Nanobodies targeting siglec-7 proteins and uses thereof

Abstract

The present disclosure relates to the field of biotechnology, in particular to a nanobody targeting Siglec-7 protein and application thereof. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region CDR sequence selected from at least one of the following: SEQ ID NO: 1-18, or an amino acid sequence having at least 80% identity thereto. The antibody or antigen-binding fragment thereof prepared in the present disclosure can specifically target and bind to Siglec-7 protein with high affinity, block the interaction between sialic acid and Siglecs, inhibit the signal transduction process, activate NK cells, and restore their recognition and killing function to tumor cells.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a nanobody targeting the Siglec-7 protein and its application, and more specifically, to an antibody or its antigen-binding fragment, a recombinant protein, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition, pharmaceutical use, and a kit for detecting Siglec-7. Background Technology

[0002] Cancer manipulates multiple immune mechanisms to ensure a favorable local microenvironment for tumor progression. Glycosylation is often considered a hallmark of cancer. Alterations in glycosylation on the surface of tumor / infected cells are, to some extent, a key feature of cancer and various infectious diseases. Siglecs, a family of lectins expressed on the surface of many immune cell subtypes, recognize sialylated glycans found on glycoproteins and glycolipids. Therefore, the interaction between sialic acid and siglecs in cancer cells / infected cells can modulate immune cell phenotypes and allow them to evade the immune system.

[0003] Siglecs contain a Sia-binding N-terminal V-group domain followed by a C2-group Ig domain. These domains are thought to act as oligomerization spacers or regulators, and most of them act as transmembrane receptors in the immune system by recognizing Sia residues. Each type of siglec generally recognizes a specific set of sialylated structures.

[0004] Siglec 7 is primarily located in human natural killer (NK) cells and monocytes. Siglec 7 regulates inhibitory signaling in NK cells. NK cells are essential innate immune cells that can directly kill unhealthy host cells, including virus-infected cells and tumor cells. NK cells participate in a specific type of cell killing called antibody-dependent cell-mediated cytotoxicity (ADCC). Unlike T cells, which must be exposed to antigen-presenting cells before recognizing tumors, NK cells can spontaneously lyse tumor cells without antigen stimulation or pre-activation.

[0005] Viral infection and tumor cells downregulate MHC-1 expression. NK cell-mediated cytotoxicity is not limited by MHC I molecules; instead, NK cells destroy cancer cells through perforin / granzyme-mediated cleavage. Siglec-7 exhibits preferred binding specificity to the Neu5Ac2,8Neu5Ac-(diSia) sequence, although certain branched 2,6-sialic acid residues (DSGb5, DSLc4) are also considered preferred binding molecules. Siglec-7 is a promising target for cancer therapy, potentially restoring the cytotoxic potential of NK cells against cancer.

[0006] Therefore, there is an urgent need in this field to develop an antibody targeting Siglec-7. Summary of the Invention

[0007] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application proposes an antibody capable of binding to the Siglec-7 protein with high affinity and high specificity.

[0008] In a first aspect, this application provides an antibody or an antigen-binding fragment thereof. According to embodiments of this application, the antibody or antigen-binding fragment comprises a heavy chain variable region (CDR) sequence selected from at least one of the following or an amino acid sequence having at least 80% identity with it: SEQ ID NO: 1-18. In some examples of this application, the antibody or antigen-binding fragment thereof can bind to the Siglec-7 protein with high affinity and specificity, blocking the interaction between sialic acid and Siglecs, inhibiting signal transduction processes, activating NK cells, and restoring their function of recognizing and killing tumor cells.

[0009] In a second aspect of this application, a recombinant protein is proposed. According to embodiments of this application, the recombinant protein comprises the antibody or its antigen-binding fragment described in the first aspect of this application. In some examples of this application, the recombinant protein can target and bind to the Siglec-7 protein, blocking the interaction between sialic acid and Siglecs, inhibiting signal transduction processes, activating NK cells, and preventing tumor invasion.

[0010] In a third aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the antibody or its antigen-binding fragment described in the first aspect of this application or the recombinant protein described in the second aspect of this application. In some examples of this application, the aforementioned antibody or its antigen-binding fragment, as well as the recombinant protein, can be expressed in large quantities in vitro using this nucleic acid molecule.

[0011] In a fourth aspect, this application provides an expression vector. According to embodiments of this application, the expression vector carries the nucleic acid molecule described in the third aspect of this application. In some examples of this application, after the expression vector is introduced into suitable recipient cells, the aforementioned antibody or its antigen-binding fragment, or recombinant protein, can be effectively expressed under the mediation of a regulatory system, thereby achieving the large-scale in vitro preparation of the antibody or its antigen-binding fragment, or recombinant protein.

