CN114426580B

CN114426580B - anti-CSF-1R antibodies, products, methods and uses thereof

Info

Publication number: CN114426580B
Application number: CN202210357137.7A
Authority: CN
Inventors: 王双; 王荣娟; 焦莎莎; 张畅; 张姣; 芦维宁; 张锦超
Original assignee: Beijing Konuoxincheng Technology Co ltd
Current assignee: Beijing Konuoxincheng Technology Co ltd
Priority date: 2022-04-07
Filing date: 2022-04-07
Publication date: 2022-06-24
Anticipated expiration: 2042-04-07
Also published as: CN114426580A

Abstract

The invention discloses an anti-human CSF-1R antibody, an antigen binding fragment, a separated polynucleotide, a vector, a host cell, a composition, a method for preparing the anti-human CSF-1R antibody or the antigen binding fragment thereof and application thereof.

Description

anti-CSF-1R antibodies, products, methods and uses thereof

Technical Field

The present invention relates to the field of biopharmaceuticals. In particular, the invention relates to novel artificially designed antibodies, in particular CSF-1R humanized antibodies or antigen binding fragments. The invention also relates to the therapeutic and diagnostic use of these antibodies that bind to CSF-1R, in particular in the treatment, prevention and/or diagnosis of CSF-1R related diseases, such as tumors.

Background

Currently, antibody therapy for cancer is gradually established and becomes one of the most successful and important strategies for treating hematologic malignancies and solid tumors. Such therapies may function by altering the function of an antigen or receptor (e.g., using agonists or antagonists), or by coupling specific drugs to antibodies that target specific antigens.

Macrophage anti-tumor is considered as another important direction besides T cell anti-tumor, and the CSF/CSF-1R signaling pathway plays an important role in the differentiation and development of monocytes/macrophages, induces TAM in the tumor microenvironment, and forms an inhibitory immune microenvironment. The research of the inhibitor of CSF-1R provides a new therapeutic direction for macrophage anti-tumor. At present, a plurality of small molecule inhibitors and 2 monoclonal antibody drugs are in clinical research stage, and the therapeutic effect of CSF-1/CSF-1R pathway inhibition on various tumors and GVHD is explored. Therefore, anti-CSF-1R antibody drug molecules are yet to be studied.

Disclosure of Invention

The invention aims to solve the problem of tumor treatment in the prior art, and particularly solves the technical problem of low curative effect of tumor targeting antibody molecules. To this end, it is an object of the present invention to prepare an antibody or antigen-binding fragment that specifically binds CSF-1R.

Colony-stimulating factor 1 receptor (CSF-1R), also known as CD115 or M-CSF-R, belongs to the type III protein tyrosine kinase receptor family, and is expressed on monocytes/macrophages, peritoneal effusion cells, plasma cell-like and conventional dendritic cells, osteoclasts. Natural ligands for the gene product include CSF1 and IL-34. CSF-1R directly affects differentiation and proliferation of tissue macrophages and osteoclasts. CSF-1R-mediated signaling is critical to the differentiation and survival of the mononuclear phagocyte system, particularly macrophages. The signal axis formed by CSF-1 and CSF-1R is closely related to the occurrence and development of tumors in various malignant tumors such as breast cancer, ovarian cancer, nasopharyngeal carcinoma and the like. The tumor cells act on CSF-1R on the surfaces of macrophages by secreting CSF-1 to activate downstream signal channels thereof, so that the CSF-1 acts on the CSF-1R to convert the CSF-1 into tumor-associated macrophages (TAM) for promoting tumor growth, the TAM can support the growth of the tumor cells by limiting T cell functions, and the CSF-1/CSF-1R is blocked to inhibit TAMs from receiving CSF-1 signals, thereby reducing the proliferation, differentiation and survival of the TAMs and relieving the effect of the TAMs in tumors. CSF1 cytokine secreted by tumor cells promotes the continuous existence of M2 phenotype, blocks CSF-1R signal channel to promote the repolarization of macrophage to M1 phenotype, and enhances the antitumor activity. Thus, according to one aspect of an embodiment of the present invention there is provided an anti-human CSF-1R antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the antigenic determinant regions VH CDR1, VH CDR2 and VH CDR3, the VH CDR1 comprising the amino acid sequence as set forth in SEQ ID NO: 12, and the VH CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 13, and the VH CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 14; the VL comprises antigenic determinants VL CDR1, VL CDR2, and VL CDR3, the VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, and the VL CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 10, and the VL CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 11, or a pharmaceutically acceptable salt thereof.

The present example discloses 1 strain of a humanized anti-CSF-1R antibody from a hybridoma. The antibody can be combined with human CSF-1R with high affinity, block the combination of CSF-1R and CSF-1 ligand, mediate CSF-1R to be internalized, and effectively block CSF-1/CSF-1R signaling pathway. According to the CSF-1R antibody or antigen-binding fragment of the embodiments, the obtained antibody can bind to human CSF-1R, and the activity of binding to human CSF-1R and the activity of inhibiting CSF-1 from binding to human CSF-1R are superior to those of a control antibody. The activity of the humanized antibody for inhibiting IL34 from being combined with human CSF-1R is obviously better than that of a control antibody. The antibody of the embodiment of the present invention has strong affinity and exhibits high specificity to human CSF-1R, and can provide an effective solution for the treatment of CSF-1R-related diseases by binding, internalizing and blocking CSF-1R.

