RU2751249C1 - Monoclonal antibody that specifically binds to csf-1r - Google Patents

Abstract

FIELD: biotechnology and medicine.SUBSTANCE: present invention relates to the field of biotechnology and medicine, namely to monoclonal antibodies or their antigen-binding fragments that specifically bind to CSF-1R (colony-stimulating factor 1 receptor). The invention also relates to nucleic acids encoding these antibodies, expression vectors, host cells and methods for their production, methods for the production of antibodies, pharmaceutical compositions containing the abovementioned antibody, and other therapeutically active compounds, methods for the treatment of diseases or disorders mediated by CSF-1R, applications of antibodies or their pharmaceutical compositions for the treatment of diseases or disorders mediated by CSF-1R, and the use of antibodies and other therapeutically active compounds for the treatment of CSF-1R-mediated diseases or disorders.EFFECT: application of antibodies or their pharmaceutical compositions for the treatment of diseases or disorders mediated by CSF-1R.59 cl, 18 dwg, 15 tbl, 29 ex

Description

The technical field to which the invention relates

The present invention relates to the field of biotechnology and medicine, namely to monoclonal antibodies or antigen-binding fragments thereof that specifically bind to CSF-1R (colony stimulating factor receptor 1). The invention also relates to nucleic acids encoding said antibodies, expression vectors, host cells and methods for their production, methods for producing antibodies, pharmaceutical compositions containing the above antibody, pharmaceutical compositions containing the above antibody and other therapeutically active compounds, methods of treating diseases or disorders mediated by CSF-1R, the use of antibodies or their pharmaceutical compositions for the treatment of diseases or disorders mediated by CSF-1R, and the use of antibodies and other therapeutically active compounds for the treatment of diseases or disorders mediated by CSF-1R.

State of the art

Colony stimulating factor 1 receptor (CSF-1R), also known as macrophage colony stimulating factor receptor (M-CSFR) or CD115, is a membrane protein that is a milestone of macrophage differentiation. Has 2 ligands: CSF-1 and IL-34. Its signaling is necessary for the differentiation of precursors of the myeloid lineage into macrophages, which may also be participants in the tumor microenvironment (TAM, tumor-associated macrophages).

Depending on the predominant activity, two groups of macrophages are distinguished: M1 and M2. M1 macrophages (also called classically activated macrophages) are responsible for the destruction of foreign agents (including tumor cells), both directly and by attracting and activating other cells of the immune system (for example, T-killers). M2 macrophages, in turn, accelerate tissue regeneration and promote wound healing.

The presence of a large number of M1 macrophages in a tumor inhibits its growth, and vice versa: M2 macrophages secrete molecules - growth factors that stimulate the division of tumor cells. It has been shown experimentally that it is M2 macrophages that usually predominate in the tumor environment. Moreover, under the action of substances secreted by tumor cells, including under conditions of increased CSF-1R expression, active M1 macrophages are "reprogrammed" into M2 macrophages, stop synthesizing antitumor cytokines (for example, IL-12, TNF) and begin release into the environment molecules that accelerate tumor growth and angiogenesis (for example, TGFb, VGF).

The tumor converts aggressive M1 macrophages into self-promoting M2 macrophages. Perceiving a tumor as a damaged tissue site, M2-macrophages start a recovery program, however, the growth factors secreted by them only increase tumor growth.

Thus, the main goal of inhibition of CSF-1R signaling is reprogramming of the tumor microenvironment (in favor of predictors of activation of the antitumor response).

Various antibodies are known in the art that specifically bind to CSF-1R (for example, WO2011140249, WO2011070024, WO2011131407, WO2011123381, WO2009112245).

Known antibody cabiralizumab, which specifically binds to CSF-1R, described in international application WO2011140249 (FIVE PRIME THERAPEUTICS INC). This antibody is in the second phase of clinical trials.

However, at the moment, there is not a single antibodies against CSF-1R, approved anywhere in the world for therapeutic use. In connection with the above, it is relevant to create new antagonistic antibodies that inhibit CSF-1R signaling, thereby reprogramming the tumor microenvironment in favor of predictors of activation of the antitumor response.

Brief description of the invention

In one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to CSF-1R, comprising:

(a) a heavy chain variable domain comprising:

(i) CDR1 with an amino acid sequence selected from the group: SEQ ID NO: 1, SEQ ID NO: 17,

(ii) CDR2 with an amino acid sequence selected from the group: SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO : 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,

(iii) CDR3 with an amino acid sequence selected from the group: SEQ ID NO: 3, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO : fifty; and

(b) a light chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 12,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 13,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that comprises:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 1,

(ii) a CDR2 with the amino acid sequence of SEQ ID NO: 2,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the isolated monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable domain that contains an amino acid sequence selected from the group: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7.

In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable domain of the amino acid sequence SEQ ID NO: 5.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain of the amino acid sequence SEQ ID NO: 6.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a light chain variable domain of the amino acid sequence SEQ ID NO: 15.

In some embodiments, an isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) a variable domain of the heavy chain containing an amino acid sequence selected from the group: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7;

(b) a light chain variable domain with the amino acid sequence of SEQ ID NO: 15.

In some embodiments, an isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) the variable domain of the heavy chain with the amino acid sequence of SEQ ID NO: 5;

(b) a light chain variable domain with the amino acid sequence of SEQ ID NO: 15.

In some embodiments, an isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) the variable domain of the heavy chain with the amino acid sequence of SEQ ID NO: 6;

(b) a light chain variable domain with the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the isolated monoclonal antibody comprises a heavy chain comprising an amino acid sequence selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11.

In some embodiments, the isolated monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9.

In some embodiments, the isolated monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the isolated monoclonal antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 16.

In some embodiments, an isolated monoclonal antibody comprises:

(a) a heavy chain containing an amino acid sequence selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11; and

(b) a light chain containing the amino acid sequence of SEQ ID NO: 16.

In some embodiments, an isolated monoclonal antibody comprises:

(a) a heavy chain containing the amino acid sequence of SEQ ID NO: 9; and

(b) a light chain containing the amino acid sequence of SEQ ID NO: 16.

In some embodiments, an isolated monoclonal antibody comprises:

(a) a heavy chain containing the amino acid sequence of SEQ ID NO: 10; and

(b) a light chain containing the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the isolated monoclonal antibody that specifically binds to CSF-1R is a full length IgG antibody.

In some embodiments, the isolated monoclonal antibody is a full length IgG antibody and is of the human IgG1, IgG2, IgG3, or IgG4 isotype.

In some embodiments, the isolated monoclonal antibody is a full length IgG antibody and is of the human IgG1 isotype.

In one aspect, the present invention provides an isolated nucleic acid that encodes any of the above-described antibody or antigen-binding fragment thereof.

In some embodiments, the isolated nucleic acid is DNA.

In one aspect, the present invention relates to an expression vector comprising any nucleic acid described above.

In one aspect, the present invention relates to a method for producing a host cell for producing any antibody or antigen-binding fragment thereof described above, comprising transforming the cell with the vector described above.

In one aspect, the present invention provides a host cell for producing any of the above-described antibody or antigen-binding fragment thereof comprising the above-described nucleic acid.

In one aspect, the present invention relates to a method for producing an antibody or antigen-binding fragment thereof as described above, comprising culturing a host cell in a culture medium under conditions sufficient to produce said antibody, if necessary, followed by isolation and purification of the resulting antibody.

In one aspect, the present invention relates to a pharmaceutical composition that contains an antibody of the present invention or an antigen-binding fragment thereof in a therapeutically effective amount in combination with one or more pharmaceutically acceptable excipients.

In one aspect, the present invention provides a pharmaceutical composition for the treatment of a disease or disorder mediated by CSF-1R, which comprises an antibody as described above or an antigen-binding fragment thereof in a therapeutically effective amount in combination with one or more pharmaceutically acceptable excipients.

In one aspect, the present invention provides a pharmaceutical composition for the treatment of a disease or disorder mediated by CSF-1R, comprising an antibody as described above or antigen-binding fragment thereof and at least one other therapeutically active compound.

In some embodiments, the other therapeutically active compound is an antibody, chemotherapeutic agent, or anti-hormonal agent.

In some embodiments, the other therapeutically active compound is a checkpoint inhibitor.

In some embodiments, the checkpoint inhibitor is selected from a PD-1 inhibitor or a CTLA-4 inhibitor.

In some embodiments, the PD-1 inhibitor is an antibody that specifically binds to PD-1.

In some embodiments, the antibody that specifically binds to PD-1 is selected from the group: prolgolimab, pembrolizumab, nivolumab.

In some embodiments, the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4.

In some embodiments, the antibody that specifically binds to CTLA-4 is ipilimumab.

In one aspect, the present invention provides a method for inhibiting the biological activity of CSF-1R in a subject in need of such inhibition, comprising administering to the subject an effective amount of an antibody or antigen binding fragment thereof as described above.

In one aspect, the present invention provides a method of treating a disease or disorder mediated by CSF-1R, comprising administering to a subject in need of such treatment an antibody or antigen binding fragment thereof described above or a pharmaceutical composition described above in a therapeutically effective amount.

In one aspect, the present invention provides a method of treating a disease or disorder mediated by CSF-1R, comprising administering to a subject in need of such treatment an antibody or antigen binding fragment thereof as described above and at least one other therapeutically active compound.

In some embodiments, the other therapeutically active compound is an antibody, chemotherapeutic agent, or anti-hormonal agent.

In some embodiments, the other therapeutically active compound is a checkpoint inhibitor.

In some embodiments, the checkpoint inhibitor is selected from a PD-1 inhibitor or a CTLA-4 inhibitor.

In some embodiments, the PD-1 inhibitor is an antibody that specifically binds to PD-1.

In some embodiments, the antibody that specifically binds to PD-1 is selected from the group: prolgolimab, pembrolizumab, nivolumab.

In some embodiments, the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4.

In some embodiments, the antibody that specifically binds to CTLA-4 is ipilimumab.

In one aspect, the present invention relates to the use of an antibody as described above, or antigen binding fragment thereof, or a pharmaceutical composition as described above, for treating a CSF-1R mediated disease or disorder in a subject in need of such treatment.

In one aspect, the present invention relates to the use of an antibody as described above or antigen-binding fragment thereof and at least one other therapeutically active compound for the treatment of a disease or disorder mediated by CSF-1R.

In some embodiments, the other therapeutically active compound is an antibody, chemotherapeutic agent, or anti-hormonal agent.

In some embodiments, the other therapeutically active compound is a checkpoint inhibitor.

In some embodiments, the checkpoint inhibitor is selected from a PD-1 inhibitor or a CTLA-4 inhibitor.

In some embodiments, the PD-1 inhibitor is an antibody that specifically binds to PD-1.

In some embodiments, the antibody that specifically binds to PD-1 is selected from the group: prolgolimab, pembrolizumab, nivolumab.

In some embodiments, the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4.

In some embodiments, the antibody that specifically binds to CTLA-4 is ipilimumab.

In some embodiments, the disease or disorder mediated by CSF-1R is selected from the group of: metastatic melanoma, metastatic bladder cancer, metastatic non-small cell lung cancer, metastatic renal cell carcinoma (light cell type), malignant neoplasms with microsatellite instability with ineffective primary therapy, locally advanced or metastatic squamous cell carcinoma of the head and neck, stomach cancer (cardio-esophageal zone), ovarian cancer, breast cancer, triple negative breast cancer, bone tumor, Hodgkin's lymphoma, glioma, glioblastoma, mesothelioma, myeloma, prostate cancer, soft tissue sarcoma.

Brief Description of Drawings

Figure 1 is a scheme for the synthesis of a combinatorial naive library of human Fab fragments.

Figure 2 is a scheme for the synthesis of a combinatorial naive library of scFv antibody fragments.

Figure 3 is a map of an expression plasmid for the production of Fab fragments

AmpR - bla gene conferring ampicillin resistance,

PBR 322 ori - replication sites,

LacI - lactose operon,

KmR - gene conferring resistance to kanamycin,

XhoI - XhoI restriction site,

NheI - NheI restriction site,

BamHI - BamHI restriction site,

NotI - NotI restriction site,

Lpelb - light chain leader peptide,

Hpelb - heavy chain leader peptide,

VK - light chain variable fragment,

CK - constant fragment of the light chain,

VH - variable fragment of the heavy chain,

CH1 - constant fragment of the heavy chain,

STOP CK - light chain stop codon,

STOP CH1 - heavy chain stop codon,

His-tag is an amino acid sequence that consists of six histidines, often at the N- or C-terminus of a protein,

Myc-tag - Myc-tag,

SeqH3b is a reverse primer.

Figure 4 is a map of an expression plasmid for scFv production

AmpR - Ampicillin resistance gene,

PBR 322 ori - replication sites,

LacI - lactose operon,

NcoI - NcoI restriction site,

NotI - NotI restriction site,

pelb - leader peptide,

His-tag is an amino acid sequence that consists of six histidines, often at the N- or C-terminus of a protein,

Myc-tag - Myc-tag,

Linker - glycine-serine linker (G4S) 3,

T7 Promoter - Bacteriophage T7 RNA polymerase promoter,

T7 Terminator - a sequence from T7 bacteriophage that provides efficient transcription termination,

T7 trans RBS - ribosome entry site.

Figure 5 is an electrophoretogram of candidates 01-011 - 01-018 under denaturing non-reducing conditions 7.5% SDS-PAGE

1. Molecular weight marker,

2. Candidate 01-011,

3. Candidate 01-012,

4. Candidate 01-013,

5. Candidate 01-014,

6. Candidate 01-015,

7. Candidate 01-016,

8. Candidate 01-017,

9. Candidate 01-018.

Figure 6 is an electrophoretogram of candidate 01-019 under denaturing non-reducing conditions 7.5% SDS-PAGE

1. Molecular weight marker,

2. Candidate 01-019.

Figure 7 is a graph showing the ability of candidate 01-016 to inhibit IL-34-dependent proliferation

01-016 - candidate 01-016.

For candidate 01-016: A (signal at the lower plateau (signal at the point where the antibody concentration is 0)) = 52098, D (signal at the upper plateau) = 4861.5, B (slope) = 1.7644, C (EC50) = 0.36792, d (standard deviation) = 332.87, r (determination coefficient) = 0.99988.

Figure 8 is a graph showing the effect of anti-CSF-1R antibody 01-016 on monocyte proliferative activity in the presence of M-CSF. CellTiter-Glo® Luminescent Cell Viability Assay Cell.

For candidate 01-016: A (signal at the lower plateau (signal at the point where the antibody concentration is 0)) = 4.2326e + 005, D (signal at the upper plateau) = 1.2845e + 005, B (slope of the curve) = 0.77816 , С (EC50) = 0.24761, d (standard deviation) = 9305.7, r (determination coefficient) = 0.99573.

Figure 9 is a graph showing the effect of anti-CSF-1R antibody 01-016 on monocyte proliferative activity in the presence of M-CSF. Apo-ONE® Homogeneous Caspase-3/7 Assay.

For candidate 01-016: A (signal at the lower plateau (signal at the point where the antibody concentration is 0)) = 26569, D (signal at the upper plateau) = 59046, B (slope) = 2.2794, C (EC50) = 0.36883, d (standard deviation) = 1161.1, r (determination coefficient) = 0.99674.

Figure 10 is a graph showing the evaluation of the effect of anti-CSF-1R antibody 01-016 on the proliferative activity of monocytes in the presence of M-CSF. Apo-ONE® Homogeneous Caspase-3/7 Assay

M1 - monocytes differentiated by the M1 pathway,

M2 - monocytes differentiated by the M2 pathway,

01-016 - M2-differentiated monocytes in the presence of 01-016 antibody.

Figure 11 is an electropherogram of antibody 01-016 under non-reducing conditions 7.5% SDS-PAGE

1. Molecular weight marker,

2. Antibody 01-016, 10 μg,

3. Antibody 01-016, 40 μg.

Figure 12 is an electropherogram of OPTIM-6_001-02 antibody under non-reducing conditions 7.5% SDS-PAGE

1. Molecular weight marker,

2. Antibody OPTIM-6_001-02, 10 μg,

3. Antibody OPTIM-6_001-02, 40 μg.

Figure 13 is an electrophoretogram of OPTIM-6_001-04 antibody under non-reducing conditions 7.5% SDS-PAGE

1. Molecular weight marker,

2. Antibody OPTIM-6_001-04, 10 μg,

3. Antibody OPTIM-6_001-04, 40 μg.

Figure 14 is an electropherogram of OPTIM-6_001-05 antibody under non-reducing conditions 7.5% SDS-PAGE

1. Molecular weight marker,

2. Antibody OPTIM-6_001-05, 10 μg,

3. Antibody OPTIM-6_001-05, 40 μg.

Figure 15 is an electropherogram of antibody 01-016 under 12% SDS-PAGE reducing conditions.

1. Molecular weight marker,

2. Antibody 01-016, 10 μg,

3. Antibody 01-016, 40 μg.

Figure 16 is an electrophoretogram of OPTIM-6_001-02 antibody under 12% SDS-PAGE reducing conditions.

1. Molecular weight marker,

2. Antibody OPTIM-6_001-02, 10 μg,

3. Antibody OPTIM-6_001-02, 40 μg.

Figure 17 is an electrophoretogram of OPTIM-6_001-04 antibody under 12% SDS-PAGE reducing conditions.

1. Molecular weight marker,

2. Antibody OPTIM-6_001-04, 10 μg,

3. Antibody OPTIM-6_001-04, 40 μg.

Figure 18 is an electropherogram of OPTIM-6_001-05 antibody under 12% SDS-PAGE reducing conditions.

1. Molecular weight marker,

2. Antibody OPTIM-6_001-05, 10 μg,

3. Antibody OPTIM-6_001-05, 40 μg.

Description of the invention

Definitions and general methods

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention will have meanings that are commonly understood by those of ordinary skill in the art.

In addition, unless the context requires otherwise, singular terms include plural terms, and plural terms include singular terms. Typically, the classifications and methods used for cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medicinal and pharmaceutical chemistry, as well as protein and nucleic acid hybridization and chemistry described herein are well known to those skilled in the art and are widely used in this field. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as is commonly done in the art, or as described herein.

The term “Ka” as used herein refers to the rate of association of a particular antibody-antigen interaction, while the term “KD” or “Kd” refers to the rate of dissociation of a particular antibody-antigen interaction.

"Binding affinity" generally refers to the strength of the cumulative non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, "binding affinity" refers to the intrinsic (intrinsic, true) binding affinity that reflects a 1: 1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X to its partner Y can usually be represented by the dissociation constant (Kd). Desirably, the Kd is about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM or less. Affinity can be measured by conventional methods known in the art, including those described herein. Low affinity antibodies generally bind slowly to the antigen and tend to dissociate easily, while high affinity antibodies generally bind antigen more quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of these methods can be used for the purposes of the present invention.

The term "koff" or "kdis" refers to the dissociation rate constant of a particular interaction between a binding molecule and an antigen. The dissociation rate constant koff can be measured by biolayer interferometry, for example with the Octet ™ system.

The term "kon" or "on-rate" refers to an association rate constant.

The term "off-rate screening" refers to a screening in which candidates are tested based on koff values alone.

The term "R ² " refers to the coefficient of determination.

The term "Response" refers to the binding signal of an antibody to an antigen.

The term "in vitro" refers to a biological object, biological process or biological response outside the body, simulated under artificial conditions. For example, in vitro cell growth should be understood as the growth of cells in an environment outside the body, for example, in a test tube, culture flask or microplate.

The term "IC ₅₀ " (50% inhibitory concentration) refers to concentrations of a drug at which a measurable activity or response, for example, the growth or proliferation of cells, such as tumor cells, is inhibited by 50%. The IC ₅₀ value can be estimated using appropriate log dose response curves, using special statistical programs for processing the curves.

The term ED50 (EC ₅₀ ) (50% effective dose / concentration) refers to concentrations of a drug at which a measurable biological effect is achieved by 50% (may include cytotoxicity).

In the present description and in the following claims, unless the context otherwise requires, the words “have”, “include” and “comprise” or variations thereof such as “has”, “having”, “includes”, “including”, “ contains "or" containing "is to be understood as including the specified whole or group of integers, but not excluding any other whole or group of integers.

Antibody

The present invention relates to a monoclonal antibody that specifically binds to CSF-1R.

The term "monoclonal antibody" or "mAb" refers to an antibody that is synthesized and isolated by a separate clonal population of cells.

An antibody of the invention is a recombinant antibody.

The term "recombinant antibody" means an antibody that is expressed in a cell or cell line containing a nucleotide sequence (nucleotide sequences) that encodes antibodies, while the specified nucleotide sequence (nucleotide sequences) is not associated with the cell in nature.

