Anti IL-34 antibodies

Abstract

The present disclosure relates to novel anti IL-34 antibodies or antigen-binding fragments thereof specifically binding cytokine IL-34 with high affinity, the method of obtaining of these antibodies and their therapeutic use.

Claims

1. An anti-IL-34 antibody or an antigen-binding fragment thereof, said antibody comprising: a light chain comprising CDR-L1 of SEQ ID NO:1, CDR-L2 of SEQ ID NO:2, and CDR-L3 of SEQ ID NO:3; and a heavy chain comprising CDR-H1 of SEQ ID NO:4, CDR-H2 of SEQ ID NO:5, and CDR-H3 of SEQ ID NO:6.

2. The antibody or an antigen-binding fragment thereof of claim 1, said antibody comprising a light chain comprising SEQ ID NO:7 and a heavy chain comprising SEQ ID NO:8.

3. The antibody or an antigen-binding fragment thereof of claim 1, said antibody being a monoclonal antibody.

4. The antibody or an antigen-binding fragment thereof of claim 1, said antibody being a chimeric antibody.

5. The antibody or an antigen-binding fragment thereof of claim 1, said antibody being a humanized antibody.

6. The antibody or an antigen-binding fragment thereof of claim 1, wherein said antigen-binding fragment is selected from the list consisting of Fv, scFv, Fab, F(ab)2, Fab, scFv-Fc, diabodies, and any antigen-binding fragment whose half-life has been increased by chemical modification with polyalkylene glycol.

7. The antibody or an antigen-binding fragment thereof of claim 1, wherein said antibody is capable of inhibiting the interaction of IL-34 with at least one of its receptors.

8. The antibody or an antigen-binding fragment thereof of claim 1, wherein said antibody is capable of inhibiting the interaction of IL-34 with at least one of the receptors selected from the group consisting of Macrophage Colony Stimulating Factor Receptor (M-CSF-R) and Receptor Protein Tyrosine Phosphatase / (RPTP/).

9. The antibody or an antigen-binding fragment thereof of claim 1, wherein the dissociation constant (KD) of said antibody is KD10.sup.11 M measured by BIAcore.

10. A pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof of claim 1, and a pharmaceutically-acceptable carrier.

11. A medicament comprising the antibody or an antigen-binding fragment thereof of claim 1.

12. A kit comprising at least the antibody or an antigen-binding fragment thereof of claim 1.

13. The kit according to claim 12, wherein said antibody or antigen-binding fragment thereof is labelled.

14. A method for production of an antibody or an antigen-binding fragment thereof of claim 1, said method comprising the steps of: a) growing a host cell comprising a vector expressing: a.1) a nucleic acid coding for the antibody or an antigen-binding fragment thereof of claim 1; a.2) a nucleic acid comprising the DNA sequences of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14; or a.3) a nucleic acid comprising a DNA sequence consisting of a pair of polynucleotides, wherein one of the polynucleotides encodes the light chain and is set forth in SEQ ID NO: 15 and the other polynucleotide encodes the heavy chain and is set forth in SEQ ID NO: 16, in an appropriate medium, and b) recovering said antibody.

15. A method for treating a disease dependent on IL-34 selected from the list consisting of an inflammatory disease, auto-immune disease, cancer, and bone disease, comprising administering to a subject in need thereof: an effective amount of the antibody or an antigen-binding fragment thereof of claim 1, or a pharmaceutical composition comprising the antibody or an antigen-binding fragment thereof of claim 1 and a pharmaceutically-acceptable carrier.

16. The method according to claim 15, wherein said inflammatory disease dependent on IL-34 is selected from the list consisting of rheumatoid polyarthritis, periodontitis, periprosthetic osteolysis, Gougerot-Sjgren syndrome, arthritis, inflammatory skin pathologies, inflammatory bowel diseases and fibrosis.

