CN118146371A - Nanometer antibody targeting Siglec-7 protein and application thereof - Google Patents

Abstract

The disclosure relates to the biotechnology field, in particular to a nanometer antibody targeting Siglec-7 protein and application thereof. Wherein the antibody or antigen binding fragment thereof comprises an amino acid sequence comprising or having at least 80% identity to a heavy chain variable region CDR sequence selected from at least one of the following: SEQ ID NO. 1-18. The antibody or the antigen binding fragment thereof prepared by the application can be used for specifically and targeted binding Siglec-7 protein with high affinity, blocking the interaction between sialic acid and Siglecs, inhibiting the signal transduction process, activating NK cells and recovering the recognition and killing functions of tumor cells.

Description

Nanobodies targeting Siglec-7 protein and their applications

Technical Field

The present application relates to the field of biotechnology. Specifically, the present application relates to a nanobody targeting Siglec-7 protein and its application. More specifically, the present application relates 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 Art

Cancer manipulates multiple immune mechanisms to ensure a permissive local microenvironment that promotes tumor progression. Glycosylation is often considered a hallmark of cancer. Alterations in glycosylation on the surface of tumor/infected cells are a key feature of cancer and different infectious diseases to a certain extent. Sialyl glycans found on glycoproteins and glycolipids are recognized by Siglecs, which are elements of the lectin family expressed on the surface of many immune cell subtypes. Therefore, the interaction of cancer/infected cells with sialic acid-bound Siglecs can modulate immune cell phenotype and enable them to evade the immune system.

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

Siglec 7 is primarily localized to human natural killer (NK) cells and monocytes. Siglec 7 regulates inhibitory signals 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 special type of cell killing called antibody-dependent cell-mediated cytotoxicity (ADCC). Unlike T cells, which must be presented with antigen-presenting cells before recognizing tumors, NK cells can spontaneously lyse tumor cells without antigen stimulation or pre-activation.

Virus-infected cells and tumor cells downregulate their MHC-I expression. NK cell-mediated cytotoxicity is not restricted by MHC I molecules, but rather NK cells destroy cancer cells through perforin/granzyme-mediated lysis. Siglec-7 has a preferred binding specificity for 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 tumor therapeutic target that can restore the killing potential of NK cells against cancer.

Therefore, there is an urgent need in the art to develop an antibody against Siglec-7.

Summary of the invention

The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present application proposes an antibody that can bind to Siglec-7 protein with high affinity and high specificity.

In the first aspect of the present application, the present application proposes an antibody or an antigen-binding fragment thereof. According to an embodiment of the present application, the antibody or its 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 therewith: SEQ ID NO: 1 to 18. In some examples of the present application, the antibody or its antigen-binding fragment can target and bind to Siglec-7 protein with high affinity and specificity, block the interaction between sialic acid and Siglecs, inhibit the signal transduction process, activate NK cells, and restore their recognition and killing functions of tumor cells.

In the second aspect of the present application, the present application proposes a recombinant protein. According to an embodiment of the present application, the recombinant protein includes the antibody or antigen-binding fragment thereof described in the first aspect of the present application. In some examples of the present application, the recombinant protein can target and bind to Siglec-7 protein, block the interaction between sialic acid and Siglecs, inhibit signal transduction process, activate NK cells, and organize tumor infiltration.

In the third aspect of the present application, the present application proposes a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the antibody or antigen-binding fragment thereof described in the first aspect of the present application or the recombinant protein described in the second aspect of the present application. In some examples of the present application, the aforementioned antibody or antigen-binding fragment thereof and recombinant protein can be expressed in large quantities in vitro by the nucleic acid molecule.

In the fourth aspect of the present application, the present application proposes an expression vector. According to an embodiment of the present application, the expression vector carries the nucleic acid molecule described in the third aspect of the present application. In some examples of the present application, after the expression vector is introduced into a suitable recipient cell, the expression of the aforementioned antibody or its antigen-binding fragment, or recombinant protein can be effectively achieved under the mediation of a regulatory system, thereby achieving in vitro mass preparation of the antibody or its antigen-binding fragment, or recombinant protein.