In one aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to CSF-1R, comprising:

(a) a heavy chain variable domain comprising:

(i) CDR1 with an amino acid sequence selected from the group: DYAMS (SEQ ID NO: 1), DYAMT (SEQ ID NO: 17),

(ii) CDR2 with an amino acid sequence selected from the group: AISWNGGSTNYADSVKG (SEQ ID NO: 2), AISWRGGSTNYADSVKG (SEQ ID NO: 18), AISWIGGSTNYADSVKG (SEQ ID NO: 19), AISWLGKGSTGNYADSVG (SEQ ID NO: 21), AISWNGGETNYADSVKG (SEQ ID NO: 22), AISWNGGITNYADSVKG (SEQ ID NO: 23), AISWNGGLTNYADSVKG (SEQ ID NO: 24), AISWNGGSTRYADSVKG (SEQIS ID NOWG: 25), 26), AISWNGGSTNYQDSVKG (SEQ ID NO: 27), AISWNGGSTNYHDSVKG (SEQ ID NO: 28), QISWNGGSTRYQDSVKG (SEQ ID NO: 29), QISWLGGSTNYQDSVKG (SEQ ID NO: 30), HISDSG ID NO: 31 SEQ ID NO: 32), HISWNGGSTRYHDSVKG (SEQ ID NO: 33), HISWIGGSTNYQDSVKG (SEQ ID NO: 34), QISWIGGSTNYQDSVKG (SEQ ID NO: 35), HISWLGGSTNYQDSVKG (SEQ ID NO: 36),

(iii) CDR3 with an amino acid sequence selected from the group: TVEVAHRRLYKYLEV (SEQ ID NO: 3), IVEVAHRRLYKYLEV (SEQ ID NO: 37), YVEVAHRRLYKYLEV (SEQ ID NO: 38), TVEVAHRRLYRLYLEV (SEQ IDEVA NOHR: 39) (SEQ ID NO: 40), TVEVAHRRLYQYLEV (SEQ ID NO: 41), TVEVAHRRLYLYLEV (SEQ ID NO: 42), TVEVAHRRLYFYLEV (SEQ ID NO: 43), TVEVAHRRLYYYLEV (SEQ ID NO: 44), TVEVAHRRLYLYKY 45), TVELAHRRLYKYLEI (SEQ ID NO: 46), TVEFAHRRLYKYLEI (SEQ ID NO: 47), TVEQAHRRLYKYLEI (SEQ ID NO: 48), TVEFSHRRLYKYLEV (SEQ ID NO: 49), TVEFAHRRLY ID NO: 50; and

(b) a light chain variable domain comprising:

(i) CDR1 with the amino acid sequence GGNNIGSKSVH (SEQ ID NO: 12),

(ii) CDR2 with the amino acid sequence GDSDRPS (SEQ ID NO: 13),

(iii) CDR3 with the amino acid sequence QVWDISSDHVV (SEQ ID NO: 14).

The definition of "isolated" ("isolated"), used to describe the various antibodies herein, means an antibody identified and isolated and / or regenerated from the cell or cell culture in which it is expressed. Contaminants (contaminants) from the natural environment are materials that typically interfere with diagnostic or therapeutic uses for a polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Typically, an isolated polypeptide is obtained from at least one purification step.

CSF-1R (colony stimulating factor 1 receptor) is a membrane protein, a control point of macrophage differentiation. Has 2 ligands: CSF-1 and IL-34. Its signaling is necessary for the differentiation of precursors of the myeloid lineage into macrophages, which may also be participants in the tumor microenvironment (TAM, tumor-associated macrophages).

Amplification of the CSF-1R gene and / or overexpression of its protein have been found in many cancers, including cervical cancer (cervical cancer), head and neck cancer, stomach cancer, BC (breast cancer), renal cell carcinoma, CRC (colorectal cancer), OC (ovarian cancer), NSCLC (non-small cell lung cancer).

The term "antibody" or "immunoglobulin" (Ig) as used herein includes whole antibodies. The term "antibody" refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. There are five types of mammalian antibody heavy chains, which are designated by Greek letters: α, δ, ε, γ and μ. (Janeway C.A., Jr. et al., Immunobiology, 5th ed., Garland Publishing, 2001). The type of heavy chain present determines the class of the antibody; these chains are found in antibodies such as IgA, IgD, IgE, IgG and IgM, respectively (Rhoades R.A., Pflanzer R.G., Human Physiology, 4th ed., Thomson Learning, 2002). The various heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε are approximately 550 amino acids. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, α and δ contain a constant region, which consists of three constant domains CH1, CH2 and CH3 (aligned) and a hinge region that confers flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, cc.89-99); the heavy chains μ and ε contain a constant region that consists of four constant domains CH1, CH2, CH3 and CH4 (Janeway C.A., Jr. et al., Immunobiology, 5th ed., Garland Publishing, 2001). In mammals, only two types of light chains are known, which are designated as lambda (λ) and kappa (κ). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The approximate length of the light chain is 211-217 amino acids. Preferably, the light chain is a kappa light (κ) -chain and the constant domain CL is preferably a C-kappa (κ).

"Antibodies" according to the invention can be antibodies of any class (eg, IgA, IgD, IgE, IgG and IgM, preferably IgG) or subclass (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1).

The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), scattered between regions that are more conserved, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs located from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

The term "antigen-binding portion" of an antibody or "antigen-binding fragment" (or simply "portion of an antibody" or "antibody fragment") as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments included in the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH 1 domains; (ii) an F (ab ') 2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CH 1 domains; (iv) an Fv fragment consisting of the VL and VH domains in a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a VH / VHH domain; and (vi) an isolated complementarity determining region (CDR). In addition, the two regions of the Fv fragment, VL and VH, are encoded by different genes, they can be combined using recombinant methods using a synthetic linker, which makes it possible to obtain them in the form of a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879 -5883). It is assumed that such single-chain molecules are also included in the term "antigennegative part" of the antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened in the same manner as intact antibodies.

The term "variable" refers to the fact that certain segments of the variable domains differ widely in sequence among antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is unevenly distributed over the 110 amino acid variable domains. In contrast, V regions are composed of invariant fragments called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" or CDRs. Each variable domain of native heavy and light chains contains four FRs, generally assuming a beta sheet configuration, linked by three hypervariable regions that form binding loops, and in some cases are part of the beta sheet structure. The hypervariable regions in each chain are held together in close proximity by the FR and, with the hypervariable regions from the other chain, contribute to the formation of the antigen binding site of antibodies (see. Kabat et al., Sequences of Proteins of Immunological Interest. 5 ^th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit various effector functions, such as the participation of an antibody in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Typically, the hypervariable region contains amino acid residues from the "complementarity determining region" or "CDR" and / or such residues from the "hypervariable loop".

Preferably, the CDR of the antigen binding region or all of the antigen binding region of the antibodies of the invention is of llama or human donor library origin, or is essentially human in origin with certain amino acid residues altered, for example substituted with different amino acid residues, in order to optimize specific antibody properties, e.g. KD, koff , IC ₅₀ , EC ₅₀ , ED50. Preferably, the antibody frameworks of the invention are of human origin or of substantially human origin (at least 80, 85, 90, 95, 96, 97, 98 or 99% human).

In some cases, it may also be preferable to alter one or more amino acid residues of the CDR regions in order to increase the binding affinity for the target epitope. This is known as "affinity maturation" and in some cases can be performed in connection with humanization, for example, in situations where humanization of an antibody leads to a decrease in the specificity or affinity of binding, and it is not possible to sufficiently improve the specificity or affinity of binding by using only reverse mutations. Various affinity maturation methods are known in the art, for example, the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA 94: 412-417 (1997), and the in vitro stepwise affinity maturation method proposed by Wu et al., Proc Natl Acad Sci USA 95: 6037 6042 (1998).

"Kabat nomenclature" or "Kabat nomenclature" is used herein to refer to a numbering system for amino acid residues that are more variable (i.e., hypervariable) than the rest of the amino acid residues in the variable regions of the heavy and light chains of an antibody (Kabat et al. Ann NY Acad Sci. 190: 382-93 (1971); Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 (1991)).

An antibody of the invention that “binds” a target antigen is an antibody that binds an antigen with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent when targeting a protein or cell or tissue expressing the antigen, and slightly cross-reacts with other proteins. According to analytical methods: sorting of fluorescently activated cells (FACS), radioimmunoprecipitation (RIA) or ELISA (ELISA), in such variants of the invention the degree of binding of the antibody to a protein that is not a “target” (with a “non-target protein”) is less than 10 % of antibody binding to a specific target protein. With respect to binding of an antibody to a target molecule, the term “specific binding” or the expression “specifically binds to” or “specific to” a particular polypeptide or epitope on a particular target polypeptide means binding that is markedly (measurably) different from a non-specific interaction.

Specific binding can be quantified, for example by determining binding of a molecule versus binding of a control molecule. For example, specific binding can be determined by competitive reaction with another molecule similar to the target, for example, with an excess of unlabeled target. In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the term “specific binding” or the expression “specifically binds to” or “specific to” a particular polypeptide or epitope on a particular target polypeptide can be characterized by a molecule having a target Kd of at least about 200 nM, or at least at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about about 2 nM, or at least about 1 nM or higher. In one embodiment, the term "specific binding" refers to binding in which a molecule binds to a particular polypeptide or epitope on a particular polypeptide, with little or no binding to any other polypeptide or epitope on the polypeptide.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that comprises:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 1,

(ii) a CDR2 with the amino acid sequence of SEQ ID NO: 2,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the selected monoclonal antibody, or antigen-binding fragment comprises a heavy chain variable domain which comprises an amino acid sequence selected from the group: QVQLQQSGGGLVQPGGSLRLSCAASEFTFDDYAMSWVRQAPGKGLEWVSAISWNGGSTNYADSVKGRFTISRDNVKNTLYLQMNSLKSEDTAVYYCASTVEVAHRRLYKYLEVWGQGTLVTVSS (SEQ ID NO: 4), QVQLVESGGGLVQPGGSLRLSCAASEFTFDDYAMSWVRQAPGKGLEWVSAISWNGGSTNYADSVKGRFTISRDNVKNTLYLQMNSLRAEDTAVYYCASTVEVAHRRLYKYLEVWGQGTLVTVSS (SEQ ID NO: 5), QVQLVESGGGLVRPGGSLRLSCAASEFTFDDYAMSWVRQAPGKGLEWVSAISWNGGSTNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCASTVEVAHRRLYKYLEVWGQGTLVTVSS (SEQ ID NO: 6), QVQLQESGGGLVQPGGSLRLSCAASEFTFDDYAMSWVRQAPGKGLEWVSAISWNGGSTNYADSVKGRFTISRDNVKNTLYLQMNSLRAEDTAVYYCASTVEVAHRRLYKYLEVWSSQGTLVTV: ID

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the isolated monoclonal antibody, or antigen-binding fragment thereof, comprises a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a light chain variable domain that contains the amino acid sequence QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYGDSDRPSGIPERFSGSNSGNTATLFGDISRVE

In some embodiments, an isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) the variable domain of the heavy chain, which contains an amino acid sequence selected from the group: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7;

(b) the variable domain of the light chain contains the amino acid sequence of SEQ ID NO: 15.

In some embodiments, an isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) the variable domain of the heavy chain, which contains the amino acid sequence of SEQ ID NO: 4;

(b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 15.

In some embodiments, an isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) the variable domain of the heavy chain, which contains the amino acid sequence of SEQ ID NO: 5;

(b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 15.

In some embodiments, an isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) the variable domain of the heavy chain, which contains the amino acid sequence of SEQ ID NO: 6;

(b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 15.

In some embodiments, an isolated monoclonal antibody, or antigen-binding fragment thereof, comprises:

(a) the variable domain of the heavy chain, which contains the amino acid sequence of SEQ ID NO: 7;

(b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the isolated monoclonal antibody comprises a heavy chain comprising an amino acid sequence selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11.

In some embodiments, the isolated monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the isolated monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9.

In some embodiments, the isolated monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 10.

In some embodiments, the isolated monoclonal antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11.

In some embodiments, the isolated monoclonal antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 16.

In some embodiments, an isolated monoclonal antibody comprises:

(a) a heavy chain containing an amino acid sequence selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11; and

(b) a light chain containing the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the isolated monoclonal antibody that specifically binds to CSF-1R is a full length IgG antibody.

In some embodiments, the isolated monoclonal antibody is a full length IgG antibody and is of the human IgG1, IgG2, IgG3, or IgG4 isotype.

In some embodiments, the isolated monoclonal antibody is a full length IgG antibody and is of the human IgG1 isotype.

In some embodiments, an isolated monoclonal antibody that specifically binds to CSF-1R is leader candidate 01-016 (or antibody 01-016).

Leader candidate 01-016 includes:

(A) a heavy chain comprising the amino acid sequence QVQLQQSGGGLVQPGGSLRLSCAASEFTFDDYAMSWVRQAPGKGLEWVSAISWNGGSTNYADSVKGRFTISRDNVKNTLYLQMNSLKSEDTAVYYCASTVEVAHRRLYKYLEVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8); and

(B) a light chain comprising the amino acid sequence QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYGDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDISSDHVVFGGGTQLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 16).

Leader candidate 01-016 includes:

(a) the heavy chain variable domain contains the amino acid sequence QVQLQQSGGGLVQPGGSLRLSCAASEFTFDDYAMSWVRQAPGKGLEWVSAISWNGGSTNYADSVKGRFTISRDNVKNTLYLQMNSLKSEDTAVYYCASKYEVAHRRL 4

(b) the light chain variable domain contains the amino acid sequence QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYGDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDISSDHVVLFGGGTQLTV

Leader candidate 01-016 includes:

(a) a heavy chain variable domain comprising:

(i) CDR1 with the amino acid sequence DYAMS (SEQ ID NO: 1),

(ii) CDR2 with the amino acid sequence AISWNGGSTNYADSVKG (SEQ ID NO: 2),

(iii) CDR3 with the amino acid sequence TVEVAHRRLYKYLEV (SEQ ID NO: 3), and

(b) a light chain variable domain comprising:

(i) CDR1 with the amino acid sequence GGNNIGSKSVH (SEQ ID NO: 12),

(ii) CDR2 with the amino acid sequence GDSDRPS (SEQ ID NO: 13),

(iii) CDR3 with the amino acid sequence QVWDISSDHVV (SEQ ID NO: 14).

Within the framework of this invention, the leader candidate 01-016 was humanized.

The term "humanization" refers to the fact that when an antibody is wholly or partially non-human, for example, a mouse or llama antibody obtained by immunizing mice or llamas, respectively, with an antigen of interest, or is a chimeric antibody based on such a mouse or llama antibody , it is possible to replace some amino acids, in particular, in the framework regions and constant domains of the heavy and light chains, in order to avoid or minimize the immune response in humans. The specificity of the interaction of an antibody with a target antigen is inherent mainly in amino acid residues located in the six CDR regions of the heavy and light chains. Therefore, the amino acid sequences within the CDRs are much more variable between individual antibodies than the sequences outside the CDRs. Since the CDR region sequences are responsible for most antibody-antigen interactions, recombinant antibodies that mimic the properties of a specific natural antibody, or more generally any specific antibody with a given amino acid sequence, can be expressed, for example, by constructing expression vectors that express CDR sequences -sites from a specific antibody and the framework sequences of another antibody. As a result, the non-human antibody can be "humanized" and the binding specificity and affinity of the parent antibody can be largely preserved. Although it is impossible to accurately predict the immunogenicity and thus the immune response against a human antibody to a particular antibody, non-human antibodies are generally more immunogenic than human antibodies. Chimeric antibodies in which foreign (eg, rodent or camel) constant regions have been replaced with sequences of human origin have shown generally lower immunogenicity than antibodies of completely foreign origin, and there is a tendency to use humanized or fully human antibodies in therapeutic antibodies. Chimeric antibodies or other antibodies of non-human origin can thus be humanized to reduce the risk of an immune response directed against the antibody in humans.

To humanize the variable domain of the heavy chain of the leader candidate 01-016, positions containing the least characteristic amino acid residues for human antibodies were selected. In the Kabat numbering scheme, their values are: 1, 5, 6, 11, 26, 74, 77, 83, 84 and 89. From the beginning of the amino acid sequence, they are numbered 1, 5, 6, 11, 26, 75, 78, 87, 88 and 93.

All of the above positions do not affect the CDRs of the leader candidate 01-016 (HCDR1 with the amino acid sequence DYAMS (SEQ ID NO: 1), HCDR2 with the amino acid sequence AISWNGGSTNYADSVKG (SEQ ID NO: 2), HCDR3 with the amino acid sequence TVEVAHRRLYKYLEV) (SEQ ID NO: 3) ).

Humanization of the light chain variable domain of the leader candidate 01-016 was not performed.

As a result of humanization of the leader candidate 01-016, the candidates OPTIM-6_001-02, OPTIM-6_001-04 and OPTIM-6_001-05 were obtained, in which the degree of humanization of the heavy chain variable domain is higher than that of the leader candidate 01-016 (see example 21).

In some embodiments, the isolated monoclonal antibody is a candidate OPTIM-6_001-02 and includes:

(A) a heavy chain comprising the amino acid sequence QVQLVESGGGLVQPGGSLRLSCAASEFTFDDYAMSWVRQAPGKGLEWVSAISWNGGSTNYADSVKGRFTISRDNVKNTLYLQMNSLRAEDTAVYYCASTVEVAHRRLYKYLEVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9); and

(B) a light chain comprising the amino acid sequence QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYGDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDISSDHVVFGGGTQLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 16).

The OPTIM-6_001-02 candidate includes:

(a) the variable domain of the heavy chain contains the amino acid sequence QVQLVESGGGLVQPGGSLRLSCAASEFTFDDYAMSWVRQAPGKGLEWVSAISWNGGSTNYADSVKGRFTISRDNVKNTLYLQMNSLRAEDTAVYYCASTVYLVHRRLY

(b) the light chain variable domain contains the amino acid sequence QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYGDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDISSDHVVLFGGGTQLTV

In some embodiments, the isolated monoclonal antibody is a candidate OPTIM-6_001-04 and includes:

(A) a heavy chain comprising the amino acid sequence QVQLVESGGGLVRPGGSLRLSCAASEFTFDDYAMSWVRQAPGKGLEWVSAISWNGGSTNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCASTVEVAHRRLYKYLEVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10); and

(B) a light chain comprising the amino acid sequence QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYGDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDISSDHVVFGGGTQLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 16).

The OPTIM-6_001-04 candidate includes:

(a) the variable domain of the heavy chain contains the amino acid sequence QVQLVESGGGLVRPGGSLRLSCAASEFTFDDYAMSWVRQAPGKGLEWVSAISWNGGSTNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCASTVYLEVAHRRLY

(b) the light chain variable domain contains the amino acid sequence QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYGDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDISSDHVVLFGGGTQLTV

In some embodiments, the isolated monoclonal antibody is a candidate OPTIM-6_001-05 and includes:

(A) a heavy chain comprising the amino acid sequence QVQLQESGGGLVQPGGSLRLSCAASEFTFDDYAMSWVRQAPGKGLEWVSAISWNGGSTNYADSVKGRFTISRDNVKNTLYLQMNSLRAEDTAVYYCASTVEVAHRRLYKYLEVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11); and

(B) a light chain comprising the amino acid sequence QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYGDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDISSDHVVFGGGTQLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 16).

The OPTIM-6_001-05 candidate includes:

(a) the variable domain of the heavy chain contains the amino acid sequence QVQLQESGGGLVQPGGSLRLSCAASEFTFDDYAMSWVRQAPGKGLEWVSAISWNGGSTNYADSVKGRFTISRDNVKNTLYLQMNSLRAEDTAVYYCASTVYLEVAHRRLY

(b) the light chain variable domain contains the amino acid sequence QAGLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYGDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDISSDHVVLFGGGTQLTV

Modification (s) of amino acid sequences of antibodies is proposed. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequences of the antibody. Any combination of deletion, insertion and substitution is performed to obtain the final construct, provided that the final construct has the desired characteristics. Changes in amino acids can also alter post-translational processes in the antibody, such as changes in the number or position of glycosylation sites.

A variant of the modification of the amino acid sequences of antibodies using amino acid substitutions. This option is the replacement of at least one amino acid residue in the antibody molecule with another residue. Sites of greatest interest for mutagenesis by substitutions include hypervariable regions or CDRs, but changes in the FR or Fc region are also contemplated. Conservative substitutions are shown in Table 1 under the heading “preferred substitutions”. If such substitutions result in a change in biological activity, then additional significant changes, referred to as “examples of substitutions” in Table A, or changes further described below in the description of amino acid classes, can be introduced, and products can be screened.