17. The method according to claim 15, wherein said cancer dependent on IL-34 is selected from the list consisting of: tumour osteolysis, bone metastases, brain cancers, lung cancer, and bone sarcomas selected from osteosarcoma and Ewing's sarcoma.

Description

FIGURE LEGENDS

(1) FIG. 1: Analysis by Agilent instrument of anti-IL-34 mRNA integrity after RNA extraction;

(2) FIG. 2: Sensogram after correction of the reference obtained from SPR interaction curves for various concentration of IL-34, showing the interaction of IL-34 with anti-IL-34 antibody;

(3) FIG. 3: Sensogram after correction of the reference obtained from SPR interaction curves for 50 nM of IL-34 and 10 nM of anti-IL-34;

(4) FIG. 4: Inhibition of proliferation/survival of isolated human CD14.sup.+ monocytes by anti-IL-34 antibodies;

(5) FIG. 5: Inhibition of IL34-induced cell signalization by anti-IL-34 antibodies;

(6) FIG. 6: (A) Human CD14+ viability measured by fluorescence Intensity and (B) Inhibition of proliferation/survival of isolated human CD14.sup.+ monocytes by murine anti-IL-34 antibodies (BT34) compared to recombinant IL-34 antibodies (chimeric antibodies clone rec-BT34);

(7) FIG. 7: (A) Western blot and western blot quantification and (B) Inhibition of IL34-induced cell signalization by murine anti-IL34 antibodies (clone BT34) compared to recombinant IL-34 antibodies (chimeric antibodies clone recBT34).

EXAMPLES

(8) 1. Sequencing DataMurine IL-34 Antibodies

(9) Summary of the Process a) Cellular culture b) Total RNA extraction c) Reverse transcription of the RNA (primer oligodT) d) Amplification of variable chains by PCR (various primer pairs) e) Cloning of the amplicons in shuttle vector f) Sequencing of the inserts g) Sequence analysis

(10) RNA Extraction

(11) The full analysis and results for mRNA extraction are shown in table 2 below.

(12) TABLE-US-00002 TABLE 2 Volume conc RNA yield RNA purity Clone Cell nb 10.sup.6 (l) ng/l [g] A.sub.260/A.sub.280.sup.2 Anti-IL-34 25 250 1020 255 2.08 BT34 clone

(13) The analysis of mRNA integrity is done using the Agilent instrument (cf. FIG. 1).

(14) Monoclonal Antibodies Sequencing from B-T34

(15) Sequencing data were analyzed on the IgBlast database. The sequences corresponding to complete variables chains of antibody are presented in the annexed sequence listing.

(16) The analysis of the sequencing data shows that this sequences coding light and heavy chain variable domain is complete and functional.

(17) 2. Chimerization of the Antibodies Anti-IL-34 of the Invention

(18) The chimeric antibodies anti-IL-34 of the invention are obtained by following protocol:

(19) The DNA sequences encoding the variable domains of the tight and heavy chains are introduced by ligature restriction in two type P115 expression vectors respectively encoding the human constant domains of the heavy chain (IgG1 isotype) and the light chain (isotype Kappa), forming a heavy chain PTT5 vector and a light chain PTT5 vector. After controlling the obtained sequence, these two vectors are amplified in order to perform their transfection into HEK-EBNA cells cultivated in serum free stirred mode conditions. The transfection is carried out by using a lipofection agent (JET PEI) and the cells are then cultivated for a week with slow stirring. The secretion of recombinant antibodies (chimeric antibodies) is then monitored using an ELISA assay (Kit FASTELISA RD-Biotech) every two days after the transfection. The cell culture is stopped after 7 days and the supernatant is collected by centrifugation. Chimeric recombinant antibodies are then purified from the supernatant by affinity chromatography on protein A (Repligen corp.). The purified antibodies are tested by SDS-PAGE electrophoresis and their specificity is firstly controlled by ELISA and then, by the bioassay which allows controlling the original murine antibody properties.

