Anti-Siglec15 Antibodies and Uses Thereof

Abstract

The present application provides anti-Siglec15 antibodies or antigen-binding fragments thereof, pharmaceutical compositions and immuno-drug conjugates comprising said antibodies or antigen-binding fragments, nucleic acid molecules encoding said antibodies or antigen-binding fragments thereof, vectors comprising said nucleic acid molecules, host cells comprising said vectors, methods for producing said antibodies or antigen-binding fragments, and biopharmaceutical uses of said antibodies or antigen-binding fragments, such as modulating immune responses, reversing the inhibition of PBMC lymphocyte proliferation by Siglec15 protein, or treating tumors associated with aberrant expression of Siglec15.

Description

[Technical field]

[0001] The present application relates generally to the field of biopharmaceuticals, and in particular to novel anti-Siglec15 antibodies or antigen-binding fragments thereof, pharmaceutical compositions comprising said antibodies or antigen-binding fragments thereof, and pharma-ceutical useful uses of said antibodies or antigen-binding fragments thereof. [Background technology]

[0002] Sialic acid-binding immunoglobulin-type lectins (Siglecs) are members of the immunoglobulin superfamily and are type I transmembrane glycoproteins with a molecular structure consisting of an extracellular domain, a transmembrane domain, and an intracellular domain. Research has revealed that Siglec family members play important roles in the activation and proliferation of immune cells and in mediating physiological and pathological processes. In addition, Siglec family members are involved in the control of immune tolerance and play important roles in the control of autoimmune diseases, inflammatory responses, and tumorigenesis.

[0003] Siglec15 is a member of the Siglec family and consists of 328 amino acid residues. Its extracellular domain (ECD) contains an immunoglobulin variable domain (Ig-like V-type Domain, IgV) and a type 2 constant domain (Ig-like C2-type Domain, IgC2), where the IgV domain is the key site for binding to Siglec15 ligands such as glycoproteins with Neu5Acα2-6GalNAcα-structure, myelin-associated glycoprotein (MAG), leucine-rich repeat containing 4C (LRRC4C) or Siglec15-antireceptor. Protein sequence analysis revealed that Siglec15 has more than 30% structural homology with the B7 gene family of T cells. Siglec15 was initially found to play an important role in physiological bone remodeling and was also involved in pathological bone loss due to estrogen deficiency. Recent studies have revealed that Siglec15 is a negative regulator of tumor immunity and is involved in tumor immunity. Siglec15 is highly expressed in many types of cancer cells and tumor-associated macrophages, and binds to an unknown receptor on T cells to suppress T cell function. It can also control immunosuppression in a manner independent of the B7-H1 (PD-L1) / PD-1 pathway, and inhibiting the immunosuppressive action of Siglec15 can restore or enhance the body's antitumor immune response.

[0004] The only anti-Siglec15 antibody drug reported for solid cancers is NC-318 from NextCure, which is currently undergoing clinical trials, and its epitope of action is currently unknown. Currently, there are no approved drugs targeting Siglec15, and few clinical programs are progressing, so there is an urgent need to provide patients with more effective Siglec15 antibodies. Summary of the Invention

[0005] In a first aspect, the present application provides an anti-Siglec15 antibody or antigen-binding fragment thereof, the anti-Siglec15 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3), the light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2) and a light chain CDR3 (LCDR3), the heavy chain variable region comprises an HCDR1 of the amino acid sequence set forth in SEQ ID NO:6, an HCDR2 of the amino acid sequence set forth in SEQ ID NO:7, and an HCDR3 of the amino acid sequence set forth in SEQ ID NO:8, and the light chain variable region comprises an LCDR1 of the amino acid sequence set forth in SEQ ID NO:9, an LCDR2 of the amino acid sequence set forth in SEQ ID NO:10, and an LCDR3 of the amino acid sequence set forth in SEQ ID NO:11; or the heavy chain variable region comprises an HCDR1 of the amino acid sequence set forth in SEQ ID NO:6, an HCDR2 of the amino acid sequence set forth in SEQ ID NO:12, and an HCDR3 of the amino acid sequence set forth in SEQ ID NO:8, and the light chain variable region comprises an LCDR1 of the amino acid sequence set forth in SEQ ID NO:13, an LCDR2 of the amino acid sequence set forth in SEQ ID NO:10, and an LCDR3 of the amino acid sequence set forth in SEQ ID NO:11; or the heavy chain variable region comprises an HCDR1 of the amino acid sequence set forth in SEQ ID NO:14, an HCDR2 of the amino acid sequence set forth in SEQ ID NO:15, and an HCDR3 of the amino acid sequence set forth in SEQ ID NO:16, and the light chain variable region comprises an LCDR1 of the amino acid sequence set forth in SEQ ID NO:17, an LCDR2 of the amino acid sequence set forth in SEQ ID NO:18, and an LCDR3 of the amino acid sequence set forth in SEQ ID NO:19; or The heavy chain variable region comprises an HCDR1 having an amino acid sequence shown in SEQ ID NO: 20, an HCDR2 having an amino acid sequence shown in SEQ ID NO: 21, and an HCDR3 having an amino acid sequence shown in SEQ ID NO: 22, and the light chain variable region comprises an LCDR1 having an amino acid sequence shown in SEQ ID NO: 23, an LCDR2 having an amino acid sequence shown in SEQ ID NO: 24, and an LCDR3 having an amino acid sequence shown in SEQ ID NO: 25, Herein, there is provided an anti-Siglec15 antibody or antigen-binding fragment thereof, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 amino acid sequences are according to the Kabat definition.

[0006] In a second aspect, the present application provides an anti-Siglec15 antibody or antigen-binding fragment thereof, wherein the anti-Siglec15 antibody or antigen-binding fragment thereof specifically binds to a polypeptide having an amino acid sequence described in any one of (a) to (e).

[0007] (a) the amino acid sequence shown in SEQ ID NO:1; (b) an amino acid sequence consisting of amino acid residues 20 to 328 of the amino acid sequence set forth in SEQ ID NO:1; (c) an amino acid sequence consisting of amino acid residues 1 to 263 of the amino acid sequence set forth in SEQ ID NO:1; (d) an amino acid sequence consisting of amino acid residues 20 to 263 of the amino acid sequence set forth in SEQ ID NO:1; (e) An amino acid sequence having one or more substitutions, deletions or additions of amino acid residues in the amino acid sequence of (a) to (d).

[0008] In a third aspect, the present application provides an anti-Siglec15 antibody or antigen-binding fragment thereof, wherein the anti-Siglec15 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence that is at least 80%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 26, 28, 30 or 32, and the light chain variable region has an amino acid sequence that is at least 80%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 27, 29, 31 or 33.

[0009] In a fourth aspect, the present application provides an anti-Siglec15 antibody or antigen-binding fragment thereof, wherein said anti-Siglec15 antibody or antigen-binding fragment thereof comprises a heavy chain variable region, said heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3); the heavy chain variable region comprises an HCDR1 of the amino acid sequence set forth in SEQ ID NO:6, an HCDR2 of the amino acid sequence set forth in SEQ ID NO:7, and an HCDR3 of the amino acid sequence set forth in SEQ ID NO:8; or the heavy chain variable region comprises an HCDR1 of the amino acid sequence set forth in SEQ ID NO:6, an HCDR2 of the amino acid sequence set forth in SEQ ID NO:12, and an HCDR3 of the amino acid sequence set forth in SEQ ID NO:8; or the heavy chain variable region comprises an HCDR1 of the amino acid sequence set forth in SEQ ID NO:14, an HCDR2 of the amino acid sequence set forth in SEQ ID NO:15, and an HCDR3 of the amino acid sequence set forth in SEQ ID NO:16; or the heavy chain variable region comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 20, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 21, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 22; The present invention provides an anti-Siglec15 antibody or antigen-binding fragment thereof, in which the HCDR1, HCDR2 and HCDR3 amino acid sequences conform to the Kabat definition.

[0010] In a fifth aspect, the present application provides an immuno-drug conjugate comprising an anti-Siglec15 antibody or antigen-binding fragment thereof according to the first to fourth aspects conjugated to a therapeutic agent.

[0011] In a sixth aspect, the present application provides a pharmaceutical composition comprising an anti-Siglec15 antibody or antigen-binding fragment thereof described in the first to fourth aspects or an immuno-drug conjugate described in the fifth aspect, together with a pharmacologically acceptable excipient, diluent or carrier.

[0012] The present application further provides the use of an anti-Siglec15 antibody or antigen-binding fragment thereof according to the first to fourth aspects, or an immuno-drug conjugate according to the fifth aspect, or a pharmaceutical composition according to the sixth aspect, for modulating an immune response in an individual, reversing inhibition of PBMC lymphocyte proliferation by Siglec15 protein, reversing inhibition of PBMC lymphocyte IFN-γ secretion by Siglec15 protein, inhibiting tumor cell proliferation in an individual, or treating a hyperproliferative disorder (e.g. a hyperproliferative disorder associated with abnormal expression of Siglec15).