Table A Original residue Replacement examples Preferred substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Asp, Lys; Arg Gin Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gin Asp Gly (G) Ala Ala His (H) Asn; Gin; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Antibody fragments

In certain circumstances, it is advisable to use fragments of antibodies rather than full antibodies. The smaller size of the fragments facilitates their rapid clearance and may facilitate better penetration into solid tumors.

Various methods have been developed to obtain antibody fragments. Traditionally, these fragments were obtained by proteolytic cleavage of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods, 24, 1992, pp. 107-117, and Brennan et al., Science, 229, 1985, pp. . 81). However, at present, these fragments can be obtained directly using recombinant host cells. Fab, Fv and scFv antibody fragments can be expressed and secreted from E. coli to facilitate the production of large quantities of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically ligated to form F (ab ') 2 fragments (Carter et al., Bio / Technology, 10, 1992, pp. 163-167). According to another approach, F (ab ') 2 fragments can be isolated directly from the culture of recombinant host cells. Fab and F (ab ') 2 fragments with increased in vivo half-lives, in which epitope-binding receptor residues are retained, are described in US Pat. No. 5,869,046. Other techniques for preparing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv) (see WO 93/16185; US 5571894 and US 5587458). Fv and scFv are the only species with intact binding sites lacking constant regions; as a result, they can be used for reduced non-specific binding when used in vivo. Fusion proteins carrying the scFv can be engineered to produce an effector protein fusion at either the N- or C-terminus of the scFv (see Antibody Engineering, ed. Borrebaeck, supra). An antibody fragment can also be a "linear antibody", such as described in US Pat. No. 5,641,870. Such linear antibody fragments can be monospecific or bispecific.

Nucleic acid molecule

The present invention also relates to isolated nucleic acid molecules, namely the sequences encoding an isolated monoclonal antibody that specifically binds to CSF-1R, according to this invention, as described herein.

The terms "nucleic acid", "nucleic acid sequence" or "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", which are used interchangeably in this description, denote a clear sequence of nucleotides, modified or not modified defining a fragment or region of nucleic acid, whether or not containing unnatural nucleotides and which is either double-stranded DNA or RNA, or single-stranded DNA or RNA, or transcription products of said DNA.

It should also be mentioned here that this invention does not relate to nucleotide sequences in their natural chromosomal environment, i. E. in a natural state. The sequences of the present invention have been isolated and / or purified, i. E. were taken directly or indirectly, for example by copying, while their environment was at least partially modified. Thus, also here should be meant isolated nucleic acids obtained by genetic recombination, for example, using host cells (host cells), or obtained by chemical synthesis.

The reference to the nucleotide sequence encompasses its compliment, unless otherwise indicated. Thus, reference to a nucleic acid having a defined sequence is to be understood as encompassing its complementary strand with its complementary sequence.

An "isolated" nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in a natural source of antibody nucleic acid. An isolated nucleic acid molecule differs from the form or set in which it naturally occurs. Thus, the isolated nucleic acid molecule differs from the nucleic acid molecule that exists naturally in cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule found in cells in which the antibody is normally expressed, for example, if the nucleic acid molecule has a different localization in the chromosome from its localization in cells in natural conditions.

In one aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence selected from SEQ ID NO: 1-50. A nucleic acid molecule can also contain any combination of the indicated nucleotide sequences.

In one aspect, the present invention relates to a nucleic acid molecule comprising a nucleotide sequence that encodes an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to CSF-1R, and comprises:

(a) a heavy chain variable domain comprising:

(i) CDR1 with an amino acid sequence selected from the group: SEQ ID NO: 1, SEQ ID NO: 17,

(ii) CDR2 with an amino acid sequence selected from the group: SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO : 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36,

(iii) CDR3 with an amino acid sequence selected from the group: SEQ ID NO: 3, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO : fifty; and

(b) a light chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 12,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 13,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 14.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof, which includes a heavy chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 1,

(ii) a CDR2 with the amino acid sequence of SEQ ID NO: 2,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain, which comprises an amino acid sequence selected from the group: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody, or antigen-binding fragment thereof, comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody, or antigen-binding fragment thereof, comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody, or antigen-binding fragment thereof, comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 6.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody, or antigen-binding fragment thereof, comprising a heavy chain variable domain that comprises the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody, or antigen-binding fragment thereof, comprising a light chain variable domain that comprises the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody, or antigen-binding fragment thereof, comprising:

(a) the variable domain of the heavy chain, which contains an amino acid sequence selected from the group: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7;

(b) the variable domain of the light chain contains the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody, or antigen-binding fragment thereof, comprising:

(a) the variable domain of the heavy chain, which contains the amino acid sequence of SEQ ID NO: 4;

(b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody, or antigen-binding fragment thereof, comprising:

(a) the variable domain of the heavy chain, which contains the amino acid sequence of SEQ ID NO: 5;

(b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody, or antigen-binding fragment thereof, comprising:

(a) the variable domain of the heavy chain, which contains the amino acid sequence of SEQ ID NO: 6;

(b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody, or antigen-binding fragment thereof, comprising:

(a) the variable domain of the heavy chain, which contains the amino acid sequence of SEQ ID NO: 7;

(b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 15.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody comprising a heavy chain comprising an amino acid sequence selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: eleven.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody comprising a heavy chain of the amino acid sequence SEQ ID NO: 8.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody comprising a heavy chain of the amino acid sequence SEQ ID NO: 9.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody comprising a heavy chain of the amino acid sequence SEQ ID NO: 10.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody comprising a heavy chain of the amino acid sequence SEQ ID NO: 11.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody comprising a light chain of the amino acid sequence SEQ ID NO: 16.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody comprising:

(a) a heavy chain containing an amino acid sequence selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11; and

(b) a light chain containing the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody comprising:

(a) a heavy chain containing the amino acid sequence of SEQ ID NO: 8; and

(b) a light chain containing the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody comprising:

(a) a heavy chain containing the amino acid sequence of SEQ ID NO: 9; and

(b) a light chain containing the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody comprising:

(a) a heavy chain containing the amino acid sequence of SEQ ID NO: 10; and

(b) a light chain containing the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a monoclonal antibody comprising:

(a) a heavy chain containing the amino acid sequence of SEQ ID NO: 11; and

(b) a light chain containing the amino acid sequence of SEQ ID NO: 16.

In some embodiments, the isolated nucleic acid is DNA.

In any of the above embodiments, the nucleic acid molecules can be isolated.

The nucleic acid molecule of this invention can be isolated from any source that produces a monoclonal antibody that specifically binds to CSF-1R. In certain embodiments of the invention, a nucleic acid molecule of this invention can be synthesized rather than isolated.

In one embodiment, nucleic acid molecules encoding VH domains (SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7) or VL (SEQ ID NO: 15) are converted to antibody genes along the entire length by insertion into an expression vector already encoding the constant domains of the heavy chain (CH) or light chain (CL), respectively, so that the VH segment is operably linked to the CH segment (s) in the vector and / or the VL segment is operatively linked to CL segment in vector. In another embodiment, nucleic acid molecules encoding VH and / or VL domains are converted to genes along the entire length of the antibody by linking, for example, ligation, a nucleic acid molecule encoding VH and / or VL domains to a nucleic acid molecule encoding CH and / or CL domains using standard molecular biology techniques. Nucleic acid molecules encoding the entire length of the heavy and / or light chain can then be expressed from the cell into which they were introduced.

Nucleic acid molecules can be used to express a recombinant monoclonal antibody that specifically binds to CSF-1R.

Vector

In one aspect, the present invention relates to an expression vector comprising the aforementioned isolated nucleic acid. The present invention provides a vector suitable for expressing any of the nucleotide sequences described herein.

The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is a plasmid, i. E. a circular double-stranded portion of DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. In some embodiments, vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into a host cell, and thus replicate with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

The present invention relates to vectors containing nucleic acid molecules that encode any of the amino acid sequences of a monoclonal antibody that specifically binds to CSF-1R, or portions thereof (e.g., the heavy chain sequences of the first and / or heavy and / or light chain of the second binding domains ) as described in this document. The present invention further relates to vectors containing nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments.

Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic viruses, cosmids, YACs, EBV derived episomes, and the like. DNA molecules can be ligated into a vector such that the sequences that control transcription and translation in the vector perform their intended function of regulating DNA transcription and translation. The expression vector and expression control sequences can be selected to be compatible with the expression host cell used. DNA molecules encoding part or all of the first and second binding domain sequences (eg, heavy and light chain sequences, where the binding domain contains the heavy and light chain sequences) can be introduced into separate vectors. In one embodiment, any combination of the above DNA molecules is introduced into the same expression vector. DNA molecules can be introduced into the expression vector by standard methods (for example, ligation of complementary restriction sites on the fragment of the antibody gene and the vector, or blunt-end ligation if there are no restriction sites).

The expression "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable prokaryotic control sequences are, for example, a promoter, optionally an operator, and a ribosome binding site. It is known that promoters, polyadenylation signals, and enhancers are present in eukaryotic cells.

A nucleic acid is “operably linked” if it is in a functional relationship with another nucleotide sequence. For example, DNA of a presequence or secretory leader is operably linked to DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; the ribosome binding site is operably linked to a coding sequence if located so that it can facilitate translation. Typically, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, are contiguous and in readout phase. However, enhancers do not have to be contiguous.

A suitable vector is one that encodes a functionally complete CH or CL sequence of a human immunoglobulin with the construction of an appropriate restriction site so that any VH or VL sequence can be easily incorporated and expressed as described above. HC- and LC-coding of genes in such vectors may contain intron sequences, which leads to an overall increase in antibody protein products by stabilizing the corresponding mRNA. Intron sequences are flanked by splice donor and splice acceptor sites that determine where RNA splicing will occur. The location of the intron sequences can be either in the variable or constant regions of the antibody chains, or in both variable and constant regions when multiple introns are used. Termination of polyadenylation and transcription can occur downstream of the native chromosome site of the encoded regions. The recombinant expression vector can also encode a signal peptide that facilitates the production of an antibody chain by a host cell. An antibody chain gene can be cloned into a vector such that the signal peptide is linked to the reading frame of the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide of a non-immunoglobulin protein).

In addition to the antibody gene chain, the recombinant expression vectors of the invention may carry regulatory sequences that control the expression of the antibody chain genes in the host cell. Those of skill in the art will understand that the design of the expression vector, including the choice of regulatory sequences, may depend on factors such as the selection of the host cell for transformation, the level of expression of the desired protein, etc. Preferred regulatory sequences for a mammalian host cell expression include viral elements for high expression of proteins in mammalian cells, such as promoters and / or enhancers derived from retroviral LTR, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g. SV40 promoter / enhancer), adenovirus (e.g., adenovirus large late promoter (AdMLP)), polyoma virus, and strong mammalian promoters such as the native immunoglobulin promoter or actin promoter. Methods for expressing polypeptides in bacterial or fungal cells, eg yeast cells, are also well known in the art.

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate vector replication in host cells (eg, origin of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced.

The term "expression control sequence" as used herein means polynucleotide sequences that are required to influence the expression and processing of the coding sequences to which they are ligated. Expression control sequences include the corresponding transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., the Kozak consensus sequence); sequences that increase protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences differs depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and transcription termination sequences; in eukaryotes, such control sequences generally include promoters and transcription termination sequences. The term "control sequences" includes at least all components that are essential for expression and processing, and may also include additional components whose presence is useful, such as leader sequences and fusion cell sequences.

Host cell

In one aspect, the present invention provides a method for producing a host cell for producing an antibody or antigen-binding fragment thereof that specifically binds to CSF-1R, comprising transforming a cell with the vector described above.

In one aspect, the present invention provides a host cell for producing an antibody or antigen-binding fragment thereof that specifically binds to a CSF-1R containing a nucleic acid as described above.

The term "recombinant host cell" (or simply "host cell") as used herein means a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells, which may include, for example, a vector in accordance with the present invention described above. The present invention also relates to host cells that include, for example, a nucleotide sequence encoding a heavy chain or antigen-binding portions thereof, a nucleotide sequence encoding a light chain or antigen-binding portions thereof, or both of a first binding domain and / or a second binding domain binding molecule according to this invention. It should be understood that "recombinant host cell" and "host cell" mean not only the specific cell claimed, but also the progeny of such a cell. Since modifications may occur in subsequent generations due to mutation or environmental influences, such offspring may not actually be identical to the parent cell, but such cells are still included within the scope of the term "host cell" as used herein.

Nucleic acid molecules encoding a monoclonal antibody that specifically binds to CSF-1R of the invention and vectors containing these nucleic acid molecules can be used to transfect a suitable mammal or its cell, plant or cell, bacterial or yeast host cell. Conversion can occur by any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, transfection with a nucleic acid-positively charged polymer complex, transfection with a nucleic acid-calcium phosphate precipitate, polybrene-mediated transfection, fusion of protoplasts, transfected with liposomal encapsulation and polynucleotide DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors.

Mammalian cell lines used as hosts for transformation are well known in the art and include many immortal cell lines available. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, hamster kidney cells (BHK), African kidney cells green monkeys (COS), human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, and a number of other cell lines. Cell lines are selected by determining which cell lines have high levels of expression and provide the desired characteristics of the produced protein. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells. When recombinant expression vectors encoding a monoclonal antibody that specifically binds to CSF-1R are introduced into mammalian host cells, antibodies are produced by culturing the host cells for a time sufficient to express the antibodies in the host cells, or, more preferably, isolate the antibodies in a nutrient medium in which host cells are grown. A monoclonal antibody that specifically binds to CSF-1R can be isolated from culture media using standard protein purification methods. Plant host cells, for example, include Nicotiana, Arabidopsis, duckweed, corn, wheat, potatoes, etc. Host bacterial cells include Escherichia and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

In addition, the level of production of a monoclonal antibody that specifically binds to CSF-1R of the present invention from a producing cell line can be enhanced by a number of known techniques. For example, the glutamine synthetase gene expression system (GS system) is common enough to enhance expression under certain conditions.

It is likely that a monoclonal antibody that specifically binds to CSF-1R from different cell lines or transgenic animals will have a different glycosylation profile. However, a monoclonal antibody that specifically binds to CSF-1R, encoded by the nucleic acid molecules described herein, or containing the amino acid sequences described herein, are part of this invention, regardless of the glycosylation state of the binding molecules, and in general, regardless of the presence or lack of post-translational modifications.

The above host cell does not refer to a host cell obtained using human embryos.

The above host cell does not refer to a host cell obtained by modifying the genetic integrity of human germline cells.

Antibody production method

In one aspect, the present invention relates to a method for producing an antibody or antigen-binding fragment thereof that specifically binds to CSF-1R, comprising culturing a host cell as described above in a culture medium under conditions sufficient to produce said antibody, optionally followed by isolation and purifying the resulting antibody.

The present invention relates to methods for producing a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CSF-1R according to this invention. One embodiment of the invention relates to a method for producing a monoclonal antibody that specifically binds to CSF-1R as defined herein, comprising obtaining a recombinant host cell capable of expressing a monoclonal antibody that specifically binds to CSF-1R by culturing said host cell under conditions suitable for expression, production of a monoclonal antibody that specifically binds to CSF-1R, and recovering the resulting monoclonal antibody that specifically binds to CSF-1R. A monoclonal antibody that specifically binds to CSF-1R obtained by such expression in such recombinant host cells is referred to herein as a “recombinant monoclonal antibody that specifically binds to CSF-1R”. The invention also provides progeny cells of such host cells and a monoclonal antibody that specifically binds to CSF-1R prepared in a similar manner.

Pharmaceutical compositions

Another aspect of the invention is a pharmaceutical composition comprising, as an active ingredient (or as the only active ingredient), a monoclonal antibody that specifically binds to CSF-1R.

In one aspect, the present invention relates to a pharmaceutical composition that contains an antibody of the present invention or an antigen-binding fragment thereof in a therapeutically effective amount in combination with one or more pharmaceutically acceptable excipients.

"Pharmaceutical composition" means a composition comprising an antibody according to the invention and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, solvents, diluents, carriers, auxiliary, dispensing and receiving agents, agents delivery agents such as preservatives, stabilizers, fillers, grinders, humectants, emulsifiers, suspending agents, thickeners, sweeteners, fragrances, flavors, antibacterial agents, fungicides, lubricants, delayed delivery regulators, the choice and ratio of which depends on the nature and method of administration and dosage ... Examples of suspending agents are ethoxylated isostearyl alcohol, polyoxyethylene, sorbitol and sorbitol ether, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures of these substances. Protection against the action of microorganisms can be provided by a variety of antibacterial and antifungal agents such as parabens, chlorobutanol, sorbic acid, and the like. The composition may also include isotonic agents, for example, sugars, polyols, sodium chloride and the like. The prolonged action of the composition can be provided by agents that slow down the absorption of the active principle, for example, aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery vehicles are water, ethanol, polyalcohols, as well as mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Examples of fillers are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate, and the like. Examples of grinders and distributing agents are starch, alginic acid and its salts, silicates. Examples of lubricants are magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol. A pharmaceutical composition for oral, sublingual, transdermal, intraocular, intramuscular, intravenous, subcutaneous, local or rectal administration of an active principle, alone or in combination with another active principle, can be administered to animals and humans in a standard administration form as a mixture with traditional pharmaceutical carriers ... Suitable unit forms of administration include oral forms such as tablets, gelatin capsules, pills, powders, granules, chewing gums and oral solutions or suspensions, sublingual and buccal administration forms, aerosols, implants, topical, transdermal, subcutaneous, intramuscular, intravenous, intranasal or intraocular administration forms and rectal administration forms.

The pharmaceutical composition may include at least one monoclonal antibody that specifically binds to CSF-1R and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients.

The term "excipient" or "excipient" is used herein to describe any component other than the compound (s) of this invention. These are substances of inorganic or organic origin used in the production process, the manufacture of drugs to give them the necessary physical and chemical properties.

The pharmaceutical composition may include at least one monoclonal antibody that specifically binds to CSF-1R and one or more additional binding molecules (eg, antibodies) that target one or more corresponding surface receptors.

In some embodiments, the compositions are designed to improve, prevent, or treat disorders that may be associated with CSF-1R.

In one aspect, the present invention provides a pharmaceutical composition for treating a disease or disorder mediated by CSF-1R, comprising an antibody or antigen-binding fragment thereof that specifically binds to CSF-1R, in a therapeutically effective amount, in combination with one or more pharmaceutically acceptable excipients ...

The term "disease or disorder mediated by CSF-1R" means all diseases or disorders that are either directly or indirectly associated with CSF-1R, including the etiology, development, progression, persistence or pathology of the disease or disorder.

"Treat", "treatment" and "therapy" refer to a method of alleviating or eliminating a biological disorder and / or at least one of its attendant symptoms. As used herein to "alleviate" a disease, disease, or condition, means reducing the severity and / or frequency of symptoms of a disease, disorder, or condition. In addition, references herein to "treatment" include references to curative, palliative and prophylactic therapy.

In one aspect, the subject of treatment or patient is a mammal, preferably a human subject. The above subject can be male or female of any age.

The term "disorder" means any condition that can be improved as a result of the treatment of the present invention. The definition of this term includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the occurrence of this disorder.

A "therapeutically effective amount" is an amount of a therapeutic agent administered during treatment that will relieve to some extent one or more of the symptoms of the disease being treated.

In some embodiments, the CSF-1R mediated disease or disorder is selected from the group: metastatic melanoma, metastatic bladder cancer, metastatic non-small cell lung cancer, metastatic renal cell carcinoma (light cell type), malignant neoplasms with microsatellite instability when ineffective main anticancer therapy, locally advanced or metastatic squamous cell carcinoma of the head and neck, stomach cancer (cardio-esophageal zone), ovarian cancer, breast cancer, triple-negative breast cancer, bone tumor, Hodgkin's lymphoma, glioma, glioblastoma, mesothelioma, myeloma, prostate cancer, soft tissue sarcoma.

The pharmaceutical compositions of the present invention and methods of making them will be undeniably obvious to those skilled in the art. The production of pharmaceutical compositions should preferably be in accordance with GMP (Good Manufacturing Practice) requirements. The composition may include a buffering composition, tonic agents, stabilizers, and solubilizers. The prolonged action of the composition can be provided by agents that delay the absorption of the active pharmaceutical ingredient, for example, aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery vehicles are water, ethanol, polyalcohols, as well as mixtures thereof, oils and injectable organic esters.

Any method of administering peptides, proteins or antibodies accepted in the art can be suitably used for a monoclonal antibody that specifically binds to CSF-1R of the present invention.

The term "pharmaceutically acceptable" means one or more compatible liquid or solid components that are suitable for administration to a mammal, preferably a human.