In addition, the antibody or antigen-binding fragment according to the above embodiment of the present invention may have the following additional technical features:

according to an embodiment of the invention, said VH comprises the amino acid sequence as set forth in SEQ ID NO: 4, or an amino acid sequence having at least 80% identity to a selected sequence; and the VL comprises the amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having at least 80% identity to a selected sequence.

According to an embodiment of the invention, said VH comprises the amino acid sequence as set forth in SEQ ID NO:6, or an amino acid sequence having at least 80% identity to a selected sequence; and the VL comprises the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence having at least 80% identity to a selected sequence. Further stated, the VH and VL sequences of the antibody or antigen-binding fragment can be as set forth in SEQ ID NO: 3. SEQ ID NO: 4. the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO:6 by insertion, deletion, mutation or modification of one or more amino acids based on the amino acid sequence shown in (6).

According to an embodiment of the invention, the antibody or antigen binding fragment thereof is a monoclonal antibody.

According to an embodiment of the invention, the antibody or antigen-binding fragment thereof is a chimeric antibody or a humanized antibody. The humanized antibody is mainly a mouse monoclonal antibody which is modified by gene cloning and DNA recombination technology and combined with a human target after modification, and the re-expressed antibody has most of amino acid sequences replaced by human sequences, basically keeps the affinity and specificity of a parent mouse monoclonal antibody, reduces the heterogeneity thereof, and is favorable for being applied to a human body, for example, the antibody or antigen binding fragment of the embodiment of the invention can be specifically combined with human CSF-1R after being humanized.

According to an embodiment of the invention, the antibody or antigen-binding fragment thereof is an antibody fragment that specifically binds human CSF-1R and is selected from the group consisting of Fv, Fab ', scFv, and F (ab') 2.

According to another aspect of the invention, there is provided an isolated polynucleotide. According to an embodiment of the present invention, the isolated polynucleotide encodes the aforementioned anti-human CSF-1R antibody or fragment thereof. According to an embodiment of the invention, the isolated polynucleotide encodes the CSF-1R antibody or antigen-binding fragment of the preceding clause. Thus, the polypeptide encoded by the polynucleotide can specifically bind to CSF-1R.

According to another aspect of the invention, the invention provides an isolated polynucleotide. According to an embodiment of the present invention, the isolated polynucleotide encodes the light chain variable region or the heavy chain variable region, or the light chain or the heavy chain of the aforementioned anti-human CSF-1R antibody or fragment thereof.

According to another aspect of the present invention, there is provided a vector. According to an embodiment of the invention, the vector comprises one or more of the polynucleotides described above.

According to an embodiment of the invention, the vector comprises the two polynucleotides described above and encodes a VL region and a VH region which together bind CSF-1R.

According to another aspect of the present invention, there is provided a pair of vectors. According to an embodiment of the invention, each vector pair comprises one of the foregoing polynucleotides, wherein the vector pairs together encode a VL region and a VH region that together bind CSF-1R.

According to another aspect of the present invention, there is provided a host cell. According to an embodiment of the invention, the host cell comprises the aforementioned vector, or the aforementioned pair of vectors.

According to another aspect of the present invention, there is provided a composition. According to an embodiment of the present invention, the composition comprises the above-mentioned anti-human CSF-1R antibody or a fragment thereof, the above-mentioned polynucleotide, the above-mentioned vector pair, or the above-mentioned host cell; and optionally pharmaceutically acceptable excipients. Therefore, the composition can be specifically combined with CSF-1R, and has good effect of inhibiting tumor cells and good drug effect.

According to another aspect of the present invention, there is provided a method for preparing an anti-human CSF-1R antibody or fragment thereof. According to an embodiment of the invention, the method comprises: 1) culturing the aforementioned host cell under suitable conditions; 2) separating and recovering the anti-human CSF-1R monoclonal antibody or fragment thereof, the anti-human CSF-1R chimeric antibody or fragment thereof, or the humanized anti-human CSF-1R antibody or fragment thereof. The invention adopts a method of separating and purifying target protein after in vitro cell culture to obtain the antibody specifically binding to CSF-1R.

According to another aspect of the invention, the invention provides a use. According to an embodiment of the invention, the use comprises: use of the above-mentioned anti-human CSF-1R antibody or fragment thereof, the above-mentioned polynucleotide, the above-mentioned vector pair, or the above-mentioned host cell and the above-mentioned composition for the preparation of a cancer diagnostic agent.

