WO2022237819A1 - 抗Siglec-15抗体及其用途 - Google Patents

抗Siglec-15抗体及其用途 Download PDF

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WO2022237819A1
WO2022237819A1 PCT/CN2022/092136 CN2022092136W WO2022237819A1 WO 2022237819 A1 WO2022237819 A1 WO 2022237819A1 CN 2022092136 W CN2022092136 W CN 2022092136W WO 2022237819 A1 WO2022237819 A1 WO 2022237819A1
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antibody
seq
antigen
siglec
binding fragment
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PCT/CN2022/092136
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French (fr)
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荆华
陈炳良
李莉
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信达生物制药(苏州)有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids

Definitions

  • the present invention relates to the field of immune regulation, and more specifically relates to anti-Siglec-15 antibody and its application.
  • Sialic acid-binding Ig-like lectins are members of the Ig superfamily.
  • Two major subclasses of Siglecs have been identified: one subclass includes CD-33 and CD33-related Siglecs such as Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-14, and Siglec-16, and CD33 and Siglec-E, Siglec-F, Siglec-G, and Siglec-H in mice.
  • the second subclass consists of Sn (sialoadhesin) (Siglec-1), CD22 (Siglec-2), MAG (myelin-associated glycoprotein) (Siglec-4) and Siglec-15, which in mammals is very conservative.
  • Siglec-15 protein is widely expressed in a variety of tumor cells.
  • immune checkpoint PD-1/PD-L1 inhibitors have emerged. These strategies mainly target tumor immune escape mechanisms.
  • the PD-1/PD-L1 pathway is activated in the tumor microenvironment, the anti-tumor immune response of effector T cells is suppressed. Treatments that block this pathway are effective in improving antitumor immune responses in a variety of tumor types.
  • Anti-PD-1/PD-L1 therapy is currently the most famous and clinically effective cancer immunotherapy, but it is only effective against 20%-30% of human solid tumors. The low response to anti-PD-1/PD-L1 therapy suggests that there are other possible pathways of immunosuppression.
  • Siglec-15 are mutually exclusive in tumor tissue, which indicates that anti-Siglec-15 antibodies may be effective in patients who do not respond to anti-PD-1/PD-L1 therapy .
  • Siglec-15 may be a complementary therapeutic target that may provide a new treatment option for those patients who do not respond to anti-PD-1/PD-L1 therapy.
  • the anti-Siglec-15 antibody provided in the prior art (such as the Siglec-15 antibody provided in WO2018/057735) has a weak binding ability to the Siglec-15 protein and cannot effectively relieve the effect of the Siglec-15 protein on the proliferation and activation of T cells. inhibition.
  • the anti-Siglec-15 antibody provided in the prior art has weaker ability to bind to Siglec-15 molecules at the cellular level and weaker inhibitory effect of releasing Siglec-15 on T cell proliferation and activation.
  • the invention provides an anti-Siglec-15 antibody which has high affinity with Siglec-15 molecule and can effectively release the inhibitory effect of Siglec-15 on T cell proliferation and activation.
  • the present inventor has carried out in-depth research and trial and error, prepared hybridoma cells capable of specifically expressing anti-Siglec-15 antibodies through hybridoma technology, and screened the hybridoma cells On this basis, the anti-Siglec-15 antibody was expressed and purified, thus completing the present invention. That is, the present invention is as follows:
  • the present invention provides an antibody or an antigen-binding fragment thereof in a first aspect, wherein the antibody or an antigen-binding fragment thereof comprises six complementarity determining regions (CDRs), and the six CDRs are selected from the group consisting of Any of the groups: (i) SEQ ID NO: 15-17 and SEQ ID NO: 19-21; (ii) SEQ ID NO: 7-9 and SEQ ID NO: 11-13; or (iii) SEQ ID NO: 11-13; ID NOs: 23-25 and SEQ ID NOs: 27-29; and, wherein the antibody or antigen-binding fragment thereof can specifically bind to the Siglec-15 protein.
  • CDRs complementarity determining regions
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region selected from the group consisting of Any of: (i) SEQ ID NO: 18 and SEQ ID NO: 22, or with SEQ ID NO: 18 and/or SEQ ID NO: 22 containing at least 50%, 60%, 70%, 80%, 85% %, 90%, 95%, 99% or higher sequence identity; (ii) SEQ ID NO: 10 and SEQ ID NO: 14, or with SEQ ID NO: 10 and/or SEQ ID NO: 14 variants thereof containing at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or higher sequence identity; (iii) SEQ ID NO: 26 and SEQ ID NO: 30, or other sequences having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 26 and/or SEQ ID NO: 30 Variants.
  • said antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, said heavy chain and light chain being selected from any one of the group consisting of: (i) SEQ ID NO : 3 and SEQ ID NO: 4, or contain at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or Variants thereof with higher sequence identity; (ii) SEQ ID NO: 1 and SEQ ID NO: 2, or contain at least 50%, 60%, 70% with SEQ ID NO: 1 and/or SEQ ID NO: 2 , 80%, 85%, 90%, 95%, 99% or higher sequence identity of its variant; (iii) SEQ ID NO: 5 and SEQ ID NO: 6, or with SEQ ID NO: 5 and / or SEQ ID NO: 6, variants thereof containing at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity.
  • the antibody described in the first aspect of the present invention is a monoclonal antibody, a chimeric antibody or a humanized antibody.
  • the resulting antibody can be purified to homogeneity.
  • methods generally used for isolation and purification of proteins can be used. For example, chromatographic columns, filters, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric point electrophoresis, and the like can be appropriately selected and combined for separation and purification of antibodies, but are not limited to these.
  • the second aspect of the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention in the preparation of anti-tumor drugs.
  • the tumor is a solid tumor; in other embodiments, the anti-tumor drug further includes an immune checkpoint inhibitor and/or an immune agonist.
  • the third aspect of the present invention provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention and a pharmaceutically acceptable carrier or excipient.
  • the fourth aspect of the present invention provides a DNA molecule, characterized in that the DNA molecule encodes the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.
  • the present invention provides a recombinant vector in the fifth aspect, characterized in that, the recombinant vector comprises the coding sequence of the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention or comprises the antibody or antigen-binding fragment thereof described in the fourth aspect of the present invention A DNA molecule as described in the aspect.
  • a "vector” is a replicon, such as a plasmid, phage, virus or cosmid, into which another segment of DNA may be inserted to effectuate replication of the inserted segment.
  • the vector can be an expression vector.
  • An "expression vector” is a vector that includes one or more expression control sequences, which are DNA sequences that control and regulate the transcription and/or translation of another DNA sequence.
  • Suitable expression vectors include, but are not limited to, plasmid and viral vectors derived from, for example, bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus, and adeno-associated virus.
  • Expression vectors can include tag sequences. Tag sequences are usually expressed as fusions to the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including the carboxyl or amino termini.
  • useful labels include, but are not limited to, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, maltose E binding protein, and protein A.
  • GFP green fluorescent protein
  • GST glutathione S-transferase
  • polyhistidine polyhistidine
  • c-myc hemagglutinin
  • maltose E binding protein and protein A.
  • the sixth aspect of the present invention provides a recombinant host cell, characterized in that the recombinant host cell comprises the recombinant vector as described in the fifth aspect of the present invention.
  • the term "host cell” is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced.
  • the seventh aspect of the present invention provides a kit for treating tumors, which is characterized in that the kit contains the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or contains the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention
  • the pharmaceutical composition described in the third aspect in some preferred embodiments, the tumor is a solid tumor.
  • the present invention provides a method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention , or administering a therapeutically effective amount of the pharmaceutical composition as described in the third aspect of the present invention.
  • the subject suffers from cancer or an infectious disease; preferably, the subject suffers from a cancer comprising cells expressing or overexpressing Siglec-15 ligand; more preferably, The cancer is a solid tumor.
  • the method further comprises administering a second therapeutic agent to the subject; preferably, the second therapeutic agent is an immune checkpoint inhibitor and/or an immune agonist.
  • the ninth aspect of the present invention provides a method for detecting or diagnosing a disease, characterized in that the method comprises: (a) using the antibody or antigen-binding fragment thereof described in the first aspect of the present invention to detect Siglec-15 expression level in the cell or tissue sample of patient, and (b) Siglec-15 expression level described in (a) is compared with control level, wherein compared with described control level, the Siglec-15 expression level of described determination An increased expression level of -15 indicates that the subject has the disease.
  • the term “comprising” or “comprising” means including stated elements, integers or steps, but not excluding any other elements, integers or steps.
  • the term “comprising” or “comprises” is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps.
  • an antibody variable region that "comprises” a particular sequence it is also intended to encompass an antibody variable region that consists of that particular sequence.
  • antibody is intended to mean an immunoglobulin molecule having a "variable region” antigen recognition site.
  • antigen-binding fragment of an antibody refers to one or more portions of an antibody that contain the complementarity determining regions ("CDRs") of an antibody and optionally include an antibody "variable region” antigen recognition site framework residues and exhibits the ability to immunospecifically bind antigen.
  • CDRs complementarity determining regions
  • Such fragments include Fab', F(ab') 2 , Fv, single chain (ScFv) and mutants thereof, naturally occurring variants, and those comprising antibody "variable region” antigen recognition sites and heterologous proteins (e.g. , fusion proteins of toxins, antigen recognition sites of different antigens, enzymes, receptors or receptor ligands, etc.).
  • variable region refers to the domains of an antibody heavy or light chain that participate in the binding of the antibody to an antigen.
  • the variable domains of the heavy chain (HC) and light chain (LC) of native antibodies generally have a similar structure, with each domain comprising four conserved framework regions (FR) and three complementarity determining regions (see, e.g. , Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co. 91 p. (2007)).
  • a single heavy chain variable region (VH) or light chain variable region (VL) domain may be sufficient to confer antigen binding specificity.
  • VH or VL domains from antibodies that bind a particular antigen can be used to isolate antibodies that bind that antigen to screen libraries of complementary VL or VH domains, respectively. See, eg, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature, 352:624-628 (1991).
  • Variable regions generally exhibit the same general structure of relatively conserved framework regions (FRs) joined by three hypervariable regions, also known as complementarity determining regions or CDRs.
  • the CDRs from the two chains of each pair, which allow binding of specific epitopes, are usually aligned by the framework regions.