The term "buffer", "buffer composition", "buffering agent" means a solution that is able to maintain a pH value due to the interaction of acid and alkaline components that make up its composition, which enables an antibody preparation that specifically binds to CSF-1R to develop resistance to changes in pH. In general, the pH values of the pharmaceutical composition are preferably between 4.0 and 8.0. As buffering agents, for example, acetate, phosphate, citrate, histidine, succinate and the like can be used. buffer solutions, but not limited to.

The terms "tonic agent", "osmolytic" or "osmotic agent" as used herein refer to an excipient that can apply the osmotic pressure of a liquid antibody preparation. An "isotonic" drug is a drug that has an osmotic pressure equivalent to that of human blood. Isotonic formulations typically have an osmotic pressure of about 250 to 350 mOsm / kg. As isotonic agents, polyols, mono- and disaccharides, amino acids, metal salts such as sodium chloride, and the like can be used, but are not limited to them.

By "stabilizer" is meant an excipient or a mixture of two or more excipients that provide the physical and / or chemical stability of the active agent. Amino acids can be used as stabilizers, for example, but not limited to arginine, histidine, glycine, lysine, glutamine, proline; surfactants such as polysorbate 20 (trade name Tween 20), polysorbate 80 (trade name Tween 80), polyethylene-polypropylene glycol and its copolymers (trade names Poloxaner, Pluronic), sodium dodecyl sulfate (SDS ), but not limited to them; antioxidants, for example, but not limited to methionine, acetylcysteine, ascorbic acid, monothioglycerol, sulfuric acid salts, and the like; chelating agents, for example, but not limited to ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate, and the like.

A pharmaceutical composition is "stable" if the active agent maintains its physical stability and / or chemical stability and / or biological activity during the stated shelf life at a storage temperature, for example, at 2-8 ° C. It is preferred that the active agent retains both physical and chemical stability as well as biological activity. The storage period is selected based on the results of stability studies with accelerated and natural storage.

The pharmaceutical composition of the present invention can be manufactured, packaged or marketed in a single unit dose or a plurality of unit dose units in a finished dosage form. As used herein, the term "unit dosage" means a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of active ingredient is usually equal to the dosage of the active ingredient that will be administered to the subject, or a convenient part of such dosage, for example, half or a third of such dosage.

The pharmaceutical compositions of the present invention are generally suitable for parenteral administration in the form of sterile medicaments intended for administration to the human body in violation of the integrity of the skin or mucous membranes, bypassing the gastrointestinal tract by injection, infusion or implantation. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intraarticular, transdermal injection or infusion; and renal dialysis infusion techniques. Intratumoral delivery, such as intratumoral injection, may also be useful. Regional perfusion is also provided. Preferred embodiments of the invention include intravenous and subcutaneous routes. Any method of administering peptides or proteins accepted in the art can be suitably used for an antibody that specifically binds to CSF-1R of the present invention.

Injectable drugs can be manufactured, packaged or sold in unit dosage form, for example, in ampoules, vials, polymer containers, pre-filled syringes, autoinjection devices, but not limited to them. Medicines for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous bases, pastes, and the like.

Another embodiment of the invention is a medicament for parenteral administration, where the pharmaceutical composition is provided in dry form, i.e., a powder or granules for dissolution in a suitable solvent (eg sterile pyrogen-free water) prior to administration. Such a drug can be obtained, for example, by lyophilization, i. E. a process known in the art as freeze drying, which includes freezing the preparation and then removing the solvent from the frozen contents.

The antibody that specifically binds to CSF-1R can also be administered intranasally or by inhalation, alone, as a mixture with a suitable pharmaceutically acceptable excipient from an inhaler, such as a pressurized aerosol container, pump, spray, nebulizer, or nebulizer, with or without a suitable propellant is used, or in the form of nasal drops or spray.

The drug for parenteral administration can be immediate or modified release. Modified release drugs include delayed, sustained, pulsed, controlled, targeted and programmed release.

Therapeutic Uses of a Monoclonal Antibody That Specifically Binds to CSF-1R

In one aspect, an antibody that specifically binds to CSF-1R is used in the treatment of disorders that are mediated with CSF-1R activity.

In one aspect, the subject of treatment or patient is a mammal, preferably a human subject. The above subject can be male or female and of any age.

In the case of a tumor (eg, cancer), a therapeutically effective amount of an antibody or antibody fragment (eg, an antibody or antibody fragment that specifically binds CSF-1R) can reduce the number of cancer cells; reduce the initial size of the tumor; inhibit (ie, to some extent slow down and, preferably, stop) the infiltration of cancer cells of peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or alleviate, to some extent, one or more of the symptoms associated with the disorder. The antibody or antibody fragment can, to some extent, prevent the growth and / or kill existing cancer cells, it can cause a cytostatic and / or cytotoxic effect. In cancer therapy, efficacy in vivo can be determined, for example, by assessing lifespan, time to disease progression (TTP), tumor response rate (RR), duration of response, and / or quality of life.

As used herein, uses or methods relating to an antibody that specifically binds to CSF-1R, one or more other therapeutic agents, is intended to mean, refer to, or include:

1) the simultaneous administration of such a combination of an antibody that specifically binds to CSF-1R and a therapeutic agent to a patient in need of treatment, when such components are formulated together in a single dosage form, from which said components are released substantially simultaneously to said patient,

2) the simultaneous administration of such a combination of an antibody that specifically binds to CSF-1R and a therapeutic agent to a patient in need of treatment, when such components are formulated separately in different dosage forms, the administration of which occurs at almost the same time to the specified patient, after whereby these components are released almost simultaneously to the specified patient,

3) sequential administration of such a combination of an antibody that specifically binds to CSF-1R and a therapeutic agent to a patient in need of treatment, when such components are formulated separately from each other in separate dosage forms that are taken sequentially in time by the specified patient with a significant time the interval between each administration, after which these components are released at practically different times to the specified patient; as well as

4) sequential administration of such a combination of an antibody that specifically binds to CSF-1R and a therapeutic agent to a patient in need of treatment, when such components are formulated together in a single dosage form, from which the release of these components occurs in a controlled manner, after which they are simultaneously, sequentially or jointly released at the same time and / or different times to the specified patient, where each part can be entered in one or different ways.

An antibody that specifically binds to CSF-1R can be administered without additional therapeutic treatment, i. E. as an independent therapy. In addition, treatment with an antibody that specifically binds to CSF-1R may include at least one additional therapeutic treatment (combination therapy). In some embodiments, an antibody that specifically binds to CSF-1R can be co-administered with another drug / cancer treatment.

In one aspect, the present invention provides a pharmaceutical composition for treating a disease or disorder mediated by CSF-1R, comprising an antibody or antigen-binding fragment thereof that specifically binds to CSF-1R and at least one other therapeutically active compound.

In some embodiments, the disease or disorder mediated by CSF-1R is selected from the group of: metastatic melanoma, metastatic bladder cancer, metastatic non-small cell lung cancer, metastatic renal cell carcinoma (light cell type), malignant neoplasms with microsatellite instability with ineffective primary therapy, locally advanced or metastatic squamous cell carcinoma of the head and neck, stomach cancer (cardio-esophageal zone), ovarian cancer, breast cancer, triple negative breast cancer, bone tumor, Hodgkin's lymphoma, glioma, glioblastoma, mesothelioma, myeloma, prostate cancer, soft tissue sarcoma.

In some embodiments, the other therapeutically active compound is an antibody, chemotherapeutic agent, or anti-hormonal agent.

A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins (eg bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin and 9-aminocamptothecin); bryostatin; callistatin; CC-1065 (including its synthetic analogs adoselesin, carzelesin and bisellesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (for example, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmicin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pankratistatin; sarcodictyin; spongystatin; nitrogen mustards such as chlorambucil, chloronaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, fenesterol, prednimustine, trophosphamide, uramustine; nitrosoureas such as carmustine, chlorosotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enedyne antibiotics (e.g., calicheamicin, e.g., calicheamicin gamma II and calicheamicin omega II (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemycin, including dynemycin A; esperamycin; as well as the chromophore of neocarcinostatin and related chromophores of chromoproteins - enediin antibiotics, aclacinomisins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, carbicin, carminomycin, carzinomycin-6 -L-norleucine, doxorubicin (including ADRIAMICIN®, morpholinodoxorubicin, cyanomorpholine-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicinHCl in injectable liposomes (DOXOL®), doxorubicin liposomal doxorubicin D-XELY®), and doxorubicin D-99 epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin , rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilone, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, karmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; adrenal suppressants such as aminoglutethimide, mitotane, trilostane; folic acid compensator such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisanthrene; edatraxate; defopamine; demecolcine; diazichon; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguason; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pyirubicin; losoxantrone; 2-ethylhydrazide; procarbazine; polysaccharide complex PSK® (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizophyran; spirogermanium; tenuazonic acid; triaziquon; 2,2 ', 2 "-trichlorotriethylamine; trichothecenes (eg, T-2 toxin, verracurin A, roridin A, and anguidin); urethane; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("ara-C"); thiotepa; a taxoid such as paclitaxel (TAXOL®), an engineered albumin-bound nanoparticle formulation of paclitaxel (ABRAXANETM) and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum-based agents such as cisplatin, oxaliplatin, and carboplatin; Vinca alkaloids, which prevent tubulin from polymerizing from the resulting microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoids such as retinoic acid including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (e.g. BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID® ) or risendronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, for example oligonucleotides, which inhibit the expression of genes in signaling pathways involved in the proliferation of aberrant cells, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; a topoisomerase 1 inhibitor (eg LURTOTECAN®); rmRH (e.g. ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosin, a COX-2 inhibitor (eg, celecoxib or etoricoxib), a proteosome inhibitor (eg, PS341); bortezomib (VELCADE®); CCI-779; typifarnib (811577); orafenib, ABT510; a Bcl-2 inhibitor such as sodium oblimersen (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the above agents; as well as combinations of two or more of the above, such as CHOP, an abbreviation for combination therapy with cyclophosphamide, doxorubicin, vincristine and prednisolone, and FOLFOX, an abbreviation for an oxaliplatin regimen (ELOXATINTM) in combination with 5-FU and leucovorin.

Also included in this definition are anti-hormonal agents that act by regulating or inhibiting the action of hormones on tumors, such as antiestrogens with a mixed agonist / antagonist profile, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, trioxifene, toremifene (FARESTON®); idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3; pure antiestrogens without agonistic properties such as fulvestrant (FASLODEX®) and EM800 (such agents can block estrogen receptor (ER) dimerization, inhibit DNA binding, enhance ER metabolism and / or lower ER levels); aromatase inhibitors including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®) and non-steroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutethimide and other aromatase inhibitors including vorozole®) (RIVISOR®) megestrol acetate (MEGASE®), fadrozole, imidazole; luteinizing hormone releasing hormone agonists including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, including progestins such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens / retinoids such as fluoxymesterone, all-trans-retinoic acid, and fenretinide; onapristone; antiprogesterones; down regulators of estrogen receptors (ERD); antiandrogens such as flutamide, nilutamide and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids or derivatives of any of the above agents; as well as combinations of two or more of the above means.

In some embodiments, the other therapeutically active compound is a checkpoint inhibitor.

The term "checkpoint inhibitor" (or checkpoint inhibitor) refers to compounds that inhibit the activity of immune checkpoints. Inhibition includes decreased function or complete blockage. Examples of inhibitory checkpoint molecules include B7-H3, B7-H4, BTLA, CTLA-4, KIR, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, TIGIT, and VISTA. In some embodiments, the immune checkpoint inhibitor is an antibody that specifically recognizes an immune checkpoint protein. A number of immune checkpoint inhibitors are known and alternative immune checkpoint inhibitors similar to these known immune checkpoint protein inhibitors may be developed in the near future. Immune checkpoint inhibitors include, but are not limited to, peptides, antibodies, nucleic acid molecules, and small molecule compounds.

Tumor-associated macrophages (TAMs) were found to express high levels of PD1, PD-L1 and PD-L2. In TAMs derived from mice treated with CSF-1R inhibitors, PD-L2 and PD1 expression levels are significantly reduced. 70% of activated CTLs have high levels of PD1 expression and CSF-1R blockade has no effect on this. In turn, CTLA-4 expression on CD8 + was significantly enhanced by CSF-1R inhibition. Thus, when CSF-1R is inhibited, it is necessary to take into account the possibility of a certain paradox. Despite the fact that CSF1 / CSF-1R blockade reprograms the microenvironment towards the antitumor response of macrophages and indirectly activates CTL, this reprogramming leads to CTLA-4 on activated cytotoxic T cells. Thus, it is obvious that anti-CSF1R drugs should be used in combination with checkpoint inhibitors (anti-CTLA4).

In some embodiments, the checkpoint inhibitor is selected from a PD-1 inhibitor or a CTLA-4 inhibitor.

In some embodiments, the PD-1 inhibitor is an antibody that specifically binds to PD-1. Examples of antibodies that specifically bind to PD-1 include pembrolizumab, nivolumab, prolgolimab, toripalimab, cemiplimab, sintilimab, and others. Most preferred are prolgolimab, pembrolizumab, nivolumab.

In some embodiments, the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4. Examples of antibodies that specifically bind to CTLA4 include ipilimumab and tremelimumab, zalifrelimab, and others. Most preferred is ipilimumab.

It is contemplated that the monoclonal antibody of this invention can be used in methods of treatment as described above, can be used in treatment as described above, and / or can be used in the manufacture of medicaments for treatment as described above.

In one aspect, the present invention provides a method of inhibiting the biological activity of CSF-1R in a subject in need of such inhibition, comprising administering to the subject an effective amount of an antibody, or antigen binding fragment thereof, that specifically binds to CSF-1R, of the present invention.

In one aspect, the present invention provides a method of treating a disease or disorder mediated by CSF-1R, comprising administering to a subject in need of such treatment, an antibody or antigen binding fragment thereof that specifically binds to the CSF-1R of this invention, or a pharmaceutical composition, described above, in a therapeutically effective amount.

In one aspect, the present invention provides a method for treating a disease or disorder mediated by CSF-1R, comprising administering to a subject in need of such treatment an antibody or antigen binding fragment thereof as described above and at least one other therapeutically active compound.

In one aspect, the present invention relates to the use of an antibody as described above, or antigen binding fragment thereof that specifically binds to CSF-1R, or a pharmaceutical composition as described above, for treating a CSF-1R mediated disease or disorder in a subject in need of such treatment.

In one aspect, the present invention relates to the use of an antibody as described above, or antigen-binding fragment thereof, which specifically binds to CSF-1R, and at least one other therapeutically active compound for the treatment of a disease or disorder mediated by CSF-1R.

In some embodiments, the disease or disorder mediated by CSF-1R is selected from the group of: metastatic melanoma, metastatic bladder cancer, metastatic non-small cell lung cancer, metastatic renal cell carcinoma (light cell type), malignant neoplasms with microsatellite instability with ineffective primary therapy, locally advanced or metastatic squamous cell carcinoma of the head and neck, stomach cancer (cardio-esophageal zone), ovarian cancer, breast cancer, triple negative breast cancer, bone tumor, Hodgkin's lymphoma, glioma, glioblastoma, mesothelioma, myeloma, prostate cancer, soft tissue sarcoma.

Doses and routes of administration

A monoclonal antibody that specifically binds to CSF-1R of the present invention will be administered in an amount effective to treat the condition in question, i. E. in doses and for periods of time necessary to achieve the desired result. The therapeutically effective amount may vary depending on factors such as the particular condition being treated, the age, sex, and weight of the patient, and whether administration of a monoclonal antibody that specifically binds to CSF-1R is self-medication or combinations with one or more additional drugs or treatments.

The dosage regimens can be adjusted to provide the optimal desired response. For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased depending on the severity of the therapeutic situation. It is particularly useful to formulate parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as dosage units for patients / subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specification for unit dosage forms of the present invention is generally dictated by and directly dependent on (a) the unique characteristics of the therapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the compounding technique of such an active compound for treating sensitivity in subjects.

Thus, those skilled in the art will understand, based on the disclosure provided herein, that dosages and dosage regimen are adjusted in accordance with methods well known in the therapeutic field. This means that the maximum tolerated dose can be readily established and the effective amount can also be determined to provide a detectable therapeutic effect for the patient, as well as the time requirements for administration of each agent to achieve a visible therapeutic effect for the patient. Thus, although some dosages and regimens of administration are given as examples in this document, these examples in no way limit the doses and regimes of administration that may be needed for a patient in the practice of using the present invention.

It should be noted that dosage values may vary depending on the type and severity of the condition to be alleviated, and may include one or more doses. In addition, it should be understood that for any particular patient, specific administration regimens should be adjusted over time according to the individual need and at the discretion of the healthcare professional who administers or monitors the administration of the compositions, and that the concentration ranges given in this description are given only by way of example and are not intended to limit the scope or practice of the claimed compositions. In addition, the dosage regimen with the compositions of this invention can be based on various factors, including the type of disease, age, weight, sex, health status of the patient, severity of the condition, route of administration, and the particular monoclonal antibody used that specifically binds to CSF-1R. Thus, the dosage regimen can vary widely, but can be determined regularly using standard methods. For example, doses can be adjusted based on pharmacokinetic and pharmacodynamic parameters, which can include clinical effects such as toxic effects or laboratory values. Thus, the present invention encompasses individual dose escalation as determined by the skilled artisan. Determination of the required dose and regimens are well known in the art and will be understood by the person skilled in the art upon reading the teachings disclosed herein.

Examples of suitable routes of administration are provided above.

It is contemplated that a suitable dose of a monoclonal antibody that specifically binds to CSF-1R according to this invention will be in the range of 0.1-200 mg / kg, preferably 0.1-100 mg / kg, including about 0.5- 50 mg / kg, for example, about 1-20 mg / kg. A monoclonal antibody that specifically binds to CSF-1R can be administered, for example, at a dose of at least 0.25 mg / kg, for example, at least 0.5 mg / kg, including at least 1 mg / kg , for example, at least 1.5 mg / kg, for example, as well as at least 2 mg / kg, for example, at least 3 mg / kg, including at least 4 mg / kg, for example, at least 5 mg / kg; and for example up to a maximum of 50 mg / kg, including up to a maximum of 30 mg / kg, for example up to a maximum of 20 mg / kg, including up to a maximum of 15 mg / kg. The administration will be repeated, usually at appropriate intervals, for example, once a week, once every two weeks, once every three weeks, or once every four weeks, and as long as deemed appropriate by the responsible physician, who may in some cases increase or reduce dose if necessary.

Diagnostic use of an antibody that specifically binds to CSF-1R

A monoclonal antibody that specifically binds to CSF-1R of the present invention is also used for diagnostic purposes (eg, in vitro, ex vivo). For example, a given monoclonal antibody, which specifically binds to CSF-1R, of the present invention can be used to detect or measure the level of CSF-1R in samples obtained from a patient, for example, a tissue sample or a sample of a body fluid such as inflammatory exudate, blood, blood serum, intestinal fluid, saliva, or urine. Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence analysis, radioimmunoassay, and immunohistology.