[0012] In a fifth aspect of this application, a recombinant cell is provided. According to embodiments of this application, the recombinant cell carries the nucleic acid molecule described in the third aspect or the expression vector described in the fourth aspect; or expresses the antibody or its antigen-binding fragment described in the first aspect or the recombinant protein described in the second aspect. In some examples of this application, under suitable conditions, this recombinant cell can effectively express the aforementioned antibody or its antigen-binding fragment, or recombinant protein within the cell.

[0013] In a sixth aspect of this application, a pharmaceutical composition is provided. According to embodiments of this application, the pharmaceutical composition comprises the antibody or its antigen-binding fragment described in the first aspect of this application, the recombinant protein described in the second aspect of this application, the nucleic acid molecule described in the third aspect of this application, the expression vector described in the fourth aspect of this application, or the recombinant cell described in the fifth aspect of this application. In some examples of this application, the pharmaceutical composition can bind to the Siglec-7 protein with high affinity and high specificity, blocking the interaction between sialic acid and Siglecs, inhibiting signal transduction processes, activating NK cells, and preventing tumor invasion.

[0014] In a seventh aspect of this application, the use of the antibody or its antigen-binding fragment described in the first aspect, the recombinant protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, the recombinant cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of a medicament for treating or preventing tumors. In some examples of this application, medicaments prepared based on the antibody or its antigen-binding fragment, the recombinant protein, the nucleic acid molecule, the expression vector, the recombinant cell, or the pharmaceutical composition can bind to the Siglec-7 protein with high affinity and high specificity, blocking the interaction between sialic acid and Siglecs, inhibiting signal transduction processes, activating NK cells, and preventing tumor invasion.

[0015] In an eighth aspect of this application, the application discloses the use of the antibody or antigen-binding fragment thereof described in the first aspect, the recombinant protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cell described in the fifth aspect in the preparation of a kit for detecting Siglec-7. In some examples of the application, kits prepared based on the antibody or antigen-binding fragment thereof, recombinant protein, nucleic acid molecule, expression vector, or recombinant cell can be used for portable detection of Siglec-7 protein.

[0016] In a ninth aspect of this application, a kit is provided. According to embodiments of this application, the kit comprises: the antibody or its antigen-binding fragment described in the first aspect, the recombinant protein described in the second aspect, the nucleic acid molecule described in the third aspect, the expression vector described in the fourth aspect, or the recombinant cells described in the fifth aspect. In some examples of this application, the kit can be used for efficient and portable detection of Siglec-7 protein.

[0017] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0019] Figure 1 This is a Siglec-7 ECD protein peak diagram from an embodiment of this application;

[0020] Figure 2 This is a schematic diagram of the SDS-PAGE electrophoresis detection results of the Siglec-7 ECD protein in an embodiment of this application;

[0021] Figure 3 This is a schematic diagram of the phage infection TG1 detection results according to an embodiment of this application;

[0022] Figure 4 This is a schematic diagram illustrating the results of the second round of phage washing for the Siglec-7 ECD phage display in an embodiment of this application.

[0023] Figure 5 This is a schematic diagram of the ELISA screening results for positive clones in an embodiment of this application;

[0024] Figure 6 This is a schematic diagram of the nanobody chromatography detection results according to an embodiment of this application;

[0025] Figure 7 This is an SDS-PAGE gel electrophoresis image of the nanobody Fc fusion protein from an embodiment of this application;

[0026] Figure 8 This is a schematic diagram showing the ELISA characterization results of the binding affinity between the nanobody and Siglec-7 according to an embodiment of this application;

[0027] Figure 9 This is a schematic diagram of the experimental results for verifying the SPR affinity of nanobodies according to an embodiment of this application. Detailed Implementation

[0028] The embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout.

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0030] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0031] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0032] In this application, the amino acid sequences of the CDRs listed above are all as shown in the IMGT definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as the Kabat rule, the Chothia rule, etc. Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined as described in any of the known schemes above. Although the scope of protection claimed in this application is based on the sequences shown in the IMGT definition rules, amino acid sequences corresponding to other CDR definition rules should also be included in the scope of protection of this application.

[0033] In this application, the abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids, with the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Aspartic acid (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: Histidine; I: Ile (isoleucine); L: Leu (leucine); K: Lysine (lysine); M: Met (methionine); F: pHe (phenylalanine); P: Proline (proline); S: Serine (serine); T: Threonine (threonine); W: Tryptophan (tryptophan); Y: Tyrosine (tyrosine); V: Valine (valine).

[0034] In this application, the term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, see, for example, Ausubel et al., eds. (1995), Current Protocols in Molecule & Larbiology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see AltschμL et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (AltschμL et al., Meth.Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST AltschμL et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0035] In this application, without substantially affecting antibody activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain sequences of the antibody or its functional fragments. These are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences described in this application can have at least 80% identity (or homology) with the reference sequences, meaning at least 80%, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with each reference sequence. The sequence consistency described in this application can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this invention are shown from the N-terminus to the C-terminus.

