Anti-granulocyte colony stimulating factor (g-CSF) receptor agonist antibody or antigen-binding fragment thereof and use thereof

Abstract

The present invention relates to a novel anti-G-CSF receptor agonist antibody and use thereof. The anti-G-CSF receptor-specific agonist antibody according to an embodiment has excellent G-CSF receptor affinity, induces molecular programs that promote the release of HSPC, and can induce balanced activity in both the bone marrow system and the lymphatic system, and thus can be used for the treatment of cancer, immune diseases, or hematologic diseases, which require HSPC induction.

Description

Antibody of a granulocyte colony-stimulating factor (G-CSF) receptor agonist or an antigen-binding fragment thereof and uses thereof

[0001] The present invention relates to a novel anti-G-CSF receptor agonist antibody and its uses.

[0002] Hematopoietic stem cells are progenitor cells found in the bone marrow that possess the ability to self-replicate and differentiate, generating all the cells that make up the blood and immune system. For patients with impaired hematopoietic function due to blood disorders or bone marrow suppression caused by aggressive chemotherapy, the transplantation of healthy hematopoietic stem cells is a treatment that promotes the reconstruction of blood cells and helps restore immune function.

[0003] Although there is an invasive method of directly harvesting hematopoietic stem cells from the bone marrow, many donors prefer a non-invasive method in which cells are transferred from the bone marrow into the bloodstream and then recovered from the peripheral blood. To achieve this, the injection of a promoter is essential to facilitate the release of hematopoietic stem cells from the bone marrow into the bloodstream; the primary mechanism of action of this promoter is to induce direct or indirect interference with molecules that anchor hematopoietic stem cells within the bone marrow microenvironment. Representative promoters include 1) molecules that block the bone marrow hematopoietic stem cell anchoring molecule axis (CXCR4-CXCl12), 2) plerixafor, and filgrastim, a recombinant granulocyte-colony stimulating factor (G-CSF) that helps promote the secretion of proteases that induce transient disruption of the bone marrow microenvironment and degradation of hematopoietic stem cell anchoring molecules.

[0004] Granulocyte colony-stimulating factor (G-CSF) is a major cytokine that drives the efflux of hematopoietic stem / progenitor cells (HSPCs) into peripheral blood. Recombinant G-CSF (filagrastim) and its glycosylated analog (lenograstim) have been established as standard therapies for PBSC mobilization, but their clinical application is somewhat limited due to limitations in pharmacokinetic characteristics and efficacy [1, 2]. Both drugs have short half-lives, requiring multiple administrations, and their efficacy still falls short of optimal levels in patients with extensive prior treatment histories or the elderly [3]. To address the issue of administration frequency, a pegylated analog (pegfilgrastim) was developed, but it has not been proven to improve mobilization efficiency [4].

[0005] Recently, mobilization strategies involving combination therapy with chemotherapy or CXCR4 antagonists (plerixafor) are increasingly being utilized, and these combination therapies can successfully collect HSPCs in some patients with poor mobilization [6]. However, these strategies also introduce additional logistical complexity, and cytotoxic drug pretreatment can degrade the quality of the mobilized grafts [7]. These challenges are further complicated by clinical heterogeneity among patients, which limits the selection and applicability of combination drugs.

[0006] However, the G-CSF / G-CSF receptor (G-CSFR) axis coordinates various molecular events to create a bone marrow environment that promotes HSPC efflux, so it remains the most effective and clinically validated pathway despite the emergence of alternative targets. The researchers have made efforts to design an antibody-based agonist capable of inducing more sustained G-CSF receptor signaling with a single administration. As a result, the present invention was completed by developing a novel variant antibody that exhibits excellent G-CSF receptor affinity, induces molecular programs that promote HSPC release, and induces balanced activity in both the myeloid and lymphoid systems.

[0007] [선행기술문헌]

[0008] [비특허문헌]

[0009] [1] Schmitt, M., et al. "Mobilization of autologous and allogeneic peripheral blood stem cells for transplantation in haematological malignancies using biosimilar G-CSF." Vox Sang, 111(2), 2016, pp. 178-186.

[0010] [2] Lisenko, K., et al. "Comparison of biosimilar filgrastim, originator filgrastim, and lenograstim for autologous stem cell mobilization in patients with multiple myeloma." Transfusion, 57(10), 2017, pp. 2359-2365.

[0011] [3] Ahn, W. K., et al. "Poor Mobilization-Associated Factors in Autologous Hematopoietic Stem Cell Harvest." Cancers (Basel), 16(10), 2024.

[0012] [4] Wen, J., et al. "A novel PEGylated form of granulocyte colony-stimulating factor, mecapegfilgrastim, for peripheral blood stem cell mobilization in patients with hematologic malignancies. " BMC Cancer, 23(1), 2023, pp. 694.

[0013] [5] Kuan, J. W., et al. "Pegylated granulocyte-colony stimulating factor versus non-pegylated granulocyte-colony stimulating factor for peripheral blood stem cell mobilization: A systematic review and meta-analysis. " J Clin Apher, 32(6), 2017, pp. 517-542.

[0014] [6] Worel, N., et al. “Plerixafor as preemptive strategy results in high success rates in autologous stem cell mobilization failure.” J Clin Apher, 32(4), 2017, pp. 224-234.

[0015] [7] Popat, U., et al. “Impairment of filgrastim-induced stem cell mobilization after prior lenalidomide in patients with multiple myeloma.” Biol Blood Marrow Transplant, 15(6), 2009, pp. 718-723.

[0016] One aspect is to provide an antibody or an antigen-binding fragment thereof that specifically binds to a granulocyte-colony stimulating factor (G-CSF) receptor.

[0017] Another aspect is to provide a gene encoding the antibody or its antigen-binding fragment; a vector comprising the gene; or a recombinant cell comprising the gene or the vector.

[0018] Another aspect is to provide an antibody-drug conjugate (ADC) comprising the above antibody or an antigen-binding fragment thereof.

[0019] Another aspect provides a pharmaceutical composition for the prevention or treatment of cancer, immune diseases or blood diseases, comprising: the antibody or its antigen-binding fragment; a gene encoding the antibody or its antigen-binding fragment; a vector comprising the gene; a recombinant cell comprising the gene or the vector; or the antibody-drug conjugate.

[0020] Another aspect provides a method for preventing or treating cancer, immune disease, or blood disease, comprising the step of administering an effective amount of the antibody or its antigen-binding fragment; a gene encoding the antibody or its antigen-binding fragment; a vector containing the gene; a recombinant cell containing the gene or the vector; or the antibody-drug conjugate to an individual in need thereof.

[0021] Another aspect provides the use of said antibody or its antigen-binding fragment for use in pharmaceutical preparations for the prevention or treatment of cancer, immune diseases or blood diseases; a gene encoding said antibody or its antigen-binding fragment; a vector comprising said gene; a recombinant cell comprising said gene or said vector; or said antibody-drug conjugate.

[0022] Another aspect provides the use of said antibody or its antigen-binding fragment for use in the manufacture of pharmaceutical preparations for the prevention or treatment of cancer, immune diseases or blood diseases; a gene encoding said antibody or its antigen-binding fragment; a vector comprising said gene; a recombinant cell comprising said gene or said vector; or said antibody-drug conjugate.

[0023] One aspect provides an antibody or an antigen-binding fragment thereof that specifically binds to a granulocyte-colony stimulating factor (G-CSF) receptor.

[0024] The antibody or its antigen-binding fragment described herein refers to a variant antibody comprising a sequence in which one or more amino acids of the CDR sequence of a wild-type 3B3 antibody that specifically binds to a G-CSF receptor are mutated. The antibody or its antigen-binding fragment described herein is a variant antibody with improved G-CSF receptor affinity and improved G-CSF agonist activity.

[0025] In this specification, "3B3 antibody" refers to a wild-type G-CSF receptor-specific antigen-binding fragment comprising L-CDR1 of SEQ ID NO. 1, L-CDR2 of SEQ ID NO. 2, L-CDR3 of SEQ ID NO. 2, H-CDR1 of SEQ ID NO. 3, H-CDR2 of SEQ ID NO. 4, and H-CDR3 of SEQ ID NO. 5. The wild-type 3B3 antibody has the full amino acid sequence of SEQ ID NO. 20.

[0026] The antibody or its antigen-binding fragment in this specification means a variant in which the amino acid corresponding to i) position 2 of the amino acid sequence of SEQ ID NO. 3, ii) position 4 of the amino acid sequence of SEQ ID NO. 4 and / or iii) position 8 of the amino acid sequence of SEQ ID NO. 5 is modified.

[0027] In this specification, "antibody," "antigen-binding region or site," or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. Since the present invention relates to a G-CSF receptor antibody that specifically binds to a G-CSF receptor, unless otherwise specifically designated, the term "antibody" used without modification in this specification may refer to an antibody that specifically binds to a G-CSF receptor.

[0028] The antibody or its antigen-binding fragment in this specification may be a whole antibody; or any antigen-binding fragment or single chain thereof. The term "whole antibody" means a structure in which two full-length light chains and two full-length heavy chains are connected by disulfide bonds. The whole antibody includes IgA, IgD, IgE, IgM, and IgG, wherein IgG is a subtype including IgG1, IgG2, IgG3, and IgG4.

[0029] In this specification, "antigen-binding fragment" refers to a fragment of the entire antibody structure that includes a portion capable of binding to an antigen.

[0030] The antibody or its antigen-binding fragment described herein may comprise any protein or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule having biological activity for binding to an antigen among the whole antibody. Examples such as a complementarity determining region (CDR) of a heavy chain or light chain or a ligand-binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein, are included but not limited thereto.

[0031] In this specification, “heavy chain (HC or CH)" means a full-length heavy chain and a fragment thereof comprising a variable region domain VH having an amino acid sequence having a sufficient variable region (VR) sequence to confer specificity to an antigen, and three constant region domains CH1, CH2 and CH3.

[0032] In this specification, “Light Chain (LC or CL)" means a full-length light chain and a fragment thereof comprising a variable region domain VL and a constant region domain CL, comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen.

[0033] In this specification, "Complementary Determining Region (CDR; i.e., CDR1, CDR2, and CDR3)" refers to a region within the variable region of an antibody that has binding specificity with an antigen. Each variable region typically has three CDR regions identified as CDR1, CDR2, and CDR3. In this specification, the CDR included in the heavy chain variable region is denoted as "H-CDR," and the CDR included in the light chain variable region is denoted as "L-CDR."

[0034] In one embodiment, the antibody or its antigen-binding fragment may comprise one or more selected from the group consisting of a monoclonal antibody, a domain antibody (dAb), a single chain antibody (scAb), a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFab fragment, a Fv fragment, a dsFv fragment, a single chain variable fragment (scFv), a scFv-Fc fragment, a single domain heavy chain antibody, a single domain light chain antibody, a variant antibody, a multimeric antibody, a minibody, a diabody, a triabody, a tetrabody, a Bis-scFv, a nanobody, a bispecific antibody, and a multispecific antibody. Preferably, the antibody of the present invention may be an antibody comprising a scFv or scFv-Fc fragment.