(20) 3. Humanization of the Antibodies Anti-IL-34

(21) During the humanization step, the murine sequences present in the chimeric antibody are analyzed for each amino acid and compared with databanks in order to mimic a human sequence. The construction of 5 to 10 humanization variants is performed by DNA neosynthesis. After synthesis of DNA encoding these variants (humanized heavy chain and humanized light chain) the sequences are subcloned by ligature restriction into PTT5 vectors described above. The vectors encoding the different variants are then transfected using the same conditions and in the same cells as previously described in order to obtain the different humanized antibody variants which are purified as described above. The characterization of these variants and the analysis of their performance are performed as for the chimeric antibodies.

(22) For clinical purpose, the humanized antibodies may be produced in CHO cells.

(23) 4. Analysis of Interaction Between Antibody Anti-IL-34 of the Invention and Recombinant Protein IL-34 and Measuring of Kd, Kon and Koff.

(24) 4.1. Methods and Materials

(25) BIACore T 100 system has been used in order to analyze the interaction between anti-IL-34 and the recombinant IL-34.

(26) The following microarrays: CM5 (CM-dextran, 1-ethyl-3-(3-diaminopropyl) carbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) have been purchased by GE Healthcare BIAcore and used according to manufacture instructions.

(27) A polyclonal ant-mouse antibody was immobilized covalently on the CM5 microarray surface, thus allowing capturing IL-34 antibody as ligand. Capture level of anti-IL-34 is comprised between 700 and 1000 RU (resonance unity).

(28) A control channel anti-IL-34 was prepared as indicated above without in order to assess nonspecific interaction on the microarray's surface.

(29) Theses interaction were performed at 25 C., in HBS-P+ buffer (HEPES 10 mM pH 7.4; NaCl 150 mM; P20 0.05%) by administrating different concentrations of IL-34 obtained by serial dilution of the protein IL-34 (Peprotech Ref 200-34, whole protein without signal peptide with His tag on C-terminal).

(30) The interaction kinetics are performed without regeneration between each administration.

(31) The regeneration is performed by administering 10 mM of Glycine-HCl buffer at pH=1.7 allowing dissociating the antibody IL-34 from immobilized polyclonal antibody.

(32) 4.2. Results

(33) The obtained results are shown by sensograms of FIG. 2. The sensograms have been analyzed via 1:1 mathematic pattern allowing adjusting the obtained values shown in table 3 below.

(34) TABLE-US-00003 TABLE 3 k.sub.a (M.sup.1S.sup.1) k.sub.d (s.sup.1) K.sub.D (M) 3.1 0.4 10.sup.+4 2.9 2.3 10.sup.7 9.4 7 10.sup.12

(35) Obtained sensograms show a slow association between IL-34 and the anti-IL-34 antibody BT34 (Ka is about 310.sup.4M.sup.1S.sup.1) and a very low dissociation (Kd<10-6s-1).

(36) Thus, these resultants clearly demonstrate that interaction between IL-34 and the anti-IL-34 antibody is very stable and consequently, that the binding activity is very high.

(37) Furthermore, obtained dissociation during the IL-34 and the anti-IL-34 antibody interaction was so weak that it was impossible to determine it by presently used tools.

(38) 5. Immobilization of the Receptor

(39) 5.1. Methods and Materials

(40) The measurement of surface plasmon resonance (SPR) was performed on BIACore T100. The microarrays CM5 (CM-dextran, 1-ethyl-3-(3-diaminopropyl) carbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) have been purchased by GE Healthcare BIAcore and used according to manufacture instructions.

(41) The ligand, M-CSF receptor (R&D-Systems ref: 329-MR) was immobilized covalently on the CM5 microarray surface. Capture level of anti-IL-34 is comprised between 600 and 800 RU.

(42) A control channel without protein was prepared as indicated previously in order to assess nonspecific interaction on the microarray's surface.

(43) Theses interaction were performed at 25 C., in HBS-P+ buffer (HEPES 10 mM pH 7.4; NaCl 150 mM; P20 0.05%).

