Anti-CSF-1R Antibody

Abstract

The present invention relates to a monoclonal antibody or 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 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 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 CSF-1R ( set fall prick Stimulate because sub 1 by 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 in a sample. The present invention also encompasses a complex comprising the monoclonal antibody or fragment thereof of the present invention and CSF-1R, such as a human CSF-1R polypeptide. Background Art

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

[0003] There are currently two known CSF-1R ligands that bind to the extracellular domain of CSF-1R. The first is CSF-1 (colony stimulating factor 1), which is found as a disulfide-linked homodimer outside the cell (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 effect of CSF-1R signaling is the differentiation, proliferation, migration and survival of hematopoietic precursor cells to the macrophage lineage. The activation of CSF-1R is mediated by its CSF-1R ligands CSF-1 and IL-34. The binding of CSF-1 to CSF-1R induces the formation of homodimers and the activation of kinases through tyrosine phosphorylation (Stanley et al., Mol. Reprod. Dev. 46 (1997) 4-10).

[0004] CSF-1R is associated with many diseases and conditions (see, for example, Cannarile, MA, et al. (2017), J Immunother Cancer 5(1): 53, Ries, CH, et al. (2014), Cancer Cell 25(6): 846-859). For example, CSF-1R plays an important role in initiating inflammation, cancer, and bone diseases. Therefore, many CSF-1R inhibitors (such as small molecule inhibitors and monoclonal antibodies) have been described and analyzed in clinical trials (reviewed in El-Gamal et al., J Med Chem. 2018, 61(13): 5450-5466).

[0005] Anti-CSF-1R antibodies are disclosed, for example, in WO2011 / 123381, WO2011 / 140249 and WO2012 / 110360.

[0006] The extracellular domain of the CSF-1 receptor (CSF-1R-ECD) contains five subdomains (D1 to D5). The biologically active homodimeric CSF-1 binds to CSF-1R in the D1 to D3 subdomains. Subdomains D4 to 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, WO 2009 / 026303 and WO 2009 / 112245 disclose anti-CSF-1R antibodies that bind to CSF-1R within the first three subdomains (D1 to D3) of the extracellular domain. WO2011 / 070024 discloses anti-CSF-1R antibodies that bind to CSF-1R within the dimerization domain (D4 to 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 also 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). The polyclonal antibodies are generated by using chemically synthesized phosphopeptides as immunogens.

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

[0010] WO2012 / 150320 describes a scaffold technology for producing antibodies. The document describes fusion polypeptides comprising one or more fragments of a peptidyl-prolyl cis / trans isomerase or FKBP domain family member, and their use in methods of antibody screening / selection, epitope mapping, and their use as immunogens for producing antibodies that specifically bind to secondary structures presented by immunogenic peptides or fusion polypeptides. WO2015 / 044083 discloses that the Thermus thermophilus SlyD FKBP domain is suitable for use in fusion polypeptides to produce antibodies.

[0011] The inventors have grafted 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 generation of monoclonal antibodies in rabbits. The results showed that these antibodies showed high antibody / receptor affinity and specificity with respect to CSF-1R, especially the antibody called "1H11". The antibody showed excellent immunohistochemical properties and can therefore be used in clinical studies of small molecules or antibodies against CSF-1R. Surprisingly, the antibody was able to detect both native and denatured CSF-1R. Summary of the invention

[0012] The present invention relates to monoclonal antibodies or fragments thereof that bind to CSF-1R (colony stimulating factor 1 receptor), in particular human CSF-1R.

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

[0014] In one embodiment of the present invention, the monoclonal antibody or fragment thereof comprises

[0015] (a) a light chain variable domain comprising:

[0016] (a1) a light chain CDR1 that differs from the light chain CDR1 having the amino acid sequence set forth in SEQ ID NO:4 (QSSESVYSNNFLS) by no more than a total of three amino acid additions, substitutions and / or deletions,

[0017] (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 no more than a total of three amino acid additions, substitutions and / or deletions, and / or

[0018] (a3) a light chain CDR3 that differs from the light chain CDR3 having the amino acid sequence set forth in SEQ ID NO:6 (AGGYDVSDDA) by no more than a total of three amino acid additions, substitutions and / or deletions,

[0019] (b) a heavy chain variable domain comprising:

[0020] (b1) a heavy chain CDR1 that differs from the heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:7 (TASGFSLSRYWMT) by no more than a total of three amino acid additions, substitutions and / or deletions,

[0021] (b2) a heavy chain CDR2 that differs from the heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:8 (RSGNTYFADWAKG) by no more than a total of three amino acid additions, substitutions and / or deletions, and / or

[0022] (b3) a heavy chain CDR3 that differs from the heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:9 (GGQNNGYDL) by no more than a total of three amino acid additions, substitutions and / or deletions,

[0023] or

[0024] (c) both the light chain variable domain as defined in (a) and the heavy chain variable domain as defined in (b).

[0025] 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 of the two tyrosine residues of the epitope, in particular both tyrosine residues, are phosphorylated.

[0026] In one embodiment, the monoclonal antibody or fragment thereof comprises

[0027] (a) a light chain variable domain comprising:

[0028] (a1) a light chain CDR1 having the sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS),

[0029] (a2) a light chain CDR2 having the sequence shown in SEQ ID NO: 5 (EASKVAS), and

[0030] (a3) a light chain CDR3 having the sequence shown in SEQ ID NO: 6 (AGGYDVSDDA),

[0031] as well as

[0032] (b) a heavy chain variable domain comprising:

[0033] (b1) a heavy chain CDR1 having the sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT),

[0034] (b2) a heavy chain CDR2 having the sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG), and

[0035] (b3) Heavy chain CDR3 having the sequence shown in SEQ ID NO: 9 (GGQNNGYDL).

[0036] The present invention also relates to the in vitro use of any monoclonal antibody or fragment thereof of the present invention for detecting CSF-1R in a sample.

[0037] The present invention further contemplates a method for detecting CSF-1R in a sample, comprising

[0038] (a) contacting a sample containing CSF-1R with the monoclonal antibody or a fragment thereof of the present invention, thereby forming a complex containing CSF-1R and the monoclonal antibody or a fragment thereof, and

[0039] (b) detecting the complex formed in step (a), thereby detecting CSF-1R in the sample.