[0036] In this application, the term "monoclonal antibody (abbreviated as MABS)" refers to an antibody that can recognize only one specific antigenic epitope. Common MABS consist of two lighter light chains and two heavier heavy chains, linked by disulfide bonds to form a tetrapeptide molecule. The amino-terminal (N-terminus) amino acid sequence of this heavy or light chain varies considerably and is called the variable region (V-terminus), while the carboxyl-terminus (C-terminus) of the heavy or light chain is relatively stable and changes very little, and is called the constant region (C-terminus). The V-termini of the L-chain and H-chain are called VL and VH, respectively. MABS can also be small molecule antibodies, which mainly include Fab antibodies, Fv antibodies, single-chain antibodies, single-domain antibodies, and the smallest recognition unit.

[0037] In this application, the term "polyclonal antibody (abbreviated as polyclonal antibody)" refers to an antibody that can recognize multiple antigenic epitopes, such as an antibody that can recognize two antigenic epitopes (abbreviated as biclonal antibody), an antibody that can recognize three antigenic epitopes, or an antibody that can recognize four antigenic epitopes. It is interpreted in a broad sense, and the specific structure is not limited, as long as it can recognize multiple antigenic epitopes.

[0038] In this application, the term "nanobody" refers only to the heavy chain variable region (VH) and the conventional CH2 and CH3 regions, which bind specifically to the antigen through the heavy chain variable region.

[0039] In one aspect of this application, an antibody or its antigen-binding fragment thereof is provided, comprising a heavy chain variable region (CDR) sequence selected from at least one of the following or an amino acid sequence having at least 80% identity with it: SEQ ID NO: 1-18. In some examples of this application, the antibody or its antigen-binding fragment is capable of binding to the Siglec-7 protein with high affinity and high specificity. The relevant amino acid sequences are shown in Table 1.

[0040] In some examples of this application, the antibody or its antigen-binding fragment includes a heavy chain variable region CDR1 sequence as shown in the amino acid sequences of SEQ ID NO:1-6 or having at least 80% identity with the amino acid sequences of SEQ ID NO:1-6. In some preferred examples of this application, the heavy chain variable region CDR1 sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:1-6. Using the heavy chain variable region CDR1 sequence shown in SEQ ID NO:1-6 results in higher antibody affinity and specificity.

[0041] In some examples of this application, the antibody or its antigen-binding fragment includes a heavy chain variable region CDR2 sequence as shown in the amino acid sequences of SEQ ID NO:7-12 or having at least 80% identity with the amino acid sequences of SEQ ID NO:7-12. In some preferred examples of this application, the heavy chain variable region CDR2 sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:7-12. Using the heavy chain variable region CDR2 sequence shown in SEQ ID NO:7-12 results in higher antibody affinity and specificity.

[0042] In some examples of this application, the antibody or its antigen-binding fragment includes a heavy chain variable region CDR3 sequence as shown in the amino acid sequences of SEQ ID NO:13-18 or having at least 80% identity with the amino acid sequences of SEQ ID NO:13-18. In some preferred examples of this application, the heavy chain variable region CDR3 sequence of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:13-18. Using the heavy chain variable region CDR3 sequence shown in SEQ ID NO:13-18 results in higher antibody affinity and specificity.

[0043] In some examples of this application, the antibody or its antigen-binding fragment further comprises a heavy chain framework region sequence, at least a portion of which is derived from at least one of alpaca-derived antibodies, shark-derived antibodies, mouse-derived antibodies, rabbit-derived antibodies, primate-derived antibodies, or mutants thereof. In a preferred example of this application, the heavy chain framework region sequence of the antibody or its antigen-binding fragment is selected from alpaca-derived antibodies. In some examples of this application, the framework region helps maintain antibody structural stability.

[0044] In some examples of this application, the antibody or its antigen-binding fragment further contains a heavy chain constant region, at least a portion of which is derived from at least one of a murine antibody, a rabbit antibody, a primate antibody, or a mutant thereof.

[0045] In some examples of this application, the antibody or its antigen-binding fragment further comprises a light chain variable region, which includes a light chain complementarity-determining region (LCDR) and a light chain framework region. At least a portion of the light chain framework region sequence is derived from at least one of a murine antibody, a rabbit antibody, a primate antibody, or a mutant thereof.

[0046] In some examples of this application, the antibody or its antigen-binding fragment further contains a light chain constant region, at least a portion of which is derived from at least one of a mouse antibody, a rabbit antibody, a primate antibody, or a mutant thereof.