(44) The microarray was regenerated before each interaction.

(45) 5.2. Results

(46) Positive Control: Interaction IL-34/M-CSF-R

(47) Different concentrations (10 nM, 20 nM and 50 nM) of IL-34 protein were administered on the microarray surface comprising the immobilized receptor M-CSF-R.

(48) An interaction signal of about 20 RU was observed with only 50 nM of IL-34.

(49) During previously performed assays-receptor capture on the microarray surface) for lower level of immobilization (about 100-200 RU), the interaction signal was observed after administration of 20 nM IL-34.

(50) The above results show that there are several type of receptors immobilized on the microarray surface.

(51) As previously mentioned, the regeneration of microarray was difficult because receptors immobilization on the microarray surface was weak.

(52) Thus, it was difficult to assess IL-34/anti-IL-34 antibody with increasing concentrations of the antibody.

(53) For this purpose, in order to perform the assays without errors, novel microarrays were performed as indicated above, for each assessed concentration of the anti-IL-34 antibody BT34.

(54) Negative Control

(55) 10 nM of the anti-IL-34 antibody were injected on the microarray surface. No signal was observed showing that there is no interaction between the anti-IL-34 antibody and the M-CSF-R receptor.

(56) Inhibiting Assay

(57) The inhibiting assay was performed in order to measure signal emitted during the interaction between IL-34 and M-CSF-R.

(58) IL-34 protein was incubated with large quantity of the anti-IL-34 antibody BT34 (50 nM/1 M). In these experimental conditions no signal was observed. This indicates that in presence of high concentrations of the anti-IL-34 antibody, IL-34 protein does not interact with its receptor.

(59) After that, 50 nM of IL-34 protein was injected on the microarray surface.

(60) As shown on FIG. 3 the obtained sensogram (about 30 RU) demonstrates that the interaction between IL-34 and its receptor is stable.

(61) After that 10 nM of anti-IL-34 antibody were injected and a signal demonstrating the interaction of IL-34 with the anti-IL-34 antibody was observed (FIG. 3).

(62) These assays demonstrate that IL-34 interact with its receptor M-CSF-R on the microarray surface. Even though the antibody is capable of interacting with IL-34 when bound to its receptor, the interaction between IL-34 and its receptor is inhibited when the antibody anti-IL-34 is incubated with IL-34 before the interaction of this protein with its receptor.

(63) Thus, these data demonstrate that the anti-IL-34 antibody epitope is distinct of the interaction site between IL-34 and its receptor and that the inhibitory effect is due to steric configuration of complex protein/antibody.

(64) Consequently, it is demonstrated that the antibodies of the invention are capable of binding the cytokine IL-34 even when this one is binded to its receptor which allow increasing the therapeutical efficiency of these antibodies.

(65) 6. Inhibitory Effect of Murine Anti-IL-34 Antibodies on Cell Proliferation

(66) 6.1. Materials and Methods

(67) Isolation of Human CD14.sup.+ Monocytes and Analysis of Cell Proliferation

(68) Human CD14.sup.+ monocytes were initially isolated from human peripheral blood donors provided by the French blood bank institute (Etablissement Franais du Sang, Nantes, France, authorization number: NTS 2000-24), by using MACS microbeads (MiltenyiBiotec, Germany). Cells were cultured in the presence of 25 ng/mL or 50 ng/mL of IL-34, with or without increased concentrations of anti-IL-34 antibodies. The effects of treatments on CD14.sup.+ survival/proliferation were determined by measuring metabolic activity using an Alamar Blue assay. Forty thousand cells per well were put into 96-well plates with -MEM and the corresponding treatments. After 3 days, Alamar Blue reagent was added and the fluorescence produced was read in the linear range (excitation 530 nm/emission 600 nm).