[0035] In this specification, the “Fc” region may comprise two heavy chain fragments comprising CH2 and CH3 domains of the antibody. These two heavy chain fragments may be combined with each other by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0036] In this specification, a “Fab fragment” may consist of one heavy chain and one light chain comprising only CH1 and variable regions. The heavy chain of the Fab molecule may not form disulfide bonds with other heavy chain molecules.

[0037] In this specification, the “Fab’ fragment” comprises, in addition to the Fab fragment, a region between the CH1 and CH2 domains of the heavy chain, which can form a disulfide bond between the two heavy chains of two molecules of the Fab’ fragment to form an F(ab’)2 molecule.

[0038] In this specification, the “F(ab’)2 fragment” comprises two heavy chains and two light chains, including a variable region, CH1, and a portion of the invariant region between the CH1 and CH2 domains as described above, thereby forming an intrachain disulfide bond between the two heavy chains. Thus, the F(ab’)2 fragment consists of two Fab’ fragments, and the two Fab’ fragments can be bonded to each other by a disulfide bond between them.

[0039] In this specification, "Fv fragment" may be an antibody that includes each variable region of the heavy chain and light chain, but does not include a constant region. "Single-chain variable fragment," "single-chain Fv," or "antibody fragment" may refer to a fusion protein containing the VH and VL domains of an antibody, and these domains exist within a single polypeptide chain. The Fv polypeptide may further include a polypeptide linker between the VH domain and the VL domain to enable scFv to form a structure intended for antigen binding. scFv-Fc may be a case where Fc is linked to scFv. Diabody may include two molecules of scFv.

[0040] In the present invention, "corresponding amino acid" refers to an amino acid residue at a corresponding position in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the amino acid residue at that position. Identifying the amino acid at the corresponding position may involve determining a specific amino acid of a sequence that references a specific sequence. In the present invention, "corresponding position" generally refers to a similar or corresponding position in the amino acid sequence of a related protein or in a reference sequence. For example, any amino acid sequence may be aligned with SEQ ID NO. 1, and based on this, each amino acid residue of said amino acid sequence may be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue of SEQ ID NO. 1. For example, through a sequence alignment algorithm known in the art, the position of the corresponding amino acid, or the position where modifications such as substitution, insertion, or deletion occur, may be identified by comparing with a query sequence (also referred to as a "reference sequence").

[0041] In the present invention, "homology" or "identity" refers to the degree of relationship with two given amino acid sequences or base sequences and may be expressed as a percentage.

[0042] In one embodiment, the amino acid variation may include substitution with a nonpolar amino acid. For example, the amino acid variation may include substitution with glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, or tryptophan. Preferably, the amino acid variation may include substitution with alanine, glycine, or isoleucine.

[0043] In one embodiment, the antibody or its antigen-binding fragment may include a substitution of the amino acid corresponding to position 2 of the amino acid sequence of SEQ ID NO. 3 with alanine.

[0044] In one embodiment, the antibody or its antigen-binding fragment may include a substitution of the amino acid corresponding to position 4 of the amino acid sequence of SEQ ID NO. 4 with glycine.

[0045] In one embodiment, the antibody or its antigen-binding fragment may include a substitution of isoleucine with an amino acid corresponding to position 8 of the amino acid sequence of SEQ ID NO. 5.

[0046] In one embodiment, the antibody or the antigen-binding fragment thereof may include a heavy chain complementarity determining region 1 (H-CDR1) comprising a substitution of an amino acid corresponding to position 2 of the amino acid sequence of SEQ ID NO. 3; a heavy chain complementarity determining region 2 (H-CDR2) comprising the amino acid sequence of SEQ ID NO. 4; and a heavy chain variable region 3 (H-CDR3) comprising a substitution of an amino acid corresponding to position 8 of the amino acid sequence of SEQ ID NO. 5.

[0047] In one embodiment, the antibody or its antigen-binding fragment may include a heavy chain complementarity determining region 1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO. 3; a heavy chain complementarity determining region 2 (H-CDR2) comprising a substitution of an amino acid corresponding to the 4th position of the amino acid sequence of SEQ ID NO. 4; and a heavy chain variable region comprising a heavy chain complementarity determining region 3 (H-CDR3) comprising the amino acid sequence of SEQ ID NO. 5.

[0048] In one embodiment, the antibody or its antigen-binding fragment may include a heavy chain complementarity determining region 1 (H-CDR1) comprising a substitution of the amino acid corresponding to position 2 of the amino acid sequence of SEQ ID NO. 3 with alanine; a heavy chain complementarity determining region 2 (H-CDR2) comprising the amino acid sequence of SEQ ID NO. 4; and a heavy chain complementarity determining region 3 (H-CDR3) comprising a substitution of the amino acid corresponding to position 8 of the amino acid sequence of SEQ ID NO. 5 with isoleucine.

[0049] In one embodiment, the antibody or its antigen-binding fragment may comprise a heavy chain complementarity determining region 1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO. 3; a heavy chain complementarity determining region 2 (H-CDR2) comprising a substitution of the amino acid corresponding to position 4 of the amino acid sequence of SEQ ID NO. 4 with glycine; and a heavy chain variable region comprising a heavy chain complementarity determining region 3 (H-CDR3) comprising the amino acid sequence of SEQ ID NO. 5.

[0050] In one embodiment, the antibody or its antigen-binding fragment may include a light chain complementary determining region 1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO. 1; a light chain complementary determining region 2 (L-CDR2) comprising the amino acid sequence of SEQ ID NO. 2; and a light chain variable region comprising a light chain complementary determining region 3 (L-CDR3) comprising the amino acid sequence of SEQ ID NO. 2.

[0051] In one embodiment, the antibody or its antigen-binding fragment may include a light chain variable region comprising a substitution of an amino acid corresponding to positions 53, 54, 66, or 100 of the amino acid sequence of SEQ ID NO. 9.

[0052] In one embodiment, the antibody or its antigen-binding fragment may include a light chain variable region comprising a substitution of amino acids corresponding to positions 53 and 66 of the amino acid sequence of SEQ ID NO. 9.

[0053] In one embodiment, the antibody or its antigen-binding fragment may include a light chain variable region comprising a substitution of amino acids corresponding to positions 54 and 100 of the amino acid sequence of SEQ ID NO. 9.

[0054] In one embodiment, the antibody or its antigen-binding fragment may comprise one or more substitutions selected from the group consisting of the following in the amino acid sequence of SEQ ID NO. 9:

[0055] Substitution of the amino acid corresponding to position 53 with glycine;

[0056] Substitution of the amino acid corresponding to position 54 with serine;

[0057] Substitution with arginine of the amino acid corresponding to position 66; and

[0058] Substitution with arginine of the amino acid corresponding to position 100.

[0059] In one embodiment, the antibody or its antigen-binding fragment may include a heavy chain variable region comprising substitutions of amino acids corresponding to positions 5, 15, 27, 50, 56, 64, 69, 70, 80, and 104 of the amino acid sequence of SEQ ID NO. 12.

[0060] In one embodiment, the antibody or its antigen-binding fragment may include a heavy chain variable region comprising substitutions of amino acids corresponding to positions 5, 27, 50, 64, 69, 70, and 104 of the amino acid sequence of SEQ ID NO. 12.

[0061] In one embodiment, the antibody or its antigen-binding fragment may include a heavy chain variable region comprising a substitution of amino acids corresponding to positions 15, 56, and 80 of the amino acid sequence of SEQ ID NO. 12.

[0062] In one embodiment, the antibody or its antigen-binding fragment may comprise one or more substitutions selected from the group consisting of the following in the amino acid sequence of SEQ ID NO. 12:

[0063] Substitution of the amino acid corresponding to position 5 with serine;

[0064] Substitution with threonine of the amino acid corresponding to position 15;

[0065] Substitution of the amino acid corresponding to position 27 with alanine;

[0066] Substitution with valine of the amino acid corresponding to position 50;

[0067] Substitution of the amino acid corresponding to position 56 with glycine;

[0068] Substitution of the amino acid corresponding to position 64 with phenylalanine;

[0069] Substitution of the amino acid corresponding to position 69 with glycine;

[0070] Substitution of the amino acid corresponding to position 70 with isoleucine;

[0071] Substitution with tyrosine of the amino acid corresponding to position 80; and

[0072] Substitution of the amino acid corresponding to position 104 with isoleucine.

[0073] In one embodiment, the antibody or its antigen-binding fragment may include a heavy chain variable region comprising an amino acid sequence of either SEQ ID NO. 13 or 14. SEQ ID NO. 13 or 14 may be a heavy chain framework region.

[0074] In one embodiment, the antibody or its antigen-binding fragment may include a light chain variable region comprising the amino acid sequence of SEQ ID NO. 10 or 11. SEQ ID NO. 11 or 12 may be a light chain framework region.

[0075] In one embodiment, the heavy chain variable region and the light chain variable region may be connected directly or via a linker.

[0076] In one embodiment, the antibody or its antigen-binding fragment may comprise or be composed of any one of the amino acid sequences of SEQ ID NOs 16, 17, 23, or 24.

[0077] The antibody or its antigen-binding fragment according to the present specification may exhibit excellent G-CSF agonist efficacy by improving affinity for G-CSF receptors. Specifically, the antibody or its antigen-binding fragment according to one embodiment may promote the release of hematopoietic stem cells by inducing proteolytic activity, changes in the chemokine gradient, and scaffold reorganization within HSPCs.

[0078] An antibody or its antigen-binding fragment according to one embodiment can induce activity in both the myeloid and lymphoid systems.

[0079] The antibody or its antigen-binding fragment described herein does not occur naturally and may be synthesized chemically or recombinantly. Specifically, it may be synthesized by linking an Fc fragment to a scFv fragment using a linker.

[0080] In one embodiment, the antibody or its antigen-binding fragment may be a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0081] The antibody or its antigen-binding fragment described herein may have a high binding affinity to both human G-CSF receptors and mouse G-CSF receptors. That is, the antibody of the present invention is applicable to both clinical and preclinical studies.

[0082] The antibody or its antigen-binding fragment described herein may include not only the sequence of an anti-G-CSF receptor antibody but also biological equivalents thereof, and may be modified within a range capable of specifically recognizing the G-CSF receptor. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion, and / or substitution of amino acid sequence residues of the antibody. Such amino acid variations are made based on the relative similarity of amino acid side chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Additionally, the antibody or its antigen-binding fragment may be modified by conjugation or binding, glycosylation, tagging, or a combination thereof.