In the fifth aspect of the present application, the present application proposes a recombinant cell. According to an embodiment of the present application, the recombinant cell carries the nucleic acid molecule described in the third aspect of the present application or the expression vector described in the fourth aspect; or expresses the antibody or antigen-binding fragment thereof described in the first aspect of the present application or the recombinant protein described in the second aspect of the present application. In some examples of the present application, the recombinant cell can effectively express the aforementioned antibody or antigen-binding fragment thereof, or recombinant protein in the cell under suitable conditions.

In the sixth aspect of the present application, the present application proposes a pharmaceutical composition. According to the embodiments of the present application, the pharmaceutical composition includes the antibody or antigen-binding fragment thereof described in the first aspect of the present application, the recombinant protein described in the second aspect of the present application, the nucleic acid molecule described in the third aspect of the present application, the expression vector described in the fourth aspect of the present application, or the recombinant cell described in the fifth aspect of the present application. In some examples of the present application, the pharmaceutical composition can bind to Siglec-7 protein with high affinity and high specificity, block the interaction between sialic acid and Siglecs, inhibit signal transduction processes, activate NK cells, and organize tumor infiltration.

In the seventh aspect of the present application, the present application proposes a 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, the recombinant cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of a drug, wherein the drug is used to treat or prevent tumors. In some examples of the present application, drugs prepared based on antibodies or antigen-binding fragments thereof, recombinant proteins, nucleic acid molecules, expression vectors, recombinant cells or pharmaceutical compositions can bind to Siglec-7 proteins with high affinity and high specificity, block the interaction between sialic acid and Siglecs, inhibit signal transduction processes, activate NK cells, and organize tumor infiltration.

In the eighth aspect of the present application, the present application proposes a 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, a kit prepared based on an antibody or antigen-binding fragment thereof, a recombinant protein, a nucleic acid molecule, an expression vector, or a recombinant cell can be used for portable detection of Siglec-7 protein.

In the ninth aspect of the present application, the present application proposes a kit. According to an embodiment of the present application, the kit includes: 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 some examples of the present application, the kit can be used for efficient and portable detection of Siglec-7 protein.

Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG1 is a peak diagram of Siglec-7 ECD protein in an embodiment of the present application;

FIG2 is a schematic diagram of the SDS-PAGE electrophoresis detection results of Siglec-7 ECD protein in the embodiment of the present application;

FIG3 is a schematic diagram of the detection results of bacteriophage infection TG1 in an embodiment of the present application;

FIG4 is a schematic diagram of the results of the second round of phage panning of Siglec-7 ECD phage display in the embodiment of the present application;

FIG5 is a schematic diagram of the results of ELISA screening of positive clones in an embodiment of the present application;

FIG6 is a schematic diagram of the results of nanoantibody chromatography detection in an embodiment of the present application;

Figure 7 is a SDS-PAGE gel electrophoresis diagram of the Nanobody Fc fusion protein of the present application example;

Figure 8 is a schematic diagram of the ELISA results of characterizing the binding affinity of nanobodies to Siglec-7 in the examples of the present application;

FIG. 9 is a schematic diagram of the results of the SPR affinity verification experiment of the nanobody of the embodiment of the present application.

DETAILED DESCRIPTION

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 throughout represent the same or similar elements or elements having the same or similar functions.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

In the present application, the amino acid sequences of the CDRs listed above are all shown in accordance with the IMGT definition rules. However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in the art by a variety of methods, such as the Kabat rule, the Chothia rule, etc. It should be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or a region thereof (such as a variable region) should be understood to cover the complementary determining region defined by any of the above-mentioned known schemes described in the present invention. Although the scope of protection requested in this application is based on the sequences shown in the IMGT definition rules, the amino acid sequences corresponding to the definition rules of other CDRs should also be included in the scope of protection of this application.