[0040] The present invention further contemplates a complex comprising a monoclonal antibody or fragment thereof of the present invention and a CSF-1R, such as a human CSF-1R polypeptide. DETAILED DESCRIPTION

[0041] As mentioned above, 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.

[0042] CSF-1R is a polypeptide belonging to the class III subfamily of receptor tyrosine kinases and is encoded by the c-fms proto-oncogene. Synonyms for CSF-1R are M-CSF receptor; macrophage colony stimulating factor 1 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 the 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). The cloning of the receptor was first described in Roussel, MF, et al., Nature 325 (1987) 549-552. CSF-1R is a single-chain, transmembrane receptor tyrosine kinase (RTK), a member of the immunoglobulin (Ig) motif family containing RTKs, characterized by the presence of five repeated Ig-like subdomains D1-D5 in 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, in particular, the kinase domain of CSF-1R.

[0043] The amino acid sequence of full-length human CSF-1R is as follows (SEQ ID NO: 10):

[0044]

[0045]

[0046] In the above sequence, the extracellular domain of human CSF-1R is underlined 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 region of the antibody identified in the studies underlying the present invention is shown in bold. The sequence of the epitope is also shown in SEQ ID NO: 1 (YKNIHLEKKY). As described in more detail below, the tyrosine residue at position 699 and / or the tyrosine residue at position 708 are phosphorylated.

[0047] Advantageously, the antibodies of the invention bind to the native form of CSF-1R, ie the non-denatured form and the denatured form of CSF-1R. Thus, the term "CSF-1R" includes both native CSF-1R and denatured CSF-1.

[0048] The antibodies or antigen-binding fragments of the present invention should have the following sequence:

[0049] Preferably, the monoclonal antibody or fragment thereof that binds to CSF-1R (colony stimulating factor 1 receptor) comprises a light chain variable domain that is, in increasing order of preference, at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to a light chain variable domain having the sequence set forth in SEQ ID NO:2; and / or a heavy chain variable domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to a heavy chain variable domain having the sequence set forth in SEQ ID NO:3. Preferably, the monoclonal antibody or fragment thereof comprises a light chain variable domain that is, in increasing order of preference, at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to a light chain variable domain having the sequence set forth in SEQ ID NO: 2; and a heavy chain variable domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to a heavy chain variable domain having the sequence set forth in SEQ ID NO: 3. For example, the monoclonal antibody or fragment thereof comprises a light chain variable domain that is, in increasing order of preference, at least 85% identical to a light chain variable domain having the sequence set forth in SEQ ID NO: 2; and a heavy chain variable domain that is at least 85% identical to a heavy chain variable domain having the sequence set forth in SEQ ID NO: 3.

[0050] 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.

[0051] The sequence of the light chain variable region is as follows (SEQ ID NO: 2):

[0052]

[0053] The sequence of the heavy chain variable region is as follows (SEQ ID NO: 3):

[0054]

[0055] Alternatively or additionally, the monoclonal antibody or fragment thereof of the invention comprises

[0056] (a) a light chain variable domain comprising:

[0057] (a1) a light chain CDR1 which differs from the light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS) by no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions,

[0058] (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 no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions, and / or

[0059] (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 no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions, and / or

[0060] (b) a heavy chain variable domain comprising:

[0061] (b1) a heavy chain CDR1 which differs from the heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT) by no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions,

[0062] (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 no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions, and / or

[0063] (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 no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions,

[0064] or

[0065] Both the light chain variable domain as defined in (a) and the heavy chain variable domain as defined in (b).

[0066] Alternatively or additionally, the monoclonal antibody or fragment thereof of the present invention comprises (a) a light chain variable domain comprising:

[0067] (a1) a light chain CDR1 which differs from the light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS) by no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions,

[0068] (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 no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions, and

[0069] (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 no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions,

[0070] as well as

[0071] (b) a heavy chain variable domain comprising:

[0072] (b1) a heavy chain CDR1 which differs from the heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT) by no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions,

[0073] (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 no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions, and

[0074] (b3) a heavy chain CDR3 which differs from the heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 9 (GGQNNGYDL) by no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions.

[0075] In one aspect, the monoclonal antibodies or fragments thereof of the invention comprise

[0076] (a) a light chain variable domain comprising:

[0077] (a1) a light chain CDR1 comprising at least 11, in particular at least 12 consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS),

[0078] (a2) a light chain CDR2 comprising at least 5, in particular at least 6 consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 5 (EASKVAS), and / or

[0079] (a3) a light chain CDR3 comprising at least 8, in particular at least 9 consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 6 (AGGYDVSDDA),

[0080] and / or

[0081] (b) a heavy chain variable domain comprising:

[0082] (b1) a heavy chain CDR1 comprising at least 11, in particular at least 12 consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT),

[0083] (b2) a heavy chain CDR2 comprising at least 11, in particular at least 12 consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG), and / or

[0084] (b3) A heavy chain CDR3 comprising at least 7, in particular at least 8 consecutive amino acids of the amino acid sequence shown in SEQ ID NO: 9 (GGQNNGYDL).

[0085] In one aspect, the monoclonal antibodies or fragments thereof of the invention comprise

[0086] (a) a light chain variable domain comprising:

[0087] (a1) a light chain CDR1 having the sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS),

[0088] (a2) a light chain CDR2 having the sequence shown in SEQ ID NO: 5 (EASKVAS), and / or

[0089] (a3) a light chain CDR3 having the sequence shown in SEQ ID NO: 6 (AGGYDVSDDA),

[0090] and / or

[0091] (b) a heavy chain variable domain comprising:

[0092] (b1) a heavy chain CDR1 having the sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT),

[0093] (b2) a heavy chain CDR2 having the sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG), and / or

[0094] (b3) Heavy chain CDR3 having the sequence shown in SEQ ID NO: 9 (GGQNNGYDL).

[0095] In one aspect, the monoclonal antibodies or fragments thereof of the invention comprise

[0096] (a) a light chain variable domain comprising:

[0097] (a1) a light chain CDR1 having the sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS),

[0098] (a2) a light chain CDR2 having the sequence shown in SEQ ID NO: 5 (EASKVAS), and

[0099] (a3) a light chain CDR3 having the sequence shown in SEQ ID NO: 6 (AGGYDVSDDA),

[0100] as well as

[0101] (b) a heavy chain variable domain comprising:

[0102] (b1) a heavy chain CDR1 having the sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT),

[0103] (b2) a heavy chain CDR2 having the sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG), and

[0104] (b3) Heavy chain CDR3 having the sequence shown in SEQ ID NO: 9 (GGQNNGYDL).

