of the present invention. In a preferred embodiment, the host that produces the antibody of the present invention is a hybridoma cell. Furthermore, the host cell can be any type of cell line that can be engineered to produce the antibody according to the present invention. For example, the host cell can be an animal cell, particularly a mammalian cell. In certain embodiments, HEK293 (human embryonic kidney cells) such as HEK293-F cells used in the Examples section, or CHO (Chinese hamster ovary) cells are used as the host cell. In another embodiment, the host cell is a non-human animal or mammalian cell.

[0066] The host cell preferably contains at least one polynucleotide encoding the antibody or fragment thereof of the present invention. For example, the host cell contains at least one polynucleotide encoding the light chain of the antibody of the present invention and at least one polynucleotide encoding the heavy chain of the antibody of the present invention. The polynucleotide(s) shall be operably linked to a suitable promoter.

[0067] The present invention further relates to a pharmaceutical composition comprising the monoclonal antibody or fragment thereof of the present invention and a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0068] The present invention provides an in vivo or in vitro method for reducing CSF-1R expression in target cells expressing CSF-1R, the method comprising administering an effective amount of the monoclonal antibody or fragment thereof or pharmaceutical composition of the present invention to the cells.

[0069] The present invention provides a method for treating or preventing a disease, the method comprising administering a therapeutically effective amount or prophylactically effective amount of a monoclonal antibody or fragment thereof, or the pharmaceutical composition of the present invention, to a subject suffering from or susceptible to the disease. In some embodiments, the disease is cancer.

[0070] The present invention provides a monoclonal antibody or a fragment thereof for use in medicine, or a pharmaceutical composition of the present invention.

[0071] The present invention provides the monoclonal antibody or a fragment thereof or a pharmaceutical composition of the present invention for use in the treatment or prevention of cancer.

[0072] The present invention provides the use of the monoclonal antibody or a fragment thereof or a pharmaceutical composition of the present invention for preparing a medicine for treating or preventing cancer.

Examples

[0073] The present invention is merely illustrated by the following examples. The above examples shall not be construed in any way as limiting the scope of the present invention. Example 1: Preparation of immunogen

[0074] TtSlyD-CSF1R and TtSlyD-sh3 ORF were synthesized by GeneArt and delivered into an ampicillin-resistant cloning vector. TtSlyD-CSF1R encodes the following amino acid sequence: >TtSlyD-CSF1R (SEQ ID NO: 11) MRSKVGQDKV VTIRYTLQVE GEVLDQGELS YLHGHRNLIP GLEEALEGRE EGEAFQAHVP AEKAYGAGSM LGPSLSPGQD PEGGVDYKNI HLEKKYVRRD SGFSSQGVDT YVEMRPVSTS SNDSFSEQDL DKEDGRPGSS GKDLDFQVEV VKVREATPEE LLHGHAHGGG SRPLLPPLPG GGSRKHHHHH HHH TtSlyD-sh3 was applied as an insert-free control protein and encodes the following amino acid sequence: >TtSlyDsh3 (SEQ ID NO: 12) MRSKVGQDKV VTIRYTLQVE GEVLDQGELS YLHGHRNLIP GLEEALEGRE EGEAFQAHVP AEKAYGAGSG SSGKDLDFQV EVVKVREATP EELLHGHAHG GGSRPLLPPL PGGGSRKHHH HHHHH

[0075] Restriction enzymes and the "Rapid DNA Ligation Kit" were obtained from Roche. Escherichia coli (E. coli) XL1-Blue supercompetent cells and BL21 Codon Plus were obtained from Stratagene. For DNA purification, the "High Pure Plasmid Isolation Kit" and the "High Pure PCR Product Purification Kit" (Roche) were used. pQE80L was used as a cloning and expression vector.

[0076] Bacteria were grown in Lysogeny broth (LB: 5 g / l yeast extract, 10 g / l tryptone, 5 g / l NaCl, pH 7.0) containing the selective antibiotic (100 μg / ml ampicillin). To improve growth during protein expression, the LB medium was replaced with super broth medium (SB: 20 g / l yeast extract, 32 g / l tryptone, 5 g / l NaCl, pH 7.0).