An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to CSF-1R, comprising:

(a) a heavy chain variable domain comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 51: DYAMXaa ₁ , where Xaa ₁ is the amino acid T or S;

(ii) CDR2 with amino acid sequence SEQ ID NO: 52: Xaa ₂ ISWXaa ₃ GGXaa ₄ TXaa ₅ YXaa ₆ DSVKG, where

Xaa ₂ is amino acid A, Xaa ₃ is amino acid N, R, I, L, or F, Xaa ₄ is amino acid S, X ₅ is amino acid N, and Xaa ₆ is amino acid A;

or

Xaa ₂ is amino acid A, Xaa ₃ is amino acid N, Xaa ₄ is amino acid E, I or L, Xaa ₅ is amino acid N and Xaa ₆ is amino acid A;

or

Xaa ₂ is amino acid A, Xaa ₃ is amino acid N, Xaa ₄ is amino acid S, Xaa ₅ is amino acid R or W, and Xaa ₆ is amino acid A;

or

Xaa ₂ is amino acid A, Xaa ₃ is amino acid N, Xaa ₄ is amino acid S, Xaa ₅ is amino acid N, and Xaa ₆ is amino acid Q or H;

or

Xaa ₂ is the amino acid Q or H, Xaa ₃ is the amino acid N, Xaa ₄ is the amino acid S, Xaa ₅ is the amino acid R, and Xaa ₆ is the amino acid Q; or where Xaa ₂ is the amino acid Q or H, Xaa ₃ is the amino acid N, Xaa ₄ is the amino acid S, Xaa ₅ is the amino acid R; and Xaa ₆ is the amino acid H;

Xaa ₂ is the amino acid Q or H, Xaa ₃ is the amino acid L, Xaa ₄ is the amino acid S, Xaa ₅ is the amino acid N, and Xaa ₆ is the amino acid Q; or where Xaa ₂ is the amino acid Q or H, Xaa ₃ is the amino acid I, Xaa ₄ is the amino acid S, Xaa ₅ is the amino acid N, and Xaa ₆ is the amino acid Q;

(iii) CDR3 with amino acid sequence SEQ ID NO: 53: Xaa ₇ VEXaa ₈ Xaa ₉ HRRLYXaa ₁₀ YLEXaa ₁₁ , where

Xaa ₇ is the amino acid T, I or Y, Xaa ₈ is the amino acid V, Xaa ₉ is the amino acid A, Xaa ₁₀ is the amino acid K and Xaa ₁₁ is the amino acid V;

or

Xaa ₇ is the amino acid T, Xaa ₈ is the amino acid V, Xaa ₉ is the amino acid A, Xaa ₁₀ is the amino acid R, N, Q, L, F, or Y, and Xaa ₁₁ is the amino acid V;

or

Xaa ₇ is the amino acid T, Xaa ₈ is the amino acid V, Xaa ₉ is the amino acid A, Xaa ₁₀ is the amino acid K, and Xaa ₁₁ is the amino acid I;

or

Xaa ₇ is the amino acid T, Xaa ₈ is the amino acid Q, L or F, Xaa ₉ is the amino acid A, Xaa ₁₀ is the amino acid K, and Xaa ₁₁ is the amino acid I;

or

Xaa ₇ is the amino acid T, Xaa ₈ is the amino acid F, Xaa ₉ is the amino acid S, Xaa ₁₀ is the amino acid K, and Xaa ₁₁ is the amino acid V;

or

Xaa ₇ is the amino acid T, Xaa ₈ is the amino acid F, Xaa ₉ is the amino acid A, Xaa ₁₀ is the amino acid K and Xaa ₁₁ is the amino acid V; and

(b) a light chain variable domain comprising:

(i) CDR1 with the amino acid sequence GGNNIGSKSVH (SEQ ID NO: 12),

(ii) CDR2 with the amino acid sequence GDSDRPS (SEQ ID NO: 13),

(iii) CDR3 with the amino acid sequence QVWDISSDHVV (SEQ ID NO: 14).

An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to CSF-1R, comprising:

(a) a heavy chain variable domain comprising:

(i) CDR1 with the amino acid sequence SEQ ID NO: 1: DYAMS or SEQ ID NO: 17: DYAMT;

(ii) CDR2 with amino acid sequence SEQ ID NO: 2: AISWNGGSTNYADSVKG or

SEQ ID NO: 54: AISWXaa ₃ GGSTNYADSVKG,

where Xaa ₃ represents the amino acid R, I, L or F;

or

SEQ ID NO: 55: AISWNGGXaa ₄ TNYADSVKG,

where Xaa ₄ represents the amino acid E, I or L;

or

SEQ ID NO: 56: AISWNGGSTXaa ₅ YADSVKG,

where Xaa ₅ represents the amino acid R or W;

or

SEQ ID NO: 57: AISWNGGSTNYXaa ₆ DSVKG,

where Xaa ₆ represents the amino acid Q or H;

or

SEQ ID NO: 58: Xaa ₂ ISWNGGSTXaa ₅ YXaa ₆ DSVKG,

where Xaa ₂ is the amino acid Q or H, Xaa ₅ is the amino acid R and Xaa ₆ is the amino acid Q; or where Xaa ₂ is the amino acid Q or H, Xaa ₅ is the amino acid R and Xaa ₆ is the amino acid H;

or

SEQ ID NO: 59: Xaa ₂ ISWXaa ₃ GGSTNYXaa ₆ DSVKG,

where Xaa ₂ is the amino acid Q or H, Xaa ₃ is the amino acid L and Xaa ₆ is the amino acid Q; or where Xaa ₂ is the amino acid Q or H, Xaa ₃ is the amino acid I and Xaa ₆ is the amino acid Q;

(iii) CDR3 with amino acid sequence SEQ ID NO: 3: TVEVAHRRLYKYLEV or

SEQ ID NO: 60: Xaa ₇ VEVAHRRLYKYLEV,

where Xaa ₇ represents the amino acid I or Y;

or

SEQ ID NO: 61: TVEXaa ₁₀ AHRRLYKYLEV,

where Xaa ₁₀ represents the amino acid R, N, Q, L, F or Y;

or

SEQ ID NO: 45: TVEVAHRRLYKYLEI,

or

SEQ ID NO: 62: TVEXaa ₈ AHRRLYKYLEXaa ₁₁ ,

where Xaa ₈ is the amino acid Q, L or F, and Xaa ₁₁ is the amino acid I;

or

SEQ ID NO: 63: TVEXaa ₈ Xaa ₉ HRRLYKYLEV,

where Xaa ₈ is an amino acid F, and Xaa ₉ is an amino acid S;

or

SEQ ID NO: 50: TVEFAHRRLYKYLEV; and

(b) a light chain variable domain comprising:

(i) CDR1 with the amino acid sequence GGNNIGSKSVH (SEQ ID NO: 12),

(ii) CDR2 with the amino acid sequence GDSDRPS (SEQ ID NO: 13),

(iii) CDR3 with the amino acid sequence QVWDISSDHVV (SEQ ID NO: 14).

Implementation of the invention

For a better understanding of the invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any form.

All publications, patents, and patent applications mentioned in this specification are incorporated by reference in this document. Although the aforementioned invention has been described in some detail by way of illustration and example in order to avoid ambiguity, it will be readily apparent to those skilled in the art based on the teachings disclosed in this invention that certain changes and modifications may be made without departing from the spirit and scope of the accompanying variations. implementation of the invention.

Materials and general methods

For general information regarding the nucleotide sequences of human immunoglobulin light and heavy chains, see: Kabat E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The amino acids of the antibody chains are numbered according to EU numbering (Edelman GM et al., Proc Natl Acad Sci USA 63, 1969, ee 78-85; Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

Recombinant DNA Techniques

For DNA manipulation, standard methods were used as described in Sambrook J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biology reagents were used according to manufacturer's instructions.

Gene synthesis

The required gene segments were obtained from oligonucleotides created by chemical synthesis. Gene segments from 300 to 4000 kb in length, which are flanked by unique restriction sites, were collected by annealing and ligation of oligonucleotides, including PCR amplification and subsequent cloning through these restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

Determination of DNA sequences

DNA sequences were determined by Sanger sequencing.

DNA and protein sequence analysis and sequence data processing

Infomax's Vector NT1 Advance suite, version 8.0, was used to create, map, analyze, annotate, and illustrate sequences.

Expression vectors

For the expression of the described antibodies and antigens, variants of expression plasmids intended for expression in prokaryotic (E. coli) cells of short-term expression in eukaryotic cells (for example, in CHO cells) were used. In addition to the antibody expression cassette, the vectors contained: a replication initiation site providing replication of the said plasmid in E. coli, genes that confer resistance in E. coli to various antibiotics (for example, ampicillin and kanamycin).

The fusion of genes containing the described antibody chains, as described below, was carried out by PCR and / or synthesis and assembly of genes using known recombination methods and procedures by connecting the corresponding segments of nucleic acids, for example, using unique restriction sites in the corresponding vectors. Subcloned nucleotide sequences were confirmed by DNA sequencing. For transient transfections, large numbers of plasmids were generated by obtaining plasmids from transformed E. coli cultures.

Example 1. Production of recombinant antigens in suspension culture of mammalian cells

Sequences of human extracellular domains CSF-1R (SEQ https://www.uniprot.org/uniprot/P07333 (19-517 a.a.)) were cloned into plasmids for protein production in mammalian cells with tags FcLama, Avi, His at restriction sites SalI / NotI. Plasmids were produced in the required quantities in E. Coli cells and purified using the Qiagen kit.

The sequence of the extracellular domain of rhesus monkey CSF-1R (SEQ https://www.ncbi.nlm.nih.gov/protein/XP_001107711.4) was cloned into a plasmid for protein production in mammalian cells with His tag at the SalI / NotI restriction sites. Plasmids were produced in the required quantities in E. coli cells and purified using the Qiagen kit.

Example 2. Production of recombinant antigens and antibodies in suspension culture of mammalian cells

Antigens and antibodies were produced in cells of a permanent cell line derived from Chinese hamster ovary cells (CHO-T line). Suspension cultivation was carried out in flasks on an orbital shaker-incubator using serum-free media manufactured by HyCell TransFx-C with the addition of 8 mM L-glutamine and 1 g / L Pluronic 68. For transient expression of cells at a concentration of 2-2.2 × 10 ⁶ cells / ml were transfected with linear polyethyleneimine (PEI MAX, Polysciences). The DNA / PEI ratio was 1: 3/1: 10. 9 days after transfection, the culture fluid was separated from the cells by filtration through a depth filter with a pore size of 0.5 / 0.22 μm.

The hCSF1R-Fc_llama antigen was isolated from the culture liquid and purified using a column with a sorbent for affinity chromatography with Protein A (Biocad ZAO). The clarified culture fluid was passed through a 20 ml column equilibrated with phosphate-buffered saline (PBS, pH 7.4). The column was then washed with 5 column volumes of PBS to wash out non-specific binding components. The bound antigen was eluted using 0.1 M glycine buffer, pH 2.5. The main protein elution peak was collected and its pH adjusted to neutrality with 1 M Tris buffer (pH 7.5). Next, the protein was converted to PBS (pH 7.4) by dialysis and 1 mM DTT was added, filtered through Millex GP 0.22 μm, transferred into tubes and stored at -70 ° C.

0.5 M Imidazole and 0.1 M NiCl2 were added to the clarified culture fluid with antigens CSF1R-Avi, hCSF1R_His and 00_RhAG_000_04_6His (Macaca mulatta CSF1R) to a content of 0.02 M Imidazole and 0.001 M NiCl2. The culture broth was then passed through a 10 ml Ni Sepharose High Performance column (GE Healthcare) equilibrated with phosphate buffered saline supplemented with 10 mM imidazole (PBS, pH 7.4). The column was then washed with 5 column volumes of PBS supplemented with 10 mM imidazole to wash out non-specific binding components. The bound antigen was eluted using PBS supplemented with 500 mM imidazole (pH 7.4). Then the protein was converted into PBS by dialysis and 1 mM DTT was added. Thereafter, the protein was applied to a HiLoad 16/600 Superdex 75 pg gel filtration column (GE Healthcare), pre-equilibrated with PBS with the addition of 1 mM DTT. The protein was eluted with PBS supplemented with 1 mM DTT, filtered through a Millex GP 0.22 μm, transferred to tubes and stored at -70 ° C.

Electrophoresis of antigens was carried out in a denaturing 10% polyacrylamide gel under non-reducing conditions without the addition of mercaptoethanol and under reducing conditions with the addition of mercaptoethanol.

Example 3. Creation of a naive phage Fab-library of human antibodies MeganLibTM

The total RNA of B-lymphocytes from individual blood samples of more than a thousand human donors was isolated using the RNeasy Mini Kit according to the proposed protocol (from QIAGEN). RNA concentration was determined using the Nanovue kit (from GE Healthcare) and the quality of the isolated RNA was checked by electrophoresis in 1.5% agarose gel.

The reverse transcription reaction was performed using the MMLV RT kit (from Evrogen) according to the recommended protocol using MMuLV reverse transcriptase and random hexameric oligonucleotides as primer.

Reverse transcription products were used as a template in a two-step polymerase chain reaction to obtain variable domain genes flanked by restriction sites using a set of oligonucleotides according to the authors' protocols [J Biol Chem. 1999 Jun 25; 274 (26): 18218-30].

The resulting DNA preparation VL-CK-VH (Fig. 1) was treated with restriction endonucleases NheI / Eco91I and ligated into the original phagemid pH5. The ligation products were transformed into electrocompetent cells of the strain SS320 E. coli, prepared according to the protocols [Methods Enzymol. 2000; 328: 333-63.]. The repertoire of the MeganLibTM combinatorial phage Fab display library consisted of 10 ¹¹ transformants. Phage Fab-library preparations were prepared according to the procedure described previously [J Mol Biol. 1991 Dec 5; 222 (3): 581-97].

Example 4. Creation of Immune Fab, scFv, VHH Libraries of Lama-Human Hybrid Antibodies (CHARM)

The total RNA of B-lymphocytes from individual blood samples immunized with recombinant CSF-1R Lama Glama antigens, which demonstrated the maximum specific serum titer, was isolated using the KingFisher Pure RNA Tissue Kit, Cat. 98040196 according to suggested protocol (from Thermo Scientific). The RNA concentration was determined using a DropSence 96 spectrophotometer (Trinean) and the quality of the isolated RNA was checked by electrophoresis in 1.5% agarose gel.

The reverse transcription reaction was performed using the GoScript Reverse Transcriptase kit, Promega, A5001 (from Promega) according to the recommended protocol using reverse transcriptase and random hexameric oligonucleotides as primer.

Reverse transcription products were used as a template in a two-step polymerase chain reaction to obtain variable domain genes flanked by restriction sites using a set of oligonucleotides according to the authors' protocols [J Biol Chem. 1999 Jun 25; 274 (26): 18218-30].

The resulting DNA preparation VL-CK-VH (Fig. 1) or VH- (G4S) 3-VL (Fig. 2) was treated with restriction endonucleases NheI / SalI; NcoI / NotI and ligated into the original pH6 phagemid or pscK1 phagemid. The ligation products were transformed into electrocompetent strain SS320 cells prepared according to the protocols [Methods Enzymol. 2000; 328: 333-63.]. The repertoire of combinatorial phage Fab, scFv, VHH-display libraries consisted of 10 ⁹ transformants. Phage preparations Fab, scFv, VHH libraries were prepared according to the procedure described previously [J Mol Biol. 1991 Dec 5; 222 (3): 581-97]. A summary table of immune library preparations is shown in Table 1.

Table 1 - Combinatorial libraries of different formats of antigen-binding sites after immunization

P / p No. Name Library variety / species Repertoire, CFU Concentration, unit / ml one 01_ChAzVL_bhp Contains combinations of the heavy chain variable domain genes of Azazel llama immunized with CSF-1R and human lambda light chains R = 1.3x10E9 C = 3.6x10E13 2 01_ChAzVK_bhp Contains combinations of the heavy chain variable domain genes of Azazel llama immunized with CSF-1R and human light chain kappa R = 2.3x10E9 C = 3.2x10E13 3 01_ChAzscFVK_bhp Contains combinations of genes of variable domains of heavy chains of Azazel llama immunized with CSF-1R and human light chain kappa linked by a polypeptide linker. R = 3.7x10E9 C = 3.6x10E13 4 01_ChAzscFVL_bhp Contains combinations of genes of variable domains of heavy chains of llama Azazel, immunized with CSF-1R and human lambda light chains, connected by a polypeptide linker. R = 5.6x10E9 C = 4.2x10E13 five 01_ChAzscFVK_bhp Contains combinations of genes of variable domains of heavy chains of Azazel llama immunized with CSF-1R and human light chain kappa linked by a polypeptide linker. R = 3.7x10E9 C = 3.6x10E13 6 01_ChAzscFVL_bhp Contains combinations of genes of variable domains of heavy chains of llama Azazel, immunized with CSF-1R and human lambda light chains, connected by a polypeptide linker. R = 5.6x10E9 C = 4.2x10E13 7. 01_ChAjscFVK_bhp Contains combinations of genes of variable domains of heavy chains of Ajax llama immunized with CSF-1R and human light chain kappa linked by a polypeptide linker. R = 2.0x10E9 C = 5.7x10E13 eight. 01_ChAjscFVL_bhp Contains combinations of genes of variable domains of heavy chains of Ajax llama immunized with CSF-1R and human lambda light chains connected by a polypeptide linker. R = 1.0x10E9 C = 2.8x10E13 nine. 01_IAzVHH_bhp Contains combinations of genes of variable domains of heavy chains of Azazel llama immunized with CSF-1R R = 8.2x10E9 C = 3.2x10E13 10. 01_IAjVHH_bhp Contains combinations of genes of variable domains of heavy chains of Ajax llama immunized with CSF-1R R = 2.2x10E9 C = 4.0x10E13

Example 5. Selection of phage antibody libraries

Specific human phage antibodies against CSF-1R were obtained from the combinatorial phage Fab display library MeganLibTM and immune CHARM libraries described in examples 3 and 4. Selection was performed on human CSF-1R by phage display method [Nat Biotechnol. 1996 Mar; 14 (3): 309-14; J Mol Biol. 1991 Dec 5; 222 (3): 581-97], but using magnetic particles and the KingFisher Flex instrument, since the use of this technique allows up to 96 different circuits and variants to be run in parallel.

Human biotinylated antigen CSF-1R (Fc, Avi-His) was directed immobilized onto the surface of streptavidin magnetic beads (NEB) at a concentration for the human phage library: 10 μg / ml for the first round, 5 μg / ml for the second, 0 , 5 and 0.2 μg / ml for the third and fourth, respectively; for immune libraries: 5 μg / ml for the first round, 1 μg / ml for the second, 0.2 for the third round. The antigen was incubated with the particles for 1 hour at room temperature on a rotator. Next, the particles were washed with PBS (pH 7.4) and the surface of the particles was blocked with a solution of 2% skim milk or 1% BSA on PBS (pH 7.4) for 1 hour. Human phage library MeganLibTM was diluted at a concentration of 2 * 10 ¹³ phage particles per ml, CHARM immune libraries were diluted at a concentration of 2 * 10 ¹² phage particles per ml in PBS (pH 7.4) with 2% skim milk and a non-target antigen with a target antigen tag and preselected with magnetic particles free of surface antigen to remove nonspecifically binding phages. The CSF-1R-coated magnetic particles were then incubated with the libraries for 1-2 hours at room temperature.

Unbound phages were removed during several washes of magnetic particles with a PBS solution (pH 7.4) with 0.1% Tween 20. The number of washes was increased from round to round (10 washes in the 1st round, 15 -m - 20, on the 4th - 25). Phages bound to the antigen on the surface of the magnetic particles were eluted from the particles with a 100 mM Gly-HCl solution (pH 2.2) for 15 min with stirring; after elution, the 1M Tris-HCl solution (pH 7.6) was neutralized. Bacteria of the E. coli TG1 strain were infected with phages, accumulated in culture, and used in the next round of selection. After three to four rounds, phagemid DNA was isolated from the E. coli TG1 culture according to the manufacturer's standard protocol (Qiagen). The enrichment of the library for target antigens and the assessment of the presence of nonspecifically binding phage particles was carried out using an enzyme-linked immunosorbent assay for polyclonal phage (ELISA).

Example 6. Immunoassay of polyclonal phage for specific and nonspecific antigens

For ELISA, the target antigen CSF-1R-Fc and the non-target antigen with Fc-fusion protein were immobilized on high-sorption plates (Greiner-Bio). Protein was added at a concentration of 1 and 5 μg / ml, respectively, in 0.1 M NaHCO3 (pH 9.0) and titrated in steps of 2 to 7 dilutions, after which, hermetically sealed plates were incubated overnight at + 4 ° C. All subsequent steps were performed according to the standard ELISA protocol using a high-performance automated platform based on the Tecan Freedom EVO 200 robotic system (from Tecan). To block nonspecific binding, a blocking buffer of 2% skim milk or 1% BSA on PBS (pH 7.4) was added to the wells of the plates. The plates were incubated for one hour at room temperature. After washing with phosphate-buffered saline containing Tween-20 (PSBT), 50 μl per well of the tested polyclonal phage was added. After washing, anti-M13 HRP-conjugated secondary antibody (from Pierce-Thermo Scientific) was added (50 μl / well) at a ratio of 1: 7500 in PSTB. After incubation for 50 minutes at room temperature, the plates were washed with PSTB three times. The colorimetric signal was developed by adding a substrate solution (H ₂ O ₂ -0.02% and TMB in CH ₃ COONa pH 5.5) for 10 minutes, then the reaction was stopped by adding 1% sulfuric acid (20 μl). The color signal was measured at 450 nm using a suitable Tecan-Sunrise plate reader (from Tecan).

After the third and fourth rounds of selection on the target antigen, the ELISA analysis of the polyclonal phage preparation showed a significant enrichment. Libraries were selected for recloning and further screening, in which the signal was observed to be more than 5 times higher at the minimum dilution of phage libraries for non-homologous control antigens.

Example 7. Cloning of antibody variable domain genes into an expression plasmid

The recloning of antibody variable domain genes into the expression plasmid pLL (Fig. 3) or pET22 (Fig. 4) from the phagemid vector after successful rounds of selection was carried out according to a standard protocol using the restriction enzyme ligase method.

Subsequently, expression vectors containing antibody fragments were transformed into E. coli strain BL21-Gold for comparative analysis of the affinity of variable antibody fragments from display libraries to antigen by ELISA using the Mabnext Flow Chart technological platform.