[0083] The antibody or antigen-binding fragment thereof that specifically binds to the G-CSF receptor described herein may include "variants" that maintain the same or corresponding activity. It is obvious that said variants may refer to polypeptides in which one or more amino acid residues are inserted, deleted, added, and / or substituted from said polypeptide sequence. That is, even if the present application is described as "an antibody or antigen-binding fragment having or containing the amino acid sequence described by a specific sequence number," an antibody or antigen-binding fragment having (or containing) an amino acid sequence in which some sequences are deleted, modified, substituted, or added may also be used in the present application, provided that it has the same or corresponding activity as the antibody or antigen-binding fragment consisting of the amino acid sequence of said sequence number. Accordingly, said antibody and / or antigen-binding fragment may include a fusion polypeptide connected to another polypeptide, provided that the desired biological activity and / or structure of said antibody and / or antigen-binding fragment is maintained. The above variant may have sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with respect to the antibody or its antigen-binding fragment while maintaining the same or corresponding activity, and for example, may have sequence identity of about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80%.

[0084] In the present invention, “amino acid” and “amino acid residue” mean natural amino acid, non-natural amino acid, or modified amino acid. Unless otherwise noted, all references to amino acid include references to both D and L stereoisomers, either generally or specifically by designation (where the structure allows for such stereoisomer forms). Natural amino acids include alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Non-natural amino acids include modified amino acid residues that are chemically modified at the N-terminal amino group or side chain, or reversibly or irreversibly chemically blocked, such as N-methylated D and L amino acids or residues in which a side chain functional group is chemically modified to another functional group.

[0085] The antibody or antigen-binding fragment thereof that specifically binds to the G-CSF receptor of this specification may be an antibody or antigen-binding fragment thereof that exhibits superior efficacy compared to wild-type G-CSF. Such antibody or antigen-binding fragment thereof may be a variant in which activity as a G-CSF receptor agonist is increased due to improved binding affinity to the G-CSF receptor, and as a result, the mobilization activity of hematopoietic stem / progenitor cells (HSPC) may be significantly superior compared to wild-type G-CSF.

[0086] Furthermore, the antibody or its antigen-binding fragment described herein may have the advantage of simultaneously activating the myeloid system and the immune system, unlike the myeloid-skewed response induced by G-CSF. Specifically, the antibody or its antigen-binding fragment may simultaneously induce the expression of lymphoid-associated chemokine receptors and ligands (Ccl6, Ccl9) along with the expression of lymphoid-associated chemokine receptors and ligands (Cxcr5, Cxcl9, Cxcl10, Ccr6), thereby enabling balanced immune activation of myeloid and lymphoid cells.

[0087]

[0088] Another aspect provides an antibody that specifically binds to the above-mentioned G-CSF receptor or a gene encoding an antigen-binding fragment thereof.

[0089] Another aspect provides a vector containing the above gene.

[0090] In this specification, the term “coding” refers to the inherent properties of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, which acts as a template for the synthesis of other polymers and macromolecules in a biological process having either a limited sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a limited sequence of amino acids, and the biological properties arising therefrom. As such, a gene encodes a protein when the transcription and translation of the mRNA corresponding to that gene in a cell or other biological system produces a protein. Both the coding strand, which is the same as the mRNA sequence and is usually provided in the sequence list, and the non-coding strand used as a template for the transcription of the gene or cDNA, may be said to encode the protein or other product of that gene or cDNA.

[0091] In this specification, "vector" refers to any nucleic acid construct capable of delivering or directing the transfer of foreign genetic material to a target cell where said polynucleotide may be replicated and / or expressed. The vector comprises the construct to be delivered. The vector may be a linear molecule or a circular molecule. The vector may be integrating or non-integrating.

[0092] In one embodiment, the vector may be any one selected from the group consisting of plasmids, cosmids, viruses, phages, recombinant expression cassettes, and transposons, but is not limited thereto; it is obvious that any vector among those commonly available that is suitable for the purpose of the present invention may be used.

[0093] It is obvious that if a gene or vector of the present invention has the same or corresponding function as the gene of the present invention, a gene sequence having a deletion, modification, substitution, or addition of a portion of the sequence may also be used in the present application.

[0094] In one embodiment, the gene may comprise a gene sequence encoding SEQ ID NO. 23 or 24, a part thereof, or a gene sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with these.

[0095]

[0096] Another aspect provides a recombinant cell containing the gene or vector above.

[0097] The recombinant cell may comprise a single vector containing a single gene encoding the antibody or antigen-binding fragment. The recombinant cell may be one into which a single vector containing genes encoding the light chain and heavy chain of the antibody or antigen-binding fragment, respectively, has been introduced; or one into which multiple vectors containing each of these genes have been introduced. Each gene or vector may be introduced into the recombinant cell independently, simultaneously, sequentially, or in reverse order.

[0098] In one embodiment, the recombinant cell may be an archaea cell, a bacterium cell, a eukaryotic cell, a eukaryotic unicellular organism, a somatic cell, a germ cell, a liver cell, a plant cell, an animal cell, a mammalian cell, a mouse cell, a non-human primate cell, or a human cell.

[0099]

[0100] Another aspect provides an antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof that specifically binds to the G-CSF receptor.

[0101] In this specification, "antibody-drug conjugate (ADC)" may refer to a substance having a structure in which a cytotoxic small molecule drug (payload) is conjugated to an antibody that binds to a specific target antigen on the surface of a cell or to an antigen-binding fragment thereof.

[0102] In one embodiment, the antibody-drug conjugate may be formed by binding a therapeutic agent (drug) to an antibody having target specificity or an antigen-binding fragment thereof through a linker. That is, the antibody-drug conjugate may include an antibody or an antigen-binding fragment thereof that specifically binds to the G-CSF receptor; a drug moiety; and a linker connecting the antibody or the antigen-binding fragment thereof and the drug moiety.

[0103] The above linker may include a cleavable linker or a non-cleavable linker. The cleavable linker may be cleaved by intracellular peptidase or protease enzymes, such as lysosome or endosome proteases, as with peptide linkers. For example, the cleavable linker may be any one selected from the group consisting of protease cleavable linkers, acid-cleavable linkers, disulfide linkers, beta-glucuronide-based (β-based) linkers, and beta-galactoside-based (β-based) linkers, but is not limited thereto. The non-cleavable linker may release a drug after the antibody is non-selectively degraded by intracellular hydrolysis.

[0104] According to one embodiment, the drug or therapeutic agent may be any one selected from the group consisting of chemotherapeutic compounds, cytotoxic compounds, immunomodulatory compounds, anticancer agents, antiviral agents, antimicrobial agents, antifungal agents, antiparasitic agents, and combinations thereof. For example, the cytotoxic compound may be any one selected from the group consisting of mitotic inhibitors, DNA alkyalting agents, topoisomerase inhibitors, and combinations thereof, but is not limited thereto. More specifically, the therapeutic agent may be MMAE_Protein A, cotinine-duocarmycin, or Herceptin, but is not limited thereto.

[0105]

[0106] Another aspect provides a pharmaceutical composition for the prevention or treatment of cancer, immune disease or blood disease comprising: the antibody or its antigen-binding fragment; a gene encoding the antibody or its antigen-binding fragment; a vector comprising the gene; a recombinant cell comprising the gene or the vector; or the antibody-drug conjugate.

[0107] In this specification, "prevention" refers to any act of administering the composition of the present invention to an individual to suppress or delay disease. For preventive benefit, the composition may be administered to a subject at risk of developing a specific disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom has not yet appeared.

[0108] In this specification, "treatment" refers to any act of administering the composition of the present invention to an individual to improve or benefit from the symptoms of a disease. As used herein, "treatment," "relief," or "improvement" may be used interchangeably. "Therapeutic benefit" means any therapeutically significant improvement of one or more diseases, conditions, or symptoms under treatment, or an effect thereon.

[0109] The pharmaceutical composition of this specification may be intended for diseases requiring hematopoietic stem cell transplantation or diseases requiring hematopoietic stem cell mobilization. The pharmaceutical composition of this specification may be suitable for patients for whom hematopoietic stem cell transplantation and mobilization are difficult, for example, elderly patients with low overall hematopoietic stem cell mobilization efficiency or patients with reduced response to existing treatments.

[0110] In this specification, "hematological disorder" refers to a structural or functional disorder occurring in the blood itself or blood components, meaning a disease in which abnormalities occur in the cells constituting the blood (red blood cells, white blood cells, platelets), plasma, blood proteins, blood coagulation system, etc., resulting in impaired normal blood function. The hematological disorder may include, without limitation, red blood cell-related disorders, blood coagulation disorders, blood cancers, and platelet disorders, as well as blood-related metabolic disorders, immune disorders, and bone marrow dysfunction disorders that occur congenitally or acquiredly. For example, the hematological disorder may include neutropenia caused by an immune disorder.

[0111] In one embodiment, the cancer may include a solid tumor or a blood cancer.

[0112] In one embodiment, the solid tumor may be a solid tumor requiring hematopoietic stem cell transplantation. For example, the solid tumor may include one or more selected from the group consisting of breast cancer, ovarian cancer, small cell lung cancer, pediatric tumor, neuroblastoma, Williams tumor, Ewing sarcoma, and germ cell tumor.

[0113] In one embodiment, the blood cancer may include one or more selected from the group consisting of leukemia, lymphoma, multiple myeloma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), Hodgkin's lymphoma (NHL), non-Hodgkin's lymphoma (NHL), and myelodysplastic syndrome (MDS).

[0114] In one embodiment, the immune disease may include an autoimmune disease, an immunodeficiency disease, or an immune hypersensitivity disease.

[0115] In one embodiment, the autoimmune disease may include one or more selected from multiple sclerosis, systemic sclerosis, Crohn's disease, systemic lupus erythematosus, and rheumatoid arthritis.

[0116] The pharmaceutical composition of the present invention may be in any form suitable for the intended method of administration. In the pharmaceutical composition of the present invention, "administration" means introducing a specific substance to a patient by any appropriate method.

[0117] The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. "Pharmaceutically effective amount" means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention, and may be adjusted according to factors including the type of disease of the patient, the severity of the disease, the type of active ingredient administered, the type of formulation, the age, gender, weight, health condition, diet, sensitivity, the time and method of administration of the drug, the combination of the composition or drugs used concurrently, and other factors well known in the medical field.

[0118] The dosage of the pharmaceutical composition for the prevention or treatment of a disease according to the present invention may be in the range of 0.01 µg / kg to 10 g / kg per day, specifically in the range of 0.01 mg / kg to 1 g / kg, depending on the patient's condition, body weight, gender, age, severity of the patient, and route of administration. Administration may be performed once a day or divided into several doses. Such dosage shall not be interpreted as limiting the scope of the present invention in any aspect.

[0119] The above dosage may vary depending on factors such as the formulation method, method of administration, patient's age, body weight, gender, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and a person skilled in the art can appropriately adjust the dosage by considering these factors. The frequency of administration may be once a day or two or more times within the range of clinically acceptable side effects, and the administration site may be one or two or more sites. The total duration of administration may range from 1 to 30 days per treatment, administered daily or at intervals of 2 to 5 days. If necessary, the same treatment may be repeated after the appropriate time. For animals other than humans, the dosage may be the same as that for humans per kg, or an amount calculated by converting the above dosage based on, for example, the ratio of organ (e.g., heart) volumes between the target animal and humans (e.g., average value).