[0013] In a seventh aspect, the present application provides the use of an anti-Siglec15 antibody or antigen-binding fragment thereof according to the first to fourth aspects, or an immuno-drug conjugate according to the fifth aspect, or a pharmaceutical composition according to the sixth aspect, in the manufacture of a medicament for modulating an immune response in an individual, reversing inhibition of PBMC lymphocyte proliferation by Siglec15 protein, reversing inhibition of PBMC lymphocyte IFN-γ secretion by Siglec15 protein, inhibiting tumor cell proliferation in an individual, or treating a hyperproliferative disorder (e.g. a hyperproliferative disorder associated with abnormal expression of Siglec15).

[0014] In an eighth aspect, the present application provides a method for modulating an immune response in an individual, reversing inhibition of PBMC lymphocyte proliferation by Siglec15 protein, reversing inhibition of PBMC lymphocyte IFN-γ secretion by Siglec15 protein, inhibiting tumor cell proliferation in an individual or treating a hyperproliferative disorder (e.g. a hyperproliferative disorder associated with aberrant expression of Siglec15), comprising administering to an individual in need thereof a therapeutically effective amount of an anti-Siglec15 antibody or antigen-binding fragment thereof described in the first to fourth aspects, an immuno-drug conjugate described in the fifth aspect or a pharmaceutical composition described in the sixth aspect.

[0015] In a ninth aspect, the present application provides an isolated polynucleotide encoding an anti-Siglec15 antibody or antigen-binding fragment according to the first to fourth aspects.

[0016] In a tenth aspect, the present application provides a vector comprising a polynucleotide encoding an anti-Siglec15 antibody or antigen-binding fragment according to the first to fourth aspects.

[0017] In an eleventh aspect, the present application provides an isolated host cell comprising the vector according to the tenth aspect.

[0018] In a twelfth aspect, the present application provides a method for producing an anti-Siglec15 antibody or antigen-binding fragment thereof according to the first to fourth aspects, the method comprising culturing a host cell according to the eleventh aspect. In some embodiments of the twelfth aspect, the method further comprises isolating the anti-Siglec15 antibody or antigen-binding fragment thereof. [Brief description of the drawings]

[0019] [Figure 1] Plasma antibody titer curves of mice protein-immunized with Siglec15-ECD-Fc and Siglec15-IgV-Fc. [Diagram 2] 1 is a graph showing the inhibitory effect of several exemplary anti-Siglec15 antibodies of the present application on the interaction of Siglec15 with LRRC4C-Hek293 cells. [Diagram 3] 1 shows ELISA measurement curves of binding of several exemplary anti-Siglec15 antibodies of the present application to human Siglec15 protein. [Figure 4] 1 shows ELISA measurement curves of binding of several exemplary anti-Siglec15 antibodies of the present application to monkey Siglec15 protein. [Diagram 5] 1 shows ELISA measurement curves of binding of several exemplary anti-Siglec15 antibodies of the present application to mouse Siglec15 protein. [Figure 6] 1 shows result curves of binding of several exemplary anti-Siglec15 chimeric antibodies of the present application and a control antibody to Hek293 cells expressing human Siglec15. [Figure 7]1 is a graph showing the reversal effect of several exemplary anti-Siglec15 chimeric antibodies of the present application and a control antibody on the inhibition of PBMC lymphocyte proliferation by Siglec15 protein. [Figure 8] 1 is a graph showing the inhibitory reversal effect on PBMC lymphocyte IFN-γ secretion by several exemplary anti-Siglec15 chimeric antibodies of the present application and a control antibody Siglec15 protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The inventors of the present application designed an antibody screening strategy based on the signal transduction mechanism of the Siglec15 molecule, and finally screened various anti-Siglec15 antibodies that exhibit excellent properties, such as higher affinity for human Siglec15 compared to control antibodies, greater recovery of Siglec15-mediated T cell activity, and greater recovery of Siglec15-mediated inhibition of T cell activity. In addition, the anti-Siglec15 antibodies of the present application exhibit the property of regulating immune responses.

[0021] definition Unless otherwise defined, all scientific and technical terms used herein have the same meaning as understood by those skilled in the art.For definitions and terms in the art, those skilled in the art are particularly referred to Current Protocols in Molecular Biology (Ausubel).Abbreviations of amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0022] While the numerical ranges and parameters set forth in the broad ranges of this application are approximations, the values ​​set forth in the specific embodiments are documented as precisely as possible. However, any value inherently contains a certain amount of error, which is attributable to the standard deviation present in their respective measurements. Moreover, all ranges disclosed herein should be understood to cover any subranges contained therein. For example, a range of "1-10" should be considered to encompass any subrange between a minimum value of 1 and a maximum value of 10 (including the endpoints), i.e., all subranges beginning with a minimum value of 1 or more (e.g., 1-6.1) and ending with a maximum value of 10 or less (e.g., 5.5-10). Any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0023] In a broad sense, an "antibody" may refer to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule, and thus includes an intact antibody, an antibody single chain, or any antigen-binding fragment of an antibody (also called an "antigen-binding portion"). When the terms "antibody" and "antigen-binding fragment / portion" are used in the same context, "antibody" may be understood as a full complement to "antigen-binding fragment / portion", both of which together correspond to the concept of "antibody" in the broad sense.

[0024] The term "full-length antibody" refers to a protein in which at least two heavy chains (H) and two light chains (L) are linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region contains three regions: CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region contains one region, CL. The VH and VL regions are further subdivided into multiple highly variable regions called complementarity determining regions (CDRs) and multiple more conservative regions called framework regions (FRs) interspersed between them. Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. These variable regions of the heavy and light chains contain binding regions that interact with antigens. The constant region of an antibody mediates the binding of the immunoglobulin to host tissues and factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. A full-length antibody can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or a subclass of the above), but an antibody need not belong to a particular class. Immunoglobulins can be assigned to different classes, depending on the amino acid sequence of the antibody in the constant region of the heavy chain. In general, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these classes can be further differentiated into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to the different immunoglobulin classes are called (, (, (, (, and (. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. Chimeric or humanized antibodies are also contemplated as antibodies according to the present application. It is well known to those skilled in the art that the complementarity determining regions (CDRs, usually CDR1, CDR2 and CDR3) are the regions of the variable region that most influence the affinity and specificity of the antibody. The CDR amino acid sequences of VH or VL are defined in two common ways, namely the Chothia definition and the Kabat definition.For a given antibody variable region amino acid sequence, the CDR amino acid sequences in the VH and VL amino acid sequences can usually be determined based on either the Chothia definition or the Kabat definition. In an embodiment of the present application, the CDR amino acid sequences are defined using Kabat. For a given antibody variable region amino acid sequence, the intermediate CDR amino acid sequences of the variable region amino acid sequence can be analyzed in various ways, for example, can be determined using the online software Abysis (HYPERLINK "http: / / www.abysis.org / " http: / / www.abysis.org / ).

[0025] As used herein, the term "antigen-binding fragment" refers in particular to antibody fragments such as Fv, Fab, F(ab')2 or Fab', or any fragment that could increase half-life by chemical modification or by incorporation into liposomes, preferably said functional fragment consists of or comprises a subsequence of the heavy or light variable chain of the source antibody, said subsequence being sufficient to retain the same binding specificity and sufficient affinity as the source antibody, and said functional fragment comprises a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the source antibody sequence. Examples of antigen-binding fragments include, but are not limited to, (1) a Fab fragment, which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F(ab')2 fragment, which may be a bivalent fragment having two Fab' fragments (i.e., a dimer of Fab) linked by a disulfide bridge at the hinge region; (3) an Fv fragment having the VL and VH regions with a single arm of an antibody.

[0026] The term "single chain antibody (scFv)" refers to a single polypeptide chain in which the VH and VL domains are linked by a peptide linker. (scFv)2 contains two VH domains linked by a peptide linker and two VL domains linked to the two VH domains via disulfide bridges.

[0027] The term "Fc fragment", "Fc structural region", "Fc portion" or similar terms refers to a portion of the constant region of an antibody heavy chain, including the hinge region, the CH2 fragment and the CH3 fragment of the constant region.

[0028] As used herein, the term "specific binding" is intended to mean a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope.

[0029] The term "humanized antibody" refers to an antibody obtained by grafting CDR sequences from other mammalian species, such as mouse strains, into human framework sequences. The human framework sequences may include modifications of other framework regions. The humanized antibody or fragment thereof according to the present application may be produced by techniques known to those skilled in the art.

[0030] The term "chimeric antibody" refers to an antibody whose variable region sequence is derived from one species and whose constant region sequence is derived from another species, for example, whose variable region sequence is derived from a mouse antibody and whose constant region sequence is derived from a human antibody. The chimeric antibody or a fragment thereof according to the present application can be produced by using a gene recombination technique. For example, the chimeric antibody can be produced by cloning a recombinant DNA comprising a promoter, a sequence encoding the variable region of a non-human, particularly mouse, monoclonal antibody according to the present application, and a sequence encoding the constant region of a human antibody. The chimeric antibody according to the present application encoded by such a recombinant gene is, for example, a mouse-human chimera, whose specificity is determined by the variable region derived from the mouse DNA and whose isotype is determined by the constant region derived from the human DNA. For the method of producing a chimeric antibody, reference can be made to, for example, the literature Verhoeyn et al. (BioEssays, 8:74, 1988).