[0105] Also preferably, the monoclonal antibody or fragment thereof that binds to CSF-1R (colony stimulating factor 1 receptor) comprises a light chain variable domain that is, in increasing order of preference, at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain having the sequence set forth in SEQ ID NO: 2; and / or a heavy chain variable domain that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain having the sequence set forth in SEQ ID NO: 3,

[0106] The light chain variable domain comprises

[0107] (a1) a light chain CDR1 which differs from the light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 4 (QSSESVYSNNFLS) by no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions,

[0108] (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 no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions, and

[0109] (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 no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions,

[0110] and

[0111] The heavy chain variable domain includes

[0112] (b1) a heavy chain CDR1 which differs from the heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 7 (TASGFSLSRYWMT) by no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions,

[0113] (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 no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions, and

[0114] (b3) a heavy chain CDR3 which differs from the heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 9 (GGQNNGYDL) by no more than a total of three, two or in particular one amino acid additions, substitutions and / or deletions.

[0115] "Percent (%) amino acid sequence identity" relative 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 a reference polypeptide sequence after aligning the candidate sequence with the reference sequence and introducing gaps (if necessary) to achieve the maximum percentage of sequence identity. Preferably, standard parameters are applied to determine the degree of sequence identity between two sequences. Preferably, the degree of identity is determined by comparing two optimally aligned sequences in a comparison window, wherein the amino acid sequence fragment in the comparison window may contain additions or deletions (e.g., gaps or overhangs) to achieve optimal alignment compared to the reference sequence (excluding additions or deletions). The percentage is calculated by determining the number of positions of the identical amino acid residues present in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences for comparison can be performed by the homology alignment algorithm of Smith and Waterman (Add. APL. Math. 2: 482 (1981)), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443 (1970)), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988)), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection. Given 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. It is best to use the default values ​​of 5.00 for the gap weight and 0.30 for the gap weight length. 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), which has been incorporated into the needle program in the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice, P., Longden, I. and Bleasby, A., Trends in Genetics 16 (6), 276-277, 2000), the BLOSUM62 scoring matrix, and a gap opening penalty of 10 and a gap widening penalty of 0.5.A preferred non-limiting example of parameters for aligning two amino acid sequences using the needle program are the default parameters including the EBLOSUM62 scoring matrix, a gap opening penalty of 10, and a gap widening penalty of 0.5.

[0116] The term "antibody" refers to an immunoglobulin or immunoglobulin-like molecule, including, but not limited to, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during the immune response of any vertebrate, such as mammals such as goats, rabbits and mice, and non-mammalian species, such as shark immunoglobulins. For example, the antibody can be a rabbit antibody. The term "antibody" includes complete immunoglobulins and antibody binding fragments that specifically bind to a target molecule (or a group of highly similar target molecules) to substantially exclude binding to other molecules. The term "antibody" also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized mouse antibodies) and complex conjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, 111.); Kuby, J., Immunology, 3rd edition, WH Freeman & Co., New York, 1997.

[0117] In particular, the term "antibody" refers to a polypeptide ligand comprising at least a light chain and a heavy chain immunoglobulin variable region, which specifically recognizes and binds to an epitope of an antigen. Antibodies are composed of heavy chains and light chains, each of which has a variable region, referred to as a variable heavy chain (VH) region and a variable light chain (VL) region. The VH region and the VL region are jointly responsible for binding to the antigen recognized by the antibody. Typically, the antibodies of the present invention have a heavy (H) chain and a light (L) chain interconnected by disulfide bonds. As used herein, the term "light chain" includes full-length light chains and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes the variable region domains VH and VL. L and constant region domain C L The variable region domain of the light chain is located at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains. The term "heavy chain" includes full-length heavy chains and fragments thereof that have sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes the variable region domain V H and three constant region domains C H 1. C H 2 and C H 3. V H The domain is at the amino terminus of the polypeptide, C H The domain is at the carboxyl terminus, where C H 3 closest to the carboxyl terminus of the peptide,

[0118] There are five major heavy chain classes (or isotypes) that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE. The heavy and light chains each contain a constant region and a variable region (these regions are also referred to as "domains"). Combined together, the heavy and light chain variable regions specifically bind to an antigen. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also referred to as a "complementarity determining region" or "CDR". The CDRs are primarily responsible for binding to an antigenic epitope. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, are numbered sequentially from the N-terminus, and are also usually identified by the chain in which a particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while a VL CDR1 is a CDR1 from the variable domain of the light chain of the antibody in which it is found. Antibodies that bind to CSF-1R will have specific VH and VL region sequences, and thus specific CDR sequences.

[0119] The antibody of the present invention or its fragment can be a single-chain antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody and a fragment thereof. For example, the antibody can be an IgG antibody such as an IgG1 antibody, an IgG2 antibody, an IgG3 antibody or an IgG4 antibody. In one embodiment, the antibody or its fragment has been recombinantly produced.

[0120] The present invention also encompasses fragments of the monoclonal antibodies of the present invention. The fragments should be immunologically functional fragments, i.e., antigen binding fragments. Thus, the monoclonal antibody fragments of the present invention should be able to bind to CSF-1R, such as human CSF-1R. Thus, as used herein, the term "immunologically functional fragment" of an antibody refers to a portion of an antibody that lacks at least some of the amino acids present in the full-length chain but is able to specifically bind to CSF-1R. Immunologically functional immunoglobulin fragments include Fab, Fab', F(ab')2 and Fv fragments. How to produce antigen binding fragments is well known in the art. For example, fragments can be produced by enzymatic cleavage of the antibodies of the present invention. In addition, fragments can be produced by synthetic or recombinant techniques. Fab fragments are preferably produced by papain digestion of antibodies, Fab' fragments are produced by pepsin digestion and partial reduction, and F(ab')2 fragments are produced by pepsin digestion. Fv fragments are preferably produced by molecular biology techniques.

[0121] Antibody fragments may also be diabodies, which are small antibody fragments with two antigen-binding sites. Diabodies preferably comprise a heavy chain variable domain connected to a light chain variable domain in the same polypeptide chain.