[0120] The route of administration of the above pharmaceutical composition may be any general route as long as the drug can reach the target tissue. For example, the administration may be oral or parenteral, and may include, but is not limited to, ocular local administration (e.g., periocular (e.g., subtenon's), subconjunctival, intraocular, intravitreal, anterior chamber, subretinal, supracorbital, and retroocular administration), intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, local administration, intranasal administration, intrapulmonary administration, rectal administration, etc. Additionally, the pharmaceutical composition of the present invention may be administered by any device capable of delivering the active ingredient to target cells. It is preferable that the route of administration of the pharmaceutical composition of the present invention be determined according to the type of disease to which it is applied.

[0121] The pharmaceutical composition of the present invention may be used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, or parenteral formulations such as suspensions, emulsions, lyophilized preparations, topical preparations, suppositories, sterile injectable solutions, and implantable preparations, which are formulated according to conventional methods. In addition to the active ingredient, the pharmaceutical composition may further include pharmaceutically acceptable excipients that can be used for formulation.

[0122] The above excipients comprise a carrier, a vehicle, a diluent, a solvent, e.g., a monohydric alcohol, e.g., ethanol, isopropanol, and a polyhydric alcohol, e.g., glycerol, and an edible oil, e.g., soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, an oily ester, e.g., ethyl oleate, isopropyl myristate; It may include one or more selected from the group consisting of binders, adjuvants, solubilizers, thickeners, stabilizers, disintegrants, lubricants, buffers, emulsifiers, wetting agents, suspending agents, sweeteners, coloring agents, flavoring agents, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, low-melting point waxes, ion exchange resins, etc., but is not limited thereto.

[0123] The above carrier is one that is commonly used in formulations and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0124] The fact that the pharmaceutical composition of the present invention is formulated in the form of a parenteral administration formulation may mean that it is administered by a method of administration such as subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. In this case, in order to formulate the pharmaceutical composition into the parenteral administration formulation, the active ingredient is mixed in water with a stabilizer or a buffer to prepare a solution or suspension, and such a solution or suspension may be prepared in a unit dosage form in an ampoule or vial.

[0125] The pharmaceutical composition of the present invention being formulated in the form of an oral administration formulation may be, for example, a tablet, a pill, a hard or soft capsule, a liquid, a suspension, an emulsifier, a syrup, a granule, an elixir, etc. Depending on the conventional composition of each formulation, such oral administration formulations may include, in addition to the active ingredient, a pharmaceutically acceptable carrier such as a diluent such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine, or a lubricant such as silica, talc, stearic acid and its magnesium or calcium salt and / or polyethylene glycol.

[0126] When the above oral formulation is a tablet, it may include a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethyl cellulose and / or polyvinylpyrrolidine, and in some cases, it may include a disintegrant such as starch, agar, alginic acid or its sodium salt, a boiling mixture and / or an absorbent, a coloring agent, a flavoring agent or a sweetener, etc.

[0127] In addition, the above pharmaceutical composition may be sterilized or further include adjuvants such as preservatives, stabilizers, hydrating agents or emulsification promoters, salts and / or buffers for osmotic pressure regulation, and further include other therapeutically useful substances, and may be formulated according to conventional methods of mixing, granulation, or coating.

[0128] The content of the antibody in the pharmaceutical composition of the present invention can be appropriately adjusted according to the purpose of use of the pharmaceutical composition, the form of the formulation, etc., and, for example, may be 0.001 to 99 weight %, 0.001 to 90 weight %, 0.001 to 50 weight %, 0.01 to 50 weight %, 0.1 to 50 weight %, or 1 to 50 weight % based on the total weight of the pharmaceutical composition.

[0129] Another aspect provides a method for preventing or treating cancer, immune disease, or blood disease, comprising the step of administering an effective amount of the antibody or its antigen-binding fragment; a gene encoding the antibody or its antigen-binding fragment; a vector containing the gene; a recombinant cell containing the gene or the vector; or the antibody-drug conjugate to an individual in need thereof.

[0130] Another aspect provides the use of the antibody or its antigen-binding fragment for use in pharmaceutical preparations for the prevention or treatment of cancer, immune diseases or blood diseases; a gene encoding the antibody or its antigen-binding fragment; a vector comprising the gene; a recombinant cell comprising the gene or the vector; or the antibody-drug conjugate.

[0131] Another aspect provides the use of the antibody or its antigen-binding fragment for use in the manufacture of pharmaceutical preparations for the prevention or treatment of cancer, immune diseases or blood diseases; a gene encoding the antibody or its antigen-binding fragment; a vector comprising the gene; a recombinant cell comprising the gene or the vector; or the antibody-drug conjugate.

[0132]

[0133] The present invention is capable of various modifications and may have various embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present invention. In describing the present invention, detailed descriptions of related prior art are omitted if it is determined that such detailed descriptions may obscure the essence of the present invention.

[0134] The anti-G-CSF receptor-specific agonist antibody according to one embodiment has excellent affinity for G-CSF receptors and induces molecular programs that promote the release of HSPC, such as proteolytic activity, changes in chemokine gradients, and skeletal reorganization within HSPC, and can induce balanced activity in both the myeloid and lymphoid systems, so it can be used as a therapeutic strategy for cancer, immune diseases, or hematological diseases requiring HSPC induction, and has high binding affinity to both humans and mice, making it suitable for clinical and preclinical use.

[0135] Figure 1 is a schematic diagram of the phage display selection process for recombinant human G-CSFR (rhG-CSFR), an ELISA result graph confirming the binding ability of selected phages for each biopanning round (top), and a single phage ELISA result graph comparing the binding activity of individual clones in 3 panning rounds (bottom).

[0136] Figure 2 is a graph (top) measuring the binding power of 3B3 and variant anti-G-CSFR antibodies to recombinant human G-CSFR (rhG-CSFR) analyzed by ELISA, and a graph (bottom) measuring the proliferation multiplication of BaF3 / G-CSFR cells stimulated with 3B3 or variant anti-G-CSFR antibodies.

[0137] Figure 3A is a graph measuring the binding power of 3B3, C9, and C10 to rhG-CSFR analyzed by ELISA.

[0138] Figure 3B shows the results of SPR analysis of 3B3, C9, and C10 antibodies against rhG-CSFR (top) or recombinant mouse G-CSFR (rmG-CSFR) (bottom).

[0139] Figure 4 shows the results of analyzing the cell surface binding of 3B3, C9, and C10 antibodies by flow cytometry.

[0140] Figure 5 is a graph showing the proliferation rate of BaF3 / G-CSFR cells (left) and KASUMI-1 cells (right) 24 hours after G-CSF or antibody treatment.

[0141] Figure 6 shows G-CSF (10 ng mL -1 This is an image showing the results of Western blot analysis of BaF3 / G-CSFR cells (left) and KASUMI-1 cells (right) after stimulating them with ) or antibodies at the indicated concentration for 30 minutes.

[0142] Figure 7 shows G-CSF (10 ng mL -1 ) or C9 (10 μg mL) -1 This is an image showing the results of a Western blot analysis of BaF3 / G-CSFR cells stimulated with antibodies for the indicated time.

[0143] Figure 8 is an image showing the change in phosphorylation multiple of signaling molecules relative to their respective total forms by quantifying the density analysis results of Figure 7.

[0144] Figure 9 shows G-CSF (10 ng mL) in PB-CD34 cells obtained from three individual donors. -1 ) or granulocytes induced by antibodies at the indicated concentration (CD45 + , CD15 + This is a graph showing the numerical values.

[0145] Fig. 10 is a schematic diagram for evaluating the HSPC mobilization ability of 3B3, C9, and C10 (top) and LSK cells (Lin) in bone marrow and peripheral blood. - , Sca-1 + , c-kit + The ratio of ) and HSC cells (CD48 + , CD150 + This is a graph (bottom) showing the ratio of ). BM stands for bone marrow, and PB stands for peripheral blood. n = 6-11 mice were used per group.

[0146] Figure 11 is a schematic diagram for evaluating the HSPC mobilization capacity and changes in peripheral blood leukocyte counts of C9. BM stands for bone marrow, PB for peripheral blood, and SPL for splenocytes, and n = 3-5 mice were used per group.

[0147] Figure 12 shows LSK cells (Lin) in the bone marrow, peripheral blood, and spleen cells of adult mice and aged mice after treatment with G-CSF or C9 antibodies. - , Sca-1 + , c-kit + The ratio of ) and HSC cells (CD48 + , CD150 + This is a graph showing the ratio of ).

[0148] Figure 13 is a graph showing the blood levels of white blood cells (WBC), monocytes, neutrophils, and lymphocytes in adult (top) and aged (bottom) mice after treatment with G-CSF or C9 antibodies.

[0149] Figure 14 is an image showing the Uniform manifold approximation and projection (UMAP) of cell clusters in the bone marrow based on a single-cell transcriptome.

[0150] Figure 15 is a bar graph showing the proportion of each cell lineage upon G-CSF or C9 treatment.

[0151] Figure 16 is a Metascape heatmap showing the top 20 enriched GO terms of DEG upregulated in response to G-CSF or C9 treatment.

[0152] Figure 17 is a heatmap showing the scaled expression of genes related to chemokine response in the DEGs of each group.

[0153] Figure 18 is a Metascape heatmap (left) of commonly enriched GO terms of DEG upregulated in bone marrow-derived neutrophils in response to G-CSF or C9 treatment, and a dot plot (right) showing the GO cellular component analysis of DEG upregulated in C9-responsive neutrophils compared to an untreated control.

[0154] Figure 19 is a Metascape heatmap of commonly enriched GO terms of DEG upregulated in bone marrow-derived monocytes / macrophages in response to G-CSF or C9 treatment.

[0155] Figure 20 is a heatmap showing the average expression of Csf3r target genes in neutrophils and monocytes / macrophages by C9.

[0156] Figure 21 is a dot plot showing the average expression of major DEGs upregulated by G-CSF or C9 in macrophages compared to an untreated control group.

[0157] Figure 22 shows the change in the number of macrophage populations in the bone marrow (left), a violin plot showing Cxcr4 expression in macrophages (middle), and a box plot showing chemokine response scores (right).

[0158] Figure 23 is a bar graph showing the change in the ratio of T cell and B cell subclusters upon G-CSF or C9 treatment.

[0159] Figure 24 is a dot plot showing the average expression of major DEGs upregulated in T cells (top) and B cells (bottom) upon treatment with G-CSF or C9.

[0160] Figure 25 is a dot plot showing the analysis of the GO biological processes of upregulated DEGs in B cells that responded to G-CSF or C9 compared to an untreated control group.