In this application, the abbreviations for amino acid residues are the standard three-letter and/or one-letter codes used in the art to refer to one of the 20 common L-amino acids, and have the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamate); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: pHe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

In this application, the term "identity" is used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in MolecμLar Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 48:443); the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:24 ... l.70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altsch μL et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altsch μL et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altsch μL et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC/Gene program provided by Intelligenetics, Mountain View, California.

In the present application, under the premise of not substantially affecting the activity of the antibody (retaining at least 95% of the activity), those skilled in the art may replace, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence of the present application to obtain a variant of the sequence of the antibody or its functional fragment. They are all considered to be included in the scope of protection of the present invention. For example, amino acids with similar properties are replaced in the variable region. The variant sequence described in the present application may have at least 80% identity (or homology) with the reference sequence, which means at least 80% with each reference sequence, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity. The sequence consistency described in the present application can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, with default parameters is used. The amino acid sequences described in the present invention are all shown in the manner of N-terminus to C-terminus.

In this application, the term "monoclonal antibody (abbreviated as MAb)" refers to an antibody that can only recognize one specific antigen epitope. Among them, common MAbs include two light chains with lighter molecular weight and two heavy chains with heavier molecular weight. The heavy chain (H chain) and the light chain (L chain) are connected by disulfide bonds to form a tetrapeptide chain molecule. The amino acid sequence of the amino terminal (N terminal) of the heavy chain or light chain varies greatly, which is called the variable region (V region). The carboxyl terminal (C terminal) of the heavy chain or light chain is relatively stable and varies little, which is called the constant region (C region); the V regions of the L chain and the 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 minimum recognition units.

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

In the present application, the term "nanobody" comprises only the heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind to the antigen via the heavy chain variable region.

In one aspect of the present application, the present application proposes an antibody or an antigen-binding fragment thereof, 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 therewith: SEQ ID NO: 1 to 18. In some examples of the present application, the antibody or its antigen-binding fragment can bind to Siglec-7 protein with high affinity and high specificity. The amino acid sequence involved is shown in Table 1.

In some examples of the present application, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region CDR1 sequence as shown in SEQ ID NOs: 1 to 6 amino acid sequences or an amino acid sequence having at least 80% identity with SEQ ID NOs: 1 to 6, respectively. In some preferred examples of the present application, the heavy chain variable region CDR1 sequence of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NOs: 1 to 6. With the heavy chain variable region CDR1 sequence as shown in SEQ ID NOs: 1 to 6, the antibody has higher affinity and specificity.

In some examples of the present application, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region CDR2 sequence as shown in SEQ ID NOs: 7 to 12 amino acid sequences or an amino acid sequence having at least 80% identity with SEQ ID NOs: 7 to 12, respectively. In some preferred examples of the present application, the heavy chain variable region CDR2 sequence of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NOs: 7 to 12. With the heavy chain variable region CDR2 sequence as shown in SEQ ID NOs: 7 to 12, the antibody has higher affinity and specificity.

In some examples of the present application, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region CDR3 sequence as shown in SEQ ID NOs: 13 to 18 amino acid sequences or an amino acid sequence having at least 80% identity with SEQ ID NOs: 13 to 18, respectively. In some preferred examples of the present application, the heavy chain variable region CDR3 sequence of the antibody or antigen-binding fragment thereof is as shown in SEQ ID NOs: 13 to 18. With the heavy chain variable region CDR3 sequence as shown in SEQ ID NOs: 13 to 18, the antibody has higher affinity and specificity.

In some examples of the present application, 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 an alpaca antibody, a shark antibody, a mouse antibody, a rabbit antibody, a primate antibody, or a mutant thereof. In a preferred example of the present application, the heavy chain framework region sequence of the antibody or its antigen-binding fragment is selected from an alpaca antibody. In some examples of the present application, the framework region helps maintain the structural stability of the antibody.

In some examples of the present application, the antibody or antigen-binding fragment thereof 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.