  • Both light and heavy chain variable regions generally comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from N-terminus to C-terminus.
  • a “complementarity determining region” or “CDR region” or “CDR” or “hypervariable region” is an antibody variable domain that is hypervariable in sequence and Structurally defined loops ("hypervariable loops") and/or regions containing antigen contact residues ("antigen contact points") are formed.
  • the CDRs are primarily responsible for binding to antigenic epitopes.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially starting from the N-terminus.
  • the CDRs located within the variable domain of an antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of an antibody light chain are referred to as LCDR1, LCDR2, and LCDR3.
  • the precise amino acid sequence boundaries of each CDR can be determined using any one or combination of a number of well-known antibody CDR assignment systems, including For example: Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al., (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S.
  • bind or “specifically bind” means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions.
  • the ability of an antibody to bind a particular antigen can be determined by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), or optical interferometry of biofilm layers (ForteBio) or other conventional binding assays known in the art.
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • FormeBio optical interferometry of biofilm layers
  • an antibody or antigen-binding fragment binds to an antigenic epitope in an in vitro assay, preferably in optical interferometry of biofilm layers using purified wild-type antigen.
  • an antibody or antigen-binding fragment is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
  • variant refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide but retains essential properties.
  • a typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Typically, the differences are limited such that the sequences of the reference polypeptide and the variant are very similar overall, and identical in many regions.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (eg, substitutions, additions and/or deletions). The substituted or inserted amino acid residues may or may not be those encoded by the genetic code.
  • Variants of a polypeptide may be naturally occurring, such as allelic variants, or may be variants not known to occur in nature.
  • percent (%) sequence identity is defined as the difference between the nucleotides or amino acids in a candidate sequence and the nucleotides in a reference nucleic acid sequence after aligning the sequences and introducing gaps, if necessary, to achieve the highest percent sequence identity achieved. or the same percentage of amino acids. Alignment for determining percent sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software . Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared, can be determined by known methods.
  • chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules, eg, an antibody having variable regions derived from a non-human antibody and human immunoglobulin constant regions.
  • humanized antibody refers to an immunoglobulin that includes human framework regions and one or more CDRs from a non-human (usually mouse or rat) immunoglobulin.
  • the non-human immunoglobulin that provides the CDRs is called the "donor” and the human immunoglobulin that provides the framework is called the “acceptor”.
  • Constant regions need not be present, but if they are, they should be substantially identical, ie at least about 85-99%, preferably about 95% or more identical to human immunoglobulin constant regions.
  • all parts of a humanized immunoglobulin are substantially identical to corresponding parts of native human immunoglobulin sequences.
  • a humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin.
  • a humanized antibody would not encompass a typical chimeric antibody because, for example, the entire variable region of a chimeric antibody is non-human.
  • therapeutically effective amount refers to an amount of a therapeutic agent sufficient to mediate clinically relevant elimination, alleviation or alleviation of such symptoms. An effect is clinically relevant if the magnitude of the effect is sufficient to affect the health or prognosis of the recipient subject.
  • a therapeutically effective amount can refer to an amount of a therapeutic agent sufficient to delay or minimize the onset of disease, eg, an amount sufficient to delay or minimize the spread of cancer.
  • a therapeutically effective amount can also refer to the amount of a therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease.
  • cancer refers to a neoplasm or tumor arising from the abnormal, uncontrolled growth of cells.
  • the technical solution of the present invention has the following beneficial effects:
  • the anti-Siglec-15 antibody provided by the invention has a unique antibody sequence
  • the anti-Siglec-15 antibody provided by the present invention can bind Siglec-15 protein with high affinity and effectively release the inhibition of Siglec-15 protein on T cell proliferation and activation;
  • the anti-Siglec-15 antibody provided by the present invention can release the inhibitory effect of Siglec-15 protein on the tumor immune response, enhance the tumor immune response, and inhibit the growth of tumor in the mouse tumor model in vivo, showing effective anti-tumor efficacy;
  • the anti-Siglet-15 antibody provided by the present invention can be used alone or in combination with drugs (including immune checkpoint inhibitors and/or immune agonists) used in various immunotherapy methods to improve the response rate of tumor patients, It can be used to treat patients with solid tumors that do not respond to PD-1 therapy.
  • drugs including immune checkpoint inhibitors and/or immune agonists
  • Figure 1 shows the binding activity of different kinds of anti-Siglec-15 antibodies to GS-CHO cells expressing human Siglec-15 protein
  • Figure 2 shows the binding activity of different kinds of anti-Siglec-15 antibodies to GS-CHO cells expressing cynomolgus Siglec-15 protein
  • Figure 3 shows the binding activity of different kinds of anti-Siglec-15 antibodies to GS-CHO cells expressing mouse Siglec-15 protein
  • Figure 4 shows the binding activity of Siglec-15 protein and CD8+T cells detected by flow cytometry
  • FIG. 5 shows that Siglec-15 protein can significantly inhibit the proliferation of T cells activated by anti-CD3 antibody
  • Figure 6 shows that different kinds of anti-Siglec-15 antibodies promote the proliferative activity of CD4+T cells
  • Figure 7 shows that different kinds of anti-Siglec-15 antibodies promote the proliferative activity of CD8+T cells
  • Figure 8 shows the binding activity of Siglec-15 protein and CD14+ monocytes detected by flow cytometry
  • Figure 9 shows that anti-Siglec-15 antibody inhibits the monocyte survival activity mediated by Siglec-15 protein
  • Figure 10 shows a schematic diagram of the curve of the tumor volume of the mice over time after injection of different types of anti-Siglec-15 antibodies in MC38 tumor-bearing mice;
  • Figure 11 shows a schematic diagram of the curve of mouse body weight over time after injection of different types of anti-Siglec-15 antibodies into MC38 tumor-bearing mice;
  • Figure 12 shows the expression of Siglec-15 protein in bone marrow-derived macrophages detected by flow cytometry
  • Figure 13 shows a schematic diagram of the curve of the tumor volume of the mice over time after injection of different types of anti-Siglec-15 antibodies in CT26 tumor-bearing mice;
  • Fig. 14 is a schematic diagram showing the curve of the body weight of CT26 tumor-bearing mice injected with different kinds of anti-Siglec-15 antibodies over time.
  • test materials, test reagents and instruments used in the examples of the present invention are all commercially available.
  • Hybridoma technology is lymphocyte hybridoma technology, also known as monoclonal antibody technology, which is a somatic cell fusion technology that fuses myeloma cells and immune lymphocytes to form monoclonal antibodies that can secrete highly specific monoclonal antibodies.
  • hybridoma technology fuses two types of cells while maintaining the main characteristics of both.
  • the two kinds of cells are antigen-immunized mouse splenocytes and mouse myeloma cells, respectively.
  • mouse spleen cells (B lymphocytes) immunized with specific antigens is that they have antibody secretion function, but they cannot be continuously cultured in vitro; mouse myeloma cells can divide and proliferate indefinitely under culture conditions, that is, they have So-called immortality. Under the action of the selection medium, only the hybrid cells fused with B lymphocytes and myeloma cells can have the ability of continuous culture, and form cell clones with both characteristics of antibody secretion and cell immortality.
  • the human Siglec-15 protein was used as an antigen to immunize mice, and then the splenocytes of the immunized mice were fused with myeloma cells to obtain hybridoma cells expressing positive antibodies.
  • the specific operation is as follows:
  • Table 1 Types of experimental animals and information about immunizations
  • Electrofusion dish preparation thoroughly soak the electric fusion dish with 70% ethanol, and dry it in a clean bench for later use.
  • Isolation of splenocytes kill the mice by cervical dislocation, disinfect the body surface with 75% alcohol for 5 minutes, then put them on the mouse dissecting board in the ultra-clean bench, lie on the left side, and fix the limbs with No. 7 needles.
  • the spleen was then transferred to another plate containing basal medium. Press the spleen with a curved needle, insert a hole in the spleen with a small needle, and squeeze it with tweezers to fully release the spleen cells to make a spleen cell suspension.
  • the cell suspension was filtered through a 70 ⁇ M cell mesh, washed once with 30 ml of basal medium, and centrifuged at 1200 rpm for 6 min.
  • Lyse red blood cells Remove the supernatant and resuspend the cells with 10ml RBC Lysis Buffer (GIBCO, A10492-01). Then add 20ml RBC Lysis Buffer. The suspension was left to stand for 5 minutes and then centrifuged at 1100 rpm for 6 minutes. After removing the supernatant, resuspend the cells with 10ml basal medium, then add 30ml basal medium, and centrifuge at 1100rpm for 6min. After removing the supernatant, cells were resuspended in 20 ml of basal medium and counted.
  • Electrofusion Resuspend mouse myeloma cells SP2/0 cells (ATCC, CRL-1581) with 20 ml of basal medium and count them.
  • the SP2/0 cells and the splenocytes described above were mixed at a ratio of 1:2 to 1:1, and centrifuged at 1000 rpm for 6 min. After removing the supernatant, the mixed cells were resuspended in 10 ml fusion buffer (BTXpress, 47-0001). Then add 15ml of fusion buffer, centrifuge at 1000rpm for 5min, and remove the supernatant. After repeating the above steps once, resuspend the cells with an appropriate amount of fusion buffer to make a cell suspension, and adjust the mixed cell density to 1 ⁇ 10 7 cells/ml.
  • the parameter settings of the electrofusion instrument are shown in Table 3 below. Add 2ml of cell suspension to each electrofusion dish for electrofusion.
  • Plating after electrofusion Place the cells in an electrofusion dish at room temperature for 5 minutes. Transfer the cells into a centrifuge tube, and dilute the cells to 1 ⁇ 10 4 -2 ⁇ 10 4 cells/ml with screening medium (the specific components and preparation methods are shown in Table 4 below). Add 100 ⁇ l of cell suspension to each well of a 96-well plate. The selection medium was replaced 7 days after fusion. Screening was performed after culture day 10 (or longer, depending on cell growth status). Hybridoma cells expressing specific anti-Siglec-15 antibodies were screened by FACS (C6 (BD Biosciences)) detection.