[0161] Figure 26 is a violin plot showing gene expression related to activation and immune regulation in B cells.

[0162] Figure 27 is a schematic diagram of the evaluation of the hematopoietic reconstitution ability of cells mobilized to C9 in mice transplanted after radiation.

[0163] Figure 28 is a graph showing the survival rate (left) and body weight change (right) of transplanted mice.

[0164] Figure 29 is a graph showing the levels of white blood cells (WBC), monocytes, neutrophils, lymphocytes, and platelets in transplanted mice.

[0165] The following examples will be explained in more detail. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0166]

[0167] Reference Example 1. Cell lines (Cells)

[0168] Mouse pre-B cell line BaF3 was obtained from Dr. Arthur J. Sykowski (Beth Israel Deaconess Medical Center, Boston, MA, US) and cultured in RPMI-1640 medium (Lonza) supplemented with 10% fetal bovine serum (FBS; Hyclone), 1% antibiotic-antifungal mixture (GenDEPOT), and 5% WEHI-3B cell condition medium (WEHI-CM) as a source of IL-3. BaF3 cells were stably transfected with the pCMV-human CSF3R plasmid (Origene) to establish a BaF3 / G-CSFR cell line stably expressing human CSF3R. Surface expression of human CSF3R was confirmed by flow cytometry using the anti-CD114-APC-conjugated antibody (130-119-007; Miltenyi Biotec).

[0169] The KASUMI-1 cell line was obtained from the American Type Culture Collection (Manassas) and cultured in RPMI-1640 medium supplemented with 10% FBS and a 1% antibiotic-antifungal mixture according to the manufacturer's recommendations. Expi293F™ cells (Gibco) were cultured in Expi293F™ expression medium (Gibco) according to the manufacturer's recommendations.

[0170]

[0171] Reference Example 2. Construction of a Random Mutant Phage Library

[0172] The gene encoding the 3B3 single-chain variable fragment (SEQ No. 19) was randomly mutated using the GeneMorph II Random Mutagenesis Kit (Agilent) via repetitive error-prone PCR according to the manufacturer's recommendations. The gene, formed by combining the resulting secondary, tertiary, and quaternary PCR products, was inserted into the pCGMT phagemid located between SfiI restriction enzyme cleavages. The cloned gene was precipitated in ethanol and resuspended in distilled water. The concentrated phagemid was transformed into XL1-Blue susceptible E. coli (Agilent), and M13KO7 helper phage (NEB) was added at a multiplicity of infection of 20. Infected cells were sterilized with tetracycline (10 μg mL⁻¹). -1 ; Sigma-Aldrich), Carbenicillin (50 μg mL) -1 ; Sigma-Aldrich), kanamycin (70 μg mL) -1The phage particles were incubated overnight at 37°C and 250 rpm under conditions containing antibiotics (Sigma-Aldrich). The generated phage particles were precipitated using polyethylene glycol / NaCl and then resuspended in Tris-buffered saline (TBS) containing 1% bovine serum albumin (BSA; BD Biosciences).

[0173]

[0174] Reference Example 3. Antibody Screening

[0175] 3B3 variant phage library(diversity ≈ 6 × 10 8Solution-phase panning for ) was performed according to methods reported in previous studies [Shin, J., et al., "Thrombopoietin receptor agonist antibody for treating chemotherapy-induced thrombocytopenia." BMC Cancer, 23(1), 2023, pp. 490.][ Lee, BY, et al., "GDNF family receptor alpha-like antagonist antibody alleviates chemotherapy-induced cachexia in melanoma-bearing mice." J Cachexia Sarcopenia Muscle, 14(3), 2023, pp. 1441-1453.]. Briefly, recombinant human G-CSFR (rhG-CSFR; R&D system) was conjugated to Dynabeads M-270 epoxy beads (Invitrogen) overnight at 37°C while maintaining end-over-end rotation. After washing the beads with 0.1% skim milk added to phosphate-buffered saline (PBS), they were blocked with phosphate-buffered saline (PBS) containing 2% skim milk powder and 0.05% Tween 20 at room temperature for 1 hour. The blocked beads and the variant phage library were co-incubated at room temperature for 2 hours while maintaining end-over-end rotation, and then washed with PBS and 0.05% Tween 20. The bound phages were eluted with 0.2 M glycine-HCl (pH 2.2), immediately neutralized with 2 M Tris-HCl (pH 8.0), and then infected with XL1-Blue transsusceptible E. coli to amplify the phage particles for the next round of panning.

[0176]

[0177] Reference Example 4. Antibody expression and purification

[0178] The scFv genes of the selected clones (C9: SEQ No. 20, C10: SEQ No. 21) were inserted into the pFuse-Fc expression vector (Invitrogen) and then transfected into Expi293F™ cells according to the manufacturer's instructions. The collected supernatant was centrifuged sequentially at 300 g for 5 minutes and at 2,500 g for 20 minutes, followed by passing through a 0.22 μm filter. The scFv-Fc type antibody was purified using an AKTAprime Plus system (Cytiva). The HiTrap protein G HP column (Cytiva) was equilibrated with 20 mM sodium phosphate, the bound antibody was eluted with 0.1 M glycine-HCl (pH 2.7), and immediately neutralized with 1.0 M Tris-HCl (pH 9.0). The purified antibody was buffered with PBS using a 30 K MWCO Amicon Ultra-4 centrifuge (Millipore) and filtered using a 0.22 μm Spin-X centrifuge filter tube (Corning).

[0179]

[0180] Reference Example 5. ELISA

[0181] rhG-CSFR or BSA was coated onto 96-well microplates overnight at 4°C. After removing uncoated antigens, each well was washed with PBS and blocked with PBS containing 5% skim milk powder at 37°C for 1 hour. Subsequently, phages or antibodies were added and incubated at 37°C for 2 hours. After washing five times with PBS, the bound phages and antibodies were detected using anti-M13 phage-HRP (1:2000; 11973-MM05T-H, Sino Biological) or anti-Human IgG Fc-HRP (1:2000; 97225; Abcam), respectively. Absorbance at 450 nm was measured using a VersaMax™ Microplate Reader (Molecular Devices).

[0182]

[0183] Reference Example 6. Surface plasmon resonance analysis

[0184] The binding kinetics of the antibodies (3B3, C9, C10) were measured using a Biacore T200 system (Cytiva). rhG-CSFR and recombinant mouse G-CSFR (R&D System) were immobilized at a level of approximately 300 response units (RUs) on a research-grade carboxyl group (COOH) sensor chip (icluebio) using 10 mM sodium acetate buffer (pH 4.0). The chip was blocked with 1 M ethanolamine (pH 8.0), and two-fold serial dilutions (512–32 nM) of each antibody were fed at a flow rate of 10 μg / mL. -1 Individual injections were performed under the conditions. Data analysis was performed using Biacore T200 software.

[0185]

[0186] Reference Example 7. Cell-based binding assay

[0187] After washing BaF3 cells and BaF3 / G-CSFR cells with PBS, they were blocked with PBS containing 1% BSA and 5% normal goat serum (SPL Laboratory) at 4°C for 1 hour. Subsequently, the cells were incubated with 3B3, C9, C10 (50 nM) or an isotype control (DDXCH01P-100; Novus Biologicals) at 4°C for 1 hour. After washing the cells with PBS containing 1% BSA, they were incubated with anti-human IgG Fc-FITC conjugated antibody (1:2000; ab97224; Abcam) at 4°C for 1 hour. Samples were analyzed using a CytoFLEX flow cytometer (Beckman Coulter), and data were processed using FlowJo v10.7 (BD Life Sciences).

[0188]

[0189] Reference Example 8. Cell proliferation analysis

[0190] BaF3 / CSF3R cells (1 × 10⁴ cells / well) or KASUMI-1 cells (5 × 10⁴ cells / well) were seeded into a white 96-well plate with 100 μl of RPMI-1640 medium (containing 0.5% FBS). After equilibrating for 4 hours, a diluted series of recombinant human G-CSF (rhG-CSF; PeproTech) or antibody was added, and the cells were incubated for 24 hours under humid conditions (37°C, 5% CO₂). Cell viability was analyzed using the CellTiter Glo® Luminescence Cell Viability Analyzer Kit (Promega). After adding 100 μl of reagent, the plate was shaken for 2 minutes and then equilibrated at room temperature for 10 minutes, and luminescence (450 nm wavelength) was measured using a Victor 3 1420 Multilabel counter (PerkinElmer).

[0191]

[0192] Reference Example 9. CCLE data mining for CSF3R expression

[0193] RNA-seq expression data for all cell lines in the Cancer Cell Line Encyclopedia (CCLE, DepMap 22Q2) were obtained via cBioPortal (https: / www.cbioportal.org / ; Broad Institute, USA). From the entire dataset (n = 1,739), only 173 cell lines classified as hematopoietic or lymphoid malignancies were filtered. CSF3R (Ensembl ID ENSG00000119535) transcriptome expression levels were tabulated and ranked to select the cell line with the highest endogenous expression. The KASUMI-1 cell line showed an absolute value of 119.5 RPKM relative to the average expression level (11.5 RPKM), and was selected for subsequent validation experiments.

[0194]

[0195] Reference Example 10. Western blot analysis

[0196] BaF3 / CSF3R cells at a final density of 4 × 10 5 cells mL -1After inoculation, the cells were serum-depleted overnight (kinetic experiments) or for 3 hours (30-minute endpoint analysis) in RPMI-1640 (containing 0.5% FBS) under humidified conditions (37°C, 5% CO₂). Subsequently, cells were stimulated with rhG-CSF or antibodies (3B3, C9, C10) according to the concentrations and times specified in each figure. The reaction was stopped by washing twice with ice-cold PBS, and the cell pellet was dissolved in Radioimmunoprecipitation Assay (RIPA) buffer (LPS Solution) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Protein concentration was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein (10 μg per lane) were heated in SDS-PAGE sample buffer for 5 minutes, electrophoresed on an 8-12% SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane (Millipore). Membranes were cleaved as necessary and blocked for 1 hour in TBS containing Tween 20 and 5% BSA, followed by incubation with the following primary antibodies (Cell Signaling Technology, unless otherwise noted) for 1 night at 4°C: JAK1(3344S), p-JAK1(Y1034 / 1035; 74129S), JAK2(3230S), p-JAK2(Y1007 / 1008; 3776S), STAT3(4904S), p-STAT3(Y705; 9145S), STAT5(94205S), p-STAT5(Y694; 9351S), ERK1 / 2(9102S), p-ERK1 / 2(Thr202 / Tyr204; 9101S), AKT(9272S), p-AKT (S473; 9271S). β-Actin (AC-15; Novus Biologicals) was used as a loading control.After washing, the membrane was incubated with HRP-conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Cell Signaling Technology) at room temperature for 1 hour. Bands were visualized using SuperSignal™ Western Blot Substrate (GE Healthcare) and captured using an Amersham™ ImageQuant 800 system (Thermo Fisher Scientific). Band density analysis was performed using ImageJ, and the phospho / total ratio was normalized to the value at 0 min.