[0077] TtSlyD-CSF1R was released from a delivery vector containing EcoRI and HindIII, ligated into the expression vector pQE80-L, and then digested with EcoRI and HindIII. After transformation of Escherichia coli XL1-Blue bacteria, plasmid DNA was obtained and used to transform Escherichia coli BL21 Codon Plus. Briefly, for recombinant expression and purification, cells were grown in SB medium at 37 °C. When the exponential phase was reached, expression of the TtSlyD mutant was induced with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) for at least 3 h. Inclusion bodies from cell pellets were resuspended in chilled sodium phosphate buffer (pH 8.0) containing 7.0 M GdmCl and stirred for 2 h until complete cell lysis. The pre-clarified lysate was applied to a Ni-NTA column and 10 - 15 column volumes of wash buffer (phosphate buffer pH 8.0, 7.0 M GdmCl, 10 mM imidazole) were applied. To avoid reactivation of co-purified protease, a protease inhibitor cocktail (complete EDTA-free, Roche) was included in the refolding buffer (phosphate buffer pH 8.0, 20 mM imidazole). A total of 20 - 25 column volumes of refolding buffer were slowly applied overnight. Before eluting the protein with a gradient of 250 mM imidazole, the inhibitor cocktail was removed with 5 - 10 column volumes of additional wash with refolding buffer. Protein-containing fractions were pooled and further purified through a size exclusion chromatography column (HiLoad™ 26 / 60 Superdex™ 75 size exclusion chromatography column, Amersham Pharmacia) in storage buffer (50 mM KH2P04 pH 6.95, 100 mM KCl, 0.5 mM EDT). Only monomer-containing fractions were recovered and evaluated for purity in an SDS denaturing gel. For Western blot analysis, Novex® NuPAGE® SDS-PAGE Gel Systems (Invitrogen) were used. Coomassie-like protein staining was performed using SimplyBlue™ Safe-Stain (Invitrogen). Protein concentration was measured using DU RPerformed using a 7400 spectrophotometer (Beckman Coulter (trademark)). The molar extinction coefficient (ε 280 ) of the fusion protein was calculated by bioinformatics. Example 2: Circular Dichroism Spectrum Analysis

[0078] Protein concentration was measured using a DU R 7400 spectrophotometer (Beckman Coulter (trademark)). The molar extinction coefficient (ε 280 ) of the fusion protein was calculated by bioinformatics.

[0079] The near-UV CD spectrum was recorded using a Jasco-720 spectropolarimeter equipped with a thermostatted cell holder set at 20 °C and converted to mean residue ellipticity. The buffer was 50 mM potassium phosphate (pH 6.95), 100 mM KCl and 0.5 mM EDTA. The spectrum was recorded at a path length of 0.2 cm between 330 and 250 nm, and the protein concentration was 500 μM. The bandwidth was 1 nm, the scanning speed was 20 nm / min at a resolution of 0.5 nm, and the response was 1 s. To improve the signal-to-noise ratio, the spectrum was measured 9 times and averaged. The far-UV CD spectrum was recorded using a Jasco-720 spectropolarimeter equipped with a thermostatted cell holder set at 20 °C and converted to mean residue ellipticity. The buffer was 10 mM potassium phosphate (pH 6.95) and 10 mM KCl. The spectrum was recorded at a path length of 0.2 cm between 250 and 190 nm, and the protein concentration was 5 μM. The bandwidth was 1 nm, the scanning speed was 20 nm / min at a resolution of 0.5 nm, and the response was 1 s. To improve the signal-to-noise ratio, the spectrum was measured 9 times and averaged.

[0080] For the thermal unfolding transition of the fusion protein, the protein was measured at 500 μM in 50 mM potassium phosphate (pH 6.95), 100 mM KCl and 0.5 mM EDTA. The heat-induced unfolding-refolding transition was recorded at 278 nm, and the path length of the cuvette was 0.2 cm. The heating and cooling rates were 1 °C / min and the response time was 4 s. To evaluate the reversibility of unfolding, the near-UV CD spectra of the fusion protein were recorded before and after the heat-induced unfolding-refolding cycle.

[0081] Protein concentration measurements were performed using a DU R 7400 spectrophotometer (Beckman Coulter (trademark)). The molar extinction coefficient (ε 280 ) of the fusion protein was calculated by bioinformatics according to Gasteiger et al 2005.