Example 8. Selection of high-affinity clones from post-selection libraries that specifically bind human CSF-1R

The production of Fabs was carried out according to the standard technique: bacterial cells of E. coli BL21-Gold were transformed with expression vectors containing Fab genes, and the subsequent addition of an inducer triggered transcription of the lac operon, which, upon cultivation of the obtained transformants, caused the expression of Fabs, which were exported to the periplasmic space of cells. Next, ELISA was performed to test the binding of Fabs to the CSF-1R-Fc_lama antigen immobilized on a substrate at a concentration of 0.2 μg / ml on plates (medium binding from Greiner bio one) in 0.1 M NaHCO ₃ (pH 9.0) (antigen immobilized overnight at 4 ° C). All subsequent steps were performed at room temperature according to a standard ELISA protocol using a high-performance automated platform based on robotic systems Genetix Qpix2xt (from Molecular Device) and Tecan Freedom EVO 200 (from Tecan). After each stage, a wash was performed with 300 μL per well of 1x FSBT in triplicate. Nonspecific binding sites were blocked on a plate with 1% skim milk in 1x FSBT; after washing, an analyte was added, which served as supernatants of E. coli cells in a volume of 60 μl per well. Detection of immune complexes was carried out using goat anti-Fab antibodies conjugated with peroxidase (from Pierce-ThermoScientific) at a dilution of 1: 7500. Staining of the substrate-chromogenic mixture was carried out by adding a substrate solution (Н ₂ О ₂ -0.02% and TMB in CH ₃ COONa (pH5.5)) in a volume of 50 μl for 15 minutes. The reaction was stopped with 25 μl H ₂ SO ₄ 1%. The color signal was measured at 450 nm using a suitable Tecan-Sunrise plate reader (from Tecan). The degree of antibody binding was proportional to the production of a color signal. Clones in which the color signal exceeded the signal from the control antibody were tested in ELISA for nonspecific binding.

Example 9. Analysis of non-specific binding of selected Fabs to other antigens

ELISA was also used to analyze the nonspecific binding of the studied Fab fragments to other antigens. The study was carried out as described above, but IL6R-Fc, PCSK9-VG-FE in 0.1 M NaHCO ₃ (pH 9.0) were used as antigens for immobilization (the antigen was immobilized overnight at 4 ° C). CSF-1R-Fc_lama, CSF-1R-his were used as a control for specific binding (antigen was immobilized overnight at 4 ° C). All subsequent steps were performed according to the standard ELISA protocol using a high-performance automated platform based on robotic systems Genetix Qpix2xt (from Molecular Device) and Tecan Freedom EVO 200 (from Tecan). Clones in which the color signal of nonspecific binding did not exceed the control signal were tested in a competitive ELISA assay to identify Fabs that block the interaction of the ligand and the receptor.

Example 10. Selection of Fab fragments by dissociation rate on ForteBio

Off-rate screening was performed using a Pall Forte Bio Octet Red 384 device. Anti-FABCH1 biosensors were rehydrated for 30 minutes in a working buffer containing 10 mM PBS (pH 7.2-7.4), 0.1% Tween- 20, 0.1% BSA. Working buffer was added to the studied samples of E. coli supernatants to a final concentration of 10%. Then, anti-FABCH1 biosensors were immersed in E. coli supernatants containing Fab fragments of candidate antibodies for 12 hours at 4 ° C. Sensors with Fab fragments immobilized on the surface were transferred into wells with a working buffer, where the baseline was prescribed (60 s). Then the sensors were transferred into wells with an analyte solution (CSF-1R-his, 30 μg / ml) for the association of the antigen-antibody complex (300 s). Then the sensors were returned to the wells containing the working buffer for the subsequent dissociation step (600 s). After each experiment, the used sensors were regenerated by placing them three times in the regeneration buffer (10 mM Gly-HCl, pH 1.7) and then used in the next experiment. The analysis of the obtained curves was carried out using the Octet Data Analysis software (version 9.0) according to the standard procedure according to the 1: 1 Local interaction model.

As a result of the analysis, the following Fab fragments were selected, which were further used to create full-length antibodies (Table 2).

Table 2 - Dissociation rate constants of Fab-fragments upon interaction with human antigen CSF-1R-his

Fab name Candidate ForteBio Response, nm Kdis, 1 / s 01_ChBin1VLSel3_MP11_H1_22 01-011 0.2641 8,35E-05 01_ChBin1VLSel3.CD.AE3_MP12_G5_5 01-012 0.5271 3.20E-04 01_ChBin1VKSel3.CD_MP10_D4_48 01-013 0.7169 3.50E-04 01_ChBin2VKSel3.AE3_MP11_A11_18 01-014 0.5573 3.53E-04 01_ChAzVLSel3.CD_MP8_A5_12 01-015 0.7516 1.20E-04 01_ChAzVLSel3.CD_MP8_C4_6 01-016 0.6284 1.89E-04 01_ChAzVLSel3.CD_MP8_D5_15 01-017 0.4196 5.27E-04 01_ChAzVKSel2.CD_MP6_A6_17 01-018 0.4369 9.66E-04 01_ChBin1VLSel3.CD.AE3_MP12_B8_24 01-019 0.9515 8.19E-05

Example 11. Obtaining genetic constructs for the synthesis of full-length antibodies

Cloning was carried out according to the standard method. PCR products containing genes of variable domains of heavy and light chains of antibodies with primers containing restriction sites were prepared. The variable domain of the heavy chain was cloned into the pEE-HCLALA IgG1 vector at the Sal1 / Nhe1 restriction sites. The light chain variable domain was cloned into the pEE-CK vector at the Sal1 / BsiW1 restriction sites. The obtained genetic constructs were transferred to the transient production of proteins in the CHO-T cell line.

Example 12. Obtaining full-length antibodies

Full-length antibodies were produced in cells of a permanent cell line obtained from Chinese hamster ovary cells (CHO-T line) by transient transfection in a Beckman NX robotic station in duplicate. Suspension cultivation was carried out in TubeSpin 50 bioreactors on an orbital shaker using serum-free media from HyCell TransFx-C supplemented with 8 mM L-Glutamine and 1 g / L Pluronic 68. For transient expression, cells at a concentration of 2-2.2 × 10 ⁶ cells / ml were transfected with linear polyethyleneimine (for example, PEI "MAX", company "Polysciences"). The DNA / PEI ratio was 1: 3/1: 10. On the 10th day of cultivation, the cell suspension was centrifuged at 2000g for 15 minutes and filtered through a filter with a pore size of 0.22 μm.

Antibodies were purified by affinity chromatography on PreDictor RoboColumn MabSelect SuRe 200 columns (GE Healthcare) using a Beckman FX robotic station. The column was equilibrated with 50 mM NaPB, 150 mM NaCl (pH 7.5), a filtered culture liquid with antibodies was applied, then the column was washed with 8 volumes of 50 mM NaPB, 150 mM NaCl, and 4 volumes of 50 mM NaPB (pH 7.5). The protein was eluted with a solution of 50 mM NaPB, 100 mM NaCl (pH 3) in 6 column volumes. The resulting samples were neutralized with 25 μl of 1M phosphate buffer (pH 8).

Polyacrylamide gel electrophoresis and size exclusion HPLC were used to control the purity of the candidates. Electrophoresis was performed in a 7.5% polyacrylamide gel under denaturing non-reducing conditions. The determination of the protein purity was carried out by the intensity of the band staining at a protein load of 10 μg per lane. Electropherograms are shown in FIG. 5 and FIG. 6. Size exclusion HPLC was performed on a Tosoh TSK-Gel G3000SWXL column in a mobile phase of 0.05M NaH2PO4, 0.3 M NaCl pH = 7.0.

Example 13. Determination of the affinity of full-length candidates for human CSF-1R on Forte Bio Octert RED 384

Experiments to study the binding affinity of antibodies to the antigen with human Avi-hCSF1R were performed on a Forte Bio Octert RED 384 device. Antibodies at a concentration of 30 μg / ml are immobilized on the surface of ProteinA biosensors (ForteBio). The analysis was carried out at 30 ° C using PBS containing 0.1% Tween-20 and 0.1% BSA as a working buffer. After the baseline was prescribed in the buffer solution, the sensors with immobilized antibodies were immersed in wells with a solution of Avi-hCSF1R at a concentration of 10 μg / ml, where the complex was associated for 300 seconds. Then the dissociation of the complex in the buffer solution is detected for 600 seconds.

The reference subtracted binding curves are analyzed using Octet Data Analysis (version 9.0) according to a standard procedure using a 1: 1 Global interaction model.

The analyzed anti-CSF-1R antibodies bind specifically to the human antigen (Table 3).

Table 3 - Kinetic constants of candidates (01-011 - 01-019) upon interaction with human avi-hCSF1R antigen

Candidate KD, M Kon, 1 / Ms Kdis, 1 / s R ² 01-011 1.68E-09 9.14E + 04 1.54E-04 0.98 01-012 3,254E-09 5.26E + 04 0.000171 0.9964 01-013 7.04E-10 9.91E + 04 6.98E-05 0.9763 01-014 1.32E-09 7.06E + 04 9.34E-05 0.9927 01-015 9.32E-10 5.64E + 04 5.26E-05 0.9955 01-016 1.21E-09 1.88E + 05 2.28E-04 0.9413 01-017 2.66E-08 4.07E + 04 1.08E-03 0.9975 01-018 1.69E-08 3.24E + 04 5.48E-04 0.9948 01-019 6.17E-10 1.07E + 05 6.58E-05 0.9765

Example 14. Analysis of interactions of full-length candidates with CSF-1R rhesus monkey and mice on a Forte Bio Octet RED 384 device

Experiments to study the binding affinity of antibodies to the antigen c 00_RhAG_000_04_6His (Macaca mulatta CSF-1R) were carried out on a Forte Bio Octert RED 384 device. Antibodies at a concentration of 30 μg / ml are immobilized on the surface of ProteinA biosensors (ForteBio). The analysis was carried out at 30 ° C using PBS containing 0.1% Tween-20 and 0.1% BSA as a working buffer. After the baseline was prescribed in the buffer solution, the sensors with immobilized antibodies were immersed in wells with a solution of c 00_RhAG_000_04_6His (Macaca mulatta CSF-1R) at a concentration of 10 μg / ml, where the complex was associated for 300 seconds. Then the dissociation of the complex in the buffer solution is detected for 600 seconds.

The reference subtracted binding curves are analyzed using Octet Data Analysis (version 9.0) according to a standard procedure using a 1: 1 Global interaction model.

Anti-CSF-1R antibodies specifically bind to macaque antigen.

Table 4 - Kinetic constants of candidates (01-011 - 01-019) when interacting with the 00_RhAG_000_04_6His antigen (Macaca mulatta CSF-1R)

Candidate Response, nm KD, M Kon, 1 / Ms Kdis, 1 / s R ² 01-011 0.9172 2.00E-08 6.68E + 04 1.34E-03 0.9887 01-012 0.5089 5.02E-08 3.64E + 04 1.82E-03 0.9951 01-013 0.9851 1.20E-09 7.14E + 04 8.60E-05 0.982 01-014 0.8173 1.79E-09 6.72E + 04 1.20E-04 0.987 01-015 0.0294 1.31E-08 1.01E + 05 1.32E-03 0.8591 01-016 0.9749 4.32E-09 2.31E + 05 9.98E-04 0.9538 01-017 0.0374 - - - - 01-017 0.0776 - - - - 01-018 0.1141 2.97E-08 2.92E + 04 8.67E-04 0.9757 01-019 0.0994 - - - - 01-019 0.279 6.40E-09 3.48E + 03 2.23E-05 0.9869

Similarly, the affinity for the mouse CSF-1R antigen was measured. None of the candidates have been shown to interact with mouse CSF-1R.

Example 15. Determination of the epitope specificity of antibodies to antigens using the Forte Bio Octet RED 384 device

The human antigen CSF-1R-his at a concentration of 15 μg / ml is immobilized on the surface of anti-penta-his biosensors (ForteBio). The analysis was carried out at 30 ° C using PBS containing 0.1% Tween-20 and 0.1% BSA as a working buffer. After the baseline was prescribed in the buffer solution, the sensors were immersed in wells with a solution of Mab1 antibodies at a concentration of 60 μg / ml where the complex is associated. Then, after saturation of the antigen with the Mab1 antibody, the sensors were immersed in a solution with competing Mab2 samples at a concentration of 15 μg / ml to determine the competition with Mab1.

If Mab2 interacts with the antigen when fully saturated with Mab 1 antibodies, we believe that the Mab2 antibody does not compete with Mab1 for binding to the antigen. Otherwise, if Mab2 does not interact with the antigen when fully saturated with Mab1 antibodies, we believe that Mab2 competes with Mab1 for binding to the antigen.

The analysis revealed that the candidates were divided into 5 groups according to epitope specificity, all of which do not compete with the control antibody Cabiralizumab (Table 5).

Table 5 - Distribution of candidates by groups during epitope binning

BIN1 BIN2 BIN3 BIN4 BIN5 BIN6 Cabiralizumab (reproduced) 01-011 01-013 01-015 01-014 01-016 01-012 01-018 01-017 01-019

Example 16. Proliferative test on the cell line TF1-CSF-1R against the background of the ligand M-CSF

The analysis of the ability to inhibit CSF-dependent proliferation for anti-CSF-1R antibodies was carried out on the TF1 (bone marrow erythroleukemia) cell line, stably expressing the human CSF-1R receptor (TF1-human CSF-1R, JSC Biocad).

Candidate anti-CSF-1R antibodies were prepared on the day of analysis. These antibodies were diluted to a concentration of 100 μg / ml in the following medium: RPMI-1640, 2mM L-glutamine, 0.5% FBS. The prepared dilutions were added at 25 μl / well to culture plates (Costar, 3599).

In a medium of the same composition, M-CSF (Peprotech) was diluted to a concentration of 50 ng / ml. Add 25 μl of the prepared solution to the wells of the culture plate. A TF1-human CSF-1R cell suspension was prepared at a concentration of 4 × 10 ⁵ cells / ml, thoroughly resuspended, and 50 μl of the cell suspension was added to culture plates. The plates were mixed on an orbital shaker and incubated with a CO ₂ incubator for 72 hours.

At the end of the incubation period, the vital dye Alamar blue (Invitrogen ™) was added and additionally incubated in a CO2 incubator until a gradient color developed. The fluorescence level was measured using an Infinite M200Pro (Tecan).

The ratio of the fluorescence level for the analyzed samples (RFU AT) to the fluorescence level of the control of the M-CSF action (M-CSFctrl) was evaluated. The results of the analysis of candidates are shown in Table 6.

Table 6 - Analysis of the ability of anti-CSF-1R antibodies to inhibit CSF-dependent proliferation

Candidate RFU AT / RFU M-CSFctrl Inhibition of CSF-dependent proliferation 01-012 1.22 Does not show 01-014 1.16 Does not show 01-015 1.22 Does not show 01-016 0.15 Exhibits 01-018 1.24 Does not show

Example 17. Analysis of the ability of anti-CSF-1R antibodies to inhibit IL-34-dependent proliferation

The analysis of the ability to inhibit IL-34-dependent proliferation for anti-CSF-1R antibodies was performed on the TF1 (bone marrow erythroleukemia) cell line, stably expressing the human receptor CSF-1R (TF1-human CSF-1R, Biocad CJSC).

Candidate anti-CSF-1R antibodies were prepared on the day of analysis. Titrations of these antibodies were prepared from a concentration of 300 μg / ml in a medium of the following composition: RPMI-1640, 2mM L-glutamine, 0.5% FBS. Add 25 μl / well of the prepared dilutions to culture plates (Costar, 3599).

In a medium of the same composition, IL-34 (Peprotech) was diluted to a concentration of 3 μg / ml. Add 25 μl of the prepared solution to the wells of the culture plate. A TF1-human CSF-1R cell suspension was prepared at a concentration of 4 × 10 ⁵ cells / ml, thoroughly resuspended, and 50 μl of the cell suspension was added to culture plates. The plates were mixed on an orbital shaker and incubated with a CO ₂ incubator for 72 hours.

At the end of the incubation period, the vital dye Alamar blue (Invitrogen ™) was added and further incubated in a CO ₂ incubator until a gradient color developed. The fluorescence level was measured using Infinite M200Pro (Tecan), the activity of anti-CSF-1R antibodies was assessed.

Candidate 01-016 exhibits the ability to inhibit IL-34-dependent proliferation of the TF1-human CSF-1R cell line. Dose-response curves are shown in FIG. 7.

Example 18. Evaluation of the effect of anti-CSF-1R antibodies on the proliferative activity of monocytes in the presence of M-CSF

Evaluation of the inhibition of M-CSF-dependent monocyte proliferation was performed using a population isolated from PBMC by positive magnetic bead selection CD14 + (CD14 MicroBeads, human, 130-050-201 MACS). Isolation was performed according to the manufacturer's instructions.

After isolating the CD14 + population, the isolated monocytes were counted in a Goryaev chamber. A cell suspension of 1 × 10 ⁶ cells / ml was prepared in RPMI-1640, 25 mM HEPES, 2 mM L-glutamine. 50 μl of cell suspension was added to the inner wells of white culture plates (Corning, 3610). The plates with the cell suspension were incubated for _{1.5-2 hours in a CO 2 incubator.}

Dilutions of candidate 01-016 in RPMI-1640, 25 mM HEPES, 2 mM L-glutamine, 5% FBS from a concentration of 200 μg / ml were prepared with a titration step of 3.

At the end of the incubation time, the medium was carefully removed, and a solution of the M-CSF ligand 200 ng / ml (Peprotech), prepared in the medium of the same composition as the solutions of candidate 01-016, was added. The ligand was added at the rate of 50 μl / well. Prepared dilutions of candidate 01-016 were added to the same wells of the culture plate at the rate of 50 μl / well. The plates were mixed on an orbital shaker and incubated in a CO ₂ incubator for 6 days.

At the end of the incubation time, detection was performed using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, dose-response curves are shown in FIG. 8) and Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega, dose-response curves are shown in FIG. 9 ) according to the manufacturer's instructions. The luminescence level was measured using Infinite M200Pro (Tecan), the activity of anti-CSF-1R antibodies was assessed.

The data obtained indicate that candidate 01-016 exhibits pro-apoptotic activity.

Example 19. Evaluation of the effect of anti-CSF-1R antibodies on differentiation of monocytes along the M2 pathway

In the experiment, the level of expression of surface markers CD80 and CD163 on monocytes differentiated using the factors M-CSF and IL10 (M2-type differentiation) was assessed. Isolation of monocytes was performed similarly to the procedure described in Example 18.

Prepared a cell suspension of monocytes 1 × 10 ⁶ cells / well in RPMI-1640, 25 mM HEPES, 2 mM L-glutamine, 5% FBS. The cell suspension was dispensed into a 24-well plate (Corning, 3524) at a volume of 1 ml / well. The plates with the cell suspension were incubated for _{1.5-2 hours in a CO 2 incubator.}

After incubation, a solution was added from the well of a culture plate in 1 ml of M-CSF 200 ng / ml solution (Peprotech) and 1 μg / ml solutions of candidate 01-016 prepared in a medium of the same composition as the cell suspension. In the case of monitoring the differentiation of monocytes along the M1 pathway, instead of M-CSF, a GM-CSF solution of the same concentration was added. The plate was incubated in a CO ₂ incubator for 6 days.

After the incubation time, the supernatant was taken from the wells and a solution of IL-10, 10 ng / ml (Peprotech) was added. A solution of lipopolysaccharide (LPS) and interferon γ with a concentration of each of the components of 40 ng / ml was added to the wells with M1 type differentiation controls. Additionally, the plates were incubated for 2 days in a CO ₂ incubator.

At the end of incubation, cytometric analysis was performed to determine the level of expression of markers CD80 and CD163. The assessment of the expression level of CD163 in the presence of candidate 01-016 is shown in FIG. 10.

The data obtained indicate that candidate 01-016 causes a decrease in the expression of markers characteristic of M2-macrophages and the appearance of a pronounced CD163-negative population.

Example 20. Humanization of a leader candidate selected from an immune library

To humanize the leader candidate 01-016, the algorithms of the YLab software complex developed by BIOCAD were used. At the first stage, the similarity of the V-segment of the variable domain of the heavy chain with the germline sequences of human and the original camelid family - llama glama, vicugna pacos, and camelus dromedarius was assessed (sequences are taken from the IMGT gene database, imgt.org). The variable domain of the light chain was not evaluated because a binder library containing fully human light chains was involved in the selection. The happiest VH segment from vicugna pacos was IGHV3-1 * 01, the similarity was 91.8%. The closest VH segment from human was IGHV3-23 * 01, the similarity was 87.6%.