In some examples of the present application, the antibody or antigen-binding fragment thereof further contains a light chain variable region, the light chain variable region including a light chain complementary determining region LCDR and a light chain framework region, wherein at least a portion of the light chain framework region sequence is derived from at least one of a mouse antibody, a rabbit antibody, a primate antibody or a mutant thereof.

In some examples of the present application, the antibody or antigen-binding fragment thereof further contains a light 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.

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

In a preferred example of the present application, the antibody is a nanobody.

In some examples of the present application, the nanobody has an amino acid sequence as shown in SEQ ID NO: 19 to 24. In some examples of the present application, it has been found through sufficient experimental verification that the nanobody having an amino acid sequence as shown in SEQ ID NO: 19 to 24 can bind to Siglec-7 with high affinity and high specificity.

In another aspect of the present application, the present application proposes a recombinant protein, including the antibody or antigen-binding fragment thereof described in any of the aforementioned aspects or examples. In some examples of the present application, the recombinant protein can bind to Siglec-7 with high affinity and high specificity.

In some examples of the present application, the recombinant protein further includes at least one selected from a biologically active protein or a fragment thereof, a biologically active polypeptide or a fragment thereof.

According to an embodiment of the present application, the biologically active protein or fragment thereof includes at least one selected from a protein tag, a protein toxin or a fragment thereof, a tumor necrosis factor or a fragment thereof, an interferon or a fragment thereof, a biological response regulator or a fragment thereof and an Fc fragment.

The so-called "protein tag" generally refers to a polypeptide or protein fused with the target protein (antibody or antigen-binding fragment), which can be used for the expression, detection, disappearance or purification of the target protein, etc., including but not limited to His tag (also known as His-Tag, sequence HHHHHH), Flag tag (also known as Flag-Tag, sequence DYKDDDDK), GST tag (also known as GST-Tag, glutathione S-transferase tag), SUMO tag and C-Myc tag, etc.

The so-called "toxin" generally refers to a substance that is toxic to the host, including protein toxins and non-protein toxins. Among them, protein toxins include but are not limited to abrin, ricin A, Pseudomonas exotoxin and diphtheria toxin. In the present application, the protein toxin is preferably a protein toxin with enzymatic activity.

The so-called "tumor necrosis factor" generally refers to a substance that can cause hemorrhagic necrosis of various tumors, including but not limited to TNF-α and TNF-β.

The so-called "interferon" generally refers to a glycoprotein that has the ability to directly kill or inhibit viruses, including but not limited to IFN-α, INF-β and IFN-γ.

The so-called "biological response modifiers" generally refer to a class of protein substances that directly or indirectly enhance the body's anti-tumor effect through the immune system, including but not limited to lymphokines, IL-2, IL-6, IL-10 and GM-CSF.

The so-called "Fc fragment" generally refers to the Fc region from IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM, including CH2, CH3 region and optionally hinge region. Preferably, IgG, IgA1, IgA2, IgD, IgE or IgM is derived from mouse, primate or alpaca.

In another aspect of the present application, the present application proposes a nucleic acid molecule encoding the aforementioned antibody or its antigen-binding fragment or recombinant protein. In some examples of the present application, the antibody or its antigen-binding fragment and the recombinant protein can be expressed in large quantities in vitro based on the nucleic acid molecule.

According to an embodiment of the present application, the nucleic acid molecule is DNA.

It should be noted that, for the nucleic acid molecules mentioned in the present invention, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, in this specification and claims, although only one strand is given in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequence in this application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

In another aspect of the present application, the present application proposes an expression vector carrying the aforementioned nucleic acid molecule. In some examples of the present application, the in vitro mass production of antibodies or antigen-binding fragments and recombinant proteins can be achieved based on the expression vector.

It should be noted that when the above-mentioned nucleic acid molecules are connected 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. Of course, these control elements can come directly from the vector itself, or they can be exogenous, that is, not from the vector itself. Of course, the nucleic acid molecules can be operably connected to the control elements. In this article, "operably connected" means connecting the exogenous gene to the vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, etc., can play their intended function of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, plasmids, bacteriophages, etc.