According to another aspect of the invention, the invention provides a use. According to an embodiment of the invention, the use comprises: use of the above-mentioned anti-human CSF-1R antibody or fragment thereof, the above-mentioned polynucleotide, the above-mentioned vector, the above-mentioned pair of vectors, the above-mentioned host cell or the above-mentioned composition for the preparation of a medicament for the treatment of cancer.

According to an embodiment of the invention, the cancer is a solid tumor.

According to an embodiment of the invention, the solid tumor comprises giant cell tumor of tendon sheath (TGCT), breast cancer, colorectal cancer, head and neck cancer.

According to the embodiment of the invention, the inventor finds that the antibody of the embodiment of the invention can bind and activate CSF-1R, has the affinity less than 2.60E-09, overcomes the defects of insufficient affinity, specificity and adverse reaction of an anti-human CSF-1R antibody in the prior art, has high stability, strong affinity, higher biological activity and good clinical anti-tumor application prospect.

For a better understanding of the present invention, certain terms are first defined. Other definitions are set forth throughout the detailed description.

The term "specific" refers to the determination of the presence or absence of a protein in a heterogeneous population of proteins and/or other organisms. Thus, under the conditions specified, a particular ligand/antigen binds to a particular receptor/antibody and does not bind in significant amounts to other proteins present in the sample.

The term "antibody" herein is intended to include full-length antibodies and any antigen-binding fragment (i.e., antigen-binding portion) or single chain thereof. Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH 3. Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region is composed of one domain CL. The VH and VL regions can also be divided into hypervariable regions, called Complementarity Determining Regions (CDRs), which are separated by more conserved Framework Regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component of the classical complement system (C1 q).

The term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "antigen-binding fragment" of an antibody (or simply antibody portion), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind antigen. It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments comprised in the "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH 1; (ii) a F (ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a hinge region disulfide bridge; (iii) an Fd fragment consisting of VH and CH 1; (iv) an Fv fragment consisting of VL and VH antibody single arms; (v) dAb fragments consisting of VH (Ward et al, (1989) Nature 341: 544-546); (vi) an isolated Complementarity Determining Region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they may be joined by recombinant methods via a synthetic linker that makes the two single protein chains in which the VL and VH regions pair to form monovalent molecules (referred to as single chain Fc (scFv); see, e.g., Bird et al, (1988) Science 242: 423-. These single chain antibodies are also intended to be included within the term meaning. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

Antigen-binding fragments of the invention include those capable of specifically binding to an antigen. Examples of antibody binding fragments include, for example, but are not limited to, Fab ', F (ab')₂Fv fragments, single chain Fv (scFv) fragments and single domain fragments.

The Fab fragment contains the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab 'fragments are generated by cleavage of the disulfide bond at the hinge cysteine of the F (ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those of ordinary skill in the art. Fab and F (ab')2 fragments lack the fragment crystallizable (Fc) region of intact antibodies, clear more rapidly from the circulation of animals, and may have less non-specific tissue binding than intact antibodies (see, e.g., Wahl et al, 1983, J. Nucl. Med. 24: 316).

As is generally understood in the art, an "Fc" region is a fragment crystallizable constant region of an antibody that does not comprise an antigen-specific binding region. In IgG, IgA and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of an antibody (CH 2 and CH3 domains, respectively). The IgM and IgE Fc regions contain three heavy chain constant domains (CH 2, CH3, and CH4 domains) per polypeptide chain.

The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or fragment thereof, and an antigen). The term "binding affinity" is used to describe monovalent interactions (intrinsic activity). The binding affinity between two molecules (e.g., an antibody or fragment thereof, and an antigen) via a monovalent interaction can be quantitatively determined by determining the dissociation constant (KD). KD can then be determined by measurement of complex formation and dissociation kinetics, for example by SPR methods. The rate constants corresponding to association and dissociation of monovalent complexes are referred to as the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. KD is linked to ka and KD by the equation KD = KD/ka. According to the above definitions, the binding affinities associated with different molecular interactions, e.g. the binding affinities of different antibodies for a given antigen, can be compared by comparing the KD values of the individual antibody/antigen complexes. Similarly, the specificity of an interaction can be evaluated by determining and comparing the KD value for the interaction of interest (e.g., a specific interaction between an antibody and an antigen) to the KD value for an interaction not of interest. The value of the dissociation constant can be determined directly by well-known methods, e.g., by standard assays that assess the binding ability of a ligand (e.g., an antibody) to a target are known in the art and include, e.g., ELISA, western blot, RIA, and flow cytometry analysis. The binding kinetics and binding affinity of the antibody can also be assessed by standard assays known in the art, such as SPR. A competitive binding assay may be performed in which the binding of an antibody to a target and the binding of another ligand of the target (e.g., another antibody) to the target are compared.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. Also, like reference numerals are used to refer to like parts throughout the drawings. In the drawings:

FIG. 1 shows a graph of the binding activity of an anti-CSF-1R antibody according to one embodiment of the invention to cell surface CSF-1R;

FIG. 2 shows a graphical representation of the results of the blocking activity of humanized antibody number 64 on CSF/CSFR1 binding according to one embodiment of the invention;

FIG. 3 is a graph showing the results of the blocking activity of humanized antibody number 64 on IL34/CSFR1 binding according to one embodiment of the present invention;

FIG. 4 shows a graph of internalization results after an anti-CSF-1R humanized antibody binds to CSF-1R-CHO cell surface CSF-1R, according to one embodiment of the invention, wherein FIG. 4A is a graph of internalization results at 4 ℃ and FIG. 4B is a graph of internalization results at 37 ℃.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.