To humanize the variable domain of the heavy chain, positions containing the least characteristic amino acid residues for human antibodies were selected. In the Kabat numbering scheme, their values are: 1, 5, 6, 11, 26, 74, 77, 83, 84 and 89. From the beginning of the amino acid sequence, they are numbered 1, 5, 6, 11, 26, 75, 78, 87, 88 and 93.

All of the above positions do not affect the CDRs of the leader candidate (HCDR1 with the amino acid sequence DYAMS (SEQ ID NO: 1), HCDR2 with the amino acid sequence AISWNGGSTNYADSVKG (SEQ ID NO: 2), HCDR3 with the amino acid sequence TVEVAHRRLYKYLEV (SEQ ID NO: 3)). Based on the most frequently occurring amino acid residues at these positions in the conditions of their surrounding sequence, possible mutations have been proposed that increase both the similarity with the VH segments of human germlines and matted human antibodies. The assessment of such positions was made on the basis of data from the NGS library of healthy donors obtained from BIOCAD in 2014 (MegaNLib, sequencing was performed using Illumina MiSeq). All variants had at least 90.7% similarity with the VH segments of human germline sequences. Also, in all cases, the similarity with human sequences was higher than with other biological species.

Example 21. Obtaining humanized antibodies

Humanized HCVR and LCVR nucleotide sequences were synthesized de novo and cloned into pSXn-HCLALA-PR, pSXn-CK-BR vectors, IgG1 format at SalI / NheI and SalI / BsiWI restriction sites, respectively.

The degrees of humanization of the variable domains of the heavy and light chains of antibodies are shown in Table 7.

Table 7 - Comparison of the degree of humanization of variable domains

Candidate Humanization degree HCVR,% Humanization degree LCVR,% 01-016 83% 93% OPTIM-6_001-02 89% 93% OPTIM-6_001-04 91% 93% OPTIM-6_001-05 86% 93%

Vectors containing nucleic acids encoding the light and heavy chains of the antibody were generated in E. coli cells and purified using the Qiagen plasmid DNA purification reagent kit.

Example 22. Creation of a stable cell line, production and purification of anti-CSF-1R humanized antibodies

Stable pools of producers of monoclonal antibodies 01-016, OPTIM-6_001-02, OPTIM-6_001-04, OPTIM-6_001-05 were obtained by transfection by electroporation using a 4D Nucleofector device (Lonza) according to the manufacturer's protocol. Suspension cell line CHO-S was used as a parental line. Transfection of the parental line was carried out with genetic constructs that contain genes encoding antibody light and heavy chains and antibiotic resistance.

The transfected parental line was cultivated in S.3.87 MM medium (synthetic medium developed by ZAO BIOCAD without FBS content) supplemented with 6 mM L-Glutamine. 48 hours after transfection and before the cells were placed in the bank, the antibiotics Puromycin dihydrochloride (Sigma) at a concentration of 9.6 μg / ml and Blasticidin S HCl (Gibco) at a concentration of 5.12 μg / ml were added to the medium.

The stable pools obtained as a result of selection are stored in a bank for further cloning in order to obtain a monoclonal cell line.

After thawing, stable pools were cultivated in S.3.87 MM medium (synthetic medium developed at ZAO BIOCAD without FBS) supplemented with 6 mM L-Glutamine and 12% Boost 4. On the 11th day of cultivation, the cell suspension was separated from the cells by filtration through a submerged filter with pore size 0.5 / 0.22 μm.

Antibodies with Fc 01-016, OPTIM-6_001-02, OPTIM-6_001-04, OPTIM-6_001-05 were isolated and purified by affinity chromatography on the bacterial protein Protein A, viral inactivation, and subsequent purification on SP Sepharose HP and Capto Adhere sorbents. Then 01-016, OPTIM-6_001-02, OPTIM-6_001-04 were transferred into 20 mM histidine buffer with the addition of 100 mM trehalose (pH 5.5), OPTIM-6_001-05 in 20 mM acetate buffer (pH 5.0) with the addition of 100 mM trehalose by dialysis, filtered (0.22 μm), transferred to tubes and stored at -70 ° C.

Electrophoresis was performed under denaturing non-reducing conditions in a 7.5% polyacrylamide gel (FIGS. 11-14) and under denaturing reducing conditions in a 12% polyacrylamide gel (FIGS. 15-18). Determination of protein purity was carried out by the intensity of band staining at a protein load of 40 μg per lane. Size exclusion HPLC was performed on a Tosoh TSK-Gel G3000SWXL column in the mobile phase 0.05M NaH ₂ PO ₄ , 0.3 M NaCl pH = 7.0.

Example 23. Determination of the affinity of full-length antibodies for Forte Bio Octert RED 384

Experiments to study the binding affinity of antibodies to the antigen with human Avi-hCSF-1R were performed on a Forte Bio Octert RED 384 device. Antibodies at a concentration of 30 μg / ml are immobilized on the surface of ProteinA biosensors (ForteBio). The analysis was carried out at 30 ° C using PBS containing 0.1% Tween-20 and 0.1% BSA as a working buffer. After the baseline was prescribed in the buffer solution, the sensors with immobilized antibodies were immersed in wells with solutions with Avi-hCSF-1R at different concentrations, where the complex was associated for 300 seconds. Then the dissociation of the complex in the buffer solution is detected for 600 seconds. The antigen concentration ranged from 5 μg / ml to 78 ng / ml.

The reference subtracted binding curves are analyzed using Octet Data Analysis (version 9.0) according to a standard procedure using a 1: 1 Global interaction model.

Anti-CSF-1R antibodies specifically bind to human antigen.

Table 8 - Kinetic constants of humanized candidates when interacting with human avi-hCSF-1R antigen

Candidate KD, M kon, 1 / Ms Kdis, 1 / s R ² 01-016 5.13E-10 3.73E + 05 1.92E-04 0.9989 OPTIM-6_001-02 5.67E-10 3.89E + 05 2.21E-04 0.9992 OPTIM-6_001-04 7.76E-10 3.85E + 05 2.99E-04 0.9991 OPTIM-6_001-05 4.85E-10 3.88E + 05 1.88E-04 0.9994

Example 24. Analysis of interactions with rhesus monkey receptors on the Forte Bio Octet RED 384 device

Experiments to study the binding affinity of antibodies to the antigen c 00_RhAG_000_04_6His (Macaca mulatta CSF-1R) were carried out on a Forte Bio Octert RED 384 device. Antibodies at a concentration of 30 μg / ml are immobilized on the surface of ProteinA biosensors (ForteBio). The analysis was carried out at 30 ° C using PBS containing 0.1% Tween-20 and 0.1% BSA as a working buffer. After the baseline was prescribed in the buffer solution, the sensors with immobilized antibodies were immersed in wells with a solution of c 00_RhAG_000_04_6His (Macaca mulatta CSF-1R) at different concentrations, where the complex was associated for 300 seconds. Then the dissociation of the complex in the buffer solution is detected for 600 seconds.

The antigen concentration ranged from 5 μg / ml to 78 ng / ml.

The reference subtracted binding curves are analyzed using Octet Data Analysis (version 9.0) according to a standard procedure using a 1: 1 Global interaction model.

Anti-CSF-1R antibodies specifically bind to macaque antigen.

Table 9 - Kinetic constants of humanized candidates when interacting with 00_RhAG_000_04_6His antigen (Macaca mulatta CSF-1R)

Candidate KD, M kon, 1 / Ms Kdis, 1 / s R ² 01-016 2.66E-09 4.11E + 05 1.09E-03 0.9978 OPTIM-6_001-02 3.09E-09 4.62E + 05 1.43E-03 0.9980 OPTIM-6_001-04 4.00E-09 4.73E + 05 1.89E-03 0.9969 OPTIM-6_001-05 2,75E-09 4.55E + 05 1,25E-03 0.9982

The affinity of these candidates for mouse CSF-1R antigen was measured in a similar manner as in Example 14. It was confirmed that humanized candidates did not interact with mouse CSF-1R antigens.

Example 25. Determination of colloidal and thermal stability by the point of protein aggregation by dynamic light scattering

To determine the aggregation temperature of the studied samples by the method of dynamic light scattering (DLS), the dependence of the particle size in the medium on temperature was obtained using a DynaPro® Plate Reader II (Wyatt Technology Corp.) device with gradual heating from 25 to 80 ° C. The results are shown in Table 10.

Table 10 - Aggregation point 01-016, OPTIM-6_001-02, OPTIM-6_001-04, OPTIM-6_001-05

Candidate Buffer solution Aggregation point 01-016 His 20mM
pH = 5.5
Tre 100 mg / ml 62.6 ± 0.5 ° C OPTIM-6_001-02 His 20mM
pH = 5.5
Tre 100 mg / ml 65.9 ± 0.5 ° C OPTIM-6_001-04 His 20mM
pH = 5.5
Tre 100 mg / ml 62.8 ± 0.5 ° C OPTIM-6_001-05 Acetate 20mM
pH = 5.0
Tre 100 mg / ml 63.8 ± 0.5 ° C

It can be concluded that the molecules 01-016, OPTIM-6_001-02, OPTIM-6_001-04, OPTIM-6_001-05 have high thermocolloidal stability (aggregation point in 20mM Acetate, pH = 5.0 and 20mM His, pH = 5, 5 buffer solutions is more than 60.0 ° C).

Example 26. Determination of thermal stability at a thermostress of 50 ° C

The test samples were installed in an air thermostat and thermostated at 50 ° C for 72 hours. After the end of heating, intact and stressed samples were transferred for analysis by SEC HPLC with a UV detector. Chromatography was performed on an Agilent 1100 HPLC system on a Tosoh TSK-Gel G3000SWXL column; detection was performed at 220 nm and 280 nm.

The resulting stability data for 01-016, OPTIM-6_001-02, OPTIM-6_001-04, OPTIM-6_001-05 when incubated at 50 ° C are presented in Table 11.

General conclusion: the samples have high colloidal and thermal stability.

Table 11 - Dependence of monomer content by exclusive HPLC for candidates

Candidate Buffer solution Conditions for the test sample E HPLC Content of aggregates,% Monomer content,% Fragment content,% 01-016 His 20mM
pH = 5.5
Tre 100 mg / ml Intact 0.730 97,738 1,375 72 h at 50 ° C 0.731 97,240 1,884 Δ * 0.001 -0.498 0.509 OPTIM-6_001-02 His 20mM
pH = 5.5
Tre 100 mg / ml Intact 1.278 97,593 1.129 72 h at 50 ° C 1,160 97,349 1,590 Δ * -0.118 -0.344 0.461 OPTIM-6_001-04 His 20mM
pH = 5.5
Tre 100 mg / ml Intact 0.572 97,712 1,716 72 h at 50 ° C 0.682 97,135 2.183 Δ * 0.110 -0.577 0.467 OPTIM-6_001-05 Acetate 20mM
pH = 5.0
Tre 100 mg / ml Intact 0.871 97,743 1,385 72 h at 50 ° C 0.738 97,538 1,723 Δ * -0.133 -0.205 0.338

* Δ - difference between the purity of the stressed sample and the purity of the intact sample (input control),%.

Example 27. Proliferative test on the cell line TF1-CSF-1R against the background of the ligand M-CSF

Received after humanization of candidate 01-016 candidate antibodies (OPTIM-6_001-02, OPTIM-6_001-04, OPTIM-6_001-05), accumulated in a stable pool, were also analyzed in the test described in example 16, in comparison with 01-016 before humanization. The results of the assessment are shown in Table 12. The obtained data indicate that humanization did not significantly affect the ability of anti-CSF-1R antibodies to inhibit CSF-dependent proliferation.

Table 12 - Evaluation of the effect of humanization of candidate 01-016 on the ability to inhibit CSF-dependent proliferation

Candidate Activity relative to 01-016 OPTIM6_001-02 114% OPTIM6_001-04 103% OPTIM6_001-05 126%

Example 28. Proliferative test on the cell line TF1-CSF-1R against the background of the ligand IL34

Obtained after humanization of candidate 01-016 candidate antibodies (OPTIM-6_001-02, OPTIM-6_001-04, OPTIM-6_001-05), accumulated in a stable pool, were also analyzed in the test described in example 17, in comparison with 01-016 before humanization. The results of the assessment are shown in Table 13. The obtained data indicate that humanization did not significantly affect the ability of anti-CSF-1R antibodies to inhibit IL-34-dependent proliferation activity.

Table 13 - Evaluation of the effect of humanization of candidate 01-016 on the ability to inhibit IL-34-dependent proliferation

Candidate Activity relative to 01-016 OPTIM6_001-02 102% OPTIM6_001-04 112% OPTIM6_001-05 135%

Example 29 Construction of a library of mutant antibodies specific for CSF-1R (human)

To create mutant antibodies specific to CSF-1R (human), structural analysis was performed based on 3D modeling using the Schrödinger Suites 2020-2 software package from Schrödinger and the CSF-1R model (PDB 4WRM using 4DKD, 4LIQ, 4WRL). Based on the 3D structure of the modeled complex, all possible point substitutions of amino acids in the contact zone with CSF-1R were carried out, with the exception of cysteine, proline, and glycine as initial amino acids and mutations. The obtained mutants with the preservation or increase of the antibody affinity and the stability of the complex were ranked and, based on the best substitutions, mutants were formed, each of which contained no more than 3 substitutions in each CDR. At the next stage, a library was formed from the obtained set of structures, including variants in which the substitutions led to an increase in the stability of the complex and to an increase in the affinity of the antibody.

The library of variants of HCDR regions (Kabat markup), obtained according to the protocol described above, includes 1, 19 and 14 variants for HCDR1 (SEQ ID NO: 17), HCDR2 (SEQ ID NO: 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) and HCDR3 (SEQ ID NO: 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50), respectively.

The library of CDR regions of antibodies, obtained according to the protocol described above, includes 34 sequences. Substitutions are presented in 10 positions. The number of unique replacements is 26. Table 13 shows the CDR sequences of the heavy chain loops of the original candidate, as well as the set of amino acid substitutions presented in each of the 34 mutant sequences.

Table 14 - Sequences of variants of HCDR regions

HCDR SEQ ID Subsequence HCDR1 original SEQ ID NO: 1 DYAMS HCDR1 mutant SEQ ID NO: 17 DYAMT HCDR2 original SEQ ID NO: 2 AISWNGGSTNYADSVKG HCDR2 mutant SEQ ID NO: 18 AISWRGGSTNYADSVKG SEQ ID NO: 19 AISWIGGSTNYADSVKG SEQ ID NO: 20 AISWLGGSTNYADSVKG SEQ ID NO: 21 AISWFGGSTNYADSVKG SEQ ID NO: 22 AISWNGGETNYADSVKG SEQ ID NO: 23 AISWNGGITNYADSVKG SEQ ID NO: 24 AISWNGGLTNYADSVKG SEQ ID NO: 25 AISWNGGSTRYADSVKG SEQ ID NO: 26 AISWNGGSTWYADSVKG SEQ ID NO: 27 AISWNGGSTNYQDSVKG SEQ ID NO: 28 AISWNGGSTNYHDSVKG SEQ ID NO: 29 QISWNGGSTRYQDSVKG SEQ ID NO: 30 QISWLGGSTNYQDSVKG SEQ ID NO: 31 HISWNGGSTRYQDSVKG SEQ ID NO: 32 QISWNGGSTRYHDSVKG SEQ ID NO: 33 HISWNGGSTRYHDSVKG SEQ ID NO: 34 HISWIGGSTNYQDSVKG SEQ ID NO: 35 QISWIGGSTNYQDSVKG SEQ ID NO: 36 HISWLGGSTNYQDSVKG HCDR3 original SEQ ID NO: 3 TVEVAHRRLYKYLEV HCDR3 mutant SEQ ID NO: 37 IVEVAHRRLYKYLEV SEQ ID NO: 38 YVEVAHRRLYKYLEV SEQ ID NO: 39 TVEVAHRRLYRYLEV SEQ ID NO: 40 TVEVAHRRLYNYLEV SEQ ID NO: 41 TVEVAHRRLYQYLEV SEQ ID NO: 42 TVEVAHRRLYLYLEV SEQ ID NO: 43 TVEVAHRRLYFYLEV SEQ ID NO: 44 TVEVAHRRLYYYLEV SEQ ID NO: 45 TVEVAHRRLYKYLEI SEQ ID NO: 46 TVELAHRRLYKYLEI SEQ ID NO: 47 TVEFAHRRLYKYLEI SEQ ID NO: 48 TVEQAHRRLYKYLEI SEQ ID NO: 49 TVEFSHRRLYKYLEV SEQ ID NO: 50 TVEFAHRRLYKYLEV

--->

LIST OF SEQUENCES

<110> CJSC "BIOCAD"

<120> Monoclonal antibody that specifically binds to CSF-1R

<160> 63

<170> BiSSAP 1.3.6

<210> 1

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR1_01

<400> 1

Asp Tyr Ala Met Ser

fifteen

<210> 2

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_01

<400> 2

Ala Ile Ser Trp Asn Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 3

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_01

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Thr Val Glu Val Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu Val

1 5 10 15

<210> 4

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> 01-016_VH

<400> 4

Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Trp Asn Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Thr Val Glu Val Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu

100 105 110

Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 5

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> OPTIM-6_001-02_VH

<400> 5

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Trp Asn Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Thr Val Glu Val Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu

100 105 110

Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 6

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> OPTIM-6_001-04_VH

<400> 6

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Trp Asn Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ser Thr Val Glu Val Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu

100 105 110

Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 7

<211> 124

<212> PRT

<213> Artificial sequence

<220>

<223> OPTIM-6_001-05_VH

<400> 7

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Trp Asn Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Thr Val Glu Val Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu

100 105 110

Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 454

<212> PRT

<213> Artificial sequence

<220>

<223> 01-016_HC

<400> 8

Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Trp Asn Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Thr Val Glu Val Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu

100 105 110

Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 9

<211> 454

<212> PRT

<213> Artificial sequence

<220>

<223> OPTIM-6_001-02_HC

<400> 9

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Trp Asn Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Thr Val Glu Val Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu

100 105 110

Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 10

<211> 454

<212> PRT

<213> Artificial sequence

<220>

<223> OPTIM-6_001-04_HC

<400> 10

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Trp Asn Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Ser Thr Val Glu Val Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu

100 105 110

Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 11

<211> 454

<212> PRT

<213> Artificial sequence

<220>

<223> OPTIM-6_001-05_HC

<400> 11

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Phe Thr Phe Asp Asp Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Trp Asn Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Thr Val Glu Val Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu

100 105 110

Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

115 120 125

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

130 135 140

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

145 150 155 160

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

165 170 175

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

180 185 190

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

195 200 205

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

210 215 220

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

225 230 235 240

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

245 250 255

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

260 265 270

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

275 280 285

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

290 295 300

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

305 310 315 320

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

325 330 335

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

340 345 350

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

355 360 365

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

370 375 380

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

385 390 395 400

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

405 410 415

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

420 425 430

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

435 440 445

Ser Leu Ser Pro Gly Lys

450

<210> 12

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_LCDR1

<400> 12

Gly Gly Asn Asn Ile Gly Ser Lys Ser Val His

1 5 10

<210> 13

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_LCDR2

<400> 13

Gly Asp Ser Asp Arg Pro Ser

fifteen

<210> 14

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_LCDR3

<400> 14

Gln Val Trp Asp Ile Ser Ser Asp His Val Val

1 5 10

<210> 15

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_VL

<400> 15

Gln Ala Gly Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Gly Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ile Ser Ser Asp His

85 90 95

Val Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu

100 105

<210> 16

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_LC

<400> 16

Gln Ala Gly Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr

35 40 45

Gly Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ile Ser Ser Asp His

85 90 95

Val Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 17

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR1_02

<400> 17

Asp Tyr Ala Met Thr

fifteen

<210> 18

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_02

<400> 18

Ala Ile Ser Trp Arg Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 19

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_03

<400> 19

Ala Ile Ser Trp Ile Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 20

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_04

<400> 20

Ala Ile Ser Trp Leu Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 21

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_05

<400> 21

Ala Ile Ser Trp Phe Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 22

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_06

<400> 22

Ala Ile Ser Trp Asn Gly Gly Glu Thr Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 23

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_07

<400> 23

Ala Ile Ser Trp Asn Gly Gly Ile Thr Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 24

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_08

<400> 24

Ala Ile Ser Trp Asn Gly Gly Leu Thr Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 25

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_09

<400> 25

Ala Ile Ser Trp Asn Gly Gly Ser Thr Arg Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 26

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_10

<400> 26

Ala Ile Ser Trp Asn Gly Gly Ser Thr Trp Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 27