[0197]

[0198] Reference Example 11. PB-CD34 + Cell separation (PB-CD34 + cell isolation)

[0199] This study utilized human-derived samples, was conducted in accordance with the Declaration of Helsinki, and received approval from the Institutional Review Board (IRB) of Kosin University Gospel Hospital after obtaining written consent from the donors (IRB Approval No.: KUGH 2022-12-6808, CD34 + Cell use). G-CSF (10 μg kg from healthy volunteers) -1 day -1 Peripheral blood mobilized for 5 days was collected by apheresis (Kosin University Gospel Hospital, Busan, South Korea). Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation without a brake at 400 g for 30 minutes using Ficoll-Paque™ PLUS (Cytiva). The PBMC layer was recovered, diluted 1:2 in ice-cold PBS containing 0.5% BSA (Merck KGaA), and washed twice at 300 g for 10 minutes each.

[0200] CD34 +Hematopoietic stem and progenitor cells were immunomagnetically selected using the MACS CD34 MicroBead UltraPure kit (Miltenyi Biotec) with an LS column of a QuadroMACS™ separator according to the manufacturer's protocol. The purified cells were counted using an ADAM-MC automated cell counter (NanoEntek) and resuspended in RPMI-1640 (Thermo Fisher Scientific) containing 10% FBS. Quality control was performed via flow cytometry using an anti-human CD34-PE (130-113-179; Miltenyi Biotec) analyzer (Beckman Coulter), and the results confirmed that the purity of the purified cells was always over 95%.

[0201]

[0202] Reference Example 12. Granulocyte differentiation analysis

[0203] PB-CD34 + Cells were suspended in serum-free expansion medium (StemCell Technologies), and the final cell density was 4 × 10⁶ 5 cells mL -1 It was made so that... In the medium, minimal granulocyte cytokine cocktail—LDL (20 μg mL) -1 ; Stem Cell Technologies), SCF (50 ng mL -1 ; PeproTech), IL-3(10 ng mL -1 ; PeproTech)― was added. Cells were treated with rhG-CSF or antibodies (3B3, C9, C10). Cells were cultured at 37°C, 5% CO₂, and humidified conditions, and half of the culture medium was replaced with fresh medium every 3 days, and the replacement medium contained a granulocyte cytokine cocktail and rhG-CSF or antibodies.

[0204] Cells were harvested on days 7 and 11 of culture, washed with PBS / 0.5% BSA, and stained with anti-CD45-FITC (130-110-631; Miltenyi Biotec) and anti-CD15-PE (130-113-485; Miltenyi Biotec) for 30 minutes at 4°C. Non-viable cells were excluded immediately before acquisition by adding 7-amino-actinomycin D (7-AAD; eBioscience). Data were collected using a CytoFLEX flow cytometer (Beckman Coulter), and gating was performed on 7-AAD singlet cells followed by CD45 + / CD15 + Double-positive cells were established. The granulocyte production volume was mL -1 CD45 + / CD15 + It was expressed as the absolute number of cells and the fold change relative to the inoculation density on day 0.

[0205]

[0206] Reference Example 13. In vivo experiment

[0207] All animal experiment procedures were approved following deliberation by the Institutional Animal Care and Use Committee (IACUC) of Inje University College of Medicine (IACUC Approval No.: 2023-005). For this study, wild-type female mice aged 12–14 weeks and wild-type C57BL / 6 mice aged 18–24 months (Orient Bio) were used. Mice were housed in ventilated microisolator cages under Special Pathogen-Free (SPF) / barrier conditions set to a 12-hour day-night rotation cycle at a temperature of 22–24°C. The experimental groups were administered rhG-CSF via subcutaneous injection for 5 consecutive days or each antibody (3B3, C9, C10) as a single dose.

[0208]

[0209] Reference Example 14. Immunostaining and Analysis of Hematopoiesis and Blood Cells

[0210] Bone marrow, spleen, and peripheral blood were collected for flow cytometry. Bone marrow cells were obtained by flushing the femur using a syringe; red blood cells were lysed with ammonium chloride Tris buffer and resuspended in FACS buffer (PBS + 4% FBS). The spleen was mechanically separated using a gentle MACS (Miltenyi Biotec), filtered through a 70 μm cell strainer, and suspended in PBS. Peripheral blood was collected via cardiac puncture or ocular blood sampling using a 1 mL syringe coated with 2.5 mM EDTA. Subsequently, plasma was removed by centrifugation at 1,500 rpm for 3 minutes.

[0211] For HSPC analysis, cells were stained with the following lineage marker antibodies (BD Bioscience): PerCP-Cy5.5 (560685), c-Kit-APC (567127), Sca-1-FITC (562058), CD48-PE-Cy7 (560731), and CD150-PE (562651). For macrophage analysis, cells were stained with CD45.2-APC, CD11b-PE (561689), F4 / 80-FITC, and Ly6G-PE-Cy5 (560602). Data were acquired using a flow cytometer and analyzed with FlowJo software. Platelet and leukocyte counts were measured at EONE Laboratories (Incheon, South Korea).

[0212]

[0213] Reference Example 15. Splenocyte transplantation assay

[0214] Splenocytes were isolated from 12–14 week old wild-type mice treated with rhG-CSF or antibody (C9). 5 × 10⁶ cells were placed in PBS. 5After resuspending canine splenocytes, they were serially transplanted through the tail vein of mice that had received 9.5 Gy irradiation.

[0215]

[0216] Reference Example 16. Single-cell RNA library construction and sequencing

[0217] To generate single-cell RNA sequencing samples, rhG-CSF (250 μg·kg⁻¹) was administered to mice. -1 ·day -1 Administer ) subcutaneously once daily for 2 consecutive days, or C9 (250 μg·kg -1A single injection of ) was administered, and PBS was administered as a control. Bone marrow cells were collected by washing the femur 32 hours after the initial administration. Cells from two mice in each group were pooled prior to fixation, and samples were prepared using the GEM Single Cell Fixed RNA Sample Preparation Kit (PN-1000414; 10x Genomics). Library preparation and sequencing were performed at Macrogen (Seoul, South Korea). The single-cell RNA library was constructed according to the Demonstrated Protocol Cell & Nuclei Fixation Chromium Fixed RNA Profiling (CG000478). This protocol includes procedures for fixation, storage, and post-storage processing using the Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit (PN-1000414; 10x Genomics). The library was constructed using Chromium X according to the Chromium Fixed RNA Profiling Reagent Kits for Multiplexed Samples User Guide (CG000527). Probes were added to fixed samples and hybridized to target RNA overnight. After probe hybridization, samples containing different probe barcodes were pooled. The pooled samples were mixed with the master mix and loaded into Next GEM Chip Q along with Single Cell Gel Beads and Partitioning Oil. Probes hybridized to target RNA were assigned unique barcodes within the droplets. After GEM recovery, the products were pre-amplified, followed by indexing via sample index PCR.The purified library was quantified by qPCR according to the qPCR Quantification Protocol Guide (KAPA), and its quality was verified using an Agilent Technologies 4200 TapeStation (Agilent Technologies). Subsequently, the library was sequenced using the NovaSeq platform (Illumina) according to the read lengths specified in the user guide.

[0218]

[0219] Reference Example 17. scRNA-seq data processing

[0220] Single-cell gene expression data were analyzed using Cell Ranger v7.2.0 (10x Genomics; https: / / support.10xgenomics.com / single-cell-gene-expression / software / pipelines / latest / what-is-cell-ranger). The raw count matrix from 10x Genomics was loaded into Seurat 4.3.0. For downstream analysis, all genes expressed in three or more cells and the raw counts of all cells in which at least 200 genes were detected were used. To remove rare cell populations with significantly high gene detection counts (potential multiplets) or low-quality or dying cells, cells with more than 6,000 genes or a mitochondrial gene ratio exceeding 5% were filtered out. After filtering, count values ​​for 14,755 genes from a total of 49,689 cells were selected for further analysis.

[0221] Gene expression values ​​were scaled and normalized for each gene across all integrated cells. Batch effects were corrected using anchors and canonical correlation analysis (CCA) from the Seurat R library. LogNormalize, a global scale normalization method, was applied by dividing the gene expression value of each cell by the total expression amount, multiplying by a scale factor (default 10,000), and then log-transforming the result. Genes with high variability were identified by examining the relationship between variability and mean expression, and dimensionality reduction analysis was performed using these genes. The mitochondrial gene content ratio was regressed for each feature, and the resulting residuals were scaled and centered. These model residuals were then used for subsequent dimensionality reduction and clustering analyses. Single-cell transcriptome data were processed and analyzed using the Seurat v5.0.1 R package.

[0222]

[0223] Reference Example 18. Cell type annotation

[0224] Clustering and UMAP analysis were performed based on statistically significant principal components. Subsequently, the most significant top cluster markers in each cluster were determined by the Wilcox rank sum test (min.pct=0.25, logfc.threshold=0.25, filtering only positive values) in comparison to all other cells. Cell types were identified using ScType. The ScType score is calculated as the cluster summary enrichment score for each cell type, and the cell type with the highest ScType score was assigned to the corresponding cluster.

[0225]

[0226] Reference Example 19. Differential gene expression and functional enrichment analysis

[0227] Differential gene expression analysis was performed by cell type, and each treatment group was compared with the control group. Differentially expressed genes (DEGs) were identified using Seurat's FindAllMarkers function (with the Wilcoxon rank-sum test applied as a default parameter), and the false discovery rate (FDR) was controlled by performing multiple comparison correction using the Benjamini-Hochberg method for the P-values. Genes with a corrected p-value less than 0ER.05 and a log fold-change of 0.3 or greater (i.e., corresponding to a change of 1.25 or more) were considered to be significantly differentially expressed.

[0228] Functional enrichment analysis was performed for each cell population using the EnrichR package to interpret the DEG list in a biological context. This analysis included Gene Ontology (GO) term enrichment in categories of biological processes, molecular functions, and cellular components. Additionally, enrichment analysis for DEGs was performed using the Metascape web tool (https: / metascape.org). Enrichment terms and pathways with an adjusted p-value of less than 0.05 were considered statistically significant.

[0229]

[0230] Reference Example 20. Gene set scoring and cell cycle analysis

[0231] The gene set score (GO: 1990868) associated with chemokine response was calculated using the AddModuleScore function of the Seurat package. The following GO path was used. Cell cycle analysis classified each cell into G1, S, or G2M phases based on the CellCycleScoring function of the Seurat package.

[0232]

[0233] Reference Example 21. Analysis of Receptor-Ligand Interactions

[0234] Csf3 / Csf3 receptor interactions were first evaluated using CellPhonDB (p<0.05). Next, NichNet was applied to DEGs of Csf3 receptor-expressing cell populations (neutrophils, monocytes, and macrophages) to rank Csf3 downstream target genes and visualize upstream target genes.