[0031] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for naturally occurring mutations that may be present in a small number of individuals. As used herein, monoclonal antibodies include in particular "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding amino acid sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the heavy and / or light chain is identical or homologous to the corresponding amino acid sequence of an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided that they exhibit the desired biological activity.

[0032] The term "bispecific antibody" refers to an antibody that has the ability to simultaneously bind two antigenic epitopes. The two antigenic epitopes may be on different antigens or on the same antigen. Bispecific antibodies can have various structural configurations. For example, bispecific antibodies may consist of two binding moieties (similar to natural antibodies, except that the two arms bind to different antigenic targets or epitopes) fused to two Fc fragments, respectively, in the form of a single chain antibody (scfv) or in the form of Fab fragments.

[0033] As used herein, the term "pharmaceutical composition" refers to a combination of at least one drug and, optionally, a pharmacologically acceptable carrier or excipient, which are combined together to achieve a particular purpose. In some embodiments, the pharmaceutical composition is composed of a combination that is temporally and / or spatially separate, provided that they are capable of acting together to achieve the purpose of the present application. For example, the components contained in the pharmaceutical composition (e.g., an antibody, a nucleic acid molecule, a combination of nucleic acid molecules, and / or an adjuvant according to the present application) may be administered to an individual as a whole or separately. When the compositions contained in the pharmaceutical composition are administered separately to an individual, the compositions may be administered to an individual simultaneously or sequentially. Preferably, the pharmacologically acceptable carrier is water, a buffered aqueous solution, an isotonic salt solution such as PBS (phosphate buffer), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or a polyalkylene glycol such as polypropylene glycol, a triglyceride, or the like. The type of pharmacologically acceptable carrier used will depend, inter alia, on whether the composition according to the present application is formulated for oral, intranasal, intradermal, subcutaneous, intramuscular or intravenous administration. The composition according to the present application may contain wetting agents, emulsifiers or buffer substances as additives. The pharmaceutical composition or pharmaceutical formulation according to the present application may be administered by any suitable route, for example, oral, nasal, intradermal, subcutaneous, intramuscular or intravenous.

[0034] As used herein, the term "therapeutically effective amount" or "effective amount" refers to a dose sufficient to show benefit to the individual to whom it is administered. The amount actually administered, as well as the rate and time course of administration, depends on the condition and severity of the thing being treated. The prescription of treatment (such as the determination of the dosage) usually takes into account the disease to be treated, the condition of the individual patient, the site of administration, the method of administration, and other factors known to the physician, and is ultimately the responsibility of and depends on the general practitioner or other physician.

[0035] As used herein, the term "individual" refers to a mammal, such as a human, but may also be other animals, such as wild animals (herons, storks, cranes, etc.), domestic animals (ducks, geese, etc.) or laboratory animals (orangutans, monkeys, rats, mice, rabbits, guinea pigs, groundhogs, ground squirrels, etc.).

[0036] The term "homology / consistency" with respect to amino acid or nucleic acid sequences is defined as the percentage of identical residues in amino acid or nucleic acid sequence variants after sequence alignment and, if necessary, introduction of null positions to achieve the maximum percentage of homology. Methods and computer programs used for alignment are well known in the art.

[0037] In general, for the preparation of monoclonal antibodies or functional fragments thereof, in particular of murine origin, reference may be made in particular to the techniques described in the manual "Antibodies" (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY, pp. 726, 1988), or for the preparation from hybridoma cells, to the techniques described by Kohler and Milstein (Nature, 256: 495-497, 1975).

[0038] In a first aspect, the present application provides an anti-Siglec15 antibody or antigen-binding fragment thereof, the anti-Siglec15 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3), the light chain variable region comprising a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2) and a light chain CDR3 (LCDR3), the heavy chain variable region comprises an HCDR1 of the amino acid sequence set forth in SEQ ID NO:6, an HCDR2 of the amino acid sequence set forth in SEQ ID NO:7, and an HCDR3 of the amino acid sequence set forth in SEQ ID NO:8, and the light chain variable region comprises an LCDR1 of the amino acid sequence set forth in SEQ ID NO:9, an LCDR2 of the amino acid sequence set forth in SEQ ID NO:10, and an LCDR3 of the amino acid sequence set forth in SEQ ID NO:11; or the heavy chain variable region comprises an HCDR1 of the amino acid sequence set forth in SEQ ID NO:6, an HCDR2 of the amino acid sequence set forth in SEQ ID NO:12, and an HCDR3 of the amino acid sequence set forth in SEQ ID NO:8, and the light chain variable region comprises an LCDR1 of the amino acid sequence set forth in SEQ ID NO:13, an LCDR2 of the amino acid sequence set forth in SEQ ID NO:10, and an LCDR3 of the amino acid sequence set forth in SEQ ID NO:11; or the heavy chain variable region comprises an HCDR1 of the amino acid sequence set forth in SEQ ID NO:14, an HCDR2 of the amino acid sequence set forth in SEQ ID NO:15, and an HCDR3 of the amino acid sequence set forth in SEQ ID NO:16, and the light chain variable region comprises an LCDR1 of the amino acid sequence set forth in SEQ ID NO:17, an LCDR2 of the amino acid sequence set forth in SEQ ID NO:18, and an LCDR3 of the amino acid sequence set forth in SEQ ID NO:19; or the heavy chain variable region comprises an HCDR1 having an amino acid sequence as shown in SEQ ID NO: 20, an HCDR2 having an amino acid sequence as shown in SEQ ID NO: 21, and an HCDR3 having an amino acid sequence as shown in SEQ ID NO: 22; the light chain variable region comprises an LCDR1 having an amino acid sequence as shown in SEQ ID NO: 23, an LCDR2 having an amino acid sequence as shown in SEQ ID NO: 24, and an LCDR3 having an amino acid sequence as shown in SEQ ID NO: 25; Herein, there is provided an anti-Siglec15 antibody or antigen-binding fragment thereof, wherein the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 amino acid sequences are according to the Kabat definition.

[0039] In some embodiments of the first aspect, the heavy chain variable region has an amino acid sequence set forth in SEQ ID NO:26, 28, 30 or 32, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence set forth in SEQ ID NO:26, 28, 30 or 32. In some embodiments of the first aspect, the light chain variable region has an amino acid sequence set forth in SEQ ID NO:27, 29, 31 or 33, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence set forth in SEQ ID NO:27, 29, 31 or 33.

[0040] In some embodiments of the first aspect, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:26 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:27.

[0041] In some embodiments of the first aspect, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:28 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:29. In some embodiments of the first aspect, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:30 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:31.

[0042] In some embodiments of the first aspect, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:32 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:33.

[0043] In a second aspect, the present application provides an anti-Siglec15 antibody or antigen-binding fragment thereof, wherein the anti-Siglec15 antibody or antigen-binding fragment thereof specifically binds to a polypeptide having an amino acid sequence described in any one of (a) to (e).

[0044] (a) the amino acid sequence shown in SEQ ID NO:1; (b) an amino acid sequence consisting of amino acid residues 20 to 328 of the amino acid sequence set forth in SEQ ID NO:1; (c) an amino acid sequence consisting of amino acid residues 1 to 263 of the amino acid sequence set forth in SEQ ID NO:1; (d) an amino acid sequence consisting of amino acid residues 20 to 263 of the amino acid sequence set forth in SEQ ID NO:1; (e) An amino acid sequence having one or more substitutions, deletions or additions of amino acid residues in the amino acid sequence of (a) to (d).

[0045] The full-length amino acid sequence of human Siglec15 is shown in SEQ ID NO: 1, with positions 1 to 19 being the signal peptide sequence, positions 20 to 263 being the extracellular structural domain (ECD), positions 40 to 158 being the IgV structural domain, and positions 168 to 251 being the IgC2 structural domain. Thus, the amino acid sequences described in (a) to (d) above constitute the full-length amino acid sequence of human Siglec15 and various human Siglec15 fragments, all of which contain the extracellular structural domain, i.e., the binding structural domain of the antibody of the present application.

[0046] In a third aspect, the present application provides an anti-Siglec15 antibody or antigen-binding fragment thereof, the anti-Siglec15 antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having a sequence identity at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% identical to the amino acid sequence set forth in SEQ ID NO:26, 28, 30, or 32. The present invention provides an anti-Siglec15 antibody or antigen-binding fragment thereof, having an amino acid sequence with 98% or 99% homology thereto, and a light chain variable region having an amino acid sequence with at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence set forth in SEQ ID NO:27, 29, 31 or 33.

[0047] In some embodiments of the third aspect, the heavy chain variable region has an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:26 and the light chain variable region has an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:27.

[0048] In some embodiments of the third aspect, the heavy chain variable region has an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:28 and the light chain variable region has an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:29.

[0049] In some embodiments of the third aspect, the heavy chain variable region has an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:30 and the light chain variable region has an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:31.

[0050] In some embodiments of the third aspect, the heavy chain variable region has an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:32 and the light chain variable region has an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:33.