In another aspect of the present application, the present application proposes a recombinant cell, comprising: carrying the aforementioned nucleic acid molecule or expression vector; or expressing the aforementioned antibody or antigen-binding fragment thereof or recombinant protein.

It should be noted that the recombinant cells described in the present application are not particularly limited and may be prokaryotic cells, eukaryotic cells or bacteriophages. The prokaryotic cells may be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells may be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, insect cells such as armyworms, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc. In certain embodiments, the recombinant cells described in the present 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.

In some examples of the present application, the recombinant cell is obtained by introducing the expression vector of claim 7 into a host cell, wherein the introduction method includes but is not limited to transfection or transformation under suitable conditions.

In some examples of the present application, "transformation" or "transfection" refers to the introduction of nucleic acids (e.g., vectors) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of the present invention and can be used for the expression and/or secretion of target proteins. Examples of suitable host cells that can be used in the present invention include immortalized hybridoma cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

In another aspect of the present application, the present application proposes a pharmaceutical composition, including the aforementioned antibody or antigen-binding fragment thereof, recombinant protein, nucleic acid molecule, expression vector or recombinant cell. In some examples of the present application, the pharmaceutical composition can bind to Siglec-7 protein with high affinity and high specificity, block the interaction between sialic acid and Siglecs, inhibit signal transduction process, activate NK cells, and organize tumor infiltration.

In some examples of the present application, the pharmaceutical composition includes a pharmaceutically acceptable carrier and an effective amount of the antibody active ingredient.

As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and/or animals and can be accepted by humans and/or animals.

As used herein, "pharmaceutically acceptable" ingredients are suitable for use in humans and/or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), i.e., substances with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The medicine of the present application contains a safe and effective amount of the active ingredient of the present application and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Usually, the drug preparation should match the mode of administration. The dosage form of the medicine of the present application is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent, sustained release agent. For example, it is prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. The medicine is preferably manufactured under sterile conditions.

The effective amount of the active ingredient described in the present application may vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). The factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's weight, the patient's immune status, the route of administration, etc. For example, depending on the urgency of the treatment situation, several divided doses may be given per day, or the dose may be reduced proportionally.

The pharmaceutically acceptable carriers described in the present application include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide substances, cellulose, nanogels, or combinations thereof. The choice of carrier should match the mode of administration, which are well known to those of ordinary skill in the art.

In another aspect of the present application, the present application proposes a 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, which is used to treat or prevent tumors. In some examples of the present 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 Siglec-7 protein with high affinity and high specificity, block the interaction between sialic acid and Siglecs, inhibit signal transduction processes, activate NK cells, and organize tumor infiltration.

In some examples of the present application, the tumors include liver cancer, lung cancer, gastric cancer, colorectal cancer, breast cancer, esophageal cancer, thyroid cancer, skin cancer, prostate cancer, kidney cancer, endometrial cancer, cervical cancer and blood system tumors, etc.

In another aspect of the present application, the present application proposes a 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, the kit can be used for immunoblotting, immunoprecipitation, and other kits involving detection using the specific binding properties of Siglec-7 antigen and antibody. These kits may contain any one or more of the following: antagonists, anti-Siglec-7 antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. Anti-Siglec-7 antibodies can be used for different types of diagnostic tests, for example, various diseases or the presence of drugs, toxins or other proteins can be detected in vitro or in vivo. For example, the serum or blood of the subject can be tested to test related diseases.

In another aspect of the present application, the present application proposes a kit, comprising: the aforementioned antibody or antigen-binding fragment thereof, recombinant protein, nucleic acid molecule, expression vector or recombinant cell. The antibody or antigen-binding fragment thereof in the kit provided by the present application can effectively bind to the Siglec-7 protein. In addition, under suitable conditions, the nucleic acid molecule, expression vector or recombinant cell can express the antibody or antigen-binding fragment thereof. Further, the kit containing the above substances can bind to Siglec-7 with high specificity and can be used to effectively detect 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 judge the status of an individual, such as after obtaining the Siglec-7 level of the individual, judging whether the Siglec-7 level is too high or lower than the normal level. The biological sample can be a cell, tissue, etc.