[0235]

[0236] Reference Example 22. Statistical Analysis

[0237] All data were expressed as the mean ± standard deviation (sd) of the specified replicate experiments. Statistical analysis was performed using GraphPad Prism 8.0 software, employing one-way and two-way ANOVAs and post-hoc tests. EC50 values ​​were calculated using non-linear regression with a 4-mediated logistic model. For scRNA-seq analysis, differences in gene expression were evaluated using the Kruskal-Wallis test, and post-hoc tests were applied to comparisons of individual gene expressions. P-values ​​obtained from pairwise tests were corrected for multiple comparisons using the Benjamini-Hochberg false discovery rate (FDR) method.

[0238]

[0239]

[0240] Example 1. Selection of candidate anti-G-CSFR agonist antibodies

[0241] As a strategy to improve the affinity of the wild-type anti-G-CSFR agonist antibody (3B3), variants were generated by performing error-prone PCR to introduce mutations across the variable region of 3B3, and clones with higher binding activity to the antigen were selected. To select variant phage clones that bind to G-CSFR, panning was performed in solution, and 96 phages were randomly selected from the results to compare their individual binding activities (Fig. 1). Among these, the top 10% of 9 clones that showed the highest binding signals were selected as preliminary candidate variants (see arrows in Fig. 1).

[0242] To select the final candidate clones, clones with duplicate sequences were excluded, and after converting to the scFv-Fc format (fragment crystallizable-fused single-chain variable fragment format), binding activity and agonist activity were compared.

[0243] As a result, C9 showed the best binding activity, and C10 at a low concentration (0.05 μg mL) -1 It also effectively induced proliferation of BaF3 cells (BaF3 / G-CSFR) expressing human G-CSFR (Fig. 2). In addition, both clones bound to recombinant G-CSFR with nanomolar Kd values ​​and exhibited cross-reactivity between humans and mice (Figs. 3a and 3b).

[0244]

[0245] Additionally, cell surface binding of antibodies 3B3, C9, and C10 was confirmed. Both clones specifically bound to Ba / F3 cells expressing human G-CSFR but not to parental Ba / F3 cells, with C9 showing the highest binding signal (Fig. 4). To gain structural insights, the variant clones were modeled and each was compared to 3B3, and no rearrangement of the overall three-dimensional structure was observed.

[0246]

[0247] Therefore, C9 and C10 were finally selected as candidate 3B3 variants. The amino acid sequences and sequence numbers of the complementarity determining regions (CDRs) of 3B3, C9, and C10 are shown in Table 1 below, and the mutated positions are indicated in bold and underlined.

[0248]

[0249] 3B3C9C10L-CDR1QGISSW(Sequence No. 1)QGISSW(Sequence No. 1)QGISSW(Sequence No. 1)L-CDR2AASAASAASL-CDR3CLQHNTYPFTF(Sequence No. 2)CLQHNTYPFTF(Sequence No. 2)CLQHNTYPFTF(Sequence No. 2)H-CDR1GGSISSGGYY(Sequence No. 3)GASISSGGYY(Sequence No. 6)GGSISSGGYY(Sequence No. 5)H-CDR2IYYSGST(Sequence No. 4)IYYSGST(Sequence No. 4)IYYGGST(Sequence No. 7)H-CDR3CARWNGVNNAFDIW(Sequence No. 5)CARWNGVINAFDIW(Sequence No. 8)CARWNGVNNAFDIW(Sequence No. 5)

[0250]

[0251] Example 2. Confirmation of G-CSF receptor binding ability and agonist activity of 3B3 variant antibody

[0252] To evaluate the effect of improved binding affinity on agent activity, proliferation and signaling responses induced by the antibody were analyzed using a cell line capable of G-CSF-dependent proliferation.

[0253] As a result, C9 and C10 were 0.51, 1.5, and 3.9 ng mL, respectively. -1 Exhibiting EC50 values, they induced proliferation of BaF3 / G-CSFR cells at concentrations lower than 3B3, and the maximum efficacy among the three antibodies remained similar (Fig. 5). Furthermore, as a result of short-term stimulation of BaF3 / G-CSFR cells or KASUMI-1 cells, C9 and C10 at concentrations lower than 3B3 (0.1, 1 μg mL⁻¹).-1 It more strongly activated G-CSFR-related downstream signaling in ) (Fig. 6). Due to the enhanced affinity, C9 had superior agonist efficacy and signaling activity compared to C10.

[0254] In addition, C9 (10 μg mL) -1 C9 induced signal transduction phosphorylation at a level similar to that of G-CSF. Time-course analysis showed that phosphorylation induced by G-CSF decreased rapidly after 1 hour, whereas signal transduction by C9 persisted throughout the observation period (Fig. 7). Density analysis revealed that C9 exhibited phosphorylation levels 4 to 20 times higher than G-CSF in various signaling pathways at 18 hours, indicating a longer-lasting signal response (Fig. 8).

[0255]

[0256] Additionally, to compare agonist activity more functionally, CD34, the major biological effect of G-CSF + The ability of antibodies to induce granulocyte differentiation from hematopoietic progenitor cells was analyzed. [Mehta, HM and Corey, SJ "G-CSF, the guardian of granulopoiesis." Semin Immunol, 54, 2021, pp. 101515.]

[0257] CD34 obtained from three independent healthy donors + In cell-based analysis, both variants and 3B3 induced granulocyte formation in a concentration-dependent manner. C9 CD45 on day 7 + CD15 +The cell count increased 64–76-fold on day 7 and 170–475-fold on day 11. C10 showed a moderate increase (52–57-fold on day 7, 225–365-fold on day 11), while 3B3 showed the lowest level of proliferation (37–40-fold on day 7, 190–255-fold on day 11) (Fig. 9). Collectively, C9 and C10 are agonist antibody variants with excellent activity, and in particular, C9 is the most potent agonist antibody variant among the 3B3 derivatives.

[0258]

[0259] Example 3. Confirmation of the mobilization ability of 3B3 variant antibody to hematopoietic stem cells (HSPC)

[0260] To determine whether differences in agent efficacy lead to functional improvement in vivo, the HSPC mobilization ability of each antibody was evaluated in mice.

[0261]

[0262] First, after a single injection of 3B3, C9, or C10 into wild-type C57BL / 6 mice, flow cytometry was performed on bone marrow (BM) and peripheral blood (PB) to [indicate] LSK cells (Lin-Sca-1 + c-Kit + ) and phenotype HSC(CD48 within the LSK population + CD150 + ) was analyzed.

[0263] As a result, all three antibodies reduced the frequency of LSK and HSC in the bone marrow, which was accompanied by an increase in the proportion of corresponding cells in the peripheral blood. C9 most effectively induced HSPC efflux, while C10 showed higher efficacy compared to 3B3 in vitro and exhibited a similar level of efficacy to 3B3 in vivo (Fig. 10).

[0264]

[0265] Next, the efficacy of C9 and G-CSF was compared in adult and aged C57BL / 6 mice to evaluate the potential of C9 as an alternative mobilization agent candidate. Specifically, C9 was administered as a single injection, and G-CSF was administered daily for 5 consecutive days (Fig. 11).

[0266] As a result, in adult mice, C9 reduced the frequency of LSK and HSC in the bone marrow (BM) more effectively than G-CSF, while simultaneously increasing the proportion of these cells in peripheral blood (PB) and spleen (SPL). In aged mice, responsiveness to both agents was low, but C9 still significantly mobilized LSK and HSC populations, whereas G-CSF failed to induce significant changes (Fig. 12).

[0267]

[0268] In addition to HSPC, G-CSF can release leukocytes produced and stored in the bone marrow into the bloodstream. To evaluate whether C9 induces a mobilization pattern similar to G-CSF, leukocyte levels in peripheral blood were monitored after treatment with C9 and G-CSF.

[0269] As a result, in adult mice, G-CSF significantly increased total peripheral blood leukocyte counts during the early stages of treatment, which was induced primarily by a short-term increase in monocytes. In contrast, C9 significantly increased total leukocyte counts during the later stages of treatment, during which lymphocytes increased and neutrophils increased more effectively compared to G-CSF (Fig. 3d). This distinct pattern was also observed in aging mice, but the magnitude of leukocyte changes was relatively smaller. In particular, while G-CSF induced lymphocyte depletion along with a decrease in total leukocyte counts during the later stages, C9 maintained lymphocyte levels and kept total leukocyte counts at a level similar to that of the untreated control group (Fig. 13).

[0270]

[0271] In summary, these observations highlight two key features of C9: (i) C9 possesses superior HSPC mobilization efficacy compared to G-CSF. (ii) C9 induces a unique mobilization pattern distinct from the myeloid-skewed response characteristic of G-CSF. Specifically, C9 prioritized lymphocyte mobilization in the late post-administration phase. These differential temporal mobilization patterns and lineage-specific mobilization characteristics imply that C9 can regulate the bone marrow microenvironment in a manner distinct from G-CSF.

[0272]

[0273] Example 4. Transcriptome changes in whole bone marrow (BM) hematopoietic cells induced by treatment with 3B3 variant antibody

[0274] To explore the changes induced by C9 in the bone marrow microenvironment, hematopoietic cells from the bone marrow were collected in the early stages before HSPC efflux began following C9 or G-CSF treatment, and single-cell RNA sequencing was performed, and cellular and molecular profiles were comprehensively analyzed. Eleven clusters were identified based on lineage-specific transcription factors and classified into five major hematopoietic lineages (HSPC, myeloid lineage, T / NK, B, erythrocyte / megakocyte) (Fig. 14).

[0275] As a result, G-CSF reduced the relative proportion of lymphoid cells and increased the myeloid cell population, consistent with previous reports. However, C9 did not significantly alter the cell composition compared to the untreated control group (Fig. 15).

[0276]

[0277] Additionally, the gene expression patterns of inflammatory response pathways were compared, and since G-CSF is known to reorganize the bone marrow chemokine network that promotes leukocyte efflux, the expression patterns of chemokine-related genes were compared.

[0278] As a result, hematopoietic cells stimulated with G-CSF or C9 antibodies upregulated inflammatory response-related pathways. Notably, only C9 induced pathways related to adaptive immune responses, which implies a more balanced immunomodulatory effect of C9 (Fig. 16).

[0279] In addition, G-CSF upregulated genes (Hif1a, Cxcr1, Ccl4) related to inflammatory regulatory factors and the recruitment of myeloid cell populations, whereas C9 induced the expression of chemokine receptors and ligands related to both myeloid (Ccl6, Ccl9) and lymphoid (Cxcl9, Cxcl10) (Fig. 17).

[0280]

[0281] In summary, G-CSF promotes inflammatory mobilization that is primarily biased toward the myeloid system, whereas C9 activates both myeloid and lymphoid programs more broadly and in a balanced manner during the initial mobilization phase.