Anti-human CSF-1R antibodies and antigen-binding fragments thereof

The invention provides antibodies and antigen-binding fragments thereof that specifically bind to CSF-1R. The anti-human CSF-1R antibody or the antigen-binding fragment thereof according to the embodiment of the invention can be specifically bound with human CSF-1R or mouse CSF-1R protein, reduce the level of tumor cells in the serum of a patient, reduce the growth of eosinophilic granulocyte and reduce cancer reaction.

The present embodiments provide anti-CSF-1R antibody 64, and humanized and chimeric antibodies thereto. The chimeric antibody has a VH and a VL derived from a mouse antibody. However, the constant domain of the chimeric antibody is from a human antibody (e.g., human IgG1, human IgG2, human IgG3, or human IgG 4), and the chimeric antibody is labeled ch 64. In addition, the embodiment of the invention also provides a humanized antibody hz 64.

The CDR sequences (Kabat definition), heavy and light chain variable regions, and heavy and light chain constant regions of some of the disclosed antibodies are shown in table 1 below.

TABLE 1

Ab	VL-CDR1	VL-CDR2	VL-CDR3	VH-CDR1	VH-CDR2	VH-CDR3	VL	VH	CL	CH
											64	9	10	11	12	13	14	3	4	-	-
ch64	9	10	11	12	13	14	3	4	7	8
											hz64	9	10	11	12	13	14	5	6	7	8

The embodiments of the invention also provide the amino acid sequences of the heavy chain variable region and the light chain variable region of the humanized antibody. Because there are different ways to humanize a mouse antibody (e.g., the sequences can be modified with different amino acid substitutions), the heavy and light chains of an antibody can have more than one form of humanized sequence. The amino acid sequence of the heavy chain variable region of humanized antibody hz64 is shown in SEQ ID NO 6. The amino acid sequence of the light chain variable region of humanized antibody hz64 is shown in SEQ ID NO. 5.

In some embodiments, an antibody may have a heavy chain variable region (VH) comprising or consisting of complementarity determining regions CDR1, 2, 3, wherein the CDR1 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence at least 80%, 85%, 90% or 95% identical to a selected VH CDR3 amino acid sequence; the VL comprises or consists of a CDR1, 2, 3 wherein the CDR1 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VL CDR2 amino acid sequence and the CDR3 region comprises or consists of an amino acid sequence having at least 80%, 85%, 90% or 95% identity to a selected VL CDR3 amino acid sequence.

"percent (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence shown in the specification, after aligning the candidate sequence with the specific amino acid sequence shown in the specification and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the invention contemplates variants of the antibody molecules of the invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, or 99% or more identity, relative to the antibody molecules and sequences thereof specifically disclosed herein. The variant may comprise conservative changes.

Nucleic acids, vectors and cells

The embodiments also provide nucleic acids comprising polynucleotides encoding polypeptides comprising an immunoglobulin heavy chain or an immunoglobulin light chain. The immunoglobulin heavy chain or immunoglobulin light chain comprises CDRs as shown in table 1, or has sequences as shown in table 1. When a polypeptide is paired with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptide binds CSF-1R (e.g., human CSF-1R).

The embodiments also provide vectors comprising the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors are introduced (i.e., such that the host cells contain the polynucleotides and/or polynucleotide-containing vectors), and production of recombinant antibody polypeptides or fragments thereof by recombinant techniques.

As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into said host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as an encoded polypeptide in a host cell into which the expression vector has been introduced. Thus, in an expression vector, a polynucleotide of interest is targeted for expression in the vector by being operably linked to regulatory elements such as a promoter, enhancer and/or polyadenylation tail, which are located at or near or on both sides of the site of integration of the polynucleotide of interest within the vector or in the genome of the host cell, such that the polynucleotide of interest will be translated in the host cell into which the expression vector is introduced.

The vector may be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and/or transduction (e.g., with a recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensing agents.

The embodiments of the present invention provide host cells transformed with the vectors described above. The host cell may be a prokaryotic or eukaryotic cell. A preferred prokaryotic host cell is E.coli (Escherichia coli). Preferably, the eukaryotic cell is selected from: protist cells, animal cells, plant cells and fungal cells. More preferably, the host cell is a mammalian cell, including but not limited to CHO and COS cells. A preferred fungal cell is Saccharomyces cerevisiae.