  • Subcloning step Prepare a 96-well plate, add 200 ⁇ l of medium to each well of the 2nd to 8th row, the medium is based on the screening medium and replace HAT with HT (Gibco, Cat#11067-030), and the rest The recipe is the same. Take 300 ⁇ l of the cells in the positive wells screened out above at a density of about 1 ⁇ 10 5 cells/ml and add them to each well in the first row. Using a row grab, take 100 ⁇ l of the cell suspension in the first row and add it to the second row, mix well and then take 100 ⁇ l and add it to the next row.
  • Hybridoma cells with anti-Siglec-15 antibody were obtained through the above operations.
  • the chimeric antibody was humanized according to the method described in Example 2, and the weights of the three antibodies constructed in this example (named hz59A3.
  • the amino acid sequences of the chain and light chain are listed in Table 5.
  • the amino acid sequence of the gray part in the amino acid sequence shown in Table 5 is the amino acid sequence of the heavy chain variable region/light chain variable region in the heavy chain/light chain; the bold part of the amino acid sequence shown in Table 5
  • the amino acid sequence is the CDR1, CDR2, and CDR3 sequences in the heavy chain variable region/light chain variable region.
  • amino acid sequence of the heavy chain variable region contained in the heavy chain of the hz59A3.3.p1 antibody is:
  • the amino acid sequence of the light chain variable region contained in the light chain of the hz59A3.3.p1 antibody is: DIVMTQSPDSLAVSLGERATINCKASQSVDYEGDSYMNWYQQKPGQPPKLLIYVASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIK (SEQ ID NO: 14), the amino acid sequences of CDR1 and CDR2 contained in the above light chain variable region are: KASQSVDYEGDSYMN (SEQ ID NO: 11), VASNLES (SEQ ID NO: 12) and QQSNEDPYT (SEQ ID NO: 13).
  • the amino acid sequence of the heavy chain variable region contained in the heavy chain of the hz28D6.3.p1 antibody is: QVQIVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLEWMGWINTETGEPTYADDFKGRFVFSLDTSVSMAYLQISSLKAEDTAVYYCARGLWEGDVWGQGTMVTVSS, CDR1 in the heavy chain variable region and CDR1 region
  • the amino acid sequences are: GYTFTDYSMH (SEQ ID NO: 15), WINTETGEPTYADDFKG (SEQ ID NO: 16) and GLWEGDV (SEQ ID NO: 17).
  • the amino acid sequence of the light chain variable region contained in the light chain of the hz28D6.3.p1 antibody is: EIVITQSPATLSSPGERATLSCSASSSISYAHWYQQKPGQAPRRWIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPWTFGQGTKLEIK (SEQ ID NO: 22); the amino acid sequences of CDR1, CDR2 and CDR3 contained in the light chain variable region are respectively are: SASSSISYAH (SEQ ID NO: 19), DTSKLAS (SEQ ID NO: 20) and HQRSSYPWT (SEQ ID NO: 21).
  • the amino acid sequence of the heavy chain variable region contained in the heavy chain of the 2-hz21C7.3 antibody is: QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWVRQAPGQGLEWMGDINPGGGYTNYNENFKGRVTMTADTISTAYMELSRLRSDDTAVYYCVSFYGDYQFDFWGQCDGTLVTVSS (included in the above CDR1 heavy chain variable region, CDR1 heavy chain: 26);
  • the amino acid sequences are: GYTFTNYWLG (SEQ ID NO: 23), DINPGGGYTNYNENFKG (SEQ ID NO: 24) and FYGDYQFDF (SEQ ID NO: 25).
  • the amino acid sequence of the light chain variable region contained in the light chain of the 2-hz21C7.3 antibody is: DIQMTQSPSSLSASVGDRVTITCSVSSSISSINLHWYQQKPEKAPKPWIYGASNLASGVPSRFSGSGSGTDYTITISSLQPEDFATYYCQQWSSFPLTFGGGTKVEIK (SEQ ID NO: 30); the amino acid sequences of CDR1, CDR2 and CDR1 contained in the light chain variable region are respectively are: SVSSSISSINLH (SEQ ID NO: 27), GASNLAS (SEQ ID NO: 28) and QQWSSFPLT (SEQ ID NO: 29).
  • the CDR1 contained in the heavy chain variable region of the above-mentioned antibody is defined according to AbM
  • the CDR2 and CDR3 contained in the heavy chain variable region of the above-mentioned antibody are defined according to Kabat
  • CDR1, CDR2 and CDR3 are defined according to Kabat.
  • the above antibodies were expressed and purified in GS-CHO cells or HEK293 cells, and the specific operations were as follows:
  • Antibody-expressing CHO-S cell lines were generated using the GS Xceed TM Gene Expression System kit (Lonza) according to the manufacturer's instructions.
  • the DNA coding sequences of the heavy chain and light chain of the antibody molecules described in Table 5 were inserted into the same pCHO1.0 plasmid, wherein the heavy chain was upstream of the light chain to obtain the corresponding recombinant pCHO1.0 plasmid.
  • the constructed recombinant pCHO1.0 plasmid was transformed into CHO-S cell line by chemical transfection method and electrotransfection method, and the antibody production was detected by ForteBio 48 hours after transfection to judge the transfection efficiency.
  • the transfected cells were subjected to two rounds of pressurized selection to obtain a pool of high-expressing antibody cells. Then expand the cell pool, express the antibody in large quantities, and collect the cell supernatant and purify the supernatant with Protein A to make the purity of the antibody >95%.
  • vectors pV120 or pcDNA3.1 were used for transient expression of antibodies in HEK293 cells.
  • the cDNAs encoding the heavy and light chains of the antibodies described in Table 5 were cloned into separate pV120 vectors or pcDNA3.1 vectors to obtain the corresponding recombinant vectors.
  • the recombinant vector carrying the heavy chain and light chain of the antibody molecule was transferred into HEK293 cells by chemical transfection.
  • the chemical transfection reagent used was PEI (purchased from Polysciences), and cultured 293HEK (Invitrogen) was transiently transfected according to the protocol provided by the manufacturer.
  • the medium was discarded and the cells were diluted to 4 x 106/ml with fresh EXPI293 medium (Gibco).
  • Cells were cultured for 7 days at 37°C, 5% CO 2 , with fresh medium added every 48 hours. After 7 days, centrifuge at 1300rpm for 20min. Take the supernatant, and purify the supernatant with Protein A to make the purity of the antibody >95%.
  • Embodiment 4 Antibody affinity determination experiment
  • Example 3 The three antibodies described in Example 3 in Table 5 and a control antibody (i.e., hz5G12, which is synthesized according to the sequence in patent WO2018057735A8) combined with human, cynomolgus monkey and mouse Siglec were determined by bio-light interferometry (ForteBio) assay. Equilibrium dissociation constant (KD) of -15 antigen.
  • KD Equilibrium dissociation constant
  • the senor was equilibrated offline for 30 minutes in assay buffer (HBS buffer, catalog number: BR-1006-69), and then detected online for 60 seconds to establish a baseline, and loaded human, cynomolgus monkey and mouse Siglec-15 antigens online to ForteBio affinity measurements were performed on AHQ sensors (ForteBio).
  • the sensor with loaded antigen was then exposed to 100 nM of the purified Siglec-15 antibody obtained according to the method described in Example 3 for 5 minutes, after which the sensor was transferred to the assay buffer for dissociation for 5 minutes for dissociation rate Measurement. Analysis of kinetics was performed using a 1:1 binding model.
  • the affinities of the 2-hz21C7.3 antibody, the hz28D6.3.p1 antibody, the hz59A3.3.p1 antibody and the control antibody hz5G12 in the experiments described by the above assays are shown in Table 6.
  • Example 5 Binding experiment of anti-Siglec-15 antibody to human, cynomolgus monkey and mouse Siglec-15 expressed on the cell surface
  • the binding ability of the three antibodies described in Example 3 in Table 5 to human, cynomolgus monkey and mouse Siglec-15 overexpressed on the surface of GS-CHO cells (Lonza) was measured based on flow cytometry assay.
  • the hIgG1 antibody is selected as a negative control
  • the hz5G12 antibody is selected as a positive control
  • the amino acid sequence of the heavy chain in the hIgG1 antibody is shown in SEQ ID NO: 31 (the specific amino acid sequence is:
  • Test results It can be seen from the results of Figures 1-3 that, compared with the control antibody, the three antibodies constructed by the present invention have higher binding activity to GS-CHO cells expressing human and cynomolgus Siglec-15; 2- The binding ability of hz21C7.3 and hz59A3.3.p1 antibodies to GS-CHO cells expressing mouse Siglec-15 is stronger than that of the control antibody, and the binding ability of hz28D6.3.p1 to GS-CHO cells expressing mouse Siglec-15 is higher than that of the control Antibodies are comparable.
  • Siglec-15 protein can bind to unknown ligands on T cells, thereby inhibiting the activity and proliferation of T cells.
  • CD8+ T cells activated by CD3/CD28beads after adding human Siglec-15-mFc fusion protein (i.e. hSiglec-15-mFc, R&D, 9227-SL-050), and then adding anti-Fc secondary antibody, it can be detected by flow cytometry To the combination of Siglec-15 protein and CD8+ T cells (see Figure 4 for details).
  • the addition of Siglec-15 protein can significantly inhibit the proliferation of T cells activated by anti-CD3 antibody (see Figure 5 for details).
  • this study added anti-CD3 antibody to CD4+T cell and CD8+T cell culture system to promote T cell proliferation, and further added Siglec-15 protein to inhibit T cell proliferation.
  • the activity of different anti-Siglec-15 antibody molecules to promote T cell proliferation can be evaluated.
  • the specific experimental process is:
  • Antibody preparation, negative control group (hIgG1 antibody) and antibody group (including hz28D6.3p1 antibody and hz59A3.3p1 antibody and positive control antibody hz5G12) were added at the same time 20 ⁇ g/ml of human Siglec-15-mFc fusion protein, 37 °C It was added to the cells after premixing for 30 minutes.
  • the cell proliferation ratio was detected by flow cytometry (BD, Celesta).
  • (6) Acquire the data and draw the cell proliferation curve, and compare the EC50 and the peak value of the curve (see Figure 6 and Figure 7 for details).
  • the hz28D6.3.p1 antibody promotes the proliferation of human CD4+T cells and CD8+T cells stronger than the positive control antibody hz5G12 (expressed as lower EC50).