[0047] In some examples of this application, the antibody includes at least one selected from polyclonal antibodies and monoclonal antibodies; or the antigen-binding fragment includes at least one selected from F(ab')2 fragment, Fab' fragment, Fab fragment, F(ab)2 fragment, Fv fragment, scFv fragment, scFv-Fc fusion protein, scFv-Fv fusion protein and minimum recognition unit.

[0048] In a preferred embodiment of this application, the antibody is a nanobody.

[0049] In some examples of this application, the nanobodies have amino acid sequences as shown in SEQ ID NO:19-24. Extensive experimental verification in some examples of this application has shown that nanobodies with amino acid sequences as shown in SEQ ID NO:19-24 can bind to Siglec-7 with high affinity and high specificity.

[0050] In another aspect of this application, a recombinant protein is proposed, comprising the antibody or its antigen-binding fragment described in any of the foregoing aspects or examples. In some examples of this application, the recombinant protein is capable of binding to Siglec-7 with high affinity and high specificity.

[0051] In some examples of this application, the recombinant protein further includes at least one selected from bioactive proteins or fragments thereof, bioactive peptides or fragments thereof.

[0052] According to embodiments of this application, the bioactive protein or fragment thereof includes at least one selected from protein tags, protein toxins or fragments thereof, tumor necrosis factor or fragments thereof, interferon or fragments thereof, biological response regulators or fragments thereof, and Fc fragments.

[0053] The term "protein tag" typically refers to a polypeptide or protein fused together with a target protein (antibody or antigen-binding fragment) for expression. It can be used for the expression, detection, detection, or purification of the target protein. Examples include, but are not limited to, His tags (also known as His-Tag, sequence HHHHHH), Flag tags (also known as Flag-Tag, sequence DYKDDDDK), GST tags (also known as GST-Tag, glutathione thiotransferase tag), SUMO tags, and C-Myc tags.

[0054] The term "toxin" generally refers to substances toxic to the host, including protein toxins and non-protein toxins. Protein toxins include, but are not limited to, abrin, ricin A, Pseudomonas exotoxin, and diphtheria toxin. In this application, the protein toxin is preferably an enzymatic protein toxin.

[0055] The term "tumor necrosis factor" usually refers to substances that can cause hemorrhagic necrosis in various tumors, including but not limited to TNF-α and TNF-β.

[0056] The term "interferon" typically refers to a glycoprotein that can directly kill or inhibit viruses. This includes, but is not limited to, IFN-α, IFN-β, and IFN-γ.

[0057] The term "biological response modulators" generally refers to a class of protein substances that enhance the body's anti-tumor effects directly or indirectly through the immune system. These include, but are not limited to, lymphokines, IL-2, IL-6, IL-10, and GM-CSF.

[0058] The term "Fc fragment" typically refers to the Fc region of IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM, including CH2, CH3 regions and optionally a hinge region. Preferably, the IgG, IgA1, IgA2, IgD, IgE, or IgM are derived from mouse, primate, or alpaca sources.

[0059] In another aspect of this application, a nucleic acid molecule is proposed that encodes the aforementioned antibody or its antigen-binding fragment or recombinant protein. In some examples of this application, the nucleic acid molecule enables the large-scale in vitro expression of the antibody or its antigen-binding fragment, as well as the recombinant protein.

[0060] According to an embodiment of this application, the nucleic acid molecule is DNA.

[0061] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned in this invention actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is also disclosed. Furthermore, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0062] In another aspect of this application, an expression vector carrying the aforementioned nucleic acid molecule is proposed. In some examples of this application, this expression vector enables the large-scale in vitro preparation of antibodies or antigen-binding fragments, and recombinant proteins.

[0063] It should be noted that when ligating the aforementioned nucleic acid molecules to a vector, the nucleic acid molecules can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. These control elements can originate directly from the vector itself or be exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecules and control elements only need to be operatively linked. In this article, "operatively linked" means ligating a foreign gene to a vector so that the control elements within the vector, such as transcriptional control sequences and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids, bacteriophages, etc.

[0064] In another aspect of this application, a recombinant cell is proposed, comprising: carrying the aforementioned nucleic acid molecule or expression vector; or expressing the aforementioned antibody or its antigen-binding fragment or recombinant protein.

[0065] It should be noted that the recombinant cells described in this application are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, Trichoderma, etc.; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described in this application are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0066] In some examples of this application, the recombinant cells are obtained by introducing the expression vector of claim 7 into host cells. The introduction methods include, but are not limited to, transfection or transformation under suitable conditions.

[0067] In some examples of this application, "transformation" or "transfection" refers to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0068] In another aspect of this application, a pharmaceutical composition is proposed, comprising the aforementioned antibody or its antigen-binding fragment, a recombinant protein, a nucleic acid molecule, an expression vector, or recombinant cells. In some examples of this application, the pharmaceutical composition is capable of binding to the Siglec-7 protein with high affinity and high specificity, blocking the interaction between sialic acid and Siglecs, inhibiting signal transduction processes, activating NK cells, and preventing tumor invasion.