[0051] In a fourth aspect, the present application provides an anti-Siglec15 antibody or antigen-binding fragment thereof, the anti-Siglec15 antibody or antigen-binding fragment thereof comprising a heavy chain variable region, the heavy chain variable region comprising a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3); the heavy chain variable region comprises an HCDR1 of the amino acid sequence set forth in SEQ ID NO:6, an HCDR2 of the amino acid sequence set forth in SEQ ID NO:7, and an HCDR3 of the amino acid sequence set forth in SEQ ID NO:8; or the heavy chain variable region comprises an HCDR1 of the amino acid sequence set forth in SEQ ID NO:6, an HCDR2 of the amino acid sequence set forth in SEQ ID NO:12, and an HCDR3 of the amino acid sequence set forth in SEQ ID NO:8; or the heavy chain variable region comprises an HCDR1 of the amino acid sequence set forth in SEQ ID NO:14, an HCDR2 of the amino acid sequence set forth in SEQ ID NO:15, and an HCDR3 of the amino acid sequence set forth in SEQ ID NO:16; or the heavy chain variable region comprises an HCDR1 having the amino acid sequence shown in SEQ ID NO: 20, an HCDR2 having the amino acid sequence shown in SEQ ID NO: 21, and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 22; Herein, there is provided an anti-Siglec15 antibody or antigen-binding fragment thereof, wherein the HCDR1, HCDR2 and HCDR3 amino acid sequences conform to the Kabat definition.

[0052] An example of an antibody according to the fourth aspect is a single domain antibody, which consists of only one heavy chain variable domain, also known as a variable domain of heavy chain antibody (VHH), also called nanobody. VHH antibodies are naturally occurring. VHH antibodies are naturally occurring light chain-deficient antibodies originally discovered in alpaca peripheral blood, which have only one heavy chain variable domain (VHH) and two conventional CH2 and CH3 domains. The cloned and expressed VHH structure has the same structural stability and antigen binding activity as the original heavy chain antibody, and is the smallest unit known to bind to a target antigen.

[0053] In some embodiments of the first to fourth aspects, the amino acid sequence of the heavy chain variable region differs by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and / or additions from the amino acid sequence set forth in SEQ ID NO: 26, 28, 30 or 32. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 26, 28, 30 or 32 may be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while maintaining similar function of the heavy chain variable region of the antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 27, 29, 31 or 33, and the resulting amino acid sequence may maintain a similar function of the heavy chain variable region of the antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted to a region other than the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 27, 29, 31 or 33, as long as the amino acid sequence after the change basically maintains a similar function of the heavy chain variable region of the antibody.

[0054] In some embodiments of the first to fourth aspects, the amino acid sequence of the light chain variable region differs by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and / or additions from the amino acid sequence set forth in SEQ ID NO: 27, 29, 31 or 33. In some embodiments, the C-terminal or N-terminal region of the amino acid sequence set forth in SEQ ID NO: 27, 29, 31 or 33 may be further truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while maintaining similar function of the light chain variable region of the antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 27, 29, 31 or 33, and the resulting amino acid sequence may maintain a similar function of the light chain variable region of the antibody. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted to a region other than the C-terminal or N-terminal of the amino acid sequence shown in SEQ ID NO: 27, 29, 31 or 33, as long as the amino acid sequence after the change basically maintains a similar function of the light chain variable region of the antibody.

[0055] In some embodiments of the first to fourth aspects, the anti-Siglec15 antibody is a full-length antibody, a single-chain antibody (scFv), or a bispecific antibody. In some embodiments of the first to fourth aspects, the antigen-binding fragment of the anti-Siglec15 antibody is a Fab, a Fab', an Fv, or an F(ab')2. In some embodiments of the first to fourth aspects, the anti-Siglec15 antibody is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments of the first to fourth aspects, the anti-Siglec15 antibody is a monoclonal antibody. In some embodiments of the first to fourth aspects, the anti-Siglec15 antibody is a bispecific antibody. In some embodiments of the first to fourth aspects, the anti-Siglec15 antibody is an IgG1, IgG2, or IgG4 isotype, i.e., the antibody comprises a heavy chain constant region of an IgG1, IgG2, or IgG4 subtype. In some specific embodiments, the anti-Siglec15 antibody is an IgG1 isotype. In some embodiments of the first through fourth aspects, the anti-Siglec15 antibody comprises a light chain constant region of the kappa or lambda subtype.

[0056] In a fifth aspect, the present application provides an immuno-drug conjugate (also referred to as an antibody-drug conjugate (ADC)) comprising an anti-Siglec15 antibody or antigen-binding fragment thereof according to any one of the first to fourth aspects conjugated to a therapeutic agent.

[0057] In some embodiments of the fifth aspect, the therapeutic agent is an anti-tumor pharmaceutical agent, such as a cytotoxic agent, an immuno-enhancing agent, or a radioisotope.

[0058] In some embodiments, the type of cytotoxic drug comprises a microtubule protein inhibitor (eg, an alkaloid), a DNA topoisomerase inhibitor, a DNA damaging agent, antimetabolite, or an antitumor antibiotic.

[0059] In some embodiments, microtubule protein inhibitors include, but are not limited to, olestatin derivatives (e.g., Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF)) or maytansine alkaloid derivatives (e.g., DM1, DM4, Ansamitocin, Mertansine, or dolastatin and its derivatives).

[0060] In some embodiments, the DNA topoisomerase inhibitor is a camptothecin analog or a DNA topoisomerase I inhibitor and its derivatives, such as DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxyrabinomycin, topotecan, retotecan, belotecan, exetecan, silyl homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-methyl-4- ... No-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, and N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide.

[0061] In some embodiments, DNA damaging agents include, but are not limited to, the calicheamicin class, the duocarmycins, and the anthracycline derivatives PBD (pyrrolobenzodiazepines).

[0062] In some embodiments, antimetabolites include but are not limited to methotrexate, 6-mercaptopurine, or 5-fluorouracil.

[0063] In some embodiments, the antitumor antibiotics include, but are not limited to, polypeptide antibiotics (e.g., actinomycin D or bleomycin) or anthraquinones (e.g., adriamycin hydrochloride or mitoxantrone hydrochloride). In some embodiments, the radioisotopes include, but are not limited to, At, I, I, Y, Re, Re, Sm, Bi, P, Co, or Lu.

[0064] In some embodiments, immune enhancing agents include but are not limited to levamisole, pidotimod, imiquimod, isoproterenol, polynosinic acid, or polynosinic acid.

[0065] In some embodiments, the anti-Siglec15 antibody of the present application is covalently attached to the drug moiety via a conjugate. In some embodiments, the conjugate is a cleavable conjugate. In some embodiments, the conjugate is cleavable under intracellular conditions. In some embodiments, the conjugate is hydrolyzable at a pH of less than 5.5. In some embodiments, the conjugate is cleavable by intracellular proteases. In some embodiments, the conjugate is a tissue protease cleavable conjugate. In some embodiments, the conjugate comprises a dipeptide. In some embodiments, the dipeptide is valine (Val)-citrulline (Cit). In some embodiments, the antibody is attached to the conjugate via a cysteine ​​sulfhydryl group of the antibody. In some embodiments, the antibody is attached to the conjugate via an amino group of the antibody, particularly the amino group of a glutamine residue. Non-limiting examples of conjugates include mc-Val-Cit-pAB, mc-Val-Cit-pABC, mc-Val-Cit, NH2-(PEG)m-Val-Cit, and NH2-(PEG)m-Val-Cit-pAB, where m is an integer from 1 to 8.

[0066] In a sixth aspect, the present application provides a pharmaceutical composition comprising an anti-Siglec15 antibody or antigen-binding fragment thereof described in the first to fourth aspects or an immuno-drug conjugate described in the fifth aspect, together with a pharmacologically acceptable excipient, diluent or carrier.

[0067] In some embodiments, the pharmaceutical compositions may further comprise one or more of: lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preservatives such as benzoic acid, sorbic acid, and calcium propionate; sweetening and / or flavoring agents, etc. In some embodiments, the pharmaceutical compositions of the present application may be formulated in the form of tablets, pills, powders, troches, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules.

[0068] In some embodiments, the pharmaceutical compositions of the present application may be administered using any physiologically acceptable mode of administration, including, but not limited to, oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like.

[0069] In some embodiments, the therapeutic pharmaceutical composition can be formulated for storage in the form of a lyophilized preparation or an aqueous solution by appropriately mixing reagents of the desired purity with pharma- ceutically acceptable carriers, excipients, etc.

[0070] In some embodiments of the sixth aspect, the pharmaceutical composition is used to modulate an immune response in an individual, to reverse inhibition of PBMC lymphocyte proliferation by Siglec15 protein, to reverse inhibition of PBMC lymphocyte IFN-γ secretion by Siglec15 protein, to inhibit tumor cell proliferation in an individual, or to treat a hyperproliferative disorder (e.g., a hyperproliferative disorder associated with aberrant expression of Siglec15). In some specific embodiments, the hyperproliferative disorder is a tumor, e.g., a tumor associated with aberrant expression of Siglec15.

[0071] In some embodiments of the sixth aspect, the pharmaceutical composition of the sixth aspect is used in combination with an anti-PD-1 or anti-PD-L1 antibody to treat a tumor.