[0122] The antibody of the present invention is preferably a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single B lymphocyte or by a cell 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 (e.g., by preparing hybrid antibody-forming cells from the fusion of myeloma cells with immune spleen cells).

[0123] In an embodiment, the antibody of the present invention is an isolated antibody. Therefore, the antibody should be a purified antibody. The purification of the antibody can be achieved by methods well known in the art such as size exclusion chromatography (SEC). Therefore, the antibody should be separated from the cell producing the antibody. In certain embodiments, the isolated antibody is purified to an antibody greater than 70% weight as measured by, for example, the Lowry method, and in some embodiments, is purified to greater than 80%, 90%, 95%, 96%, 97%, 98% or 99% weight. In a preferred embodiment, the isolated antibody according to the present invention is purified to a purity greater than 90%, as determined by SDS-PAGE under reducing conditions using Coomassie blue staining for protein detection.

[0124] The monoclonal antibodies or fragments thereof of the present invention shall specifically bind to a CSF-1R polypeptide, such as a human CSF-1R polypeptide. The expressions "binding" and "specifically binding" are well understood and are used to indicate that the antibody (or fragment thereof) does not significantly bind to other biomolecules.

[0125] Preferably, the antibody (or fragment thereof) of the present invention should specifically bind to the intracellular domain of the CSF-1R polypeptide, in particular the kinase domain. The epitope of the monoclonal antibody or fragment thereof preferably comprises the sequence shown in SEQ ID NO: 1 (YKNIHLEKKY). SEQ ID NO: 1 corresponds to amino acids 699 to 708 of human CSF-1R (see SEQ ID NO: 10). Thus, the monoclonal antibody or fragment thereof of the present invention should specifically bind to this region. Preferably, at least one of the two tyrosine residues of the epitope is phosphorylated, i.e. the tyrosine residue at position 699 or the tyrosine residue at position 708. More preferably, both tyrosine residues of the epitope are phosphorylated, i.e. the tyrosine residue at position 699 and the tyrosine residue at position 708. Thus, it is envisaged that the antibody or fragment thereof of the present invention binds to the human CSF-1R polypeptide, wherein the tyrosine residue at position 699 and / or the tyrosine residue at position 708 are phosphorylated.

[0126] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Thus, the term preferably refers to a portion of a CSF-1R polypeptide (e.g. a human CSF-1R polypeptide) capable of specific binding by an antibody (or fragment thereof) of the present invention. Epitopes generally consist of chemically active surface macrogroups of molecules such as amino acids and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former but not the latter is lost in the presence of denaturing solvents.

[0127] In a preferred embodiment, the antibody of the present invention or its fragment is connected to a detectable marker. Detectable markers as described herein are preferably non-naturally attached to the antibody or its antigen-binding fragment. Therefore, the detectable marker is preferably heterologous to the antibody. Suitable labels are any labels that can be detected by suitable detection methods. In one embodiment, the detectable marker is an enzyme, biotin, a radioactive label, a fluorescent label, a chemiluminescent label, an electrochemiluminescent label, a gold label or a magnetic label.

[0128] Enzyme labels include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, and luciferase. Substrates for these enzymes are well known in the art. Suitable substrates for detection include diaminobenzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate). Suitable enzyme-substrate combinations can produce colored reaction products, fluorescence or chemiluminescence, which can be measured according to methods known in the art. Fluorescent labels, for example, include 5-carboxyfluorescein, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, Cy2, Cy3, and Cy5, fluorescent proteins such as GFP (green fluorescent protein), Texas Red, and Alexa dyes. Radioactive labels, for example, 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 photographic film or phosphorimager. Magnetic labels, for example, include paramagnetic and superparamagnetic labels. Chemiluminescent labels used may include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts or oxalate esters.

[0129] The definitions and explanations given above preferably apply mutatis mutandis to the following.

[0130] The antibodies or antigen-binding fragments thereof of the invention can be used in methods related to the localization and / or quantification of CSF-1R polypeptides. For example, the antibodies or fragments thereof allow the determination of the amount of CSF-1R polypeptide in a sample, for use in diagnostic methods, or for imaging CSF-1R polypeptides.

[0131] Therefore, the present invention relates to a method for detecting CSF-1R in a sample, comprising

[0132] (a) contacting a sample containing CSF-1R with any one of the monoclonal antibodies or fragments thereof of the present invention, thereby forming a complex containing CSF-1R and the monoclonal antibody or fragment thereof, and

[0133] (b) detecting the complex formed in step (a), thereby detecting CSF-1R in the sample.

[0134] The "subject" referred to herein is preferably a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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 aspects of the method, the sample is obtained from a subject diagnosed with, suspected of having, or at risk of having cancer.

[0135] 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 in a subject. Biological samples of the present disclosure include, for example, but not limited to, whole blood, plasma, semen, saliva, tears, urine, feces, sweat, oral cavity, skin, cerebrospinal fluid, and hair. Biological samples can also be obtained from biopsies of internal organs or cancers.

[0136] In one embodiment, the sample comprises cancer cells. Cancer cells are cells that have undergone malignant transformation, which makes them morbid to the patient. As used herein, the term "cancer cell" includes not only primary cancer cells, but also any cells derived from cancer cell ancestors. This includes metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells.

[0137] Preferably, the sample containing cancer cells is a biopsy containing cancer cells.Thus, the sample is obtained from a subject suffering from cancer.

[0138] According to the present invention, the sample is obtained from a subject contacted with a candidate compound for treating cancer. Therefore, before obtaining the sample to be tested, the candidate compound has been administered to the subject, in particular a subject suffering from cancer. Alternatively, the sample has been contacted with a candidate compound for treating cancer.

[0139] In one embodiment, the candidate compound for treating cancer is a CSF-1R inhibitor, 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.

[0140] In one embodiment, the CSF-1R inhibitor is selected from xidartinib, PLX7486, ARRY-382, JNJ-40346527, BLZ945, Emactuzumab, AMG820 and IMC-CS4 (see Cannarile et al., 2017 Journal for Immunotherapy of Cancer. 5(1): 53).

[0141] In one embodiment of the above method, the detection of CSF-1R is a quantitative detection of CSF-1R. Thus, the amount of CSF-1R in the sample is determined. The determined amount of CSF-1R can be compared to the amount of CSF-1R in a control biological sample. Thus, it may be possible, for example, to assess whether the amount of CSF-1R in the test sample is increased or decreased compared to the control sample.