Methods of making anti-CSF-1R antibodies

Isolated fragments of human CSF-1R may be used as immunogens to generate antibodies using standard techniques for polyclonal and monoclonal antibody preparation. Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein may be conjugated to an agent that is immunogenic in the species to be immunized. The animal may be injected multiple times with the antigenic peptide or protein.

Immunogens are typically used to produce antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations may contain, for example, recombinantly expressed polypeptides or chemically synthesized polypeptides (e.g., fragments of human CSF-1R). The formulation may further comprise an adjuvant, such as freund's complete or incomplete adjuvant, or similar immunostimulant.

Pharmaceutical compositions and uses and methods of treatment

The embodiments also provide pharmaceutical compositions containing at least one (e.g., one, two, three, or four) of the antibodies or antigen-binding fragments described in the embodiments. Two or more (e.g., two, three, or four) of any of the antibodies or antigen-binding fragments described herein can be present in a pharmaceutical composition in any combination. The pharmaceutical compositions may be formulated in any manner known in the art.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. The pharmaceutically acceptable carrier may further comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which increase the shelf-life or effectiveness of the antibody.

In one aspect, the invention provides the use of an anti-human CSF-1R antibody or antigen-binding fragment thereof as hereinbefore described in the manufacture of a medicament for the treatment of cancer, for example tenosynostocytomegaly (TGCT), breast cancer, colorectal cancer, head and neck cancer.

The present invention provides that one or more antibodies or antigen-binding fragments thereof can be used for a variety of therapeutic purposes. In one aspect, the present disclosure provides methods for treating cancer in a subject, methods of reducing the risk of cancer progression. In some embodiments, the treatment can stop, slow, or inhibit the progression of the cancer. In some embodiments, the treatment can result in a reduction in the number, severity, and/or duration of one or more symptoms of cancer in the subject.

In one aspect, the disclosure features methods that include administering to a subject in need thereof (e.g., a subject having, or identified as having, or diagnosed with, cancer), e.g., tenosynostocytomegaly (TGCT), breast cancer, colorectal cancer, head and neck cancer, a therapeutically effective dose of an antibody or antigen-binding fragment thereof disclosed herein.

In some embodiments, the compositions and methods disclosed herein can be used to treat a patient at risk for cancer. A patient having cancer can be identified by a variety of methods known in the art.

The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.

Example 1: preparation of mouse chimeric antibody against CSF-1R hybridoma

1. Preparation of control antibodies

According to patent CN 107011438A, the gene sequence of the antibody Cabirralizumab (FPA-008) from Five prime company is synthesized. Respectively cloning the light and heavy chain sequences into a eukaryotic transient expression vector containing a human IgG4 light and heavy chain constant region to obtain control antibody light chain and heavy chain expression plasmids, transferring the control antibody light chain and heavy chain expression plasmids into escherichia coli for amplification, separating to obtain a large number of plasmids containing the control antibody light chain and heavy chain, and respectively transferring the light and heavy chain plasmids of the control antibody into HEK293 cells for recombinant expression by using the plasmids according to the operation instruction of a transfection reagent 293fectin (Cat: 12347019, Gibco). 5-6 days after cell transfection, culture supernatant is taken and purified by a ProA affinity chromatography column to obtain a control antibody. The light chain and heavy chain variable region sequences are shown in SEQ ID NO.1 and SEQ ID NO. 2.

SEQ ID NO.1 FPA-008-VK

EIVLTQSPATLSLSPGERATLSCKASQSVDYDGDNYMNWYQQKPGQAPRLLIYAASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHLSNEDLSTFGGGTKVEIK

SEQ ID NO.2 FPA-008-VH

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDNYMIWVRQAPGQGLEWMGDINPYNGGTTFNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARESPYFSNLYVMDYWGQGTLVTVSS

2. Acquisition of murine hybridoma antibody No. 64

Balb/c mice were immunized with human CSF-1R recombinant protein (SEQ ID NO: NP-005202.2, 1-506 aa), and cell fusion and hybridoma preparation were performed. Analyzing the hybridoma culture supernatant by ELISA, selecting specific positive clones, and obtaining specific positive clone numbers: 64, extracting hybridoma cell total RNA using TRIzol kit (Cat: 15596026, Invitrogen); reverse transcribing hybridoma cell total RNA to cDNA using M-MuLV reverse transcriptase (Cat: M0253S, NEB); amplification of antibody light chain variable region IgVL (kappa) and heavy chain variable region V using degenerate primers and Phusion kit (Cat: E0553L, NEB)_HA sequence; purifying PCR amplification products by using a gel recovery kit (Cat: AP-GX-250, Axygen); connecting the amplified PCR product to a T vector according to the instruction of a T vector cloning kit (Cat: ZC205, Conn. biol.) and transforming competent cells of escherichia coli, amplifying strains, extracting plasmids and then carrying out DNA sequencing to obtain the variable region sequence of the monoclonal antibody. The sequencing result shows that the variable region amino acid sequence of the light chain of the clone 64 mouse antibody is shown in SEQ ID NO.3, and the variable region sequence of the heavy chain is shown in SEQ ID NO. 4. The light chain variable region and heavy chain variable region genes are introduced into enzyme cutting sites through PCR, and are respectively cloned into eukaryotic transient expression vectors containing human-kappa light chain constant region (SEQ ID NO: 7) and human IgG4 heavy chain constant region (SEQ ID NO: 8) coding genes at the upstream, so as to obtain human-mouse chimeric light chain (pKN 019-64L) and human-mouse chimeric heavy chain (pKN 034-64H) expression plasmids, and the light chain plasmids and the heavy chain plasmids of the chimeric antibody are respectively transferred into HEK293 cells for recombination expression according to the operation instructions of a transfection reagent 293fectin (Cat: 12347019, Gibco). 5-6 days after cell transfection, culture supernatant is taken and purified by a ProA affinity chromatography column to obtain a ch64 protein sample.