[0069] In some examples of this application, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an effective amount of the antibody active ingredient.

[0070] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0071] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0072] The drug of this application contains a safe and effective amount of the active ingredient of this application and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration; the dosage form of the drug of this application is an injection, an oral formulation (tablet, capsule, oral liquid), a transdermal formulation, or a sustained-release formulation. For example, it is prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The drug is preferably manufactured under aseptic conditions.

[0073] The effective amount of the active ingredient described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0074] Pharmaceutically acceptable carriers described in this application include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0075] In another aspect of this application, the application proposes the use of the aforementioned antibody or its antigen-binding fragment, recombinant protein, nucleic acid molecule, expression vector, recombinant cell, or pharmaceutical composition in the preparation of a drug for the treatment or prevention of tumors. In some examples of this application, drugs prepared based on antibodies or their antigen-binding fragments, recombinant proteins, nucleic acid molecules, expression vectors, recombinant cells, or pharmaceutical compositions can bind to the Siglec-7 protein with high affinity and high specificity, blocking the interaction between sialic acid and Siglecs, inhibiting signal transduction processes, activating NK cells, and preventing tumor invasion.

[0076] In some examples of this application, the tumors include liver cancer, lung cancer, stomach cancer, colorectal cancer, breast cancer, esophageal cancer, thyroid cancer, skin cancer, prostate cancer, kidney cancer, endometrial cancer, cervical cancer, and hematologic malignancies.

[0077] In another aspect of this application, the application discloses the use of the aforementioned antibody or its antigen-binding fragment, recombinant protein, nucleic acid molecule, expression vector, or recombinant cell in the preparation of a kit for detecting Siglec-7. In some examples of the application, this kit can be used in immunoblotting, immunoprecipitation, and other kits involving the detection of Siglec-7 antigen and antibody-specific binding properties. These kits may contain any one or more of the following: antagonist, anti-Siglec-7 antibody, or drug reference material; protein purification column; immunoglobulin affinity purification buffer; cell assay diluent; instructions or literature, etc. Anti-Siglec-7 antibodies can be used in various types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, they can be used to test for related diseases by detecting the serum or blood of a subject.

[0078] In another aspect, this application provides a kit comprising: the aforementioned antibody or its antigen-binding fragment, a recombinant protein, a nucleic acid molecule, an expression vector, or recombinant cells. The antibody or its antigen-binding fragment in the kit provided by this application can effectively bind to the Siglec-7 protein. Furthermore, under suitable conditions, the nucleic acid molecule, expression vector, or recombinant cells can all express the antibody or its antigen-binding fragment. Further, the kit containing the above substances can bind with Siglec-7 with high specificity and can be used for the effective detection of Siglec-7. The kit can be used for scientific research, such as qualitative or quantitative detection of Siglec-7 in biological samples, and can also be used to assess individual status, such as determining whether an individual's Siglec-7 level is too high or too low than normal after obtaining the individual's Siglec-7 level. The biological sample can be cells, tissues, etc.

[0079] Table 1

[0080]

[0081]

[0082]

[0083]

[0084] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0085] Example 1: Expression and purification of Siglec-7 ECD protein

[0086] The following specific methods were used to express and purify the Siglec-7 ECD protein.

[0087] Primers were designed to amplify the Siglec-7 ECD gene fragment using human cDNA as a template. The chosen vector was pTT5. The pTT5-Siglec-7 ECD plasmid was constructed using Gibson assembly.

[0088] The extracted plasmids were transfected into HEK293F cells using PEI (polyethyleneimine) transfection reagent. Parameters and dosages are shown in Table 2. The mammalian expression system was transfected using the pTT5-Siglec-7 ECD plasmid.

[0089] Table 2. Parameters and dosage of the transfection system

[0090] Polyethyleneimine 1.2 ml (1 mg / ml) plasmid 0.4 mg DMEM 30 ml

[0091] Preheat DMEM to 37°C in a water bath before transfection. Place 15 ml of DMEM in two 50 ml tubes, add the plasmid to one tube and PEI to the other. Incubate both tubes at room temperature for 12 minutes. Then transfer the PEI (in DMEM) to the plasmid tube and incubate at room temperature for 16 minutes. HEK293F cells were grown at a count of 2 million / ml and a total volume of 400 ml. After plasmid-PEI incubation, it was dropwise transferred to 400 ml of cells and incubated at 37°C for 20 minutes without shaking. Subsequently, the cells were cultured in a shaker at 120 rpm and 5% CO2 for four days. The cells were centrifuged at 4347 rcf for 30 minutes, and the supernatant was passed through 0.45 μm and 0.22 μm filters, respectively. The filtered sample was loaded onto a peristaltic pump and the protein was purified using affinity chromatography (protein A column). The pTT5 vector carries an Fc(IgG) tag, which can specifically bind to the resin of the Protein A column, thereby enriching the target protein in the supernatant onto the column.