[0072] In a seventh aspect, the application provides the use of an anti-Siglec15 antibody or antigen-binding fragment thereof according to the first to fourth aspects, or an immuno-drug conjugate according to the fifth aspect, in the manufacture of a medicament for modulating an immune response in an individual, reversing inhibition of PBMC lymphocyte proliferation by Siglec15 protein, reversing inhibition of PBMC lymphocyte IFN-γ secretion by Siglec15 protein, inhibiting tumor cell proliferation in an individual, or treating a hyperproliferative disorder (e.g. a hyperproliferative disorder associated with aberrant expression of Siglec15). In some specific embodiments, the hyperproliferative disorder is a tumor, e.g. a tumor associated with aberrant expression of Siglec15.

[0073] In some embodiments of the seventh aspect, the medicament further comprises an anti-PD-1 or anti-PD-L1 antibody.

[0074] In an eighth aspect, the present application provides a method for modulating an immune response in an individual, reversing inhibition of PBMC lymphocyte proliferation by Siglec15 protein, reversing inhibition of PBMC lymphocyte IFN-γ secretion by Siglec15 protein, inhibiting tumor cell proliferation in an individual or treating a hyperproliferative disorder (e.g. a hyperproliferative disorder associated with aberrant expression of Siglec15), comprising administering to an individual in need thereof a therapeutically effective amount of an anti-Siglec15 antibody or antigen-binding fragment thereof according to the first to fourth aspects, an immuno-drug conjugate according to the fifth aspect or a pharmaceutical composition according to any one of claims 12 to 15. In some specific embodiments, the hyperproliferative disorder is a tumor, e.g. a tumor associated with aberrant expression of Siglec15.

[0075] In some embodiments of the eighth aspect, the method further comprises administering to the individual in need thereof an anti-PD-1 or anti-PD-L1 antibody.

[0076] In embodiments of the sixth to eighth aspects, the anti-Siglec15 antibodies of the present application can be used to induce, increase or enhance T cell proliferation by reducing or preventing binding of Siglec-15 to its counter-receptor on T cells to induce a T cell response. Since upregulation of the immune system is particularly required in the treatment of cancer and chronic infections, the anti-Siglec15 antibodies of the present application can be used to treat such diseases.

[0077] The anti-Siglec15 antibodies, immuno-drug conjugates or pharmaceutical compositions of the present application can be used to treat or prevent various cancers or other abnormal proliferative diseases, including, but not limited to, cancers such as bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer and skin cancer; squamous cell carcinoma; leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma and Burkitt's lymphoma. tumors of the lymphoid hematopoietic system, including myelogenous leukemia, acute and chronic myeloid leukemia, promyelocytic leukemia; tumors of the mesenchymal system, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nerves, including astrocytoma, neuroblastoma, glioma and neuroblastoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, chromatin, keratoacanthoma, seminoma, follicular carcinoma and teratocarcinoma of the thyroid.

[0078] The anti-Siglec15 antibodies, immuno-drug conjugates or pharmaceutical compositions of the present application can be used to treat infectious diseases and infectious disorders. Typically, the method includes stimulating or enhancing an immune response to an infection-initiating agent, reducing or preventing the progression of an infectious disease, or a combination thereof, by administering to a subject an amount of a Siglec-15 binding molecule. The method can reduce one or more symptoms of an infectious disease. An infectious disease or disease can be caused by a bacteria, virus, protozoa, helminth or other microbial pathogen that invades cells and is attacked by cytotoxic T lymphocytes. Infectious diseases and illnesses include acute infections and chronic infections. Acute infections are usually short-lived infections. During acute microbial infections, immune cells begin to express immunomodulatory receptors. Thus, in some embodiments, the method includes enhancing an immunostimulatory response to an acute infection.

[0079] In a ninth aspect, the present application provides an isolated polynucleotide encoding an anti-Siglec15 antibody or antigen-binding fragment according to the first to fourth aspects. In some embodiments, the polynucleotide is operably linked to a regulatory sequence, which is recognizable by a host cell transformed with the vector.

[0080] In a tenth aspect, the present application provides a vector (e.g., an expression vector) comprising a polynucleotide encoding an anti-Siglec15 antibody or antigen-binding fragment according to the first to fourth aspects. The choice of expression vector will depend on the choice of host cell and can be selected to have the desired expression and regulatory properties in the selected host cell.

[0081] An "expression vector" is a vector that contains one or more expression control sequences, and an "expression control sequence" is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence.

[0082] The nucleic acid in the vector may be operably linked to one or more expression control sequences. As used herein, "operably linked" means incorporated into a genetic construct such that the expression control sequence effectively controls the expression of the target coding sequence. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence that consists of a region of a DNA molecule within 100 nucleotides upstream of a transcription start site (usually near the start site of RNA polymerase II). For a coding sequence to be under the control of a promoter, the translation start site of the peptide translation reading frame must be located 1 to about 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of timing, location, and level. Unlike promoters, enhancers can act even when located at different distances from the transcription site. Enhancers may also be located downstream of the transcription start site. A coding sequence is "operably linked" to and "under the control" of an expression control sequence in a cell when RNA polymerase transcribes the coding sequence into mRNA, which can be translated into the protein encoded by the coding sequence.

[0083] Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, phages, baculoviruses, tobacco mosaic viruses, herpes viruses, cytomegaloviruses, retroviruses, pox viruses, adenoviruses and adeno-associated viruses. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (LaJolla, CA) and Invitrogen Life Technologies (Carlsbad, CA).

[0084] The expression vector may contain a tag sequence. The tag sequence is typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including the carboxyl or amino terminus. Examples of useful tags include, but are not limited to, Fc fragment, polyhistidine, green fluorescent protein (GFP), glutathione S-transferase (GST), c-myc, hemagglutinin, FlagTM tag (Kodak, New Haven, CT), maltose E binding protein, and protein A. In some embodiments, the nucleic acid molecule encoding the Siglec-15 fusion polypeptide is present in a vector that includes a nucleic acid encoding one or more structural regions of the constant region of an Ig heavy chain, the structural regions corresponding to, for example, the amino acid sequences of the hinge, CH2, and CH3 regions of the human immunoglobulin Cγ1 chain (Fc fragment).

[0085] In an eleventh aspect, the present application provides an isolated host cell comprising a carrier according to the tenth aspect.

[0086] In some embodiments, the host cell may be a prokaryotic host cell, a eukaryotic host cell, or a phage. Prokaryotic host cells may be, for example, Escherichia coli, Bacillus subtilis, Streptomyces, or Aspergillus chimaericus. Eukaryotic host cells include fungi such as Pasteurella, Saccharomyces cerevisiae, fission yeast, and Xylaria, insect cells such as Meadow slime, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0087] In a twelfth aspect, the present application provides a method for producing an anti-Siglec15 antibody or antigen-binding fragment thereof according to the first to fourth aspects, the method comprising culturing a host cell according to the eleventh aspect. In some embodiments of the twelfth aspect, the method further comprises isolating the anti-Siglec15 antibody or antigen-binding fragment thereof.

[0088] It should be understood that the above detailed description is intended only to enable those skilled in the art to more clearly understand the contents of the present application, and is not intended to be limiting in any way. Those skilled in the art may make various modifications and variations to the described embodiments. EXAMPLES

[0089] It should be noted that the following embodiments are merely examples and are not intended to limit the scope of the present application.

[0090] Example 1: Preparation of Siglec15 antigen and measurement protein The inventors of the present application used Uniprot (Q6ZMC9) to obtain the amino acid sequences of the Siglec15 antigen for immunization and the Siglec15 protein for detection. The full length of the IgV region or extracellular region of Siglec15 was recombined with an IgG antibody heavy chain human IgG1Fc fragment or seven histidines (Histidine, His), respectively, and cloned into the pCDNA3.1 vector (Nanjing Kingsray Biotechnology Co, Ltd.). The above plasmids were transfected into Hek293F mammalian cells using the PEI transfection method, and the cell supernatant was collected and purified 7 days after transfection. These plasmids were named Siglec15-IgV-Fc, Siglec15-ECD-Fc, and Siglec15-ECD-His, respectively, and their amino acid sequences are shown below.

[0091] The full-length amino acid sequence of human Siglec15 (sequence number 1) is as follows, with positions 1-19 being the signal peptide sequence, positions 20-263 being the extracellular structural domain (italics, bold), positions 40-158 being the IgV structural domain (single underlined), and positions 168-251 being the IgC2 structural domain (double underlined).

[0092] TIFF0007653532000001.tif51170

[0093] The amino acid sequence of Siglec15-ECD-Fc (sequence number 2) is as follows, with positions 1-19 representing the signal peptide, positions 20-263 representing the Siglec15 extracellular structural domain (ECD) (shown in italics and bold), and positions 264-494 representing the Fc tag.