SEQ ID NO. 364-VL (VL-CDRs 1, 2 and 3: SEQ ID NO.9, 10 and 11)

dikmtqspssmyaslgervtitckasqdinsylswfqqkpglspktliyranrmrvgvpsrfsgsgsgqdysltisslefedmgiyyclqyddfpytfgggtkleik

SEQ ID NO. 464-VH (VH-CDRs 1, 2 and 3: SEQ ID NO.12, 13 and 14)

dvqlqesgpglvkpsqslsltctvtgysitsdyawnwirqfpgnklewmgditysggtkynpslksrisitrdtsknqfflqlssvttedtatyycanvnyyamdywgqgtsvtvss

3. Chimeric antibody identification

Affinity analysis was performed on the chimeric antibody ch64 using fortebio. Antibodies were diluted to 4ug/mL in PBS buffer and flowed over the surface of an AHC probe (Cat: 18-0015, PALL) for 120 s. Human CSF-1R-his recombinant protein was used as mobile phase at a concentration of 60nM each. The binding time was 100s and the dissociation time was 300 s. After the experiment, blank control response values were deducted, and the software was run for 1: 1 Langmuir binding pattern was fitted and kinetic constants for antigen-antibody binding were calculated. The affinity data of the chimeric antibody is shown in Table 2, and the affinity of ch64 is better than that of the control antibody FPA-008.

TABLE 2 chimeric antibody affinity assay

Sample ID	KD (M)	kon(1/Ms)	kdis(1/s)
				ch64	2.27E-09	6.02E+04	1.37E-04
FPA-008	2.79E-09	9.87E+04	2.76E-04

Example 2: humanization of anti-CSF-1R antibodies

The humanized antibody hz64 was prepared by subjecting a mouse antibody to humanization treatment, synthesizing the humanized antibody variable region gene, constructing a humanized antibody recombinant expression vector by the method of example 1, and expressing and purifying the antibody. The sequences of the hz64 variable region are shown in SEQ ID number 5 and SEQ ID number 6 below, and the affinity of the hz64 antibody determined by Foretibo is shown in Table 3. hz64 retains affinity comparable to that of the chimeric antibody.

SEQ ID number 5 hz64-VL (VL-CDRs 1, 2 and 3: SEQ ID NO.9, 10 and 11):

diqmtqspsslsasvgdrvtitckasqdinsylswfqqkpgkapktliyranrmrvgvpsrfsgsgsgqdytltisslqpedfatyyclqyddfpytfgggtkveik

SEQ ID number 6 hz64-VH (VH-CDRs 1, 2 and 3: SEQ ID NO.12, 13 and 14):

evqlqesgpglvkpsqtlsltctvsgysitsdyawnwirqhpgkglewigditysggtkynpslksrvtisrdtsknqfslklssvtaadtavyycanvnyyamdywgqgttvtvss

TABLE 3 humanized antibody affinity assay

Sample	KD (M)	kon(1/Ms)	kdis(1/s)
				ch64	2.17E-09	2.55E+05	5.54E-04
hz64	2.60E-09	2.53E+05	6.59E-04

Example 3: binding Activity of anti-CSF-1R antibody to cell surface CSF-1R

The CSF-1R binding activity of the antibody to the cell surface was analyzed using rhCSF-1R-CHO cells stably expressing CSF-1R. Using 5E5 cells bound to different concentrations of anti-CSF-1R antibody, respectively, the antibody was diluted in 8 gradients starting at 30. mu.g/ml in 3-fold gradients. Incubating at 4 ℃ in the dark for 60min, washing with PBS, adding FITC-labeled goat anti-human antibody (sigma, F9512) diluted at 1:200, incubating at 4 ℃ in the dark for 30min, washing with PBS, resuspending in 200 μ L of PBS, and detecting with flow cytometry. The binding activity of hz64 and control antibody FPA-008 is plotted in FIG. 1, and EC50 data is shown in Table 4, showing that humanized antibody hz64 has comparable binding ability to control antibody FPA-008.