[0092] After loading, the protein column was eluted using an AKTA Prime Plus purification system. 0.2M Tris-HCl (pH 7) was added to the protein collection tube, and the program was run to elute the protein from 0% B solution to 100% B solution. Liquid A used was PBS, and liquid B was 0.1M acetic acid solution, used to elute bound proteins. The harvested protein peaks are shown in the figure. Figure 1 As shown.

[0093] To immunize alpacas and generate specific nanobodies, the antigen needed to be as pure as possible; therefore, purified protein (Siglec-7-TEV-Fc) was digested. TEV protease was added to the collected protein and stored overnight at 4°C. The next day, the digested protein was passed through Protein A and Ni-NTA columns to completely remove undigested protein, Fc, and TEV enzyme, respectively. The effluent was collected, concentrated, and then subjected to SDS-PAGE electrophoresis. The results are as follows: Figure 2 As shown, this indicates that high-purity Siglec-7 ECD protein was obtained.

[0094] Example 2: Construction of phage display library

[0095] Adult male alpacas were immunized four times with the Siglec-7 ECD protein prepared in Example 1. The alpacas received 0.3 mg for the first immunization, followed by 0.4 mg for the next three immunizations, with a two-week interval between immunizations. Lymphocytes were collected after the fourth immunization. Total RNA was extracted using the Omega Biotek kit, and genomic DNA was removed using DNaSe. Total RNA was reverse transcribed into cDNA using the PrimeScript™ First-Strand cDNA Synthesis Kit (Takara, 6110A). A specific gene fragment of VHH was amplified using VHH primers, and then VHH was cloned into the phage plasmid pR2 using the Beyotime Seamless Cloning Kit (D7010M) via Gibson assembly. The VHH amplification primers and pR2 plasmid sequence amplification primers are shown in Table 1.

[0096] Using Gibson assembly (GA), 1.5 pmol of pR2 and 6 pmol of amplified VHH were mixed with an appropriate volume of ddH2O and the cloning master mixture (Beyotime). The reaction was carried out in a 50°C water bath for 1 hour. The ligation product was recovered using a PCR product recovery kit and then stored on ice. The GA product was mixed with 500 μl of TG1 electrocompetent cells (Biosearch 60502-1) and transferred to an ice-cold electroporation cuvette with a 0.2 cm gap (the cuvette was pre-soaked in 75% ethanol for half an hour and then placed in a fume hood under sterile conditions). The voltage was set to 2.5 kV / cm and held for 5 ms in a BTX ECM 299 system. The electroporated TG1 cells were then recovered in 2 ml of recovery medium at 37°C and 180 rpm for 1 hour. 0.2 μl and 2 μl of the 2 ml (preparative dilution) were plated on 10 cm LB / Amp / 2% Glucose plates and incubated at 37°C for 13 hours. Then, the colonies are counted to determine the library size.

[0097] Phage library amplification: Remaining TG1 cells were seeded onto 150 mm LB / Amp / 2% G plates and incubated for 13 h. Then, a mixture of 2TY and glycerol (20%) was added, and colonies were scraped and collected in centrifuge tubes. The tubes were vortexed and rapidly frozen in liquid nitrogen, then stored at -80°C for future use. 100 μl of the library culture and 10¹² pfu of helper phage KM13 (purchased from the MRC Molecular Biology Laboratory) were added to 100 mL of 2TY medium (containing 2% glucose and 100 μl ampicillin). The culture was incubated at 37°C with shaking until the bacteria reached the logarithmic growth phase (OD600, 0.5 to 0.6), and then incubated for 45 minutes for infection.

[0098] Take 50 ml, centrifuge, remove the supernatant, resuspend the precipitate in 100 mL of 2TY, add 0.1% glucose, 100 μl ampicillin and 100 μl kanamycin, and incubate at 180 rpm and 25 °C for about 16 h. Concentrate the phage by PEG precipitation, resuspend in PBS, and store on ice.

[0099] Example 3: Phage panning

[0100] Antigen coating: A concentration of 0.1 mg / ml antigen (Siglec-7, prepared in Example 1) was prepared, and a total of 100 μl of antigen was added to each well (96-well immunoassay plate). PBS was added only as a control, and the plate was incubated overnight at 4°C. Next, the plate was washed three times with PBST and 280 μl MPBS (280 μl total PBS) (PBS containing 5% skim milk was added to each well, and the plate was blocked at room temperature for 2 h).