[0094] TIFF0007653532000002.tif72170

[0095] The amino acid sequence of Siglec15-IgV-Fc (SEQ ID NO: 3) is as follows, in which positions 1-19 are the signal peptide, positions 40-158 are the IgV region of Siglec15 (shown in italics and bold), and positions 166-396 are the Fc tag:

[0096] TIFF0007653532000003.tif56170

[0097] Siglec15-ECD-His (SEQ ID NO: 4) is used as a detection reagent in the present application and has the following amino acid sequence, in which positions 1-19 are the signal peptide, positions 20-263 are the Siglec15 extracellular structural domain (ECD) (shown in italics and bold), and positions 264-270 are the histidine tag:

[0098] TIFF0007653532000004.tif40170

[0099] Example 2. Purification of recombinant proteins or antibodies 2.1 Purification of His-tagged recombinant proteins The precipitate was removed by high-speed centrifugation, and the cell expression supernatant was collected. The nickel column was pre-equilibrated with HBS buffer (10 mM HEPES pH 7.5, 500 mM NaCl) containing 10 mM imidazole, 2 to 5 times the column volume was rinsed, and the supernatant sample was loaded onto the nickel column. The column was rinsed with HBS buffer containing 10 mM imidazole until the A280 measurement value fell to the baseline. Next, the chromatography column was washed with HBS buffer containing 20 mM imidazole to remove nonspecifically bound heteroproteins, and the effluent was collected. The target protein was then eluted with HBS buffer containing 300 mM imidazole, the elution peak was collected, and the collected eluate was exchanged for HBS buffer. The Siglec15-ECD-His protein obtained by nickel column purification was used for screening and performance testing of the antibody of the present application.

[0100] 2.2 Purification of Fc fusion proteins or antibodies The cell expression supernatant was centrifuged at high speed to remove the precipitate, and the supernatant was collected. The Protein A / G column was pre-equilibrated with PB buffer, and 2 to 5 times the column volume was rinsed, and the supernatant sample was loaded onto the column. The column was rinsed with PB buffer until the A280 measurement value fell to the baseline. The target protein was eluted by passing it through 1M Tris-HCL neutralization buffer pre-spiked with 0.1M glycine solution at pH 2.7 to pH 9.0, and the collected eluate was exchanged for PBS buffer. Siglec15-ECD-Fc and Siglec15-IgV-Fc, which have Fc fragments, were used as immunogens or for performance testing of the present antibody. The fusion Fc fragment promotes the expression and secretion of the target protein in mammals, and also facilitates the purification of the fusion protein because the Fc fragment can specifically bind to Protein A, and furthermore, the Fc fragment can be recognized and bound by an anti-human secondary antibody in animals, so it can be used to demonstrate the effect of antibody inhibition.

[0101] Example 3: Preparation of anti-human Siglec15 hybridoma monoclonal antibody Healthy BALB / c female mice aged 6–8 weeks were selected and immunized with Siglec15-ECD-Fc and Siglec15-IgV-Fc proteins, respectively. For the primary immunization, a mixture of Freund's complete adjuvant and immune protein was emulsified and injected subcutaneously into the back and groin of the mice at 100 μg / mouse at a 1:1 ratio of immunogen to adjuvant. Before the primary immunization, a small amount of blood was collected from the eye of the mouse as a negative control for serum antibody titer testing. 7–10 days after the primary immunization, the mice were boosted with a mixture of Freund's incomplete adjuvant and immune protein emulsion by the same route as the primary immunization. Thereafter, booster immunizations were performed 2–3 times at 7–10-day intervals with 50 μg / mouse at a 1:1 ratio of immunogen to adjuvant. Peripheral serum was collected from the mice and tested by enzyme-linked immunosorbent assay (ELISA) as described below. Mice immunized with the Siglec15-ECD-Fc and Siglec15-IgV-Fc proteins shown in Figure 1 had serum titers of 360,000 or more, and mice with high plasma antibody titers were selected for cell fusion. Three days before fusion, 50 μg of immunogen without adjuvant was injected into the tail vein or abdominal cavity as shock immunization. On the day of fusion, mouse spleens were collected and prepared into single cell suspensions using RPMI1640 basic medium (GIBCO), and SP2 / 0 cells and spleen cells were mixed at a ratio of 1:3 to 1:5 and fused by electric fusion. The mice were cultured in HAT selection medium for 5 to 7 days, then switched to HT medium and placed in a 37 °C CO2 incubator for further culture.

[0102] ELISA is used to measure the antibody titer or screen the monoclonal antibody. The specific method is as follows: (1) Coating: Siglec15-ECD-His protein was dissolved in antigen encapsulation buffer (pH 9.6, 0.05 M carbonate buffer) to a final concentration of 1 μg / mL, and coated on a 96-well polystyrene reader plate at 4° C. overnight. (2) Closing: After washing the plate three times with PBST buffer, PBS solution containing 2% BSA was added to each well and incubated at 37°C for 2 hours, after which the plate was washed three times with PBST buffer. (3) Primary antibody incubation: Mouse serum (or hybridoma cell secreted supernatant, purified antibody) was diluted in a series of concentration gradients containing 0.2% BSA and added to the enzyme plate. Eye serum taken from mice before the first immunization was used as a negative control, and the blank was incubated in PBS containing 0.2% BSA at 37°C for 2 hours. The plate was washed three times with PBST buffer. (4) Secondary antibody incubation: Horseradish peroxidase-labeled goat anti-human IgG was added and incubated at 37° C. for 1 hour, and the plate was washed three times with PBST buffer. (5) Color development: Mix TMB substrate color development solutions A and B, add 100 μL per well, and react at room temperature for 10 minutes in the dark. (6) Termination: 100 μL of 2M H2SO4 was added to each well to terminate the color reaction. (7) Measurement: The absorbance value of each well of the enzyme plate was measured at 450 nm using the enzyme marker.

[0103] Example 4. Screening and characterization of hybridoma cell lines After the hybridoma cells were grown to a certain amount, the Siglec15-His protein was encapsulated in a 96-well enzyme-labeled plate, and the hybridoma supernatants were screened by ELISA using mouse anti-Siglec15 serum as a positive control to select positive clones. By detecting the positive clone supernatants that inhibited the binding assay of Siglec15-ECD-Fc to LRRC4C-Hek293 cells, it was revealed that antibodies 3-10B2, 1-15D1, 1-7F4, 3-8B9, 14A7, 13E4, 4G5, 9H7, 1-10E5 and 14C4 inhibited the binding of Siglec15-ECD-Fc to LRRC4C-Hek293 cells, and the experimental results were shown in Figure 2 below. The present inventors subjected these 10 cell lines to subclonal screening by the limiting dilution method, and finally stored the obtained monoclonal positive cell lines separately. The supernatants of the hybridoma cells were collected and purified by Protein G.

[0104] Binding of Siglec15-ECD-Fc to LRRC4C-Hek293 cells was detected by single-site blocking flow cytometry as follows:

[0105] Using the Lipofectamine system, human LRRC4C (amino acid sequence shown in SEQ ID NO: 5) plasmid was transfected into Hek293 cells, and after 48 h, G418 (800 μg / mL) was added and screened for 10 to 14 days to obtain cell lines stably expressing LRRC4C. The cells were harvested, washed once with PBS buffer, blocked with PBS containing 2% FBS for 1 h at room temperature, and hybridoma cell supernatant was added, 50 μl / well; immediately after, diluted Siglec15-ECD-Fc protein (2 μg / ml) was added, 50 μl / well; IgG from mouse was used as a negative control. The cells were incubated at 4 °C for 50 min, then washed three times with PBS buffer containing 0.2% FBS, then diluted with Alexa Fluor 488-labeled goat anti-human IgG in PBS containing 2% FBS, incubated at 4 °C for 50 min protected from light, washed three times with PBS buffer containing 0.2% FBS; finally, the cells were resuspended in 200 μL PBS. Analysis was performed by flow cytometry. FITC fluorescence intensity was the abscissa and cell number was the ordinate, voltage parameters were adjusted so that the blank control was at the origin of the coordinate axis, and the average fluorescence reading of 10,000 cells was recorded for each sample. The percentage of hybridoma cell supernatant that inhibited Siglec15-ECD-Fc protein binding to LRRC4C was calculated as inhibition percentage = (negative control reading - test supernatant reading - blank control reading) / (negative control reading - blank control reading).