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_11

<400> 27

Ala Ile Ser Trp Asn Gly Gly Ser Thr Asn Tyr Gln Asp Ser Val Lys

1 5 10 15

Gly

<210> 28

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_12

<400> 28

Ala Ile Ser Trp Asn Gly Gly Ser Thr Asn Tyr His Asp Ser Val Lys

1 5 10 15

Gly

<210> 29

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_13

<400> 29

Gln Ile Ser Trp Asn Gly Gly Ser Thr Arg Tyr Gln Asp Ser Val Lys

1 5 10 15

Gly

<210> 30

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_14

<400> 30

Gln Ile Ser Trp Leu Gly Gly Ser Thr Asn Tyr Gln Asp Ser Val Lys

1 5 10 15

Gly

<210> 31

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_15

<400> 31

His Ile Ser Trp Asn Gly Gly Ser Thr Arg Tyr Gln Asp Ser Val Lys

1 5 10 15

Gly

<210> 32

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_16

<400> 32

Gln Ile Ser Trp Asn Gly Gly Ser Thr Arg Tyr His Asp Ser Val Lys

1 5 10 15

Gly

<210> 33

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_17

<400> 33

His Ile Ser Trp Asn Gly Gly Ser Thr Arg Tyr His Asp Ser Val Lys

1 5 10 15

Gly

<210> 34

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_18

<400> 34

His Ile Ser Trp Ile Gly Gly Ser Thr Asn Tyr Gln Asp Ser Val Lys

1 5 10 15

Gly

<210> 35

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_19

<400> 35

Gln Ile Ser Trp Ile Gly Gly Ser Thr Asn Tyr Gln Asp Ser Val Lys

1 5 10 15

Gly

<210> 36

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_20

<400> 36

His Ile Ser Trp Leu Gly Gly Ser Thr Asn Tyr Gln Asp Ser Val Lys

1 5 10 15

Gly

<210> 37

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_02

<400> 37

Ile Val Glu Val Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu Val

1 5 10 15

<210> 38

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_03

<400> 38

Tyr Val Glu Val Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu Val

1 5 10 15

<210> 39

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_04

<400> 39

Thr Val Glu Val Ala His Arg Arg Leu Tyr Arg Tyr Leu Glu Val

1 5 10 15

<210> 40

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_05

<400> 40

Thr Val Glu Val Ala His Arg Arg Leu Tyr Asn Tyr Leu Glu Val

1 5 10 15

<210> 41

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_06

<400> 41

Thr Val Glu Val Ala His Arg Arg Leu Tyr Gln Tyr Leu Glu Val

1 5 10 15

<210> 42

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_07

<400> 42

Thr Val Glu Val Ala His Arg Arg Leu Tyr Leu Tyr Leu Glu Val

1 5 10 15

<210> 43

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_08

<400> 43

Thr Val Glu Val Ala His Arg Arg Leu Tyr Phe Tyr Leu Glu Val

1 5 10 15

<210> 44

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_09

<400> 44

Thr Val Glu Val Ala His Arg Arg Leu Tyr Tyr Tyr Leu Glu Val

1 5 10 15

<210> 45

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_10

<400> 45

Thr Val Glu Val Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu Ile

1 5 10 15

<210> 46

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_11

<400> 46

Thr Val Glu Leu Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu Ile

1 5 10 15

<210> 47

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_12

<400> 47

Thr Val Glu Phe Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu Ile

1 5 10 15

<210> 48

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_13

<400> 48

Thr Val Glu Gln Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu Ile

1 5 10 15

<210> 49

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_14

<400> 49

Thr Val Glu Phe Ser His Arg Arg Leu Tyr Lys Tyr Leu Glu Val

1 5 10 15

<210> 50

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_15

<400> 50

Thr Val Glu Phe Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu Val

1 5 10 15

<210> 51

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR1_common

<220>

<221> Xaa

<222> (5) .. (5)

<223> Amino acid at position 5: Xaa = Thr or Ser

<400> 51

Asp Tyr Ala Met Xaa

fifteen

<210> 52

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_common

<220>

<221> Xaa

<222> (1) .. (1)

<223> Amino acid at position 1: Xaa = Ala or Gln or His

<220>

<221> Xaa

<222> (5) .. (5)

<223> Amino acid at position 5: Xaa = Asn or Arg or Ile or Leu or Phe

<220>

<221> Xaa

<222> (8) .. (8)

<223> Amino acid at position 8: Xaa = Ser or Gly or Ile or Leu

<220>

<221> Xaa

<222> (10) .. (10)

<223> Amino acid at position 10: Xaa = Asn or Arg or Trp

<220>

<221> Xaa

<222> (12) .. (12)

<223> Amino acid at position 12: Xaa = Ala or Gln or His

<400> 52

Xaa Ile Ser Trp Xaa Gly Gly Xaa Thr Xaa Tyr Xaa Asp Ser Val Lys

1 5 10 15

Gly

<210> 53

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_common

<220>

<221> Xaa

<222> (1) .. (1)

<223> Amino acid at position 1: Xaa = Thr or Ile or Tyr

<220>

<221> Xaa

<222> (4) .. (4)

<223> Amino acid at position 4: Xaa = Val or Phe

<220>

<221> Xaa

<222> (5) .. (5)

<223> Amino acid at position 5: Xaa = Ala or Ser

<220>

<221> Xaa

<222> (11) .. (11)

<223> Amino acid at position 11: Xaa = Lys or Arg or Asn or Gln or Leu or Phe or Tyr

<220>

<221> Xaa

<222> (15) .. (15)

<223> Amino acid at position 15: Xaa = Val or Ile

<400> 53

Xaa Val Glu Xaa Xaa His Arg Arg Leu Tyr Xaa Tyr Leu Glu Xaa

1 5 10 15

<210> 54

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_common

<220>

<221> Xaa

<222> (5) .. (5)

<223> Amino acid at position 5: Xaa = Arg or Ile or Leu or Phe

<400> 54

Ala Ile Ser Trp Xaa Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 55

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_common

<220>

<221> Xaa

<222> (8) .. (8)

<223> Amino acid at position 8: Xaa = Glu or Ile or Leu

<400> 55

Ala Ile Ser Trp Asn Gly Gly Xaa Thr Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 56

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_common

<220>

<221> Xaa

<222> (10) .. (10)

<223> Amino acid at position 10: Xaa = Arg or Trp

<400> 56

Ala Ile Ser Trp Asn Gly Gly Ser Thr Xaa Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 57

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_common

<220>

<221> Xaa

<222> (12) .. (12)

<223> Amino acid at position 12: Xaa = Gln or His

<400> 57

Ala Ile Ser Trp Asn Gly Gly Ser Thr Asn Tyr Xaa Asp Ser Val Lys

1 5 10 15

Gly

<210> 58

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_common

<220>

<221> Xaa

<222> (1) .. (1)

<223> Amino acid at position 1: Xaa = Gln or His

<220>

<221> Xaa

<222> (10) .. (10)

<223> Amino acid at position 10: Xaa = Arg

<220>

<221> Xaa

<222> (12) .. (12)

<223> Amino acid at position 12: Xaa = Gln or His

<400> 58

Xaa Ile Ser Trp Asn Gly Gly Ser Thr Xaa Tyr Xaa Asp Ser Val Lys

1 5 10 15

Gly

<210> 59

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR2_common

<220>

<221> Xaa

<222> (1) .. (1)

<223> Amino acid at position 1: Xaa = Gln or His

<220>

<221> Xaa

<222> (5) .. (5)

<223> Amino acid at position 5: Xaa = Leu or Ile

<220>

<221> Xaa

<222> (12) .. (12)

<223> Amino acid at position 12: Xaa = Gln

<400> 59

Xaa Ile Ser Trp Xaa Gly Gly Ser Thr Asn Tyr Xaa Asp Ser Val Lys

1 5 10 15

Gly

<210> 60

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_common

<220>

<221> Xaa

<222> (1) .. (1)

<223> Amino acid at position 1: Xaa = Ile or Tyr

<400> 60

Xaa Val Glu Val Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu Val

1 5 10 15

<210> 61

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_common

<220>

<221> Xaa

<222> (4) .. (4)

<223> Amino acid at position 4: Xaa = Arg or Asn or Gln or Leu or Phe or Tyr

<400> 61

Thr Val Glu Xaa Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu Val

1 5 10 15

<210> 62

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_common

<220>

<221> Xaa

<222> (4) .. (4)

<223> Amino acid at position 4: Xaa = Gln or Leu or Phe

<220>

<221> Xaa

<222> (15) .. (15)

<223> Amino acid at position 15: Xaa = Ile

<400> 62

Thr Val Glu Xaa Ala His Arg Arg Leu Tyr Lys Tyr Leu Glu Xaa

1 5 10 15

<210> 63

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> anti-CSF-1R_HCDR3_common

<220>

<221> Xaa

<222> (4) .. (4)

<223> Amino acid at position 4: Xaa = Phe

<220>

<221> Xaa

<222> (5) .. (5)

<223> Amino acid at position 5: Xaa = Ser

<400> 63

Thr Val Glu Xaa Xaa His Arg Arg Leu Tyr Lys Tyr Leu Glu Val

1 5 10 15

<---

Claims (82)

1. An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to CSF-1R, comprising: (a) a heavy chain variable domain comprising: (i) CDR1 with an amino acid sequence selected from the group: SEQ ID NO: 1, SEQ ID NO: 17, (ii) CDR2 with an amino acid sequence selected from the group: SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO : 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, (iii) CDR3 with an amino acid sequence selected from the group: SEQ ID NO: 3, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO : fifty; and (b) a light chain variable domain comprising: (i) CDR1 with the amino acid sequence of SEQ ID NO: 12, (ii) CDR2 with the amino acid sequence of SEQ ID NO: 13, (iii) CDR3 with the amino acid sequence of SEQ ID NO: 14. 2. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable domain comprises: (i) CDR1 with the amino acid sequence of SEQ ID NO: 1, (ii) a CDR2 with the amino acid sequence of SEQ ID NO: 2, (iii) CDR3 with the amino acid sequence of SEQ ID NO: 3. 3. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the variable domain of the heavy chain contains an amino acid sequence selected from the group: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7. 4. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 5. 5. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 6. 6. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 15. 7. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1, where: (a) the variable domain of the heavy chain contains an amino acid sequence selected from the group: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7; (b) the variable domain of the light chain contains the amino acid sequence of SEQ ID NO: 15. 8. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 7, where: (a) the variable domain of the heavy chain contains the amino acid sequence of SEQ ID NO: 5; (b) the variable domain of the light chain contains the amino acid sequence of SEQ ID NO: 15. 9. An isolated monoclonal antibody or antigen-binding fragment thereof according to claim 7, where: (a) the variable domain of the heavy chain contains the amino acid sequence of SEQ ID NO: 6; (b) the variable domain of the light chain contains the amino acid sequence of SEQ ID NO: 15. 10. An isolated monoclonal antibody according to claim 1, comprising a heavy chain containing an amino acid sequence selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11. 11. The isolated monoclonal antibody of claim 10, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 9. 12. The isolated monoclonal antibody of claim 10, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 10. 13. An isolated monoclonal antibody according to claim 1, comprising a light chain comprising the amino acid sequence of SEQ ID NO: 16. 14. An isolated monoclonal antibody according to claim 1, comprising: (a) a heavy chain containing an amino acid sequence selected from the group: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11; and (b) a light chain containing the amino acid sequence of SEQ ID NO: 16. 15. An isolated monoclonal antibody according to claim 14, comprising: (a) a heavy chain containing the amino acid sequence of SEQ ID NO: 9; and (b) a light chain containing the amino acid sequence of SEQ ID NO: 16. 16. An isolated monoclonal antibody according to claim 14, comprising: (a) a heavy chain containing the amino acid sequence of SEQ ID NO: 10; and (b) a light chain containing the amino acid sequence of SEQ ID NO: 16. 17. Isolated monoclonal antibody according to any one of paragraphs. 1-9, where the antibody that specifically binds to CSF-1R is a full length IgG antibody. 18. The isolated monoclonal antibody of claim 17, wherein the full length IgG antibody is of the human IgG1, IgG2, IgG3, or IgG4 isotype. 19. The isolated monoclonal antibody of claim 18, wherein the full length IgG antibody is of the human IgG1 isotype. 20. Isolated nucleic acid that encodes an antibody or antigen-binding fragment according to any one of claims. 1-19. 21. The isolated nucleic acid of claim 20, wherein the nucleic acid is DNA. 22. Expression vector containing a nucleic acid according to any one of paragraphs. 20-21. 23. A method of obtaining a host cell for obtaining an antibody or its antigennegative fragment according to any one of paragraphs. 1-19, comprising transforming the cell with the vector of claim 22. 24. A host cell for producing an antibody or antigen-binding fragment thereof according to any one of claims. 1-19, containing a nucleic acid according to any one of paragraphs. 20, 21. 25. A method of obtaining an antibody or antigen-binding fragment according to any one of paragraphs. 1-19, which consists in culturing a host cell according to claim 24 in a culture medium under conditions sufficient to obtain said antibody, if necessary, followed by isolation and purification of the resulting antibody. 26. A pharmaceutical composition for the treatment of a disease or disorder mediated by CSF-1R, containing an antibody or antigen-binding fragment thereof according to any one of claims. 1-19 in a therapeutically effective amount in combination with one or more pharmaceutically acceptable excipients. 27. The pharmaceutical composition of claim 26, wherein the disease or disorder mediated by CSF-1R is selected from the group: metastatic melanoma, metastatic bladder cancer, metastatic non-small cell lung cancer, metastatic renal cell carcinoma (light cell type), malignant neoplasms with microsatellite instability with ineffectiveness of the main anticancer therapy, locally advanced or metastatic squamous cell carcinoma of the head and neck, stomach cancer (cardio-esophageal zone), ovarian cancer, breast cancer, triple negative breast cancer, bone tumor, Hodgkin's lymphoma, glioma , glioblastoma, mesothelioma, myeloma, prostate cancer, soft tissue sarcoma. 28. A pharmaceutical composition for the treatment of a disease or disorder mediated by CSF-1R, containing an antibody or antigen-binding fragment thereof according to any one of claims. 1-19 and at least one other therapeutically active compound. 29. The pharmaceutical composition of claim 28, wherein the disease or disorder mediated by CSF-1R is selected from the group: metastatic melanoma, metastatic bladder cancer, metastatic non-small cell lung cancer, metastatic renal cell carcinoma (light cell type), malignant neoplasms with microsatellite instability with ineffectiveness of the main anticancer therapy, locally advanced or metastatic squamous cell carcinoma of the head and neck, stomach cancer (cardio-esophageal zone), ovarian cancer, breast cancer, triple negative breast cancer, bone tumor, Hodgkin's lymphoma, glioma , glioblastoma, mesothelioma, myeloma, prostate cancer, soft tissue sarcoma. 30. The pharmaceutical composition according to any one of paragraphs. 28, 29, wherein the other therapeutically active compound is an antibody, chemotherapeutic agent, or anti-hormonal agent. 31. The pharmaceutical composition according to any one of paragraphs. 19, 20, where the other therapeutically active compound is a checkpoint inhibitor. 32. The pharmaceutical composition of claim 31, wherein the checkpoint inhibitor is selected from a PD-1 inhibitor or a CTLA-4 inhibitor. 33. The pharmaceutical composition of claim 32, wherein the PD-1 inhibitor is an antibody that specifically binds to PD-1. 34. The pharmaceutical composition of claim 33, wherein the antibody that specifically binds to PD-1 is selected from the group: prolgolimab, pembrolizumab, nivolumab. 35. The pharmaceutical composition of claim 32, wherein the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4. 36. The pharmaceutical composition of claim 35, wherein the antibody that specifically binds to CTLA-4 is ipilimumab. 37. A method of inhibiting the biological activity of CSF-1R in a subject in need of such inhibition, comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof according to any one of claims. 1-19. 38. A method of treating a disease or disorder mediated by CSF-1R, comprising administering to a subject in need of such treatment, an antibody or antigen-binding fragment thereof according to any one of claims. 1-19 or a pharmaceutical composition according to any one of claims. 26-36 in a therapeutically effective amount. 39. A method for treating a disease or disorder according to claim 38, wherein the disease or disorder mediated by CSF-1R is selected from the group: metastatic melanoma, metastatic bladder cancer, metastatic non-small cell lung cancer, metastatic renal cell carcinoma (light cell type) , malignant neoplasms with microsatellite instability if the main antitumor therapy is ineffective, locally advanced or metastatic squamous cell carcinoma of the head and neck, stomach cancer (cardio-esophageal zone), ovarian cancer, breast cancer, triple negative breast cancer, bone tumor, lymphoma Hodgkin's, glioma, glioblastoma, mesothelioma, myeloma, prostate cancer, soft tissue sarcoma. 40. A method of treating a disease or disorder mediated by CSF-1R, comprising administering to a subject in need of such treatment, an antibody or antigen-binding fragment thereof according to any one of claims. 1-19 and at least one other therapeutically active compound. 41. A method for treating a disease or disorder according to claim 40, wherein the disease or disorder mediated by CSF-1R is selected from the group: metastatic melanoma, metastatic bladder cancer, metastatic non-small cell lung cancer, metastatic renal cell carcinoma (light cell type) , malignant neoplasms with microsatellite instability if the main antitumor therapy is ineffective, locally advanced or metastatic squamous cell carcinoma of the head and neck, stomach cancer (cardio-esophageal zone), ovarian cancer, breast cancer, triple negative breast cancer, bone tumor, lymphoma Hodgkin's, glioma, glioblastoma, mesothelioma, myeloma, prostate cancer, soft tissue sarcoma. 42. A method of treating a disease or disorder according to any one of claims. 40, 41, where the other therapeutically active compound is an antibody, chemotherapeutic agent, or anti-hormonal agent. 43. A method of treating a disease or disorder according to any one of claims. 40, 41, where the other therapeutically active compound is a checkpoint inhibitor. 44. A method for treating a disease or disorder according to claim 43, wherein the checkpoint inhibitor is selected from a PD-1 inhibitor or a CTLA-4 inhibitor. 45. A method for treating a disease or disorder according to claim 44, wherein the PD-1 inhibitor is an antibody that specifically binds to PD-1. 46. A method for treating a disease or disorder according to claim 45, wherein the antibody that specifically binds to PD-1 is selected from the group: prolgolimab, pembrolizumab, nivolumab. 47. A method for treating a disease or disorder according to claim 44, wherein the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4. 48. A method for treating a disease or disorder according to claim 47, wherein the antibody that specifically binds to CTLA-4 is ipilimumab. 49. The use of an antibody or antigen-binding fragment according to any one of claims. 1-19 or a pharmaceutical composition according to any one of claims. 26-36 for treating a CSF-1R mediated disease or disorder in a subject in need of such treatment. 50. Use according to claim 49, wherein the disease or disorder mediated by CSF-1R is selected from the group: metastatic melanoma, metastatic bladder cancer, metastatic non-small cell lung cancer, metastatic renal cell carcinoma (light cell type), malignant neoplasms with microsatellite instability with ineffectiveness of the main antitumor therapy, locally advanced or metastatic squamous cell carcinoma of the head and neck, stomach cancer (cardio-esophageal zone), ovarian cancer, breast cancer, triple negative breast cancer, bone tumor, Hodgkin's lymphoma, glioma, glioblastoma, mesothelioma, myeloma, prostate cancer, soft tissue sarcoma. 51. The use of an antibody or antigen-binding fragment according to any one of paragraphs. 1-19 and at least one other therapeutically active compound for the treatment of a disease or disorder mediated by CSF-1R. 52. Use according to claim 51, wherein the disease or disorder mediated by CSF-1R is selected from the group: metastatic melanoma, metastatic bladder cancer, metastatic non-small cell lung cancer, metastatic renal cell carcinoma (light cell type), malignant neoplasms with microsatellite instability with ineffectiveness of the main antitumor therapy, locally advanced or metastatic squamous cell carcinoma of the head and neck, stomach cancer (cardio-esophageal zone), ovarian cancer, breast cancer, triple negative breast cancer, bone tumor, Hodgkin's lymphoma, glioma, glioblastoma, mesothelioma, myeloma, prostate cancer, soft tissue sarcoma. 53. Application according to any one of paragraphs. 51, 52, wherein the other therapeutically active compound is an antibody, chemotherapeutic agent, or anti-hormonal agent. 54. Application according to any one of paragraphs. 51, 52, where the other therapeutically active compound is a checkpoint inhibitor. 55. The use of claim 54, wherein the checkpoint inhibitor is selected from a PD-1 inhibitor or a CTLA-4 inhibitor. 56. The use of claim 55, wherein the PD-1 inhibitor is an antibody that specifically binds to PD-1. 57. The use of claim 56, wherein the antibody that specifically binds to PD-1 is selected from the group of prolgolimab, pembrolizumab, nivolumab. 58. The use of claim 55, wherein the CTLA-4 inhibitor is an antibody that specifically binds to CTLA-4. 59. The use of claim 58, wherein the antibody that specifically binds to CTLA-4 is ipilimumab.

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