[0142] In an alternative embodiment of the above method, the detection of CSF-1R is an immunohistochemical detection of CSF-1R. Thus, by labeling the test sample with the antibody (or antigen-binding fragment thereof) of the present invention in a manner that can be visualized on a microscope, CSF-1R is identified in the context of intact cells. By identifying CSF-1R in the context of a tissue environment or a cellular environment, the spatial relationship between the biomarker and other morphological or molecular features of the cell or tissue sample can be elucidated, which may reveal information that is not apparent in other molecular or cellular techniques.

[0143] For detection of CSF-1R, the antibodies (or fragments thereof) of the invention may comprise a detectable label as described elsewhere herein.

[0144] 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.

[0145] The terms "sample" and "subject" have been defined above. The sample is a cancer cell or cancer tissue. These definitions apply accordingly. As described above, the sample may have been contacted with a candidate compound for treating cancer. Alternatively, the sample has been obtained from a subject contacted with the candidate compound.

[0146] 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 binds to CSF-1R (eg, human CSF-1R) in the complex.

[0147] In one embodiment of the methods and uses of the present invention, a native form of CSF-1R (e.g. human CSF-1R) is detected. Thus, undenatured CSF-1R is detected. In an alternative embodiment, denatured CSF-1R is detected. Thus, the sample may have been subjected to at least one pretreatment step resulting in denaturation of CSF-1R. For example, the sample may have been heated, or at least one denaturant may have been added to the sample.

[0148] The present invention further relates to host cells producing antibodies of the present invention or their antigen-binding fragments. In a preferred embodiment, the host producing antibodies of the present invention is a hybridoma cell. In addition, the host cell can be any type of cell system, which can be engineered to produce antibodies according to the present invention. For example, the host cell can be an animal cell, particularly a mammalian cell. In one embodiment, HEK293 (human embryonic kidney cell) is used as the HEK 293-F cell in the example section, or a CHO (Chinese hamster ovary) cell is used as a host cell. In another embodiment, the host cell is a non-human animal or a mammalian cell.

[0149] The host cell preferably comprises at least one polynucleotide encoding an antibody of the present invention or a fragment thereof. For example, the host cell comprises at least one polynucleotide encoding an antibody light chain of the present invention and at least one polynucleotide encoding an antibody heavy chain of the present invention. The polynucleotide should be operably linked to a suitable promoter.

[0150] The present invention further provides 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.

[0151] The present invention provides an in vivo or in vitro method for reducing CSF-1R expression in a target cell expressing CSF-1R, the method comprising administering an effective amount of the monoclonal antibody or fragment thereof or the pharmaceutical composition of the present invention to the cell.

[0152] The present invention provides a method for treating or preventing a disease, the method comprising administering a therapeutically or prophylactically effective amount of a monoclonal antibody or fragment thereof or a pharmaceutical composition of the present invention to a subject suffering from or susceptible to the disease. In some embodiments, the disease is cancer.

[0153] The present invention provides the monoclonal antibody or fragment thereof or pharmaceutical composition of the present invention for use in medicine.

[0154] The present invention provides the monoclonal antibody or fragment thereof or pharmaceutical composition of the present invention for treating or preventing cancer.

[0155] The present invention provides use of the monoclonal antibody or fragment thereof or the pharmaceutical composition of the present invention in preparing a drug for treating or preventing cancer.

[0156] The accompanying drawings show:

[0157] Figure 1 Circular dichroism analysis. Warming up / down experiments with the TtSlyD-CSF1R immunogen. TtSlyD-CSF1R is stable until at least 60°C and reconstitutes upon cooling.

[0158] Figure 2 Circular dichroism analysis of the immunogen TtSlyD-CSF1R before and after heating / cooling. Reconstitution of TtSlyD-CSF1R after heat denaturation.

[0159] Figure 3 Biacore Sensorgram showing the concentration-dependent kinetics of captured antibody 1H11 binding to phosphorylated TtSlyD-CSF1R as analyte in solution, overlay of analyte concentrations and the Langmuir fit model (black straight line). D 0.2nM, k a 5.78E+051 / Ms,k d 1.22E-04 1 / s.

[0160] Figure 4 Biacore Sensorgram shows an overlay of 300 nM single analyte concentration injections of four analytes in solution. The highest signal amplitude was found using the analyte TtSlyD-CSF1R (dashed line), and the second highest signal response was found using the analyte pY723 phosphorylated peptide CSF1R[716-729] (bold black line). Negative control analytes were TtSlyDsh3 and non-phosphorylated peptide CSF1R[716-729] (grey), with only background binding signal levels. Antibody 1H11 showed phosphotyrosine-specific epitope binding properties.

[0161] Figure 5 CelluSpots TM Epitope mapping technique. Right: Duplicate glass slides with CSF-1R 15-mer peptide. Left: CSF-1R peptide sequence given as single amino acid code. pY indicates phosphorylated tyrosine:

[0162] SEQ ID NO: 13: PEGGVD-pY-KNIHLEKK,

[0163] SEQ ID NO: 14: EGGVD-pY-KNIHLEKK-pY

[0164] SEQ ID NO15: GGVD-pY-KNIHLEKK-pY-V

[0165] SEQ ID NO16: GVD-pY-KNIHLEKK-pY-VR

[0166] SEQ ID NO17: VD-pY-KNIHLEKK-pY-VRR

[0167] SEQ ID NO18: D-pY-KNIHLEKK-pY-VRRD

[0168] SEQ ID NO19: pY-KNIHLEKK-pY-VRRDS

[0169] SEQ ID NO20: KNIHLEKK-pY-VRRDSG

[0170] SEQ ID NO 21: NIHLEKK-pY-VRRDSGF

[0171] The 1H11 epitope sequence was determined by single amino acid step analysis of the CSF-1R sequence.

[0172] Figure 6 IHC double staining. Antibody 1H11 showed very strong staining compared to other antibodies isolated in the studies underlying the present invention (not shown) which showed only weak staining.

[0173] Figure 7 Western blot analysis of selected antibodies. (Lane 1: Western blot standard; Lane 2: NIH3T3 wt; Lane 2 NIH 3T3+CSF1 K4; Lane 4: NIH 3T3+CSF1 K8, Lane 5: Protein standard). Antibody 1H11 showed the strongest staining and overexposed the blot. Other antibodies tested showed only weak staining.