[0106] The full length amino acid sequence of human LRRC4C is as follows (SEQ ID NO:5): MLNKMTLHPQQIMIGPRFNRALFDPLLVVLLALQLLVVAGLVRAQTCPSVCSCSNQFSKVICVRKNLREVPDGISTNTRLLNLHENQIQIIKVNSFKHLRHLEILQLSRNHIRTIEIGAFNGLANLNTLELFDNRLTTIPNGAFVYLSKLKELWLRNNPI ESIPSYAFNRIPSLRRLDLGELKRLSYISEGAFEGLSNLRYLNLAMCNLREIPNLTPLIKLDELDLSGNHLSAIRPGSFQGLMHLQKLWMIQSQIQVIERNAFDNLQSLVEINLAHNNLTLLPHDLFTPLHHLERIHLHHNPWNCNCDILWLSWWIKDMA PSNTACCARCNTPPNLKGRYIGELDQNYFTCYAPVIVEPPADLNVTEGMAAELKCRASTSLTSVSWITPNGTVMTHGAYKVRIAVLSDGTLNFTNVTVQDTGMYTCMVSNSVGNTTASATLNVTAATTTPFSYFSTVETMEPSQDEARTTDNNVGPTP VVDWETTNVTTSLTPQSTRSTEKTFTIPVTDINSGIPGIDEVMKTTKIIIGCFVAITLMAAVMLVIFYKMRKQHHRQNHHAPTRTVEIINVDDEITGDTPMESHLPMPAIEHEHLNHYNSYKSPFNHTTTVNTINSIHSSVHEPLLIRMNSKDNVQETQI

[0107] Example 5: Cross-species recognition properties of anti-Siglec15 antibodies To evaluate the cross-species recognition properties of the antibodies, purified monoclonal antibodies 1-15D1, 1-7F4, 14A7 and 1-10E5 were tested for cross-recognition with human, cynomolgus monkey and murine Siglec15. Enzyme plates were coated with His-tagged human, cynomolgus monkey and murine Siglec15 proteins, and 5000ng / ml, 1666.7ng / ml, 555.6ng / ml, 185.2ng / ml, 61.7ng / ml, 20.6ng / ml, 6.9ng / ml, 2.3ng / ml, 0.8ng / ml, 0.25ng / ml and 0.1ng / ml of 1-15D1, 1-7F4, 14A7 and 1-10E5 antibodies, respectively, were added and tested by ELISA. As a result, the 1-15D1, 1-7F4, 14A7 and 1-10E5 antibodies bound to human (Figure 3) and monkey (Figure 4) Siglec15 proteins. The 14A7 and 1-10E5 antibodies bound to not only human and monkey Siglec15 proteins but also mouse Siglec15 proteins (Figure 5). EC 50 The values ​​are shown in Table 1 and FIGS.

[0108] [Table 1]

[0109] Example 6. Production, expression and purification of human-mouse chimeric anti-Siglec15 antibody 3 × 10 using the Trizol method 6 From 5×10 6 Total RNA was extracted from the hybridoma cells and reverse transcribed into cDNA according to the GoScriptTM Reverse Transcription System experimental instructions. Then, fragments of the heavy and light chain variable regions (V-regions) of mouse immunoglobulins were amplified by PCR and sequenced. The final sequences of the heavy and light chain CDR regions of clones 1-10E5, 1-7F4, 1-15D1, and 14A7 (Table 2) and the amino acid sequences of the variable regions were obtained.

[0110] [Table 2]

[0111] 1-15D1 heavy chain variable region amino acid sequence (SEQ ID NO:26) EVQLQQSGPELVKPGASVKMSCKASGYTFTTYVMHWVKQKPGQGLEWIGYINPYNDNTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCGSGYDAMDYWGQGTSVTVSS

[0112] 1-15D1 light chain variable region amino acid sequence (SEQ ID NO:27) DIQMAQSPASLSVSVGETVTITCRTSENIYSHLAWYQQKQGKSPQLLVYSATNLADGVPSRFNGSGSGTQYSLKINSLQSEDFGSYYCQHFWGTPWTFGGGTKLEIK

[0113] 1-7F4 heavy chain variable region amino acid sequence (SEQ ID NO:28) EVQLQQSGPELVKPGASVKMSCKASGYTFTTYVMHWVKQKPGQGLEWIGYINPYNDKTKYNEKFKGKATLTSDKSSSTAYMEVSRLTSEDSAVYYCGSGYDAMDYWGQGTSVTVSS

[0114] 1-7F4 light chain variable region amino acid sequence (SEQ ID NO:29) DIQMTQSPASLSVSVGETVTITCRASENIYSHLAWYQQKQGKSPQLLIYSATNLADGVPSRFNGSGSGTQYSLKINSLQSEDFGSYYCQHFWGTPWTFGGGTKLEIK

[0115] 1-10E5 heavy chain variable region amino acid sequence (SEQ ID NO: 30) DVKLVESGGGL VKPGGSLKLSCVVSGLTFSRYTMSWVRQTPEKRLEWVATISSGGNSTYYPDSVKGRFTISRDDAKDTLYLQMSSLKSEDTAMYYCTNYGYFFDYWGQGTTLTVSS

[0116] 1-10E5 light chain variable region amino acid sequence (SEQ ID NO:31) DIQMTQSPASLSASVGETTVTITCRASENIYSFLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYDRPLTFGAGTKLELK

[0117] 14A7 heavy chain variable region amino acid sequence (SEQ ID NO:32) DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDFAWNWIRQFPGNKLEWMGYIRFSGSTTYKPTLKSRISITRDTSKNQFFLQLNSVTTEDSATYYCAREDYDGLFDYWGQGTTLTVTS

[0118] 14A7 light chain variable region amino acid sequence (SEQ ID NO:33) DVLMTQTPLSLPVSLGDHASISCRSSQTILHSNGNTYLDWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK

[0119] Based on the sequence information of the variable regions of the genes encoding the above four types of monoclonal antibodies, a human-mouse chimeric antibody expression vector was constructed in which the antibody isotype is human IgG1 and asparagine (N) at position 297 is mutated to alanine (A) (referred to as hIgG1 in this application). DNA fragments containing the light chain variable and constant regions (κ) and the heavy chain variable and constant regions were synthesized and inserted into the pCDNA3.1 expression vector to form expression plasmids. This eukaryotic expression vector was transiently introduced into Hek293 cells and cultured for 5 to 7 days. The cell supernatant was collected by centrifugation, and the collected culture supernatant was purified using a Protein A column to obtain the target antibodies named 1-7F4-IgG1, 1-15D1-IgG1, 1-10E5-IgG1, and 14A7-IgG1, respectively.

[0120] Example 7: Measurement of affinity of chimeric antibodies to Siglec15 protein by ForteBio The control antibody selected in this example is antibody 5G12, which has the sequence of the CDR region from Chinese Patent Application Publication CN110035769A, which was conjugated to hIgG1 (containing the N297A mutation) to produce 5G12-IgG1 as shown in Example 6. The control antibody 5G12 is the anti-Siglec15 drug antibody NC-318 from Nesquel (NextCure), which was predicted by the inventors' analysis.

[0121] In this experiment, a HIS1K biosensor binding ligand (Sigle15-ECD-His protein) was used, followed by binding of the analyte. Human Sigle15-ECD-His protein was diluted to 5 μg / ml and immobilized on the biosensor to a binding response threshold of 0.3 nm. After a baseline step of 120 seconds, the sensor was immersed in anti-Siglec15 antibodies 1-7F4-IgG1, 1-15D1-IgG1, and 1-10E5-IgG1 analytes, which were diluted in a 2-fold gradient with 0.02% PBST buffer, respectively, with an initial antibody concentration of 100 nM and a 7-step concentration gradient. Human IgG1 (hIgG1) (B109801, Baiying Biotechnology Ltd.) was used as a reference control, and 5G12-IgG1 was used as a positive control for the affinity analysis assay. The ligand was bound to the analyte for 120 seconds and dissociated for 300 seconds. The raw data was imported into the analysis software, the zero concentration control was subtracted, the reference channel was subtracted to eliminate the volume effect, and the affinity data of the antigen and antibody was calculated from the binding curve by regression on the analysis software. The results are shown in Table 3. The candidate antibodies recognized the target antigen with high affinity, and all had significantly lower KD values ​​than the control antibody 5G12.

[0122] [Table 3]

[0123] Example 8: Binding properties of anti-Siglec15 chimeric antibodies to Hek293 cell line expressing Siglec15 The ability of anti-Siglec15 chimeric antibodies to bind to human Siglec15 expressed on the cell surface was evaluated using the human Siglec15 overexpressing cell line Hek293. The human-mouse chimeric antibodies were diluted in PBS buffer to concentrations of 10,000ng / ml, 5,000ng / ml, 1,666.7ng / ml, 555.6ng / ml.2, 61.7ng / ml, and 20.6ng / ml, respectively, with 5G12-IgG1 as a positive control. The antibodies were incubated with Hek293-siglec15 cells at 4°C for 1 hour, the cells were washed three times with PBS buffer, and subsequently labeled with Alexa Fluor 488-conjugated goat anti-human IgG antibody for 50 minutes on ice protected from light, the cells were washed three times with PBS buffer, and the fluorescence intensity of the cells was read by flow cytometry. As shown in FIG. 6, the antibodies 1-10E5-IgG1, 1-7F4-IgG1 and 1-15D1-IgG1 were able to bind to Siglec15 expressed on the Hek293 cell line in a dose-dependent manner, and all of them bound significantly stronger than the control antibody 5G12.

[0124] Example 9: Reversal of Siglec15-mediated T cell proliferation inhibition by anti-Siglec15 antibody We measured the reversal of Siglec15-mediated inhibition of human total PBMC T cell proliferation by recombinant chimeric antibodies. On the day of measurement, frozen PBMCs were thawed, washed with RPMI complete medium and counted. PBMCs (3×10 5 Cells / well) were seeded into 96-well plates that had previously contained anti-human CD3 antibody (OKT3, 50 ng / mL; eBioScience). Premixed Siglec15-ECD-Fc fusion protein (5 μg / ml) and the antibody to be tested (20 μg / ml) were then added to the designated wells. hIgG1 was used as a negative control (isotype) and 5G12-IgG1 as a positive control. Cells were incubated at 37°C for 72 hours and CellTiter-Glo (Promega) was added to detect cell viability.