  • the activity of hz59A3.3.p1 antibody in promoting the proliferation of human CD4+T cells and CD8+T cells was equivalent to that of the positive control antibody hz5G12 (appeared to be close to EC50).
  • Example 7 Anti-Siglec-15 antibody inhibits Siglec-15 protein-mediated monocyte survival activity
  • Siglec-15 protein can bind to an unknown ligand on monocytes, thereby promoting the survival of monocytes.
  • CD14+ monocytes after adding human Siglec-15-mFc fusion protein (i.e. hSiglec-15-mFc), and then adding anti-Fc secondary antibody, the binding activity of Siglec-15 protein to CD14+ monocytes can be detected by flow cytometry (See Figure 8 for details).
  • Siglec-15 protein can bind monocytes and significantly promote the survival of CD14+ monocytes. Based on this principle, this study added Siglec-15 protein to CD14+ monocytes to promote the survival of monocytes, and further added anti-Siglec-15 antibody to inhibit the survival of monocytes. In this system, the activity of different anti-Siglec-15 antibodies to inhibit monocyte survival can be evaluated.
  • the specific experimental process is:
  • Anti-Siglec-15 antibody can significantly inhibit the survival of monocytes induced by Siglec-15 protein, hz59A3.3.p1 and hz28D6.3.p1 can completely inhibit the survival of monocytes at high concentrations, and hz21C7.3 inhibits the survival of monocytes
  • the cell survival activity was weaker than that of hz59A3.3.p1 and hz28D6.3.p1, and comparable to that of the control antibody hz5G12.
  • Example 8 Anti-tumor efficacy of anti-Siglec-15 antibody in MC38 tumor-bearing mouse model
  • Siglec-15 is highly expressed in human tumor tissues.
  • this example uses MC38 cells overexpressing Siglec-15 to evaluate the anti-tumor activity of the antibody.
  • the specific experimental steps are as follows: MC38 cells overexpressing Siglec-15 (Innoventbio, hereinafter referred to as MC38-Siglec-15 cells) were routinely subcultured, and 4 ⁇ g/ml of puromycin (Gibico, A11138-02) was added during the culture process.
  • mice On the 5th day after tumor cell inoculation, the mice were divided into serpentine groups according to the tumor size (8 mice in each group), administered on the 5th, 8th, 11th, and 14th days after inoculation, respectively, and the tumor volume and body weight of the mice were monitored twice a week. Monitoring ended after 24 days.
  • Body weight was measured using an electronic balance.
  • Example 9 Anti-tumor efficacy of anti-Siglec-15 antibody in CT26 tumor-bearing humanized mouse model
  • Siglec-15 protein The expression profile of Siglec-15 protein is very different between human and mouse. Siglec-15 protein is highly expressed in human tumor tissues and myeloid cells inside tumors, but not in mouse tumors. To mimic the patient tumor microenvironment in a mouse model, this study first induced bone marrow cells to become macrophages. The induced macrophages were detected by flow cytometry for the expression of Siglec-15 protein.
  • the specific experimental process is:
  • CT26 cells (ATCC, CRL-2638) were routinely subcultured. Before inoculation, trypsinize and collect cells by centrifugation, and disperse cells to 7.5 ⁇ 10 5 cells/ml with PBS. Resuspend bone marrow-derived macrophages in PBS to 7.5 ⁇ 10 5 cells/ml. Subcutaneously inoculate 0.2ml of CT26 cell suspension and 0.2ml of bone marrow-derived macrophage suspension (that is, 1.5 ⁇ 10 5 CT26 tumor cells and 1.5 ⁇ 10 5 bone marrow-derived macrophages/mouse) into BALB/c mice in the right abdominal region of the mouse.
  • mice On the first day after tumor cell inoculation, the mice were randomly divided into groups (8 mice in each group), administered on days 0, 4, 7, 11, and 14 after inoculation, and the tumor volume and body weight of the mice were monitored twice a week. Monitoring ended after 21 days.
  • Tumor volume measurement: the maximum long axis (L) and maximum width axis (W) of the tumor were measured with a vernier caliper, and the tumor volume was calculated according to the following formula: V L ⁇ W 2 /2.
  • Body weight was measured using an electronic balance.
  • the results of tumor inhibition rate are shown in Figure 13: compared with the negative control hIgG1 antibody, 10mg/kg hz28D6.3.p1 antibody, hz59A3.3.p1 antibody, 2-hz21C7.3 antibody and positive control antibody (i.e. hz5G12 antibody) can Significantly inhibited the growth of CT26 tumors; compared with the positive control antibody, hz28D6.3.p1 antibody and 2-hz21C7.3 antibody had better tumor inhibitory effect than the positive control antibody; at the same time, the body weight of the mice was detected, and the results are shown in Figure 14. In the group of mice administered with the drug, there was no significant decrease in body weight.

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Abstract

一种抗Siglec-15抗体及其用途。所述抗体或其抗原结合片段包含六个互补决定区(CDR)，所述抗体能够以高亲和力结合Siglec-15蛋白并有效解除Siglec-15蛋白对T细胞增殖和激活的抑制，并且所述抗体在小鼠体内肿瘤模型中可以解除Siglec-15蛋白对肿瘤免疫反应的抑制作用、增强肿瘤免疫反应、抑制肿瘤的生长，显示出了有效的抗肿瘤药效。