[0174] Examples

[0175] The present invention is illustrated by the following examples only. The examples are not to be construed in any way as limiting the scope of the present invention.

[0176] Example 1: Preparation of immunogen

[0177] TtSlyD-CSF1R and TtSlyD-sh3 ORFs were synthesized by GeneArt and delivered in an ampicillin-resistant cloning vector. TtSlyD-CSF1R encoded amino acid sequence:

[0178] >TtSlyD-CSF1R (SEQ ID NO: 11)

[0179]

[0180]

[0181] TtSlyD-sh3 was used as a no-insert control protein and encodes the amino acid sequence:

[0182] >TtSlyDsh3 (SEQ ID NO: 12)

[0183]

[0184] Restriction enzymes and "Rapid DNA Ligation Kit" were purchased from Roche. E. coli XL1-Blue Supercompetent Cell and BL21 Codon Plus were purchased from Stratagene. For purification of DNA, "High Pure Plasmid Isolation Kit" and "High Pure PCR Product Purification Kit" (Roche) were used. pQE80L was used as cloning and expression vector.

[0185] Bacteria were grown in Lysogeny Broth (LB: 5 g / l yeast extract, 10 g / l tryptone, 5 g / l NaCl, pH 7.0) and selected antibiotics (100 μg / ml ampicillin). To improve growth during protein expression, 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).

[0186] TtSlyD-CSF1R was released from the delivery vector with EcoRI and HindIII and ligated into the expression vector pQE80-L, which was digested with EcoRI and HindIII. After transformation of E. coli XL1-Blue bacteria, plasmid DNA was obtained and transformed into E. coli BL21 Codon Plus. In brief, for recombinant expression and purification, cells were grown in SB medium at 37°C. When the exponential phase was reached, 0.5 mM isopropanol-β-D-thiogalactoside (IPGT) was used to induce the expression of TtSlyD variants for at least 3 hours. The inclusion bodies from the cell pellet were resuspended in chilled sodium phosphate buffer (pH 8.0) containing 7.0 M GdmCl and stirred for 2 hours until the cells were completely lysed. The pre-cleared lysate was added to the Ni-NTA column and 10-15 column volumes of wash buffer (phosphate buffer pH 8.0, 7.0 M GdmCl, 10 mM imidazole) were added. To avoid reactivation of co-purifying proteases, a protease inhibitor cocktail (completely EDTA-free, Roche) was added to 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. The inhibitor cocktail was removed, and the protein was further washed with refolding buffer for 5-10 column volumes before eluting in a 250 mM imidazole gradient. The protein-containing fractions were combined and passed through a size exclusion chromatography column (HiLoad ELISA) in storage buffer (50 mM KH2PO4 pH 6.95, 100 mM KCl, 0.5 mM EDTA). TM 26 / 60Superdex TM The fractions containing only the monomer were recovered and evaluated for purity on a SDS denaturing gel. SDS-PAGE Gel Systems (Invitrogen) was used for Western blot analysis. SimplyBlue TM Commassie-like protein staining was performed using Safe-Stain (Invitrogen). R 7400 spectrophotometer (Beckman Coulter TM ) was used to measure protein concentration. The molar extinction coefficient (ε 280 )

[0187] Example 2: Circular dichroism spectroscopy analysis

[0188] Using DU R 7400 spectrophotometer (Beckman Coulter TM) was used to measure protein concentration. The molar extinction coefficient (ε 280 ).

[0189] Near-UV CD spectra were recorded using a Jasco-720 spectropolarimeter with a thermostatic cell holder set at 20 °C and converted to mean residual ellipticity. The buffer was 50 mM potassium phosphate (pH 6.95), 100 mM KCl and 0.5 mM EDTA. Spectra were recorded between 330 and 250 nm with a path length of 0.2 cm and a protein concentration of 500 μM. The bandwidth was 1 nm, the scan speed was 20 nm / min, the resolution was 0.5 nm and the response was 1 s. To improve the signal-to-noise ratio, the spectra were measured nine times and averaged. Far-UV CD spectra were recorded using a Jasco-720 spectropolarimeter with a thermostatic cell holder set at 20 °C and converted to mean residual ellipticity. The buffer was 10 mM potassium phosphate (pH 6.95) and 10 mM KCl. Spectra were recorded between 250 and 190 nm with a path length of 0.2 cm and a protein concentration of 5 μtM. The bandwidth was 1 nm, the scanning speed was 20 nm / min, the resolution was 0.5 nm, and the response was 1 s. To improve the signal-to-noise ratio, the spectrum was measured nine times and averaged.

[0190] 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 thermally induced unfolding-refolding transition was recorded at 278 nm with a path length of 0.2 cm in the cuvette. The heating and cooling rates were 1 °C / min and the response time was 4 s. To assess the reversibility of unfolding, near-UV CD spectra of the fusion protein were recorded before and after the thermally induced unfolding-refolding cycle.

[0191] Using DU R 7400 spectrophotometer (Beckman Coulter TM ) was used to measure protein concentration. The molar extinction coefficient (ε 280 ) Calculated by bioinformatics according to Gasteiger et al. 2005.

[0192] Fluorescence spectra were recorded using a Cary Eclipse fluorescence spectrophotometer (Varian) 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 path length of 1 cm. Protein concentrations were 10-30 μM. The bandwidth was 5 nm, the scan speed was 120 nm / min, the resolution was 1 nm, and the response was 0.5 s.

[0193] For the thermal unfolding transition of the fusion protein, the same concentration and buffer were used. The full spectrum of the thermally induced unfolding transition was recorded with the same specifications as for the single measurement. The heating interval was set to 5 °C and the stabilization period between temperature changes was set to 10 minutes. To assess the reversibility of unfolding, the fluorescence spectra of the fusion protein were recorded before and after the thermally induced unfolding-refolding cycle.