(69) 6.2. Results

(70) In order to test whether the antibody murine anti IL-34 (clone BT34) is capable of inhibiting IL-34 dependent cell proliferation, increasing amounts of the antibody were added to IL-34 stimulated monocytes. As shown in FIG. 4, this antibody at a concentration of ca. 5 lag/mL was capable of preventing the proliferation of about 70% of the CD14+ cells. The same level of inhibition was observed with higher concentrations of antibody. In contrast, other II-34 antibodies (20D1 and 26D9) failed to yield such a potent effect. Finally, a third anti-IL-34 control antibody prevented the proliferation of about 70% of the monocytes at a low concentration (ca. 5 g/mL). However, the percentage of inhibition decreased when higher concentrations of antibody were used, raising doubts about the utility of this antibody. On the other hand, BT34 led to the same level of inhibition, regardless of whether 5 or 25 g/mL are used.

(71) Thus, the BT34 anti-IL-34 antibody can be used successfully for preventing and/or treating proliferative diseases such as cancer.

(72) 7. Inhibitory Effect of Murine Anti-IL-34 Antibodies on Cell Signalization

(73) 7.1 Materials and Methods

(74) Western Blot Analysis

(75) The M-CSFR expressing cells treated or not with 50 ng/mL of human IL-34 with or without 2 mg/mL of anti-Il-34 antibodies (clones 1D8, 2E8, BT34, 10E5, 17G6, 20D1, 26D9, 2867) for 10 minutes, were collected in a RIPA buffer (10 mM Tris pH8, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.1% SDS containing a cocktail of protease and phosphatase inhibitors: 1 mM sodium orthovanadate (Na.sub.2VO.sub.4), 1 mM phenylmethylsulforyl fluoride (PMSF), 10 mM sodium fluoride (NaF), 10 mM N-ethylmaleimide (NEM), 2 g/ml leupeptin and 1 g/ml pepstatine). The protein concentration was determined using a BCA (bicinchoninic acid) protein assay (Sigma Aldrich). 40 g of total protein extracts were prepared in a Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001% bromophenol blue) and then separated by SDS-polyacrylamide gel electrophoresis. After electrophoretic transfer, the immobilon-P membranes (Millipore, Molsheim, France) were blotted with the primary antibodies. Primary antibodies directed against human P-MCSFR (Tyr-723), P-Erk1/2, and the total form of proteins were purchased from Cell Signalling (Ozyme, Saint Quentin Yvelines, France). The membranes were then probed with secondary antibodies coupled with horseradish peroxidase. Antibody binding was visualised with a Pierce enhanced chemiluminescence (ECL) kit (ThermoSientific, Illkirch, France). The luminescence detected with a Charge Couple Device (CCD) camera was quantified using the GeneTools programme (Syngene, Cambridge, United Kingdom).

(76) 7.2. Results

(77) As shown on FIG. 5, IL-34-dependent Tyr-phosphorylation of the MCSF receptor is blocked by the BT34 antibodies. Likewise, activation of ERK 1/2 is prevented by this antibody. These results are consistent with the capacity of BT34 to inhibit the IL-34-mediated proliferation of cells.

(78) 8. Inhibitory Effect of Murine Anti-IL-34 Antibodies on Cell Proliferation Compared to the Inhibitory Effect of Recombinant Anti-IL-34 (Chimeric Antibody recBT34)

(79) 8.1. Materials and Methods

(80) Isolation of Human CD14.sup.+ Monocytes and Analysis of Cell Proliferation

(81) Human CD14.sup.+ monocytes were initially isolated from human peripheral blood donors provided by the French blood bank institute (Etablissement Franais du Sang, Nantes, France, authorization number: NTS 2000-24), by using MACS microbeads (MiltenyiBiotec, Germany). Cells were cultured in the presence of IL-34, with recombinant anti-IL-34 antibody (chimeric antibody recBT34) and with murine anti-IL-34 antibody (clone BT34). The effects of treatments on CD14.sup.+ survival/proliferation were determined by measuring metabolic activity using an Alamar Blue assay. Forty thousand cells per well were put into 96-well plates with -MEM and the corresponding treatments. After 3 days, Alamar Blue reagent was added and the fluorescence produced was read in the linear range (excitation 530 nm/emission 600 nm).