Description

抗Siglec-15抗体及其用途
优先权和相关申请
本申请要求2021年5月13日提交的名称为“抗Siglec-15抗体及其用途”的中国专利申请202110522398.5的优先权，该申请包括附录在内的全部内容作为参考并入本申请。
技术领域
本发明涉及免疫调节领域，更具体地涉及抗Siglec-15抗体及其用途。
背景技术
唾液酸结合性Ig样凝集素(“Siglec”)是Ig超家族的成员。已经鉴定了Siglec的两个主要亚类：一个亚类包括CD-33和CD33相关Siglec诸如人类中的Siglec-5、Siglec-6、Siglec-7、Siglec-8、Siglec-9、Siglec-10、Siglec-11、Siglec-14和Siglec-16，以及小鼠中的CD33和Siglec-E、Siglec-F、Siglec-G和Siglec-H。第二亚类由Sn(唾液酸粘附素)(Siglec-1)、CD22(Siglec-2)、MAG(髓磷脂相关糖蛋白)(Siglec-4)和Siglec-15构成，其在哺乳动物中是非常保守的。
Siglec-15蛋白广泛表达于多种肿瘤细胞。近十年来，出现了以免疫检查点PD-1/PD-L1抑制剂为代表的一种新的免疫治疗方法，这些策略主要针对肿瘤免疫逃逸机制。当PD-1/PD-L1通路在肿瘤微环境中被激活时，效应T细胞的抗肿瘤免疫反应被抑制。阻断这一途径的治疗可有效改善多种肿瘤类型的抗肿瘤免疫反应。抗PD-1/PD-L1治疗是目前最著名和临床最有效的癌症免疫治疗方法，但它仅对20％-30％的人实体瘤有效。抗PD-1/PD-L1治疗低响应表明，还有其他可能的免疫抑制途径。研究发现，PD-L1和Siglec-15的表达是在肿瘤组织中互斥的，这预示着抗Siglec-15的抗体有可能在抗PD-1/PD-L1治疗无响应的患者身上是有效的。Siglec-15可能是一个补充治疗靶点，可以为那些抗PD-1/PD-L1治疗无响应的患者提供新的治疗选择。
现有技术中提供的抗Siglec-15抗体(如WO2018/057735中提供的Siglec-15抗体)与Siglec-15蛋白的结合能力较弱、无法有效解除Siglec-15蛋白对T细胞的增殖和激活的抑制作用。
发明内容
发明要解决的问题
针对现有技术中提供的抗Siglec-15抗体在细胞水平与Siglec-15分子结合能力较弱以及解除Siglec-15对T细胞增殖和激活的抑制作用较弱等缺陷。本发明提供了一种与Siglec-15分子具有高亲和力、并且能有效解除Siglec-15对T细胞增殖和激活的抑制作用的抗Siglec-15抗体。
用于解决问题的方案
本发明人鉴于上述现有技术中存在的问题，进行了深入的研究、反复试验，通过杂交瘤技术制备能够特异性表达抗Siglec-15抗体的杂交瘤细胞，并对所述杂交瘤细胞进行筛选，在此基础上对抗Siglec-15抗体进行表达、纯化，从而完成了本发明。即本发明如下所述：
本发明在第一方面提供了一种抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含六个互补决定区(CDR)，所述六个CDR选自由以下项组成的组中的任一种：(i)SEQ ID NO：15～17和SEQ ID NO：19～21；(ii)SEQ ID NO：7～9和SEQ ID NO：11～13；或(iii)SEQ ID NO：23～25和SEQ ID NO：27～29；并且，其中所述抗体或其抗原结合片段能够与Siglec-15蛋白特异性结合。
在一些具体的实施方式中，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区和轻链可变区选自由以下项组成的组中的任一种：(i)SEQ ID NO：18和SEQ ID NO：22，或者与SEQ ID NO：18和/或SEQ ID NO：22含有至少50％、60％、70％、80％、85％、90％、95％、99％或更高序列同一性的其变体；(ii)SEQ ID NO：10和SEQ ID NO：14，或者与SEQ ID NO：10和/或SEQ ID NO：14含有至少50％、60％、70％、80％、85％、90％、95％、99％或更高序列同一性的其变体；(iii)SEQ ID NO：26 和SEQ ID NO：30，或者与SEQ ID NO：26和/或SEQ ID NO：30含有至少50％、60％、70％、80％、85％、90％、95％、99％或更高序列同一性的其变体。
在另一些具体的实施方式中，所述抗体或其抗原结合片段包含重链和轻链，所述重链和轻链选自由以下项组成的组中的任一种：(i)SEQ ID NO：3和SEQ ID NO：4，或者与SEQ ID NO：3和/或SEQ ID NO：4含有至少50％、60％、70％、80％、85％、90％、95％、99％或更高序列同一性的其变体；(ii)SEQ ID NO：1和SEQ ID NO：2，或者与SEQ ID NO：1和/或SEQ ID NO：2含有至少50％、60％、70％、80％、85％、90％、95％、99％或更高序列同一性的其变体；(iii)SEQ ID NO：5和SEQ ID NO：6，或者与SEQ ID NO：5和/或SEQ ID NO：6含有至少50％、60％、70％、80％、85％、90％、95％、99％或更高序列同一性的其变体。
在一些优选的实施方式中，本发明在第一方面中所述的抗体为单克隆抗体、嵌合抗体或人源化抗体。
可以将得到的抗体纯化至均一。抗体的分离、纯化可以使用通常用于蛋白质的分离、纯化方法。例如，可以适当选择和组合色谱柱、过滤器、超滤、盐析、透析、制备用聚丙烯酰胺凝胶电泳、等电点电泳等来进行抗体的分离、纯化，但是不限于这些。
本发明在第二方面提供了利用本发明在第一方面中所述的抗体或其抗原结合片段在制备治疗抗肿瘤药物中的用途。
在一些具体的实施方式中，所述肿瘤为实体瘤；在另外一些实施方式中，所述抗肿瘤药物中进一步包含免疫检查点抑制剂和/或免疫激动剂。
本发明在第三方面提供了一种药物组合物，其包含如本发明在第一方面中所述的抗体或其抗原结合片段以及药学上可接受的载体或赋形剂。
本发明在第四方面提供了一种DNA分子，其特征在于，所述DNA分子编码如本发明在第一方面中所述的抗体或其抗原结合片段。
本发明在第五方面提供了一种重组载体，其特征在于，所述重组载体包含如本发明在第一方面中所述的抗体或其抗原结合片段的编码序列或者包 含如本发明在第四方面中所述的DNA分子。
如本文所用，“载体”是复制子，诸如质粒、噬菌体、病毒或粘粒，其中可以插入另一个DNA区段以便实现插入区段的复制。载体可以是表达载体。“表达载体”是包括一个或多个表达控制序列的载体，“表达控制序列”是控制和调节另一DNA序列的转录和/或翻译的DNA序列。
合适的表达载体包括但不限于源自例如噬菌体、杆状病毒、烟草花叶病毒、疱疹病毒、巨细胞病毒、逆转录病毒、痘苗病毒、腺病毒和腺相关病毒的质粒和病毒载体。表达载体可包括标签序列。标签序列通常表达为与编码的多肽的融合体。此类标签可以插入多肽内的任何位置，包括羧基或氨基末端。有用标签的示例包括但不限于绿色荧光蛋白(GFP)、谷胱甘肽S-转移酶(GST)、聚组氨酸、c-myc、血凝素、麦芽糖E结合蛋白和蛋白A。
本发明在第六方面提供了一种重组宿主细胞，其特征在于，所述重组宿主细胞包含如本发明在第五方面中所述的重组载体。术语“宿主细胞”旨在包括可向其中引入重组表达载体的原核和真核细胞。
本发明在第七方面提供了一种治疗肿瘤的试剂盒，其特征在于，该试剂盒中含有如本发明在第一方面中所述的抗体或其抗原结合片段，或者含有如本发明在第三方面中所述的药物组合物；在一些优选的实施方式中，所述肿瘤为实体瘤。
本发明在第八方面提供了一种治疗有需要的受试者的方法，所述方法包括向受试者施用治疗有效量的如本发明在第一方面中所述的抗体或其抗原结合片段，或者施用治疗有效量的如本发明在第三方面中所述的药物组合物。
在一些具体的实施方式中，所述受试者患有癌症或感染性疾病；优选的，所述受试者患有包含表达或者过度表达Siglec-15配体的细胞的癌症；更优选的，所述癌症为实体瘤。
在另一些具体的实施方式中，所述方法还包含向所述受试者施用第二治疗剂；优选的，所述第二治疗剂为免疫检查点抑制剂和/或免疫激动剂。
本发明在第九方面提供了一种检测或诊断疾病的方法，其特征在于，所 述方法包括：(a)使用根据本发明在第一方面中所述的抗体或其抗原结合片段测定受试者的细胞或组织样品中的Siglec-15表达水平，以及(b)将(a)中所述Siglec-15表达水平与对照水平进行比较，其中与所述对照水平相比，所述测定的Siglec-15表达水平增加表明所述受试者患有所述疾病。
发明定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大于5％的上限的范围内的数字数值。
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
如本文中所用，属于“抗体”旨在表示具有“可变区”抗原识别位点的免疫球蛋白分子。术语抗体的“抗原结合片段”是指抗体的一个或多个部分，所述一个或多个部分含有抗体互补决定区(“CDR”)和任选地包括抗体“可变区”抗原识别位点的构架残基，并且表现出免疫特异性结合抗原的能力。此类片段包括Fab′、F(ab′) 2、Fv、单链(ScFv)及其突变体、天然存在的变体，及包括抗体“可变区”抗原识别位点和异源蛋白(例如，毒素、不同抗原的抗原识别位点、酶、受体或受体配体等)的融合蛋白。
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重或轻链的结构域。天然抗体的重链(HC)和轻链(LC)的可变结构域通常具有相似的结构，其中每个结构域包含四个保守的框架区(FR)和三个互补决定区(参见，例如，Kindt等Kuby Immunology，6th ed.，W.H.Freeman and Co.91 页(2007))。单个重链可变区(VH)或轻链可变区(VL)结构域可以足以给予抗原结合特异性。此外，可以使用来自与特定抗原结合的抗体的VH或VL结构域来分离结合所述抗原的抗体，以分别筛选互补VL或VH结构域的文库。参见，例如Portolano等，J.Immunol.150：880-887(1993)；Clarkson等，Nature，352：624-628(1991)。
可变区通常表现出由三个高变区连接的相对保守的构架区(FR)的相同的一般结构，所述高变区也被称为互补决定区或CDR。通常通过构架区定位(align)来自每对的两条链的CDR，所述CDR使得可结合特异性表位。两条轻链和重链可变区从N-末端到C-末端通常包含结构域FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。
“互补决定区”或“CDR区”或“CDR”或“高变区”(在本文中与超变区“HVR”可以互换使用)，是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人，(1989)Nature 342：877-883，Al-Lazikani等人，“Standard conformations for the canonical structures of immunoglobulins”，Journal of Molecular Biology，273，927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人，Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services，National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering) 的North CDR定义(North等，“A new clustering of antibody CDR loop confirmations，Journal ofMolecular Biology，406，228-256(2011)”)。