[0194] Example 3: Phosphorylation and purification of TtSIyD-CSF1R

[0195] Buffer components were purchased from Merck, Roche, and Sigma. Src (1-530) active kinase was purchased from Upstate (Millipore). 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) according to the manufacturer's instructions, aliquoted, and stored at -80°C. SDS-PAGE Gel Systems (Invitrogen) was used for western blot analysis. To detect phosphorylated proteins, several pan-pTyr primary antibodies were used: P-Tyr-4G10 (Millipore), P-Tyr-100, and P-Tyr-102 (Cell Signaling) and HRP-conjugated goat anti-mouse IgG (Invitrogen) as secondary antibodies. TM MP (Bio-Rad) analysis results, the membrane was incubated with Lumi-Light PLUS Western Blotting Substrate (Roche).

[0196] 4.5 mg of TtSlyD-CSF1R was diluted to a concentration of 60 μM with 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. Afterwards, Mg was chelated by using EDTA. 2+ To remove the buffer and contaminating proteins, the solution was loaded onto the HiLoad TM 16 / 60Superdex TM A 200 size exclusion chromatography column (Amersham, Pharmacia) was used with sterile filtered PBS pH 6.95 as the sheath solvent. The fractions containing TtSlyD-CSF1R were recovered and purified using Ultracell TM The protein was concentrated using a 10k Amicon (Millipore). 81% of the initial protein was recovered.

[0197] Example 4: Rabbit monoclonal antibody production

[0198] Rabbits were immunized subcutaneously with 100 μg of antigen every 30 days. Serum was collected from day 45 and tested for antibody titers against the antigen. After 2 months, the immunogen titer exceeded 200.000. Peripheral blood was drawn monthly 5 to 6 days after the boost. The blood was treated with citrate to avoid clotting and was processed fresh on the same day. Peripheral blood mononuclear cells (PBMC) are necessary to obtain antibody-producing B cells or growth factor-secreting macrophages. PBMCs were obtained from peripheral rabbit blood and antigen-specific monoclonal antibodies were produced as described in Seeber et al. (2014), PLoS One. 2014 Feb 4; 9 (2). 5.10e7PMBC / ml was resuspended in FACS buffer (PBS + 0.1% BSA) with 250nM biotinylated antigen. After incubation at 4 ° C for 15-20 minutes, the cells were washed with 40ml PBS and resuspended to 10e8 PMBC / ml in labeling buffer (PBS+2mM EDTA). 10% volume of streptavidin beads (MACS Miltenyi Biotec) were added and incubated at 4 ° C for 15-20 minutes. The cells were washed with 40ml PBS and resuspended to 2.10e8PMBC / ml in MACS buffer (PBS+2mM EDTA+0.5% BSA). The suspension was loaded into a pre-equilibrated MS column (MACS Mitenyi Biotec), washed with three volumes of MACS buffer, and the bound cells were recovered in 1ml MACS buffer. In order to distinguish cell types, the recovered cells were stained with fluorescent antibodies for rabbit IgG (AbD Serotec), and single cell sorting was performed on IgG positive cells using FACSAria I cell sorter (BD Biosciences). As described by Seeber et al., cells were incubated in B cell culture medium for one week. One week later, the supernatants of the clones were tested for IgG production and antigen specificity using the HitELISA technique. Positive clones were selected and stored at -80°C with RNA lysis buffer. HitELISA was performed using cell culture supernatants and purified mAbs. ELISA plates (Roche) were coated with 100-250ng / ml antigen in carbonate buffer pH 9.6. The biotinylated antigen was directly bound to the streptavidin-coated plate (Roche) using the same antigen concentration. After washing, the plate was blocked with incubation buffer (IB: 1% BSA 0.05% Tween-PBS). The plate was incubated with cell supernatant or purified mAb diluted with IB. After washing, the plate was incubated with HR-conjugated F(ab′)2 fragment goat anti-rabbit IgG (Dianova) for 1 hour and 100μl ABTS solution (Roche) was added for color development. The optical density was measured at the appropriate wavelength using an ELISA plate reader. IgG concentration sandwich ELISA.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. Subsequent ELISAs were performed similarly to those described above.

[0199] Use RNeasy R Plus Mini Kit (Qiagen R ) B cell mRNA was purified from positive single memory B cell cultures frozen at -80°C. Reverse transcription was performed using Transcription Universal cDNA Master (Roche). The cDNA plates were stored at -20°C until further use. IgH, Igλ and Igκ variable genes were independently amplified by PCR using appropriate primers, starting from 2μl cDNA as a template, using the Expand High Fidelity PCR System (Roche). 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) to reconnect by sequence and ligation independent cloning (SLIC). The recombinant plasmids were transformed using standard digestion and sequencing protocols and tested for correct insertion clones.

[0200] Using 293-Free TM Transfection Reagent (Novagen R ) The paired heavy chain and light chain TIPE plasmids were transiently co-transfected into serum-free FreeStyle TM 293 expression medium (Gibco R Full-length IgG mAb was produced in FreeStyle 293-F cells (Invitrogen) grown in vitro. Cells were cultured for one week at 37°C / 5-8% CO2 with continuous shaking at 180 rpm. Supernatants were collected by centrifugation and stored at -20°C.

[0201] Example 5: Interaction Analysis

[0202] The kinetics of the rabbit antibody and TtSlyD-CSF1R binding specificity were kinetically evaluated using a Biacore B3000 instrument (GE Healthcare). CM5 series sensors were installed into the system and standardized 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 gel, Sigma #86524). The system was operated at 25°C. 10000RU GAR<F(ab)2> (Relative units of goat anti-rabbit F(ab)2 / Jackson Laboratories, cat.No.100018) was immobilized on all flow cells using EDC / NHS chemistry according to the manufacturer's instructions. The sensors were saturated with 1 M ethanolamine. The binding activity of the antibody to the analyte was tested kinetically. The analytes in solution were two 2kDa peptides CSF1R (716-729) with Y723 phosphorylated and non-phosphorylated, a 14kDa TtSlyDsh3 control protein with an unrelated insertion domain of CSF1R, and a 21kDa TtSlyD-CSF1R protein. Cell culture HEK supernatant diluted 1:2 in sample buffer was injected at 10ul / min for 2 minutes to capture the antibody. The flow rate was set to 100μl / min. Analytes were injected at different concentration steps of 0nM, 1.1nM, 3.7nM, 11.1nM, 33.1nM, 100nM and 300nM for 2 minutes. Dissociation was monitored for 5 minutes. The antibody was captured using Biaevaluation software and a flow cytometer with R MAX The kinetic characteristics were monitored and evaluated by a local binary Langmuir fitting model. Acidic regeneration of the sensor surface was achieved three times by injecting 10 mM glycine pH 1.7 at 30 μl / min for 60 s.