[0082] Fluorescence spectra were recorded using a Cary Eclipse fluorescence spectrophotometer (Varian) equipped with a thermostatic cell holder set at 20 °C. The buffer was 50 mM potassium phosphate (pH 6.95), 100 mM KCl and 0.5 mM EDTA. Samples were excited at 280 / 290 / 295 nm and spectra were recorded between 300 - 425 nm with a 1 cm path length. The protein concentration was 10 - 30 μM. The bandwidth was 5 nm, the scanning speed was 120 nm / min with a resolution of 1 nm, and the response was 0.5 s.

[0083] For the thermal unfolding transition of the fusion protein, the same concentration and buffer were used. The full spectrum was recorded for the heat-induced unfolding transition with the same specifications as for the single measurement. The heating interval was set at 5 °C and the stabilization period between temperature changes was set at 10 min. To evaluate the reversibility of unfolding, the fluorescence spectra of the fusion protein were recorded before and after the heat-induced unfolding-refolding cycle. Example 3: Phosphorylation and purification of TtSlyD-CSF1R

[0084] Buffer components were obtained from Merck, Roche, and Sigma. Src(1-530) active kinase was purchased from Upstate (Millipore). According to the manufacturer's instructions, Src kinase was diluted (20 mM MOPS-NaOH pH 7.0, 1 mM EDTA, 0.01% Brij-30, 5% glycerol, 0.1% β-mercaptoethanol, and 1 mg / ml BSA), aliquoted, and stored at -80 °C. For Western blot analysis, Novex® NuPAGE® SDS-PAGE Gel Systems (Invitrogen) were used. To detect phosphorylated proteins, several pan-pTyr primary antibodies: P-Tyr-4G10 (Millipore), P-Tyr-100 and P-Tyr-102 (Cell Singaling), and HRP-conjugated goat anti-mouse IgG (Invitrogen) as the secondary antibody were used. Results were analyzed by ChemiDoc™ MP (Bio-Rad), and the membrane was incubated with Lumi-Light PLUS Western blotting substrate (Roche).

[0085] 4.5 mg of TtSlyD-CSF1R was diluted to a concentration of 60 μM in reaction buffer (10 mM MOPS-NaOH pH 7.0, 50 mM NaCl, 0.3 mM EDTA, 0.001% Brij-30, 0.5% glycerol, 10 mM MgAc, 0.1 mM ATP, 0.25 mM orthovanadate, and 0.1 mg / ml BSA). 4 units of Src (1.97 U / μg) were added, and the reaction was incubated at 30 °C for 2 hours. Then, EDTA was used to chelate Mg 2+The reaction was stopped by chelating the cation. To remove the buffer and contaminating proteins, the solution was loaded onto a HiLoad™ 16 / 60 Superdex™ 200 size exclusion chromatography column (Amersham, Pharmacia) using sterile filtered PBS pH 6.95 as the sheath solution. The TtSlyD-CSF1R-containing fractions were collected and the protein was concentrated using an Ultracell™ 10k Amicon (Millipore). 81% of the initial protein was recovered. Example 4: Production of Rabbit Monoclonal Antibodies

[0086] Rabbits were each subcutaneously immunized with 100 μg of antigen for 30 days. Serum was collected from the 45th day, and the antibody titer against the antigen was tested. Two months later, the immunogenic titer exceeded 200,000. Peripheral blood was collected monthly 5 - 6 days after the boost. The blood was treated with citrate to avoid coagulation and freshly processed on the same day. Peripheral blood mononuclear cells (PBMCs) were required to obtain either antibody - producing B cells or macrophages against their secreted growth factors. PBMCs were obtained from peripheral rabbit blood, and antigen - specific monoclonal antibodies were generated as described in Seeber et al. (2014), PLoS One. 2014 Feb 4;9(2). 5.10e7 PMBC / ml was resuspended in FACS Puffer (PBS + 0.1% BSA) together with 250 nM biotinylated antigen. After incubation at 4°C for 15 - 20 minutes, the cells were washed with 40 ml of PBS and resuspended in labeling buffer (PBS + 2 mM EDTA) at 10e8 PMBC / ml. 10% volume of streptavidin beads (MACS Miltenyi Biotec) was added and they were incubated at 4°C for 15 - 20 minutes. The cells were washed with 40 ml of PBS and resuspended in MACS buffer (PBS + 2 mM EDTA + 0.5% BSA) at 2.10e8 PMBC / ml. The suspension was loaded onto a pre - equilibrated MS column (MACS Mitenyi Biotec), washed with 3 volumes of MACS buffer, and the bound cells were recovered in 1 ml of MACS buffer. To identify the cell type, the recovered cells were stained with a fluorescent antibody against rabbit IgG (AbD Serotec), and IgG - positive cells were sorted into single cells using a FACSAria I cell sorter (BD Biosciences). As described in Seeber et al., the cells were incubated in B - cell medium for one week. After one week, the supernatant of the clone was tested for IgG production and antigen specificity using the HitELISA technique. Positive clones were selected and stored at - 80°C using RNA lysis buffer. HitELISA was performed using cell culture supernatants and purified mAb. ELISA plates (Roche) were coated with 100 - 250 ng / ml of antigen in carbonate buffer (pH 9.6).The biotinylated antigen was directly bound to streptavidin-coated plates (Roche) using the same antigen concentration. After washing, the plates were blocked with incubation buffer (IB: 1% BSA 0.05% Tween-PBS). The plates were incubated with cell supernatants or purified mAbs diluted in IB. After washing, the plates were incubated with an HRP-conjugated F(ab’)2 fragment goat anti-rabbit IgG (Dianova) for 1 hour, and the plates were developed by adding 100 μl of ABTS solution (Roche). The optical density was measured at an appropriate wavelength using an ELISA microplate reader. IgG concentration sandwich ELISA. The ELISA plates were coated with 3 μg / ml of goat anti-rabbit IgG in carbonate buffer (pH 9.6). After washing, the plates were blocked with IB. The plates were incubated with cell supernatants diluted in IB. The subsequent ELISA was performed as described above.