术语“结合”或“特异性结合”意指结合作用对抗原是选择性的，并且可以与不想要的或非特异的相互作用区别开。抗体与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)、表面等离子共振法(SPR)或生物膜层光学干涉技术(ForteBio)或本领域已知的其它常规结合测定法测定。作为本发明的实施例，指抗体或抗原结合片段在体外测定法中，优选地在采用纯化的野生型抗原的生物膜层光学干涉测量中与抗原表位结合。在某些实施方案中，在抗体或抗原结合片段优选地识别蛋白质和/或大分子的复杂混合物中其靶抗原时，将抗体或抗原结合片段称作特异性结合抗原。
术语“变体”是指与参考多肽或多核苷酸不同但保持基本特性的多肽或多核苷酸。多肽的典型变体在氨基酸序列方面与另一参考多肽不同。通常，差异是有限的，使得参考多肽和变体的序列总体上非常相似，并且在许多区域中是相同的。变体和参考多肽可以在氨基酸序列方面因一个或多个修饰(例如，取代、添加和/或缺失)而不同。取代或插入的氨基酸残基可以是或可以不是由遗传密码编码的氨基酸残基。多肽的变体可以是天然存在的，诸如等位基因变体，或者可以是不知道会天然存在的变体。
术语“序列同一性百分比(％)”定义为在比对序列和引入空位(如有必要)以达到实现最高序列同一性百分比之后，候选序列中核苷酸或氨基酸与参考核酸序列中的核苷酸或氨基酸相同的百分比。用于测定序列同一性百分比的比对可以通过本领域技术范围内的各种方式实现，例如，使用公共可用的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。用于测量比对的适当参数，包括在所比较的序列的全长上实现最大限度比对所需的任何算法，可以通过已知方法确定。
术语“嵌合抗体”是这样的分子，在所述分子中抗体的不同部分源自不同的免疫球蛋白分子，例如抗体具有源自非人抗体的可变区和人免疫球蛋白恒定区。
术语“人源化抗体”是指包括人构架区和一个或多个来自非人(通常为小鼠或大鼠)免疫球蛋白的CDR的免疫球蛋白。提供CDR的非人免疫球蛋白称为“供体”而提供构架的人免疫球蛋白称为“受体”。不需要存在恒定区，但如果它们存在，则它们应该与人免疫球蛋白恒定区基本上相同，即至少约85-99％，优选约95％或更多相同。因此，可能除CDR外，人源化免疫球蛋白的所有部分与天然人免疫球蛋白序列的相应部分基本上相同。人源化抗体是包括人源化轻链和人源化重链免疫球蛋白的抗体。例如，人源化抗体将不涵盖典型嵌合抗体，因为例如嵌合抗体的整个可变区是非人的。
术语“治疗有效量”是指治疗剂的足以介导此类症状的临床相关性消除、减轻或缓解的量。如果效应幅度足以影响受体受试者的健康或预后，则所述效应是临床相关的。治疗有效量可以指治疗剂的足以延迟或最小化疾病发作的量，例如足以延迟或最小化癌症扩散的量。治疗有效量还可以指在疾病的治疗或管理中提供治疗益处的治疗剂的量。
术语“癌症”是指由细胞异常不受控制的生长产生的赘生物或肿瘤。
发明的效果
由本发明的技术方案可见，本发明的技术方案与现有技术相比，具有以下有益效果：
(1)本发明提供的抗Siglec-15抗体具有独特的抗体序列；
(2)本发明提供的抗Siglec-15抗体能够以高亲和力结合Siglec-15蛋白并有效解除Siglec-15蛋白对T细胞增殖和激活的抑制；
(3)本发明提供的抗Siglec-15抗体在小鼠体内肿瘤模型中可以解除Siglec-15蛋白对肿瘤免疫反应的抑制作用、增强肿瘤免疫反应、抑制肿瘤的生长，显示出了有效的抗肿瘤药效；
(4)本发明提供的抗Siglet-15抗体可以单独使用或与各种免疫治疗方法中使用的药物(包括免疫检查点抑制剂和/或免疫激动剂)联用来提高肿瘤患者的响应率，可用于治疗对PD-1治疗不响应的实体瘤患者。
为了让本发明的上述和其他目的、特征和优点能更明显易懂，下面特举 较佳实施例，并配合说明书附图，作详细说明如下：
附图说明
图1示出了不同种类的抗Siglec-15抗体与表达人Siglec-15蛋白的GS-CHO细胞的结合活性；
图2示出了不同种类的抗Siglec-15抗体与表达食蟹猴Siglec-15蛋白的GS-CHO细胞的结合活性；
图3示出了不同种类的抗Siglec-15抗体与表达小鼠Siglec-15蛋白的GS-CHO细胞的结合活性；
图4示出了流式细胞术检测Siglec-15蛋白与CD8+T细胞的结合活性；
图5示出了Siglec-15蛋白可显著抑制抗-CD3抗体激活的T细胞增殖；
图6示出了不同种类的抗Siglec-15抗体促进CD4+T细胞的增殖活性；
图7示出了不同种类的抗Siglec-15抗体促进CD8+T细胞的增殖活性；
图8示出了流式细胞术检测Siglec-15蛋白与CD14+单核细胞的结合活性；
图9示出了抗Siglec-15抗体抑制Siglec-15蛋白介导的单核细胞存活活性；
图10示出了MC38荷瘤小鼠注射不同种类的抗Siglec-15抗体后，小鼠的肿瘤体积随时间变化的曲线示意图；
图11示出了MC38荷瘤小鼠注射不同种类的抗Siglec-15抗体后，小鼠体重随时间变化的曲线示意图；
图12示出了流式细胞术检测Siglec-15蛋白在骨髓源性巨噬细胞中表达情况；
图13示出了CT26荷瘤小鼠注射不同种类的抗Siglec-15抗体后，小鼠的肿瘤体积随时间变化的曲线示意图；
图14示出了CT26荷瘤小鼠注射不同种类的抗Siglec-15抗体后，小鼠体重随时间变化的曲线示意图。
具体实施方式
本发明所列举的具体实施例只作为本发明的范例，本发明并不限制于下 文所描述的具体实施例。对于本领域技术人员而言，任何对下文所述的实施例进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。
本发明实施例中所使用的试验材料、试验试剂和仪器均可市购获得。
实施例1：抗体的制备
(1)杂交瘤细胞的制备
杂交瘤技术即淋巴细胞杂交瘤技术，又称为单克隆抗体技术，其是将骨髓瘤细胞和免疫淋巴细胞融合、形成能分泌具有高度特异性的单克隆抗体的体细胞融合技术。具体来说，杂交瘤技术通过融合两种细胞而同时保持两者的主要特征。这两种细胞分别是经抗原免疫的小鼠脾细胞和小鼠骨髓瘤细胞。被特异性抗原免疫的小鼠脾细胞(B淋巴细胞)的主要特征是它具有抗体分泌功能，但不能在体外连续培养；小鼠骨髓瘤细胞则可在培养条件下无限分裂、增殖，即具有所谓的永生性。在选择培养基的作用下，只有B淋巴细胞与骨髓瘤细胞融合的杂交细胞才能具有持续培养的能力，形成同时具备抗体分泌功能和保持细胞永生性两种特征的细胞克隆。
本实验通过将人Siglec-15蛋白作为抗原免疫小鼠，再将经过免疫的小鼠的脾细胞和骨髓瘤细胞融合，获得能够表达阳性抗体的杂交瘤细胞。具体操作如下：
杂交瘤细胞融合：
实验动物及免疫信息如表1所示：
表1：实验动物的种类及免疫接种的相关信息
Figure PCTCN2022092136-appb-000001
电融合皿准备：用70％乙醇彻底浸泡电融合皿，并于超净台中吹干备用。
分离脾细胞：颈脱位将小鼠处死，用75％酒精消毒体表5min，随即放入超净台内小鼠解剖板上，左侧卧位，用7号针头固定四肢。无菌打开腹腔取 出脾脏，用基础培养基(配置方法如下表2)洗涤，并仔细去掉周围附着的结缔组织。随后将脾脏转移到另一个盛有基础培养基的平皿中。以弯头针头压住脾脏，用小针头在脾脏上插孔，并用镊子挤压，使脾细胞充分释放，制成脾细胞悬液。细胞悬液经70μM细胞筛网过滤后用30ml基础培养基洗一遍，1200rpm离心6min。
表2基础培养基的成分及其配制方法
Figure PCTCN2022092136-appb-000002
裂解红细胞：去除上清，用10ml RBC裂解缓冲液(GIBCO，A10492-01)重悬细胞。然后再加入20ml RBC裂解缓冲液。悬液静置5min后1100rpm离心6min。去上清后用10ml基础培养基重悬细胞，然后再加入30ml基础培养基，1100rpm离心6min。去除上清后，细胞重悬于20ml基础培养基中并计数。
电融合：用20ml基础培养基重悬小鼠骨髓瘤细胞SP2/0细胞(ATCC，CRL-1581)并计数。将SP2/0细胞和上文所述脾细胞以1∶2～1∶1的比例混合，1000rpm离心6min。去除上清后将混合的细胞重悬于10ml融合缓冲液(BTXpress，47-0001)中。再加入15ml融合缓冲液，1000rpm离心5min，去除上清。重复上述步骤一遍后，用适量融合缓冲液重悬细胞、制成细胞悬液，调整混合细胞密度至1×10 7个细胞/ml。电融合仪的参数设置如下表3所示。每个电融合皿中加入2ml细胞悬液进行电融合。
表3电融合仪参数
Figure PCTCN2022092136-appb-000003
电融合后铺板：细胞于电融合皿中室温静置5min。将细胞转移入离心 管中，用筛选培养基(具体成分及配制方法如下表4所示)稀释细胞至1×10 4～2×10 4个细胞/ml。96孔板中每孔加入100μl细胞悬液。融合后第7天更换筛选培养基。培养第10天(或更久，根据细胞生长状态)后进行筛选。通过FACS(C6(BD Biosciences))检测筛选出表达特异性抗Siglec-15抗体的杂交瘤细胞。
表4筛选培养基的成分及其配制方法
Figure PCTCN2022092136-appb-000004
阳性杂交瘤细胞亚克隆
根据细胞结合以及亲和力测定的结果，选择阳性杂交瘤细胞进行亚克隆。亚克隆步骤：准备一块96孔板，第2至第8排每孔加入200μl培养基，该培养基是在筛选培养基的基础上将HAT更换成HT(Gibco，Cat#11067-030)，其余配方相同。将上述融合筛选出的阳性孔的细胞，按约1×10 5个/ml的密度，分别取300μl加入到第一排的每个孔中。用排抢，将第1排细胞悬液取100μl加入第2排，充分混匀后取100μl加入下一排。重复上述步骤，直至最后一列体积变为300μl；静置96孔板15min，显微镜下观察计数。取100个细胞对应的体积加入20ml如上所述的基础培养基中，并混匀铺板，每孔200μl。两天后显微镜下观察，判断并标记出单克隆孔。待每孔细胞汇合度达到50％以上时，同上述高通量的筛选方法检测，挑出目标阳性孔，进行细胞冻存。
试验结果：通过上述操作获得抗Siglec-15抗体的杂交瘤细胞。
实施例2：嵌合抗体的人源化
嵌合抗体的人源化过程具体如下：
①确定CDR环结构；
②在人种系序列数据库为重链和轻链的每个V/J区域找到最接近的同源序列；
③筛选与重链轻链最匹配的人种系以及最低量的回复突变；
④将嵌合抗体的CDR区构建至人的骨架区上；
⑤使用序列和结构特征，确定骨架区中起到维持CDR功能的氨基酸位置；
⑥在确定为重要的序列位置进行回复突变(返回到输入氨基酸类型)；
⑦优化风险位点的氨基酸。
实施例3：抗体的表达及纯化
按照实施例2所述的方法对嵌合抗体进行人源化，本实施例中构建的3个抗体(分别命名为hz59A3.3.p1、2-hz21C7.3和hz28D6.3.p1)的重链和轻链的氨基酸序列在表5中列出。
表5单克隆抗体中重链和轻链的氨基酸序列组成
Figure PCTCN2022092136-appb-000005
Figure PCTCN2022092136-appb-000006