Table 1

The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

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

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

Primers were designed to amplify the Siglec-7 ECD gene fragment using human cDNA as a template, and the selected vector was pTT5. Gibson assembly was used to construct the pTT5-Siglec-7 ECD plasmid.

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

Table 2 Parameters and dosage of transfection system

Polyethyleneimine 1.2 ml (1 mg/ml) Plasmids 0.4 mg DMEM 30 ml

DMEM was preheated in a 37°C water bath before transfection. Two 50ml tubes were used to hold 15ml DMEM, and then the plasmid was added to one of the tubes, and PEI was added to the other tube. The two tubes were left standing at room temperature for 12 minutes. Then PEI (in DMEM) was transferred to the plasmid tube and incubated at room temperature for 16 minutes. HEK293F cells were grown with a count of 2 million/ml and a total volume of 400ml. After the plasmid-PEI incubation, it was transferred dropwise to 400ml cells, and then left standing in a 37°C incubator for 20 minutes without shaking. Subsequently, the cells were cultured in a shaking incubator at 120rpm, 5% CO ₂ for four days. The cells were centrifuged at 4347rcf for 30 minutes, and then the supernatant was passed through 0.45μm and 0.22μm filters respectively. The filtered sample was loaded onto a peristaltic pump, and affinity chromatography (protein A column) was used to purify the protein. 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.

After loading, the protein column was eluted on the 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 to elute the bound protein. The harvested protein peak is shown in Figure 1.

In order to immunize alpacas and produce specific nanobodies, the antigen needs to be as pure as possible, and for this purpose, the purified protein (Siglec-7-TEV-Fc) was digested. TEV protease was added to the collected protein and stored at 4°C overnight. 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 electrophoresed using SDS-PAGE. The results are shown in Figure 2, indicating that a highly pure Siglec-7 ECD protein was obtained.

Example 2: Construction of phage display library

Adult male alpacas were immunized 4 times with the Siglec-7 ECD protein prepared in Example 1. The alpaca was immunized with 0.3 mg for the first time and 0.4 mg for the subsequent 3 times. The immunization interval was two weeks. Blood was drawn and lymphocytes were extracted after the fourth immunization. Total RNA was extracted using the OmegaBiotek kit, and genomic DNA was removed using DNAse. Total RNA was reverse transcribed into cDNA using the PrimeScript TM First-Chain cDNA Synthesis Kit (Takara, 6110A). VHH primers were used to amplify specific gene fragments of VHH, and then the Beyotime Seamless Cloning Kit (D7010M) was used to clone VHH into the phagemid pR2 through Gibson assembly. Among them, VHH amplification primers and pR2 plasmid sequence amplification primers are shown in Table 1;

Using Gibson assembly (GA), 1.5 pmol of pR2 and 6 pmol of amplified VHH were mixed with an appropriate volume of ddH ₂ O and a cloning master mix (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 a 0.2 cm gap ice-precooled electroporation cuvette (the cuvette was pre-soaked in 75% ethanol for half an hour and then in a fume hood under sterile conditions), and the voltage was set to 2.5 kV/cm in a BTX ECM 299 system for 5 ms. The electro-transformed TG1 cells were then recovered in 2 ml of recovery medium at 37°C, 180 rpm for 1 hour. 0.2 μl and 2 μl (prepared dilution) of the 2 ml were plated on a 10 cm LB/Amp/2% Glucose plate and kept in a 37°C incubator for 13 hours. The colonies were then counted to determine the library size.