[0101] Phage inoculation: with PBST (0.1% Tween 20 + PBS). 1×10 11 Phage libraries (per well) were added to MPBS, 100 μl per well, and the immunoplate was then incubated on a shaker at 60 rpm for 1 hour. 100 μl of 0.5 mg / mL trypsin was added to each well, and the plate was digested on a shaker at room temperature (60 rpm) for 1 hour, during which the phage bindings in the wells were eluted.

[0102] Phage infection of TG1: Infect 1 mL of logarithmically growing TG1 bacteria with 10 μl of the phage obtained in the previous step, and incubate at 37°C for 45 minutes. Spread 10 μl of the diluted solution onto 100 mm LB / 2% G / Amp plates. Figure 3 The remaining phage solution was used to infect 4 mL of logarithmic growth phase TG1 bacteria, placed in a 37°C water bath for 45 minutes, plated on a 150 mm LB / 2% G / Amp plate, and incubated overnight at 37°C.

[0103] Scraping: Add 4 ml of 2TY (20% glycerol) and scrape off the colonies from the 150 mm LB plate in the previous step. Collect the library in centrifuge tubes, quickly freeze in liquid nitrogen, and store at -80°C.

[0104] The second round of biological screening involved amplifying the library using the same method as the first round and infecting it with KM13. The only difference between the two rounds was the amount of phage added and the number of washes. The phage addition amount for the library was 1 × 10⁻⁶. 8 After adding PFU and the phage library, the number of washes was increased to 20-30. 1 mL of logarithmically growing TG1 bacteria was infected with 10 μl of the phage obtained in the second round and incubated at 37°C for 45 minutes. 10 μl of the diluted solution was then spread onto 100 mm LB / 2% G / Amp plates. Figure 4As shown, after two rounds of screening, phages targeting Siglec-7 ECD were significantly enriched.

[0105] Example 4: Screening positive clones using monoclonal phage ELISA

[0106] Preparation of monoclonal phages: Add 100 μl of 2TY / 2% G / Amp to each well of a 96-well cell culture plate, inoculate a single phage into each well, and then incubate at 37°C and 250 rpm for 6 hours. Take another 96-well culture plate, add 200 μl of 2TY / 2% G / Amp to each well, and inoculate 10 μl of the bacterial culture from the first plate into the corresponding well of the second plate, then incubate at 37°C and 250 rpm for 1.5 hours. To prepare the KM13 solution, mix 8 μl of KM13 with 1 ml of 2TY, then add 5 μl of the KM13 dilution to each well, and incubate at 37°C for 45 minutes. Centrifuge the plate, remove the supernatant, and invert to dry. Add 200 μl of 2TY / 0.1% G / Amp / Kan to each well, and incubate at 25°C and 250 rpm for 16 hours. The next day, the plate was centrifuged, and 150 μl from each well (which will be used as the primary antibody against the antigen during the screening process) was transferred to a new plate and stored at 4°C.

[0107] Antigen coating: Add 0.2 μg Siglec-7 per well (total volume 100 μl, dissolved in PBS) to a 96-well plate. A blank control (no antigen coating) is used. Incubate the plate overnight at 4°C. The next day, wash three times with PBST (0.1% Tween 20), then block with MPBS (5% skim milk) at room temperature for 3 hours. After three washes, add primary antibody (monoclonal phage prepared in the previous step) and incubate at 100 rpm for 1 hour at room temperature. Wash five times, then add secondary antibody HRP-M13 (diluted 1:8000 with 5% milk). Add 100 μl of secondary antibody to each well and incubate at room temperature for 1 hour. Wash four times. Then, under dark conditions, add 100 μl of TMB chromogenic substrate to each well and incubate at room temperature for 10 minutes (until blue color develops). Finally, add 50 μl of 1M H2SO4 per well to terminate the reaction. Measure the OD450 value using a microplate reader. The results are as follows: Figure 5 As shown. Positive clones were sent for sequencing, and six nanobodies were identified based on the CDR region (especially CDR3).

[0108] Example 5: Purification of Nanobodies

[0109] The nanobody sequence was cloned into pTT5 and then purified using the same method described in Example 1 for purifying Siglec-7. The chromatogram of the harvest is shown below. Figure 6 As shown, the SDS-PAGE results are as follows: Figure 7As shown, this demonstrates the acquisition of nanobody proteins.