[0087] B cell mRNA was purified from positive single memory B cell cultures frozen at -80 °C using the RNeasy R Plus Mini Kit (Qiagen R ). Reverse transcription was performed using Transcription Universal cDNA Master (Roche). The cDNA plates were stored at -20 °C until further use. The IgH, Igλ, and Igκ variable genes were independently amplified by PCR starting from 2 μl of cDNA as a template using the Expand High Fidelity PCR System (Roche) with appropriate primers. The purified single amplification band and the plasmid containing the IgG constant region were digested with T4 DNA polymerase (Roche) to generate 5’ overhangs, followed by RecA treatment (NE Biolabs) and religated by sequence- and ligation-independent cloning (SLIC). The recombinant plasmids were transformed and tested for correct insert cloning using standard digestion and sequencing protocols.

[0088] 293-Free (trademark) Transfection Reagent (Novagen R ) was used to produce full-length IgG mAb by transient co-transfection of paired heavy and light chain TIPE plasmids into FreeStyle 293-F cells (Invitrogen) grown in serum-free FreeStyle (trademark) 293 Expression Medium (Gibco R Invitrogen). Cells were cultured at 37 °C / 5 - 8% CO2 for 1 week with continuous shaking at 180 rpm. The supernatant was collected by centrifugation and stored at -20 °C. Example 5: Interaction analysis

[0089] Using a Biacore B 3000 instrument (GE Healthcare), the kinetic and binding specificity of rabbit antibodies against TtSlyD-CSF1R were kinetically evaluated. A CM5 series sensor was attached to the system and normalized in HBS-ET buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w / v Tween 20) according to the manufacturer's instructions. The sample buffer was the system buffer supplemented with 1 mg / ml CMD (carboxymethyl dextran, Sigma #86524). The system was operated at 25 °C. 10000 RU of GAR<F(ab)2> (goat anti-rabbit F(ab)2 in relative units / Jackson Laboratories, catalog number 100018) was immobilized onto all flow cells using EDC / NHS chemistry according to the manufacturer's instructions. The sensor was saturated with 1 M ethanolamine. The binding activity of the antibody against the analyte was kinetically tested. The analytes in solution were two 2 kDa peptides CSF1R(716 - 729) where Y723 was phosphorylated and non-phosphorylated, a 14 kDa TtSlyDsh3 control protein with a CSF1R-unrelated insert domain, and a 21 kDa TtSlyD-CSF1R protein. The antibody was captured by a 2-minute injection of 10 μl / min of cell culture HEK supernatant diluted 1:2 in sample buffer. The flow rate was set at 100 μl / min. The analytes were injected for 2 minutes at different concentration steps of 0 nM, 1.1 nM, 3.7 nM, 11.1 nM, 33.1 nM, 100 nM and 300 nM. Dissociation was monitored for 5 minutes. The kinetic signature was monitored and evaluated using the binary Langmuir fitting model with Biaevaluation Software and R MAX local. Acidic regeneration of the sensor surface was achieved using three consecutive injections of 10 mM glycine pH 1.7 at 30 μl / min for 60 seconds each. Example 6: Linear epitope mapping