Figure PCTCN2022092136-appb-000007
表5中所示氨基酸序列中灰色部分的氨基酸序列为所述重链/轻链中的重链可变区/轻链可变区的氨基酸序列；表5中所示氨基酸序列中粗体部分的氨基酸序列为所述重链可变区/轻链可变区中的CDR1、CDR2、CDR3序列。
具体来说，(1)hz59A3.3.p1抗体在重链中包含的重链可变区的氨基酸序列为：
QVQLVQSGAEVKKPGASVKVSCKASGYTITDFYMDWVRQAPGQGLEWMGRLNPSSGDSSYNQNFKGRVTMTVDTSTSTVYMELSSLRSEDTAVYYCARWAIARPDYWGQGTLVTVSS(SEQ ID NO：10)；在上述重链可变区中包含的CDR1、CDR2和CDR3的氨基酸序列分别为：GYTITDFYMD(SEQ ID NO：7)、RLNPSSGDSSYNQNFKG(SEQ ID NO：8)和WAIARPDY(SEQ ID NO：9)。hz59A3.3.p1抗体在轻链中包含的轻链可变区的氨基酸序列为：DIVMTQSPDSLAVSLGERATINCKASQSVDYEGDSYMNWYQQKPGQPPKLLIYVASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGGGTKVEIK(SEQ ID NO：14)，在上述轻链可变区中包含的CDR1、CDR2和CDR3的氨基酸序列分别为：KASQSVDYEGDSYMN(SEQ ID NO：11)、VASNLES(SEQ ID NO：12)和QQSNEDPYT(SEQ ID NO：13)。
(2)hz28D6.3.p1抗体在重链中包含的重链可变区的氨基酸序列为：QVQIVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLEWMGWINTETGEPTYADDFKGRFVFSLDTSVSMAYLQISSLKAEDTAVYYCARGLWEGDVWGQGTMVTVSS(SEQ ID NO：18)；在上述重链可变区中包含的CDR1、CDR2和CDR3的氨基酸序列分别为：GYTFTDYSMH(SEQ ID NO：15)、WINTETGEPTYADDFKG(SEQ ID NO：16)和GLWEGDV(SEQ  ID NO：17)。hz28D6.3.p1抗体在轻链中包含的轻链可变区的氨基酸序列为：EIVITQSPATLSLSPGERATLSCSASSSISYAHWYQQKPGQAPRRWIYDTSKLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCHQRSSYPWTFGQGTKLEIK(SEQ ID NO：22)；在上述轻链可变区中包含的CDR1、CDR2和CDR3的氨基酸序列分别为：SASSSISYAH(SEQ ID NO：19)、DTSKLAS(SEQ ID NO：20)和HQRSSYPWT(SEQ ID NO：21)。
(3)2-hz21C7.3抗体在重链中包含的重链可变区的氨基酸序列为：QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWLGWVRQAPGQGLEWMGDINPGGGYTNYNENFKGRVTMTADTSISTAYMELSRLRSDDTAVYYCVSFYGDYQFDFWGQGTLVTVSS(SEQ ID NO：26)；在上述重链可变区中包含的CDR1、CDR2和CDR3的氨基酸序列分别为：GYTFTNYWLG(SEQ ID NO：23)、DINPGGGYTNYNENFKG(SEQ ID NO：24)和FYGDYQFDF(SEQ ID NO：25)。2-hz21C7.3抗体在轻链中包含的轻链可变区的氨基酸序列为：DIQMTQSPSSLSASVGDRVTITCSVSSSISSINLHWYQQKPEKAPKPWIYGASNLASGVPSRFSGSGSGTDYTITISSLQPEDFATYYCQQWSSFPLTFGGGTKVEIK(SEQ ID NO：30)；在上述轻链可变区中包含的CDR1、CDR2和CDR3的氨基酸序列分别为：SVSSSISSINLH(SEQ ID NO：27)、GASNLAS(SEQ ID NO：28)和QQWSSFPLT(SEQ ID NO：29)。
其中，在上述抗体的重链可变区中包含的CDR1根据AbM定义，在上述抗体的重链可变区中包含的CDR2和CDR3根据Kabat定义；在上述抗体的轻链可变区中包含的CDR1、CDR2和CDR3根据Kabat定义。
根据试验的需要，分别在GS-CHO细胞或HEK293细胞中对上述抗体进行表达和纯化，具体操作如下：
(1)GS-CHO细胞中的表达和纯化：
根据制造商的说明书，使用GS Xceed TM Gene Expression System试剂盒(Lonza)产生表达抗体的CHO-S细胞系。
首先将表5中所述抗体分子的重链和轻链的DNA编码序列插入到同一个pCHO1.0质粒中，其中重链在轻链的上游，得到相应的重组pCHO1.0质粒。之后采用化学转染法和电转染法将构建的重组pCHO1.0质粒转入CHO-S细胞系中，转染48小时之后利用ForteBio检测抗体产量以判断转染效率。转染后的细胞经过两轮加压筛选得到高表达抗体的细胞池(pool)。之后扩增细胞池，大量表达抗体，并收集细胞上清用ProteinA纯化上清液，使抗体的纯度＞95％。
(2)HEK293细胞中的表达和纯化：
对于HEK293细胞中抗体的瞬时表达，使用载体pV120或pcDNA3.1。首先将编码表5中所述抗体的重链和轻链的cDNA克隆到单独的pV120载体或pcDNA3.1载体中，得到相应的重组载体。使用化学转染的方法将带有抗体分子重链和轻链的重组载体转入HEK293细胞中。采用的化学转染试剂为PEI(购自Polysciences)，按照生产商提供的方案瞬时转染培养的293HEK(Invitrogen)。转染后，弃去培养基，并用新鲜的EXPI293培养基(Gibco)把细胞稀释到4×10 6/ml。在37℃，5％CO 2的条件下培养细胞7天，每48小时流加新鲜培养基。7天后，1300rpm离心20min。取上清液，用Protein A纯化上清液，使抗体的纯度＞95％。
实施例4：抗体亲和力测定实验
采用生物光干涉测量(ForteBio)测定法测定实施例3在表5中所述3个抗体和一个对照抗体(即hz5G12，该抗体是根据专利WO2018057735A8中序列合成)结合人、食蟹猴和鼠Siglec-15抗原的平衡解离常数(KD)。
ForteBio亲和力测定按照现有的方法(具体参见Estep，P等人，“High throughput solution Based measurement of antibody-antigen affinity and epitope binning”，《MAbs》，2013.5(2)：p.270-278)进行。
简言之，传感器在分析缓冲液(HBS buffer，货号：BR-1006-69)中线下平衡30分钟，然后线上检测60秒建立基线，在线加载人、食蟹猴和鼠Siglec-15抗原至AHQ传感器(ForteBio)上进行ForteBio亲和测量。再将具有 加载了抗原的传感器暴露于100nM的根据实施例3所述方法获得的经纯化的Siglec-15抗体中作用5分钟，之后将传感器转移至分析缓冲液解离5分钟用于解离速率测量。使用1∶1结合模型进行动力学的分析。在通过以上测定法所述的实验中，2-hz21C7.3抗体、hz28D6.3.p1抗体、hz59A3.3.p1抗体及对照抗体hz5G12的亲和力如表6所示。
表6人源化后4个抗体分子及对照抗体的亲和力
Figure PCTCN2022092136-appb-000008
由表6的结果可见，相对于对照抗体而言，本发明上述3个抗体与人、小鼠和食蟹猴Siglec-15抗原均显示高亲和力结合。
实施例5：抗Siglec-15抗体与表达于细胞表面的人、食蟹猴及小鼠Siglec-15的结合实验
基于流式细胞术测定法测量实施例3在表5中所述3个抗体与过表达于GS-CHO细胞(Lonza)表面的人、食蟹猴及小鼠Siglec-15的结合能力。本实施例中选择hIgG1抗体作为阴性对照，选择hz5G12抗体作为阳性对照；其中，所述hIgG1抗体中重链的氨基酸序列如SEQ ID NO：31所示(具体氨基酸序列为：
EVRLLESGGGLVQPGGSLRLSCAASGFTFSNYAMGWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTTSRDDSKNALYLQMNSLRAEDTAVYYCARGGPGWYAADVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK)；所述hIgG1抗体中轻链的氨基酸序列如SEQ ID NO：32所示(具体氨基酸序列为：
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQADLPAFAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC)。通过比较不同抗体与过表达于GS-CHO细胞表面的人、食蟹猴及小鼠Siglec-15的结合曲线来测定其结合能力。具体实验过程为：
(1)复苏与传代而获得生长状态良好的GS-CHO细胞。(2)使用不同浓度的候选抗体(起始浓度300nM，三倍稀释)与所述GC-CHO细胞于4℃孵育30分钟。PBS清洗三次后，使用PE标记的抗人IgG的荧光二抗(Biolegend，409304)与之4℃孵育30分钟。PBS再次清洗三次后，使用100μl PBS重悬GS-CHO细胞。(3)使用流式细胞仪(BD，Celesta)测定PE荧光通道中位荧光度值。(4)比较其EC50及曲线峰值，所述抗体与过表达人、食蟹猴和小鼠Siglec-15的GS-CHO细胞结合活性分别如图1、2和3所示。
试验结果：由图1-图3的结果可见，相对于对照抗体而言，本发明构建的3个抗体与表达人、食蟹猴Siglec-15的GS-CHO细胞的结合活性更高；2-hz21C7.3和hz59A3.3.p1抗体与表达小鼠Siglec-15的GS-CHO细胞结合能力强于对照抗体，hz28D6.3.p1与表达小鼠Siglec-15的GS-CHO细胞结合能力与对照抗体相当。
实施例6：抗Siglec-15抗体促进CD4+T细胞增殖实验
Siglec-15蛋白可以与T细胞上的未知配体结合，从而抑制T细胞的活性和增殖。在CD3/CD28beads激活的CD8+T细胞中，加入人Siglec-15-mFc融合蛋白(即hSiglec-15-mFc，R&D，9227-SL-050)后，再加入抗Fc二抗后可以流式检测到Siglec-15蛋白与CD8+T细胞结合(具体参见图4)。在人外周血CD8+T细胞中，加入Siglec-15蛋白可显著抑制抗-CD3抗体激活的T细胞增殖(具体参 见图5)。
基于这一原理，本研究在CD4+T细胞和CD8+T细胞培养体系中加入抗CD3抗体促进T细胞增殖，进一步加入Siglec-15蛋白抑制T细胞增殖。在此体系中，可以评价不同抗Siglec-15抗体分子促进T细胞增殖的活性。具体实验过程为：