[0203] Example 6: Linear epitope mapping

[0204] Using CelluSpots TM Peptide-based epitope mapping was performed as described by Intavis, Cologne Germany, http: / / www.intavis.com. Epitope mapping was performed with overlapping fixed peptide fragment libraries (length: 15 amino acids) corresponding to the sequence of the human CSF1R KID domain. Each peptide synthesized was shifted by one amino acid, i.e. it overlapped with the preceding and following peptides by 14 amino acids, respectively. To prepare the peptide arrays, the Intavis CelluSpots TMIn this method, peptides are synthesized on modified cellulose discs using an automated synthesizer (Intavis MultiPep RS) and dissolved after synthesis. The solution of individual peptides covalently linked to the macromolecular cellulose is then spotted onto a coated microscope slide. TM The synthesis was performed stepwise on amino-modified cellulose disks in 384-well synthesis plates using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. In each coupling cycle, the corresponding amino acid was activated with a solution of DIC / HOBt in DMF. Between coupling steps, unreacted amino groups were capped with a mixture of acetic anhydride, diisopropylethylamine and 1-hydroxybenzotriazole. After the synthesis was completed, the cellulose disks were 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 solubilized with a mixture of TFA, TFMSA, TIS and water, precipitated with diisopropyl ether and resuspended in DMSO. The peptide solution was then spotted onto Intavis CelluSpots using the Intavis slide spotting robot. TM On a glass slide.

[0205] For linear epitope analysis, slides prepared as described above were processed using the BenchMark XT Automated Slide Preparation System (Ventana). These slides were developed using the OptiView DAB IHC Detection Kit and the OptiView Amplification Kit (Ventana) according to standardized protocols. Briefly, slides were "wet loaded" in the system and blocked with phosphate-buffered saline (PBS) containing 1% BSA for 32 minutes. Antibodies were diluted to 1 μg / ml in Antibody Diluent (Ventana) and manually added to the slides for 1 hour at room temperature; the amplification instrument and amplification polymer reagents were each incubated for 8 minutes without counterstaining. To analyze colorimetric staining, the ChemiDoc Analyzer (BioRAD) was used.

[0206] When high signal is required, slides are manually treated with chemiluminescent reagents. Briefly, slides are washed with ethanol, then washed with Tris-buffered saline (TBS; 50 mM Tris, 137 mM NaCl, 2.7 mM KCl, pH 8), and then blocked with 5 mL 10x Western Blocking Reagent (Roche Applied Science), TBS containing 2.5 g sucrose, 0.1% Tween 20 for 16 hours at 4°C. Slides are washed with TBS and 0.1% Tween 20, then incubated with TBS and 0.1% Tween 20 containing 1 μg / mL of the corresponding antibody for 2 hours at ambient temperature, followed by washing with TBS + 0.1% Tween 20. For detection, slides are incubated with anti-rabbit / anti-mouse secondary HR antibodies (1: 20,000 in TBS-T), then incubated with the chemiluminescent substrate luminol, and observed with LumiImager (Roche Applied Science). ELISA-positive SPOTs were quantified and the antibody-binding epitopes were identified by assigning corresponding peptide sequences.

[0207] Immunohistochemistry

[0208] Immunohistochemistry was performed using the BenchMark XT automated slide preparation system (Ventana). Slides were developed using the iVIEW DAB detection kit (Ventana) according to a standardized protocol. Briefly, slides were dewaxed with CellConditioning 1 (CC1, Ventana) and blocked with phosphate-buffered saline (PBS) containing 1% BSA for 32 minutes. Antibodies were diluted to 1 μg / ml in antibody diluent (Ventana) and manually applied to slides for 1 hour at room temperature.

Claims

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 consisting of the sequence shown in SEQ ID NO: 2, and a heavy chain variable domain consisting of the sequence shown in SEQ ID NO: 3, wherein the epitope of the monoclonal antibody or the antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 1 (YKNIHLEKKY).

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein CSF1-R is human CSF-1R.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein at least one of the two tyrosine residues of the epitope is phosphorylated. The monoclonal antibody or antigen-binding fragment thereof according to claim 3 , wherein both tyrosine residues of the epitope are phosphorylated.

5. 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) a light chain variable domain comprising: (a1) a light chain CDR1 consisting of the sequence shown in SEQ ID NO:4 (QSSESVYSNNFLS), (a2) a light chain CDR2 consisting of the sequence shown in SEQ ID NO:5 (EASKVAS), and (a3) a light chain CDR3 consisting of the sequence shown in SEQ ID NO:6 (AGGYDVSDDA), as well as (b) a heavy chain variable domain comprising: (b1) a heavy chain CDR1 consisting of the sequence shown in SEQ ID NO:7 (TASGFSLSRYWMT), (b2) a heavy chain CDR2 consisting of the sequence shown in SEQ ID NO: 8 (RSGNTYFADWAKG), and (b3) Heavy chain CDR3, which consists of the sequence shown in SEQ ID NO: 9 (GGQNNGYDL).

6. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 in the preparation of a kit for detecting CSF-1R in a sample.

7. The use according to claim 6, wherein (a) the sample has been contacted with a candidate compound for treating cancer, or wherein the sample has been obtained from a subject who has been contacted with the candidate compound, (b) the sample is a cancer cell or cancer tissue, (c) the subject is a mammalian subject, (d) the detection of CSF-1R is a quantitative detection of CSF-1R, and / or (e) The detection of CSF-1R is immunohistochemical detection of CSF-1R.

8. The use according to claim 7, wherein the mammalian subject is a human subject.

9. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 in the preparation of a kit for performing a method for detecting CSF-1R in a sample, the method comprising (a) contacting a sample containing CSF-1R with the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, thereby forming a complex containing CSF-1R and the monoclonal antibody or antigen-binding fragment thereof, and (b) detecting the complex formed in step (a), thereby detecting CSF-1R in the sample. 10 . A complex comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 , and CSF-1R.

11. The use according to claim 6 or 7 or 8, the use according to claim 9 or the complex according to claim 10, wherein the CSF-1R comprises a phosphorylated tyrosine residue at a position corresponding to position 699 of human CSR-1R and / or comprises a phosphorylated tyrosine residue at a position corresponding to position 708 of human CSR-1R.

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