[0125] As a result, as shown in FIG. 7, the recombinant antibodies 1-15D1-IgG1, 1-7F4-IgG1 and 1-10E5-IgG1 reversed the suppression of human T cells by Siglec15 compared to the negative control antibody hIgG1, and all were superior to the control antibody 5G12.

[0126] Example 10: Reversal of the suppression of IFN-γ secretion by T cells via Siglec15 by anti-Siglec15 antibodies In this study, to characterize the activity of anti-Siglec-15 antibodies, we evaluated their activation effect on PBMCs by detecting the amount of IFN-γ secreted by human-derived PBMCs after activation in an in vitro immune stimulation assay. PBMCs were isolated from mixed peripheral blood of healthy humans, washed in RPMI complete medium, and counted. Total PBMCs (3 × 10 5 Siglec15-ECD-Fc fusion protein (5 μg / ml) and the antibody to be tested at a concentration of 20 μg / ml were then added to the indicated wells. hIgG1 was used as a negative control (isotype) and 5G12-IgG1 was used as a positive control. Cells were incubated at 37°C for 24 h and the supernatant was centrifuged to detect the release of IFN-γ.

[0127] As a result, as shown in Figure 8, the recombinant antibodies 1-15D1-IgG1, 1-7F4-IgG1, and 1-10E5-IgG1 effectively restored Siglec15-mediated inhibition of IFN-γ secretion by T cells, and all were significantly more effective than the control antibody 5G12.

[0128] Example 11: Epitope classification of anti-Siglec15 antibodies Antigenic epitope classification of 1-15D1-IgG1, 1-7F4-IgG1, 1-10E5-IgG1, and 5G12-IgG1 was detected by Octet epitope pairing. Human Siglec15-ECD-His protein was loaded onto the HIS1K sensor at 5ug / ml, followed by exposure to primary and secondary antibodies, respectively. The binding signal of the secondary antibody was used to determine whether the two antibodies recognized the same epitope, and the data was processed using ForteBio's data analysis software 7.0. Additional binding of the secondary antibody after primary antibody binding (60%-100%) indicated an unoccupied epitope (non-competition), additional partial binding of the secondary antibody after primary antibody binding (10%-60%) indicated a partially occupied epitope (partial competition), and no binding (less than 10%) indicated an inhibited epitope (competition). As a result, it was found that 1-7F4-IgG1 recognized the same epitope as 1-15D1-IgG1, 1-10E5-IgG1 partially overlapped with 1-7F4-IgG1 and 1-15D1-IgG1, and 1-10E5-IgG1, 1-7F4-IgG1 and 1-15D1-IgG1 recognized epitopes completely different from those of 5G12-IgG1.

[0129] The above description is merely an exemplary embodiment, and does not limit the combination of features necessary to carry out the present application. Furthermore, the titles provided are not intended to limit the various embodiments of the present application. For example, the terms "comprising", "including", and "including" are not intended to be limiting. Furthermore, unless otherwise specified, the absence of a count word modifier includes the plural, and "or" means "and / or". Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. All disclosures and patents referred to in this application are incorporated herein by reference. Many modifications and variations of the methods and compositions described herein will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the present application. Although the present application is described by certain preferred embodiments, it should be understood that the application claimed for protection should not be unduly limited to these specific embodiments. Indeed, those variations of the described modes for carrying out the present application that are apparent to those of ordinary skill in the relevant art are intended to be within the scope of the appended claims.

[0130] This application claims priority to Chinese Patent Application No. 202110211997.5, filed on February 25, 2021, the entire contents of which are incorporated herein by reference and for all purposes.

Claims

1. An anti-Siglec15 antibody or antigen-binding fragment thereof, The anti-Siglec15 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2) and a heavy chain CDR3 (HCDR3), and the light chain variable region comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2) and a light chain CDR3 (LCDR3); the heavy chain variable region comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO:6, an HCDR2 having the amino acid sequence set forth in SEQ ID NO:7, and an HCDR3 having the amino acid sequence set forth in SEQ ID NO:8, and the light chain variable region comprises an LCDR1 having the amino acid sequence set forth in SEQ ID NO:9, an LCDR2 having the amino acid sequence set forth in SEQ ID NO:10, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:11; or the heavy chain variable region comprises an HCDR1 having an amino acid sequence as shown in SEQ ID NO:6, an HCDR2 having an amino acid sequence as shown in SEQ ID NO:12, and an HCDR3 having an amino acid sequence as shown in SEQ ID NO:8; the light chain variable region comprises an LCDR1 having an amino acid sequence as shown in SEQ ID NO:13, an LCDR2 having an amino acid sequence as shown in SEQ ID NO:10, and an LCDR3 having an amino acid sequence as shown in SEQ ID NO:11; wherein the amino acid sequences of HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3 are according to the Kabat definition: An anti-Siglec15 antibody or an antigen-binding fragment thereof.

2. The heavy chain variable region has an amino acid sequence set forth in SEQ ID NO: 26 or 28, or an amino acid sequence having at least 90%, 95%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 26 or 28. The anti-Siglec15 antibody or antigen-binding fragment thereof according to claim 1.

3. The light chain variable region has an amino acid sequence set forth in SEQ ID NO:27 or 29, or an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO:27 or 29. The anti-Siglec15 antibody or antigen-binding fragment thereof according to claim 1.

4. the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO:26 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO:27; or The heavy chain variable region has the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region has the amino acid sequence shown in SEQ ID NO:

29. The anti-Siglec15 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. The heavy chain variable region has an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:26 or 28, and the light chain variable region has an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to the amino acid sequence shown in SEQ ID NO:27 or 29. The anti-Siglec15 antibody or antigen-binding fragment thereof described in claim 1. Claim 6: The heavy chain variable region has an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:26, and the light chain variable region has an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:27; or The anti-Siglec15 antibody or antigen-binding fragment thereof according to claim 5, wherein the heavy chain variable region has an amino acid sequence having at least 90%, at least 95% or at least 99% identity with the amino acid sequence set forth in SEQ ID NO:28, and the light chain variable region has an amino acid sequence having at least 80%, at least 90%, at least 95% or at least 99% identity with the amino acid sequence set forth in SEQ ID NO:

29.

7. the anti-Siglec15 antibody is a full-length antibody or a single-chain antibody (scFv); and / or The antigen-binding fragment of the anti-Siglec15 antibody is Fab, Fab', Fv or F(ab'). 2 and / or the anti-Siglec15 antibody is a murine, chimeric, or humanized antibody; and / or the anti-Siglec15 antibody is a monoclonal antibody; and / or the anti-Siglec15 antibody is of the IgG1, IgG2 or IgG4 isotype; and / or The anti-Siglec15 antibody comprises a light chain constant region of the κ or λ subtype; An anti-Siglec15 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6.

8. An immuno-drug conjugate comprising the anti-Siglec15 antibody or antigen-binding fragment thereof of any one of claims 1 to 7 conjugated to a therapeutic agent.

9. The therapeutic agent is an antitumor drug. The immuno-drug complex according to claim 8.

10. A pharmaceutical composition comprising an anti-Siglec15 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or an immuno-drug complex according to claim 8 or 9, and a pharmacologically acceptable excipient, diluent or carrier.

11. for use in modulating an immune response in an individual, reversing the inhibition of PBMC lymphocyte proliferation by Siglec15 protein, reversing the inhibition of IFN-γ secretion from PBMC lymphocytes by Siglec15 protein, inhibiting tumor cell proliferation in an individual, or treating a hyperproliferative disorder associated with aberrant expression of Siglec15; The pharmaceutical composition according to claim 10.

12. The hyperproliferative disorder associated with abnormal expression of Siglec15 is a tumor associated with abnormal expression of Siglec15. The pharmaceutical composition according to claim 11.

13. The pharmaceutical composition is used to treat tumors in combination with an anti-PD-1 or anti-PD-L1 antibody. The pharmaceutical composition according to any one of claims 10 to 12.

14. Use of an anti-Siglec15 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or an immuno-drug conjugate according to claim 8 or 9, in the manufacture of a medicament for modulating an immune response in an individual, reversing inhibition of PBMC lymphocyte proliferation by Siglec15 protein, reversing inhibition of IFN-γ secretion from PBMC lymphocytes by Siglec15 protein, inhibiting tumor cell proliferation in an individual, or treating a hyperproliferative disorder associated with abnormal expression of Siglec15.

15. The hyperproliferative disorder associated with abnormal expression of Siglec15 is a tumor associated with abnormal expression of Siglec15.

15. The use according to claim 14.

16. The medicament further comprises an anti-PD-1 or anti-PD-L1 antibody.

16. The use according to claim 14 or 15.

17. An isolated polynucleotide encoding the anti-Siglec15 antibody or antigen-binding fragment of any one of claims 1 to 7.

18. A vector comprising a polynucleotide encoding the anti-Siglec15 antibody or antigen-binding fragment of any one of claims 1 to 7.

19. 20. An isolated host cell comprising the vector of claim 18.

20. A method for producing the anti-Siglec15 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising:

20. A method comprising culturing the host cell of claim 19, optionally further comprising isolating the anti-Siglec15 antibody or antigen-binding fragment thereof.

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