(82) 8.2. Results

(83) As shown on FIGS. 6 (A and B) the recombinant anti-IL-34 (rec-BT34) antibodies markedly inhibited human CD14+ cell viability and with more efficiency than the murine IL-34 antibodies (clone BT34).

(84) Thus, the recombinant anti-IL-34 antibody can also be used successfully for preventing and/or treating proliferative diseases such as cancer.

(85) 9. Inhibitory Effect of Murine Anti-IL-34 Antibodies (Clone BT34) Compared to the Inhibitory Effect of the Recombinant IL-34 Antibody (Chimeric Antibody recBT34) on Cell Signalization

(86) 9.1. Materials and Methods

(87) Western Blot Analysis

(88) The M-CSFR expressing cells treated with 50 ng/mL of human IL-34 with 0.4 g/ml or 2 g/ml of murine anti-Il-34 antibody (clone BT34) and with of 0.4 g/ml or 2 g/ml recombinant anti IL-34 antibody (rec-BT34) for 10 minutes, were collected in a RIPA buffer (10 mM Tris pH8, 1 mM EDTA, 150 mM NaCl, 1% NP40, 0.1% SDS containing a cocktail of protease and phosphatase inhibitors: 1 mM sodium orthovanadate (Na.sub.2VO.sub.4), 1 mM phenylmethylsulforyl fluoride (PMSF), 10 mM sodium fluoride (NaF), 10 mM N-ethylmaleimide (NEM), 2 g/ml leupeptin and 1 g/ml pepstatine). The protein concentration was determined using a BCA (bicinchoninic acid) protein assay (Sigma Aldrich). 40 g of total protein extracts were prepared in a Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001% bromophenol blue) and then separated by SDS-polyacrylamide gel electrophoresis. After electrophoretic transfer, the immobilon-P membranes (Millipore, Molsheim, France) were blotted with the primary antibodies. Primary antibodies directed against human P-MCSFR (Tyr-723), P-Erk1/2, and the total form of proteins were purchased from Cell Signalling (Ozyme, Saint Quentin Yvelines, France). The membranes were then probed with secondary antibodies coupled with horseradish peroxidase. Antibody binding was visualised with a Pierce enhanced chemiluminescence (ECL) kit (ThermoSientific, Illkirch, France). The luminescence detected with a Charge Couple Device (CCD) camera was quantified using the GeneTools programme (Syngene, Cambridge, United Kingdom).

(89) 9.2. Results

(90) As shown on FIGS. 7 (A and B), IL-34-dependent Tyr-phosphorylation of the MCSF receptor is blocked by all anti-IL-34 antibodies. Likewise, activation of ERK 1/2 is prevented by this antibody. These results are consistent with the capacity of anti-IL-34 antibodies to inhibit the IL-34-mediated proliferation of cells.

(91) FIGS. 7 (A and B) also shows that the recombinant anti-IL-34 antibody (chimeric antibody recBT34) inhibits hIL-34 induced signalization in MCSFR overexpressing HEK293 in a similar manner compared to murine anti-IL-34 antibody BT34.