(1)平底板中包被0.05μg/ml的抗-CD3抗体(Biolegend，317325)，4℃过夜。(2)复苏PBMC细胞，使用cell-trace CFSE活细胞染料(Thermo，C34554)对细胞进行标记。(3)将细胞稀释至6×10 6/ml，铺到平底板，37℃孵育1小时。(4)配制抗体，阴性对照组(hIgG1抗体)和抗体组(包括hz28D6.3p1抗体和hz59A3.3p1抗体及阳性对照抗体hz5G12)同时加入20μg/ml的人Siglec-15-mFc融合蛋白，37℃预混30分钟后将其加入到细胞中。(5)培养4天后，用流式细胞仪(BD，Celesta)检测细胞增殖比例。(6)获取数据并绘制细胞增殖曲线，比较其EC50及曲线峰值(具体参见图6和图7)。
由上面的实验结果可以得出如下结论：hz28D6.3.p1抗体促进人CD4+T细胞及CD8+T细胞增殖活性强于阳性对照抗体hz5G12(表现为EC50更低)。hz59A3.3.p1抗体促进人CD4+T细胞及CD8+T细胞增殖活性与阳性对照抗体hz5G12相当(表现为EC50接近)。
实施例7：抗Siglec-15抗体抑制Siglec-15蛋白介导的单核细胞存活活性
Siglec-15蛋白可以与单核细胞上的未知配体结合，从而促进单核细胞的存活。在CD14+单核细胞中，加入人Siglec-15-mFc融合蛋白(即hSiglec-15-mFc)后，再加入抗Fc二抗后可以流式检测到Siglec-15蛋白与CD14+单核细胞的结合活性(具体参见图8)。
由此可见，Siglec-15蛋白可结合单核细胞并显著促进CD14+单核细胞存活。基于这一原理，本研究在CD14+单核细胞中加入Siglec-15蛋白促进单核细胞存活，进一步加入抗Siglec-15抗体抑制单核细胞存活。在此体系中，可以评价不同抗Siglec-15抗体抑制单核细胞存活活性。具体实验过程为：
(1)复苏CD14+单核细胞，调整细胞密度为2×10 6/ml，将细胞加入到 96孔平底板中。(2)配制抗体，阴性对照组(hIgG1抗体)和抗体组(包括2-hz21C7.3抗体、hz28D6.3.p1抗体、hz59A3.3.p1抗体和hz5G12阳性对照抗体)同时加入20μg/ml的人Siglec-15-mFc融合蛋白，37℃预混30分钟后将其加入到单核细胞中。(3)单核细胞放置培养箱中培养7天。(4)向单核细胞中加入CCK8试剂(同仁化学，CK04)检测细胞存活。(5)获取数据并绘制细胞存活曲线，比较其IC50及曲线谷值(具体参见图9)。
试验结果：抗Siglec-15抗体可显著抑制Siglec-15蛋白诱导的单核细胞存活，hz59A3.3.p1、hz28D6.3.p1在高浓度可完全抑制单核细胞存活，hz21C7.3抑制单核细胞存活活性弱于hz59A3.3.p1和hz28D6.3.p1，与对照抗体hz5G12相当。
实施例8：抗Siglec-15抗体在MC38荷瘤小鼠模型体内的抗肿瘤药效
Siglec-15在人肿瘤组织高表达。为评估抗Siglec-15抗体的药效，本实施例使用过表达Siglec-15的MC38细胞评估抗体的抗肿瘤活性。具体实验步骤为：将过表达Siglec-15的MC38细胞(Innoventbio，下文称为MC38-Siglec-15细胞)进行常规传代培养，培养过程中加入4μg/ml的puromycin(Gibico，A11138-02)。接种前，胰酶(Gibico，25200-072)消化并离心收集MC38-Siglec-15细胞，以PBS(上海生工，E607008-0500)分散细胞至7.5×10 6个/ml，每只小鼠皮下接种0.2ml细胞悬液(即1.5×10 6/只小鼠)至C57小鼠右侧腹部区域中。
给药：
肿瘤细胞接种第5天后根据肿瘤大小蛇形分组(每组8只小鼠)，分别在接种后第5、8、11、14天给药，每周2次监测小鼠瘤体积与体重。监测至24天后结束。肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W 2/2。采用电子天平测定体重。
统计结果与分析：
肿瘤抑制率结果如图10所示：与阴性对照hIgG1抗体相比，hz28D6.3.p1 抗体、hz59A3.3.p1抗体及阳性对照抗体(即hz5G12抗体)均可显著抑制肿瘤生长(具体参见图10)。其中，hz28D6.3.p1抗体治疗组有三只小鼠肿瘤消退，hz59A3.3.p1抗体治疗组有四只小鼠肿瘤消退，阳性对照抗体组有三只小鼠肿瘤消退。整个实验过程未观察到小鼠体重降低(具体参见图11)。
实施例9：抗Siglec-15抗体在CT26荷瘤人源化小鼠模型体内的抗肿瘤药效
Siglec-15蛋白的表达谱在人和鼠之间具有很大的差异性。Siglec-15蛋白高表达于人的肿瘤组织和肿瘤内部的髓系细胞中，而在小鼠的肿瘤中无表达。为了在小鼠模型中模拟病人肿瘤微环境，本研究首先将骨髓细胞诱导为巨噬细胞。诱导得到的巨噬细胞使用流式细胞术检测Siglec-15蛋白的表达情况。具体实验过程为：
(1)取BALB/c小鼠骨髓细胞。(2)体外加入20ng/ml M-CSF(R&D，416-ML-500)诱导3天，3天后换液，继续加入20ng/ml M-CSF和20ng/ml IL-10(R&D，417-ML-10)诱导4天后得到骨髓源性巨噬细胞。(3)使用AF467(invitrogen，A20186)标记的anti-siglec-15抗体(hz5G12抗体)与上述骨髓源性巨噬细胞于4℃孵育30分钟。PBS清洗三次后，使用150μl PBS重悬骨髓源性巨噬细胞。(3)使用流式细胞仪(BD)检测。结果如图12所示，在所检测的骨髓源性巨噬细胞中，有16.7％的细胞表面表达Siglec-15分子(具体参见图12)。
将CT26细胞(ATCC，CRL-2638)进行常规传代培养。接种前胰酶消化并离心收集细胞，以PBS分散细胞至7.5×10 5个/ml。PBS重悬骨髓源性巨噬细胞至7.5×10 5个/ml。皮下接种0.2ml CT26细胞悬液和0.2ml的骨髓源性巨噬细胞悬液(即1.5×10 5CT26肿瘤细胞和1.5×10 5骨髓源性巨噬细胞/只小鼠)至BALB/c小鼠右侧腹部区域中。
给药：
肿瘤细胞接种第1天后随机分组(每组8只小鼠)，分别在接种后第0、4、7、11、14天给药，每周2次监测小鼠瘤体积与体重。监 测至21天后结束。肿瘤体积测定：采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W 2/2。采用电子天平测定体重。
统计结果与分析：
肿瘤抑制率结果如图13所示：与阴性对照hIgG1抗体对比，10mg/kg hz28D6.3.p1抗体、hz59A3.3.p1抗体、2-hz21C7.3抗体及阳性对照抗体(即hz5G12抗体)可明显抑制CT26肿瘤生长；与阳性对照抗体相比，hz28D6.3.p1抗体及2-hz21C7.3抗体抑制肿瘤效果优于阳性对照抗体；同时对小鼠体重进行检测，结果如图14所示，在给药的小鼠组中，体重无明显下降。

Claims (16)

  1. 一种抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含六个互补决定区(CDR)，所述六个CDR选自由以下项组成的组中的任一种：(i)SEQ ID NO：15～17和SEQ ID NO：19～21；(ii)SEQ ID NO：7～9和SEQ ID NO：11～13；或(iii)SEQ ID NO：23～25和SEQ ID NO：27～29；并且，其中所述抗体或其抗原结合片段能够与Siglec-15蛋白特异性结合。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区和轻链可变区选自由以下项组成的组中的任一种：(i)SEQ ID NO：18和SEQ ID NO：22，或者与SEQ ID NO：18和/或SEQ ID NO：22含有至少50％、60％、70％、80％、85％、90％、95％、99％或更高序列同一性的其变体；(ii)SEQ ID NO：10和SEQ ID NO：14，或者与SEQ ID NO：10和/或SEQ ID NO：14含有至少50％、60％、70％、80％、85％、90％、95％、99％或更高序列同一性的其变体；(iii)SEQ ID NO：26和SEQ ID NO：30，或者与SEQ ID NO：26和/或SEQ ID NO：30含有至少50％、60％、70％、80％、85％、90％、95％、99％或更高序列同一性的其变体。
  3. 根据权利要求1或2所述的抗体或其抗原结合片段，其特征在于，所述抗体或其抗原结合片段包含重链和轻链，所述重链和轻链选自由以下项组成的组中的任一种：(i)SEQ ID NO：3和SEQ ID NO：4，或者与SEQ ID NO：3和/或SEQ ID NO：4含有至少50％、60％、70％、80％、85％、90％、95％、99％或更高序列同一性的其变体；(ii)SEQ ID NO：1和SEQ ID NO：2，或者与SEQ ID NO：1和/或SEQ ID NO：2含有至少50％、60％、70％、80％、85％、90％、95％、99％或更高序列同一性的其变体；(iii)SEQ ID NO：5和SEQ ID NO：6，或者与SEQ ID NO：5和/或SEQ ID NO：6含有至少50％、60％、70％、80％、85％、90％、95％、99％或更高序列同一性的其变体。
  4. 根据权利要求1～3中任一项所述的抗体或其抗原结合片段，其特征在于，所述抗体为单克隆抗体、嵌合抗体或人源化抗体。
  5. 利用权利要求1～4中任一项所述的抗体或其抗原结合片段在制备治疗 抗肿瘤药物中的用途。
  6. 根据权利要求5所述的用途，其特征在于，其中所述肿瘤为实体瘤。
  7. 根据权利要求5或6所述的用途，其特征在于，其中所述抗肿瘤药物中进一步包含免疫检查点抑制剂和/或免疫激动剂。
  8. 一种药物组合物，其特征在于，所述药物组合物包含如权利要求1～4中任一项所述的抗体或其抗原结合片段以及药学上可接受的载体或赋形剂。
  9. 一种DNA分子，其特征在于，该DNA分子编码如权利要求1～4中任一项所述的抗体或其抗原结合片段。
  10. 一种重组载体，其特征在于，该重组载体包含如权利要求1～4中任一项所述的抗体或其抗原结合片段的编码序列或者包含如权利要求9所述的DNA分子。
  11. 一种重组宿主细胞，其特征在于，该重组宿主细胞包含如权利要求10所述的重组载体。
  12. 一种治疗肿瘤的试剂盒，其特征在于，该试剂盒中含有如权利要求1～4中任一项所述的抗体或其抗原结合片段，或者含有如权利要求8所述的药物组合物；优选的，所述肿瘤为实体瘤。
  13. 一种治疗有需要的受试者的方法，其特征在于，所述方法包括向受试者施用治疗有效量的如权利要求1～4中任一项所述的抗体或其抗原结合片段，或者施用治疗有效量的如权利要求8所述的药物组合物。
  14. 根据权利要求13所述的方法，其特征在于，所述受试者患有癌症或感染性疾病；优选的，所述受试者患有包含表达或者过度表达Siglee-15配体的细胞的癌症；更优选的，所述癌症为实体瘤。
  15. 根据权利要求13或14所述的方法，其特征在于，还包含向所述受试者施用第二治疗剂；优选的，所述第二治疗剂为免疫检查点抑制剂和/或免疫激动剂。
  16. 一种检测或诊断疾病的方法，其特征在于，所述方法包括：(a)使用根据权利要求1～4中任一项所述的抗体或其抗原结合片段测定受试者的细胞 或组织样品中的Siglec-15表达水平，以及(b)将(a)中所述Siglec-15表达水平与对照水平进行比较，其中与所述对照水平相比，所述测定的Siglec-15表达水平增加表明所述受试者患有所述疾病。
PCT/CN2022/092136 2021-05-13 2022-05-11 抗Siglec-15抗体及其用途 WO2022237819A1 (zh)

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