[0090] Peptide-based epitope mapping was performed as described and commercially available from Intavis (Cologne, Germany, http: / / www.intavis.com) using CelluSpot™ technology. Epitope mapping was performed with a library of overlapping immobilized peptide fragments (length: 15 amino acids) corresponding to the sequence of the human CSF1R KID domain. Each synthesized peptide was shifted by only one amino acid, i.e., 14 amino acids overlapped with the previous and the next peptide, respectively. For the preparation of the peptide array, Intavis CelluSpot™ technology was used. In this approach, peptides were synthesized using an automated synthesizer (Intavis MultiPep RS) on a modified cellulose disk that is dissolved after synthesis. Subsequently, a solution of the individual peptides covalently bound to the polymer cellulose was spotted onto a coated microscope slide. CelluSpot™ synthesis was carried out stepwise using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on an amino-modified cellulose disk in a 384-well synthesis plate. In each coupling cycle, the corresponding amino acid was activated with a solution of DIC / HOBt in DMF. During the coupling step, unreacted amino groups were capped with a mixture of acetic anhydride, diisopropylethylamine, and 1-hydroxybenzotriazole. When the synthesis was complete, the cellulose disk was transferred to a 96-well plate and treated with a mixture of trifluoroacetic acid (TFA), dichloromethane, triisopropylsilane (TIS), and water for side-chain deprotection. After removal of the cleavage solution, the cellulose-bound peptides were dissolved with a mixture of TFA, TFMSA, TIS, and water, precipitated with diisopropyl ether, and resuspended in DMSO. Subsequently, the peptide solution was spotted onto an Intavis CelluSpot™ slide using an Intavis slide spotting robot.

[0091] For linear epitope analysis, the slides prepared as described above were treated using a BenchMark XT automated slide preparation system (Ventana). The slides were developed according to a standardized protocol using an OptiView amplification kit (Ventana) in addition to an OptiView DAB IHC detection kit. Briefly, the slides were "wet filled" into the system and blocked with 1% BSA in phosphate buffered saline (PBS) for 32 minutes. The antibody was diluted to 1 μg / ml with an antibody diluent (Ventana) and manually applied to the slides for 1 hour at room temperature, and both the Amplifier and Amplification Multimer reagents were incubated for 8 minutes each without counterstaining. A ChemiDoc Analyzer (BioRAD) was used to analyze the colorimetric staining.

[0092] When high signals were desired, the slides were manually treated with chemiluminescent reagents. Briefly, the slides were washed with ethanol and then with Tris buffered saline (TBS; 50 mM Tris, 137 mM NaCl, 2.7 mM KCl, pH 8) before blocking with 5 mL of 10× Western blocking reagent (Roche Applied Science), 2.5 g sucrose, and 0.1% Tween 20 in TBS at 4°C for 16 hours. The slides were washed with TBS and 0.1% Tween 20, then incubated with 1 μg / mL of the corresponding antibody in TBS and 0.1% Tween 20 for 2 hours at ambient temperature, followed by washing with TBS + 0.1% Tween 20. For detection, the slides were incubated with an anti-rabbit / anti-mouse secondary HRP antibody (1:20000 in TBS-T), followed by incubation with a chemiluminescent substrate luminol and visualization with a LumiImager (Roche Applied Science). ELISA positive SPOTs were quantified and the antibody-binding epitopes were identified by assignment of the corresponding peptide sequences. Immunohistochemistry

[0093] Immunohistochemistry was performed using the BenchMark XT automated slide preparation system (Ventana). The slides were developed using the iVIEW DAB Detection Kit (Ventana) according to a standardized protocol. Briefly, the slides were deparaffinized with Cell Conditioning 1 (CC1, Ventana) and blocked with 1% BSA in phosphate-buffered saline (PBS) for 32 minutes. The antibody was diluted to 1 μg / ml with Antibody Diluent (Ventana) and manually applied to the slides for 1 hour at room temperature.