[0282]

[0283] Example 5. Transcriptome and Functional Pathway Analysis of a Bone Marrow (BM) Derived Csf3r-Expressing Myeloid Population Treated with 3B3 Variant Antibody

[0284] To identify the cellular contributors and transcriptional programs that form the bone marrow (BM) microenvironment formed by C9, we focused on major populations (neutrophils, monocytes, and macrophages) expressing Csf3r.

[0285] In neutrophils, both C9 and G-CSF commonly upregulated genes related to neutrophil degranulation (Snap23, Fpr2, S100a8) and genes related to inflammatory signaling (Il1f9, Tlr13). This supports the common induction of neutrophil priming (Fig. 18, left). Genes upregulated by C9 were enriched in granule-related pathways (azurophilic granules and tertiary granules) involved in transient niche disintegration, including proteases (Fig. 18, right). These changes support the idea that C9 contributes to the formation of a protease-friendly bone marrow environment, similar to G-CSF. In the monocyte / macrophage population, both agents increased gene expression related to biological stimulus responses (Nfkbia, Trem1) and inflammation (Tlr6, Ccr1, Ywhaz) (Fig. 19).

[0286]

[0287] We predicted the target genes of the G-CSF / G-CSFR axis and investigated their expression patterns to verify the agonist activity of C9.

[0288] As a result, the predicted targets in neutrophils included Stat3 and G-CSF-inducing markers (Cxcr2, Plscr1). Genes commonly increased by both agents were associated with neutrophil maturation (Cxcr2, Plscr1), cytoskeletal organization (Flot1, Tpm4, Twf1), and immunomodulation (CD47, Hmox1, Mxd1). G-CSF preferentially induced proteases (Ctsb, Ctsl) and Stat3, whereas C9 upregulated Stat1 and inflammatory regulatory factors (Thbd, Acsl1) (Fig. 20, top).

[0289] In macrophage / monocyte populations, both agents induced genes related to metabolic inflammation (Plscr1) and inflammation resolution (Gadd45b), but showed differences in regulating different gene sets. G-CSF activated Stat3, Ctsl, and stress response genes (Ler3, Grina, Bcl6, Chil), whereas C9 promoted Stat1, genes related to cytoskeletal reorganization (Ccdc88a, Rin2, Tns3), and genes related to immunomodulation (Hmox1, Nampt) (Fig. 20, bottom).

[0290] These common transcriptional changes observed across the G-CSFR expression population support the agonist activity of C9, and the differential gene expression profile compared to G-CSF supports the potential for C9 to differently regulate the bone marrow environment and immune composition.

[0291]

[0292] Next, we focused on and analyzed macrophages that regulate adhesion molecules and chemokine expression. By a well-known mechanism, G-CSF promotes HSPC mobilization by temporarily depleting these cells [Christopher, MJ, et al. “Expression of the G-CSF receptor in monocytic cells is sufficient to mediate hematopoietic progenitor mobilization by G-CSF in mice.” J Exp Med, 208(2), 2011, pp. 251-260.].

[0293] As a result, C9 induced gene expression associated with anti-inflammatory signals (Tsc22d3, Nfkbia, Dusp1) and glucocorticoid responses (Fkbp5, Ddit4), which can induce an anti-inflammatory response along with a decrease in adhesion molecule expression. Conversely, G-CSF activated a regulated inflammatory response (Myd88, Sema6b, Ifitm1,2, Il4ra, Soc3, Cd244a, Tgfbi, Steap4) containing both pro-inflammatory and anti-inflammatory genes (Fig. 21).

[0294]

[0295] Additionally, to determine whether these transcriptome profiles lead to changes in the bone marrow microenvironment, we classified macrophages into two subgroups and analyzed changes in their numbers.

[0296] As a result, C9 reduced the total number of macrophages compared to G-CSF in the early stages (Fig. 22, left). In addition, macrophages stimulated with C9 showed higher chemokine response scores and increased Cxcr4 expression compared to cells stimulated with G-CSF (Fig. 22, right).

[0297]

[0298] Example 7. Transcriptome changes in T / B cells by treatment with 3B3 variant antibody

[0299] Disruption of the bone marrow (BM) microenvironment by G-CSF indirectly adversely affects the maintenance of the lymphatic system. To confirm this, transcriptomic changes in T-cell and B-cell populations were analyzed in the early stages after treatment.

[0300] As a result, C9 stimulation preserves the Treg population and, compared to the untreated control group, effector-memory and tissue-resident CD8 + It increased T cell frequency, which was in contrast to the lymphatic depletion observed in G-CSF (Fig. 23).

[0301]

[0302] These observations were also supported by transcriptional changes. T cells stimulated with G-CSF upregulated stress-related and proliferation-inhibiting genes (Nos2, Ifitm1, Steap4, Lipg), whereas C9 selectively increased the expression of genes associated with anti-inflammatory signals (Tsc22d3, Zfp36l2) and cell survival pathways (Il7r, Jak1) (Fig. 24, top). Similar differences were observed in the B cell population. While G-CSF significantly depleted B-lineage cells and induced inflammation-related programs, C9 increased the proportion of precursor and mature B cells (Fig. 24, right). Transcriptome analysis of B cells stimulated with C9 revealed an abundance of activation and differentiation pathways, suggesting that C9 can support early B cell development in the bone marrow (Fig. 25).

[0303]

[0304] Furthermore, these B cells exhibited transcriptional signatures associated with antigen processing and T-cell interactions, including the upregulation of the BCR signaling pathway, MHC class II pathway genes (H2-Aa), and T-cell interaction-related genes (Cd40, Icosl). Notably, this transcriptional program is consistent with the results of a previously performed whole bone marrow DEG analysis. That analysis showed an abundance of pathways related to adaptive immunity activation and antigen presentation via MHC class II (Fig. 16). In particular, the co-upregulation of inhibitory co-stimulatory molecules (Cd200, Btla) implies that in C9-stimulated bone marrow, these B cells can contribute to forming a balanced immune environment by enhancing adaptive immune capabilities while simultaneously suppressing excessive activation (Fig. 26).

[0305]

[0306] Example 8. Reconstitution ability and lymphocyte recovery by 3B3 variant antibody-mobilizing cells

[0307] To evaluate the reconstitution ability of HSPCs mobilized by C9, whole splenocytes containing LSK cells and HSCs from donor mice treated with C9 or G-CSF were transplanted into lethally irradiated mice (Fig. 27).

[0308] As a result, the transplanted mice had their survival extended by more than 50 days and their body weight recovered (Fig. 28), which indicates that the functional integrity of the HSPC mobilized by C9 was preserved.

[0309] One month after transplantation, peripheral blood leukocyte counts recovered to levels similar to those of healthy controls, but there were slight differences in lineage composition. Mice transplanted with G-CSF-mobilizing cells showed increased monocyte counts, while mice transplanted with C9-mobilizing cells showed relatively higher lymphocyte counts (Fig. 29).

[0310]

[0311] These results show that while both C9 and G-CSF support hematopoietic reconstitution, C9 can promote relatively greater lymphocyte recovery compared to G-CSF.

[0312]

[0313] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. A granulocyte-colony stimulating factor (G-CSF) receptor-specific antibody or an antigen-binding fragment thereof comprising one or more amino acid variations selected from the group consisting of the following: - Substitution of an amino acid corresponding to position 2 of the amino acid sequence of SEQ ID NO. 3; - Substitution of an amino acid corresponding to the 4th position of the amino acid sequence of SEQ ID NO. 4; and - Substitution of the amino acid corresponding to position 8 of the amino acid sequence of SEQ ID NO.

5.

2. An antibody or an antigen-binding fragment thereof, wherein the amino acid variation is a substitution with a nonpolar amino acid.

3. The antibody or its antigen-binding fragment, wherein the amino acid variation is a substitution with alanine, glycine, or isoleucine.

4. The antibody or its antigen-binding fragment, wherein the amino acid variation comprises one or more amino acid variations selected from the group consisting of: - Substitution with alanine of the amino acid corresponding to position 2 of the amino acid sequence of SEQ ID NO. 3; - Substitution with glycine of the amino acid corresponding to position 4 of the amino acid sequence of SEQ ID NO. 4; and - Substitution of the amino acid corresponding to position 8 with isoleucine.

5. The antibody or its antigen-binding fragment comprising a heavy chain variable region comprising (i) or (ii) of claim 1: (i) Heavy chain Complementary Determining Region 1 (H-CDR1) comprising a substitution of an amino acid corresponding to position 2 of the amino acid sequence of SEQ ID NO. 3; Heavy chain Complementary Determining Region 2 (H-CDR2) comprising the amino acid sequence of SEQ ID NO. 4; and Heavy chain Complementary Determining Region 3 (H-CDR3) comprising a substitution of an amino acid corresponding to position 8 of the amino acid sequence of SEQ ID NO. 5; and (ii) heavy chain complementarity determining region 1 (H-CDR1) comprising the amino acid sequence of SEQ ID NO. 3; heavy chain complementarity determining region 2 (H-CDR2) comprising a substitution of an amino acid corresponding to the 4th position of the amino acid sequence of SEQ ID NO. 4; and heavy chain complementarity determining region 3 (H-CDR3) comprising the amino acid sequence of SEQ ID NO.

5.

6. An antibody or an antigen-binding fragment thereof, comprising: a light chain complementary determining region 1 (L-CDR1) comprising the amino acid sequence of SEQ ID NO. 1; a light chain complementary determining region 2 (L-CDR2) comprising the amino acid sequence of the sequence AAS; and a light chain variable region comprising a light chain complementary determining region 3 (L-CDR3) comprising the amino acid sequence of SEQ ID NO. 2:

7. The heavy chain variable region comprising an amino acid sequence of either SEQ ID NO. 13 or 14 in Claim 1; and An antibody or its antigen-binding fragment comprising a light chain variable region containing the amino acid sequence of SEQ ID NO. 10 or 11.

8. The antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment comprises a scFv or scFv-Fc fragment.

9. The antibody or antigen-binding fragment thereof comprising the amino acid sequence of SEQ ID NO. 19 or 20, in Claim 1.

10. The antibody or antigen-binding fragment thereof having agonist activity toward a G-CSF receptor, in claim 1.

11. The antibody or its antigen-binding fragment according to claim 1, wherein the antibody or its antigen-binding fragment is capable of inducing the release of hematopoietic stem cells.

12. A gene encoding the antibody of any one of claims 1 to 11 or the antigen-binding fragment thereof.

13. A vector containing the gene of claim 12.

14. The gene of claim 12; or a recombinant cell comprising a vector containing said gene.

15. A pharmaceutical composition for the prevention or treatment of cancer, immune disease, or hematological disease comprising an antibody of any one of claims 1 to 11 or an antigen-binding fragment thereof, a gene encoding said antibody or an antigen-binding fragment thereof, a vector comprising said gene, or a recombinant cell comprising said gene or a vector comprising said gene.

Images