TABLE 4 analysis of the binding Activity of anti-CSF-1R antibodies to cell surface CSF-1R

	hz64	FPA-008
			EC50(μg/ml)	0.133	0.125

Example 4: blocking Activity of anti-CSF-1R antibodies against recombinant CSF-1 and CSF-1R

Wrapping a plate: human CSF1-His concentration of 0.2. mu.g/ml; 100 μ l per well; overnight at 4 ℃; and (3) sealing: 5% BSA in PBS, 37 degrees, 120min, PBST washing plate 4 times; adding a primary antibody: preparing CSF-1R-mFc with the concentration of 0.15 mu g/mL and Anti-CSF-1R antibody (the initial concentration is 20 mu g/mL, 3 times of serial dilution and 12 gradients); mixing Anti-CSF-1R antibody and CSF-1R-mFc 60 μ l each, shaking slightly, mixing well, 37 deg.C, 10min, adding 100 μ l per well, and coating; adding a secondary antibody: HRP-anti-mouse IgG (1:5000, Cat.115-035-071, Jackson Immuno Research) was washed 4 times with PBST at 37 ℃ for 45 min; TMB (Cat: ME142, Beijing Taitianhe biology) substrate color development for 15 min; and (4) terminating: terminating the reaction with 2M HCL; reading: the absorbance of the plate was read and recorded at a wavelength of 450 nm. The blocking activity of ch64 and hz64 on CSF-1/CSF-1R binding is shown in FIG. 2, and the IC50 value is shown in Table 5, and the results show that both ch64 and hz64 can inhibit CSF-1 and CSF-1R binding in a dose-dependent manner.

TABLE 5 blocking Activity of anti-CSF-1R antibodies against recombinant CSF-1/CSF-1R binding (IC 50)

	ch64	hz64	FPA-008
				IC50（nM）	0.28	0.22	0.34

Example 5: blocking Activity of anti-CSF-1R antibodies on the binding of recombinant IL34 to CSF-1R

Wrapping a plate: the concentration of human IL34 was 0.2. mu.g/ml; 100 μ l per well; overnight at 4 ℃; and (3) sealing: 5% BSA in PBS, 37 degrees, 120min, PBST washing plate 4 times; adding a primary antibody: preparing CSF-1R-mFc with the concentration of 0.15 mu g/mL and Anti-CSF-1R antibody (the initial concentration is 20 mu g/mL, 3 times of serial dilution and 12 gradients); mixing Anti-CSF-1R antibody and CSF-1R-mFc 60 μ l each, shaking slightly, mixing well, 37 deg.C, 10min, adding 100 μ l per well, and coating; adding a secondary antibody: HRP-anti-mouse IgG (1:5000, Cat.115-035-071, Jackson Immuno Research) was washed 4 times with PBST at 37 ℃ for 45 min; TMB (Cat: ME142, Beijing Taitianhe biology) substrate color development for 15 min; and (4) terminating: terminating the reaction with 2M HCL; reading: the absorbance of the plate was read and recorded at a wavelength of 450 nm.

The results show (FIG. 3) that both ch64 and hz64 were able to partially inhibit the binding of IL-34 to CSF-1R, suggesting that antibody hz64 may have a different antigen binding epitope than the control antibody.

Example 6: anti-CSF-1R antibodies induce internalization of cell surface CSF-1R

CSF-1R-CHO cells expressing human CSF-1R were seeded at a density of 2E4 cells/well in 96-well cell culture plates and cultured for 24 hours. Cells were washed 1 time with PBS and the supernatant was discarded. Use Mix-n-Stain CF 488A (Cat: MX488AS100, Sigma) to mark hz64, obtain hz64-CF488, dilute hz64-CF488 to 10 μ g/mL with RPMI 1640 (containing 10% FBS), add to cells, put a group in 37 ℃ electric heating constant temperature incubator, put a group in 4 ℃ refrigerator AS negative control; the negative control was incubated for 1 hour and then washed 3 times with PBS, observed with a fluorescence microscope and photographed, and the experimental group was incubated for 5 hours at 37 ℃ and then observed with a fluorescence microscope and photographed.

The internalization pattern of hz64 after binding to CSF-1R-CHO cell surface CSF-1R is shown in FIG. 4, wherein FIG. 4A is observed at 4 ℃ and FIG. 4B is observed at 37 ℃, and the experimental results show that hz64-CF488 can see uniform fluorescence on the cell surface at 4 ℃ indicating that the antibody is bound to the cell surface, and the fluorescence labeled antigen-antibody complex internalizes into the cell at 37 ℃ and is distributed in a punctate manner in the fluorescence signal cytoplasm.