REFERENCES

(92) Cronk J C, Kipnis J. Microgliathe brain's busy bees. F1000Prime Rep. (2013); 5:53. Dai X. M., G. R. Ryan, A. J. Hapel, M. G. Dominguez, R. G. Russell, S. Kapp, V. Sylvestre, E. R. Stanley, Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects, Blood 99 (2002) 111-120. Felix J., J. Elegheert, I. Gutsche, A. V. Shkumatov, Y. Wen, N. Bracke, E. Pannecoucke, I. Vandenberghe, B. Devreese, D. I. Svergun, E. Pauwels, B. Vergauwen, S. N. Savvides, Human IL-34 and CSF-1 establish structurally similar extracellular assemblies with their common hematopoietic receptor, Structure 21 (2013) 528-539. Foucher E. D., S. Blanchard, L. Preisser, E. Garo, N. Ifrah, P. Guardiola, Y. Delneste, P. Jeannin, IL-34 Induces the Differentiation of Human Monocytes into Immunosuppressive Macrophages, Antagonistic Effects of GM-CSF and IFN, PloS One 8 (2013) e56045. Lin H., E. Lee, K. Hestir, C. Leo, M. Huang, E. Bosch, R. Halenbeck, G. Wu, A. Zhou, D. Behrens, D. Hollenbaugh, T. Linnemann, M. Qin, J. Wong, K. Chu, S. K. Doberstein, L. T. Williams, Discovery of a Cytokine and Its Receptor by Functional Screening of the Extracellular Proteome, Science 320 (2008) 807-811. Liu H., C. Leo, X. Chen, B. R. Wong, L. T. Williams, H. Lin, X. He, The mechanism of shared but distinct CSF-1R signaling by the non-homologous cytokines IL-34 and CSF-1, Biochim. Biophys. Acta 1824 (2012) 938-945. Ma X., W. Y. Lin, Y. Chen, S. Stawicki, K. Mukhyala, Y. Wu, F. Martin, J. F. Bazan, Needternan S B, Wunsch C D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mol Blot. 48(1970):443-53. Pearson W R, Lipman D J. Improved tools for biological sequence comparison. Proc Natl Acad Sci USA. 85(1988):2444-8. Ries C H, Cannarite M A, Hoves S2, Benz J, Wartha K, Runza V, Rey-Giraud F, Pradel L P, Feuerhake F, Klaman I, Jones T, Jucknischke U, Scheibtich S, Katuza K, Gorr I H, Walz A, Abiraj K, Cassier P A, Sica A, Gomez-Roca C, de Visser K E, Italiano A, Le Tourneau C, Delord J P, Levitsky H, Bay J Y, Rttinger D. Targeting tumor-associated macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy. Cancer Celt 25(2014):846-59. Roth P., E. R. Stanley, The biology of CSF-1 and its receptor, Curr. Top. Microbiol. Immunol. 181 (1992) 141-167. Sgaliny A I, Mohamadi A, Dizier B, Lokajczyk A, Brion R, Lanel R, Amiaud J, Charrier C, Boisson-Vidal C, Heymann D. Interleukin-34 promotes tumour progression and metastatic process in osteosarcoma through induction of angiogenesis and macrophage recruitment. Int J Cancer. 2014 in press. Smith, T. F. and Waterman, M. S. Comparison of biosequences, Adv. Appl. Math., 2 (1981) 482-489. Stanley E R, Chitu V. CSF-1 receptor signaling in myeloid cells. Cold Spring Harb Perspect Biol (2014) 2; 6. Starovasnik M. A., Structural Basis for the Dual Recognition of Helical Cytokines IL-34 and CSF-1 by CSF-1R, Structure 20 (2012) 676-687. Tatusova T A, Madden T L. BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences FEMS Microbiol Lett. 174 (1999) 247-50. Verhoeyen M, Riechmann L. Engineering of antibodies. Bioessays. 8 (1988):74-8. Wang Y, Colonna M. Interkeukin-34, a cytokine crucial for the differentiation and maintenance of tissue resident macrophages and Langerhans cells. Eur J Immunol (2014) 44:1575-81. Wei S., S. Nandi, V. Chitu, Y. G. Yeung, W. Yu, M. Huang, L. T. Williams, H. Lin, E. R. Functional overlap but differential expression of CSF-1 and IL-34 in their CSF-1 receptor-mediated regulation of myeloid cells. J Leukoc Biol. 88 (2010) 495-505. Ye J, Ma N, Madden T L, Ostell J M. IgBLAST: an immunoglobulin variable domain sequence analysis toot, Nucleic Acids Res. 41 (Web Server issue) (2013) W34-40. WO 2013/119716.