Claims

**Claim 1** A monoclonal antibody or an antigen-binding fragment thereof that binds to CSF-1R (Colony stimulating factor 1 receptor), wherein the monoclonal antibody or the antigen-binding fragment thereof comprises a light chain variable domain having at least 90% identity with the light chain variable domain having the sequence shown in SEQ ID NO: 2, and the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain variable domain having at least 90% identity with the heavy chain variable domain having the sequence shown in SEQ ID NO: 3, the light chain variable domain of the monoclonal antibody or the antigen-binding fragment thereof comprises a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4 (QSSSEVYSNNFLS), a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5 (EASKVAS), and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6 (AGGYDVSDDDA), and the heavy chain variable domain of the monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT), a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG), and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 9 (GGQNNGYDl), a monoclonal antibody or an antigen-binding fragment thereof. **Claim 2** The monoclonal antibody or the antigen-binding fragment thereof according to claim 1, wherein CSF1-R is human CSF-1R. **Claim 3** The monoclonal antibody or the antigen-binding fragment thereof according to claim 1 or 2, wherein the epitope of the monoclonal antibody or the antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 1 (YKNIHLKKY). **Claim 4** The monoclonal antibody or the antigen-binding fragment thereof according to claim 3, wherein at least one of the two tyrosine residues of the epitope is phosphorylated. **Claim 5** The monoclonal antibody or the antigen-binding fragment thereof according to claim 4, wherein both tyrosine residues of the epitope are phosphorylated. **Claim 6** The monoclonal antibody or its antigen-binding fragment thereof comprises a light chain variable domain having a sequence at least 95% identical to the sequence shown in SEQ ID NO: 2, and the monoclonal antibody or its antigen-binding fragment thereof comprises a heavy chain variable domain having a sequence at least 95% identical to the sequence shown in SEQ ID NO:

3. The monoclonal antibody or its antigen-binding fragment thereof according to any one of claims 1 to 5.

7. The monoclonal antibody or its antigen-binding fragment thereof comprises a light chain variable domain having a sequence at least 98% identical to the sequence shown in SEQ ID NO: 2, and the monoclonal antibody or its antigen-binding fragment thereof comprises a heavy chain variable domain having a sequence at least 98% identical to the sequence shown in SEQ ID NO:

3. The monoclonal antibody or its antigen-binding fragment thereof according to any one of claims 1 to 6.

8. A monoclonal antibody or its antigen-binding fragment thereof that binds to CSF-1R (Colony stimulating factor 1 receptor), (a) A light chain variable domain comprising the following: (a1) A light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4 (QSSSEVYSNNFLS), (a2) A light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 5 (EASKVAS), and (a3) A light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 6 (AGGYDVSDDDA), and (b) A heavy chain variable domain comprising the following: (b1) A heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT), (b2) A heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG), and (b3) A heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 9 (GGQNNGYDL) The monoclonal antibody or its antigen-binding fragment thereof comprising.

9. In vitro use of the monoclonal antibody or its antigen-binding fragment thereof according to any one of claims 1 to 8 for the detection of CSF-1R in a sample.

10. (a) The sample has been contacted with a candidate compound for the treatment of cancer or the sample is obtained from a subject contacted with the candidate compound, (b) The sample is cancer cells or cancer tissue, (c) The subject is a mammalian subject such as a human subject (d) the detection of the CSF-1R is a quantitative detection of the CSF-1R, and / or (e) the detection of the CSF-1R is an immunohistochemical detection of the CSF-1R, The in vitro use according to claim 9. **Claim 11** A method for detecting CSF-1R in a sample, comprising: (a) contacting a sample containing CSF-1R with the monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8, thereby forming a complex comprising CSF-1R and the monoclonal antibody or an antigen-binding fragment thereof; and (b) detecting the complex formed in step (a), thereby detecting the CSF-1R in the sample. **Claim 12** A complex comprising the monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 8 and CSF-1R. **Claim 13** The in vitro use according to claim 9 or 10, wherein the CSF-1R comprises a phosphorylated tyrosine residue at a position corresponding to position 699 of human CSR-1R and / or a phosphorylated tyrosine residue at a position corresponding to position 708 of human CSR-1R. **Claim 14** The method according to claim 11, wherein the CSF-1R comprises a phosphorylated tyrosine residue at a position corresponding to position 699 of human CSR-1R and / or a phosphorylated tyrosine residue at a position corresponding to position 708 of human CSR-1R. **Claim 15** The complex according to claim 12, wherein the CSF-1R comprises a phosphorylated tyrosine residue at a position corresponding to position 699 of human CSR-1R and / or a phosphorylated tyrosine residue at a position corresponding to position 708 of human CSR-1R.

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