The antibody hz64 obtained from the CSF-1R antibody or antigen binding fragment of the embodiments of the invention can bind to human CSF-1R, and has better activity of binding to human CSF-1R and better activity of inhibiting CSF-1 from binding to human CSF-1R than the control antibody. The activity of the humanized antibody for inhibiting IL34 from being combined with human CSF-1R is obviously better than that of a control antibody. The anti-CSF-1R antibody according to the embodiments of the present invention has strong affinity and exhibits high specificity to human CSF-1R, and can provide an effective solution for the treatment of CSF-1R-related diseases by binding, internalizing and blocking CSF-1R.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

While embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Sequence listing

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Claims

1. An anti-human CSF-1R antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein,

the VH comprises the antigenic determinants VH CDR1, VH CDR2 and VH CDR3, the amino acid sequence of VH CDR1 is as set forth in SEQ ID NO: 12, the amino acid sequence of the VH CDR2 is shown in SEQ ID NO: 13, the amino acid sequence of the VH CDR3 is shown in SEQ ID NO: 14 is shown in the figure;

the VL comprises the antigenic determinants VL CDR1, VL CDR2 and VL CDR3, the amino acid sequence of VL CDR1 is as set forth in SEQ ID NO: 9, and the amino acid sequence of the VL CDR2 is shown as SEQ ID NO: 10, and the amino acid sequence of the VL CDR3 is shown as SEQ ID NO: shown at 11.

2. The anti-human CSF-1R antibody or antigen-binding fragment thereof of claim 1, wherein the VH has an amino acid sequence as set forth in SEQ ID NO: 4 is shown in the specification; and the amino acid sequence of the VL is shown in SEQ ID NO: 3, respectively.

3. The anti-human CSF-1R antibody or antigen-binding fragment thereof of claim 1, wherein the VH has the amino acid sequence of SEQ ID NO:6 is shown in the specification; and the amino acid sequence of the VL is shown in SEQ ID NO: 5, respectively.

4. The anti-human CSF-1R antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

5. The anti-human CSF-1R antibody or antigen-binding fragment thereof of claim 4, wherein the antibody or antigen-binding fragment thereof is a chimeric antibody.

6. The anti-human CSF-1R antibody or antigen-binding fragment thereof of claim 4, wherein the antibody or antigen-binding fragment thereof is a humanized antibody.

7. The anti-human CSF-1R antibody or antigen-binding fragment thereof of claim 5, wherein the antibody or antigen-binding fragment thereof is an antibody fragment that specifically binds human CSF-1R and is selected from the group consisting of Fv, Fab ', scFv, and F (ab')₂。

8. An isolated polynucleotide encoding an anti-human CSF-1R antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

9. An isolated polynucleotide encoding the light and heavy chains of an anti-human CSF-1R antibody or antigen-binding fragment thereof according to any one of claims 1-7.

10. An isolated polynucleotide encoding the VL and VH of an anti-human CSF-1R antibody or antigen-binding fragment thereof of any one of claims 1-7.

11. A vector comprising one or more polynucleotides according to any one of claims 8-10.

12. The vector of claim 11, wherein said polynucleotides encode said light and heavy chains of said antibody or antigen-binding fragment thereof of claim 2 or 3.

13. The vector of claim 11, wherein said polynucleotide encodes the VL and VH of the antibody or antigen-binding fragment thereof of claim 2 or 3.

14. A vector pair, wherein both vectors of said vector pair comprise two polynucleotides encoding a light chain and a heavy chain, respectively, which together encode the antibody or antigen-binding fragment thereof of claim 2 or 3.

15. A vector pair, wherein both vectors of said vector pair comprise two polynucleotides encoding VL, VH, respectively, which together encode the antibody or antigen-binding fragment thereof of claim 2 or 3.

16. A host cell comprising the polynucleotide of any one of claims 8-10, the vector of any one of claims 11-13, or the pair of vectors of any one of claims 14-15.

17. A composition, comprising:

the anti-human CSF-1R antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, the polynucleotide according to any one of claims 8 to 10, the vector according to any one of claims 11 to 13, the pair of vectors according to any one of claims 14 to 15, or the host cell according to claim 16; and

optionally pharmaceutically acceptable excipients.

18. A method of making an anti-human CSF-1R antibody or antigen-binding fragment thereof, comprising:

1) culturing the host cell of claim 16 under suitable conditions;

2) separating and recovering the anti-human CSF-1R antibody or antigen-binding fragment thereof.

19. Use of the anti-human CSF-1R antibody or the antigen-binding fragment thereof according to any one of claims 1 to 7, the polynucleotide according to any one of claims 8 to 10, the vector according to any one of claims 11 to 13, the pair of vectors according to any one of claims 14 to 15, the host cell according to claim 16, or the composition according to claim 17 for the preparation of a cancer diagnostic agent.

20. Use of an anti-human CSF-1R antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, a polynucleotide according to any one of claims 8 to 10, a vector according to any one of claims 11 to 13, a pair of vectors according to any one of claims 14 to 15, a host cell according to claim 16, or a composition according to claim 17 in the manufacture of a medicament for the treatment of cancer.

21. The use of claim 19 or 20, wherein the cancer is a solid tumor.

22. The use of claim 21, wherein the solid tumor is tenosynostocytomegalic tumor (TGCT), breast cancer, colorectal cancer, or head and neck cancer.