[0110] Example 6: ELISA characterization of the binding affinity between nanobodies and Siglec-7

[0111] Add 0.2 μg Siglec-7 per well (total volume 100 μl, dissolved in PBS) to a 96-well plate. Set up a blank control (no antigen coating). Incubate the plate overnight at 4°C. The next day, wash three times with PBST (0.1% Tween 20), then block with MPBS (5% skim milk) at room temperature for 2 hours. Wash three times, then add primary antibody (using a concentration of 10). 3 nM to 10 -4 Each nanobody (nM) was incubated at 80 rpm for 1 h at room temperature. Then, it was washed 5 times with PBST (0.1%) and incubated with secondary antibody (mouse anti-human IgG-Fc, sinobiological, 10702-MM01T) at a 1:8000 dilution at room temperature and 80 rpm for 1 h. After washing 5 times with 0.1% PBST, 100 μl of TMB chromogenic substrate was added to each well in the dark and incubated at room temperature for 10 min (until blue color appeared). The reaction was then terminated by adding 50 μl of 1M H2SO4 per well. The OD450 value was measured using a microplate reader, and the results are as follows: Figure 8 As shown, the six nanobodies prepared above exhibit high binding affinity to the Siglec-7 protein.

[0112] Example 7: Single-cycle kinetics and affinity SPR

[0113] Each nanobody was prepared at a concentration of 0.1 μg / ml, and 75 μl of each nanobody was injected into the Biacore 8K system, which was then immobilized on a Cytiva CM5 sensor chip. Once the sensing pattern was standardized, protein concentrations ranging from 50 nM to 3.12 nM were injected into each channel. Binding parameters, such as..., were determined using single-cycle kinetics. Figure 9 As shown in Table 3, the corresponding values ​​indicate that the six nanobodies prepared above have a high affinity for binding to the Siglec-7 protein.

[0114] Table 3

[0115] NB ID Ka(1 / Ms) Kd(1 / s) KD(M) 1C2 <![CDATA[8.30×10 5 ]]> <![CDATA[3.12×10 -4 ]]> <![CDATA[3.76×10 -10 ]]> 1E3 <![CDATA[3.07×10 5 ]]> <![CDATA[8.23×10 -6 ]]> <![CDATA[2.68×10 -11 ]]> 2A4 <![CDATA[9.37×10 5 ]]> <![CDATA[6.27×10 -4 ]]> <![CDATA[6.70×10 -10 ]]> 1G6 <![CDATA[1.84×10 5 ]]> <![CDATA[1.76×10 -5 ]]> <![CDATA[9.56×10 -11 ]]> 1H8 <![CDATA[9.23×10 5 ]]> <![CDATA[3.72×10 -4 ]]> <![CDATA[4.03×10 -10 ]]> 1F10 <![CDATA[6.69×10 5 ]]> <![CDATA[9.60×10 -5 ]]> <![CDATA[1.43×10 -10 ]]>

[0116] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0117] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof targeting the Siglec-7 protein, characterized in that, include: Heavy chain variable region CDR1 as shown in the amino acid sequence of SEQ ID NO: 6, heavy chain variable region CDR2 as shown in the amino acid sequence of SEQ ID NO: 12, and heavy chain variable region CDR3 as shown in the amino acid sequence of SEQ ID NO:

18.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further contains a heavy chain framework region sequence, at least a portion of which is derived from at least one of alpaca-derived antibodies, shark-derived antibodies, mouse-derived antibodies, rabbit-derived antibodies, primate-derived antibodies, or mutants thereof.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, At least a portion of the heavy chain framework region sequence is derived from an alpaca-derived antibody.

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The antibody is a nanobody.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The nanobody has an amino acid sequence as shown in SEQ ID NO:

24.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or its antigen-binding fragment as described in any one of claims 1 to 5.

7. The nucleic acid molecule according to claim 6, characterized in that, The nucleic acid molecule in question is DNA.

8. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 6 or 7.

9. The expression vector according to claim 8, characterized in that, The expression vector is a eukaryotic expression vector or a prokaryotic expression vector.

10. The expression vector according to claim 9, characterized in that, The expression vector is a plasmid expression vector.

11. A recombinant cell, characterized in that, include: Carrying the nucleic acid molecule as described in claim 6 or 7 or the expression vector as described in any one of claims 8 to 10; or Express the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5.

12. The recombinant cell according to claim 11, characterized in that, The recombinant cells are obtained by introducing the expression vector according to any one of claims 8 to 10 into host cells.

13. The recombinant cells according to claim 11 or 12, characterized in that, The recombinant cells are mammalian cells.

14. A pharmaceutical composition, characterized in that, include: The antibody or its antigen-binding fragment according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6 or 7, the expression vector according to any one of claims 8 to 10, or the recombinant cell according to any one of claims 11 to 13.

15. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6 or 7, the expression vector according to any one of claims 8 to 10, or the recombinant cell according to any one of claims 11 to 13 in the preparation of a kit for detecting Siglec-7.

16. A reagent kit, characterized in that, include: The antibody or its antigen-binding fragment according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6 or 7, the expression vector according to any one of claims 8 to 10, or the recombinant cell according to any one of claims 11 to 13.

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