Phage library amplification: The remaining TG1 cells were plated on 150 mm LB/Amp/2% G plates and incubated for 13 h, then a mixture of 2TY and glycerol (20%) was added for scraping, colonies were collected in centrifuge tubes, vortexed and quickly frozen in liquid nitrogen, and then stored at -80 ° C for future use. 100 μl of library bacterial solution and 10 12 pfu of helper phage KM13 (purchased from MRC Molecular Biology Laboratory) were added to 100 mL 2TY medium (containing 2% glucose, 100 μl ampicillin). The culture was shaken at 37 ° C until the logarithmic growth phase of bacterial growth (OD600, 0.5 to 0.6), and allowed to stand for 45 minutes for infection.

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

Example 3: Phage panning

Antigen coating: 0.1 mg/ml antigen (Siglec-7, prepared in Example 1) concentration, a total of 100 μl antigen was added to each well (96-well immunoplate), only PBS was added as a control, and placed at 4°C overnight. Next, the plate was washed 3 times with PBST and 280 μl MPBS (280 μl) (PBS containing 5% skim milk was added to each well, and blocked for 2 h at room temperature.

I. Phage inoculation: Use PBST (0.1% Tween 20 + PBS). Add 1×10 ¹¹ library phages (per well) into MPBS, add 100 μl to each well, and then incubate the immunoplate on a shaker at 60 rpm for 1 hour. Add 100 μl of 0.5 mg/mL trypsin to each well, and digest for 1 hour on a shaker at room temperature (60 rpm), and the phage binding in the well is eluted.

Phage infection of TG1: 10 μl of the phage obtained in the previous step was used to infect 1 mL of TG1 bacteria in the logarithmic growth phase, and kept in a 37°C water bath for 45 minutes. 10 μl of the dilution was taken and spread on a 100 mm LB/2% G/Amp plate (Figure 3). The remaining phage solution was used to infect 4 mL of TG1 bacteria in the logarithmic growth phase, placed in a 37°C water bath for 45 minutes, spread on a 150 mm LB/2% G/Amp plate, and cultured at 37°C overnight.

Scrape: Add 4 ml 2TY (20% glycerol) and scrape the colonies on the 150 mm LB plate in the previous step. Collect the library in a centrifuge tube, quickly freeze in liquid nitrogen and store at -80°C.

Second round of bio-panning: The above library was amplified in the same way as the first round and infected with KM13. The only difference between the two rounds was the amount of phage added and the number of washes. The amount of library phage added was 1×10 ⁸ pfu, and the number of washes was changed to 20-30 times after adding the library phage. 10μl of phage obtained in the second round was used to infect 1mL of TG1 bacteria in the logarithmic growth phase and kept in a 37°C water bath for 45 minutes. 10μl of the dilution was taken and applied to a 100mm LB/2%G/Amp plate, as shown in Figure 4, indicating that after two rounds of panning, phages targeting Siglec-7 ECD were significantly enriched.

Example 4: Screening of positive clones by monoclonal phage ELISA

Preparation of monoclonal phage: 100 μl 2TY/2% G/Amp was added to each well of a 96-well cell culture plate, and a single clone was inoculated into each well, and then cultured at 37°C and 250 rpm for 6 hours. Another 96-well culture plate was taken, and 200 μl 2TY/2% G/Amp was added to each well, and 10 μl of the bacterial solution from the first plate was inoculated into the corresponding well of the second plate, and then incubated at 37°C and 250 rpm for 1.5 hours. To prepare the KM13 solution, 8 μl KM13 was mixed with 1 ml 2TY, and then 5 μl of the KM13 dilution was added to each of the above wells and incubated at 37°C for 45 minutes. The plate was centrifuged, the supernatant was removed and inverted to dry. 200 μl 2TY/0.1% G/Amp/Kan was added to each well and incubated at 25°C and 250 rpm for 16 hours. The next day, the plates were centrifuged and 150 μl from each well (which will be used as primary antibody to the antigen during screening) was transferred to a new plate and stored at 4°C.

Coating antigen: Add 0.2μg Siglec-7/well (total volume 100μl, dissolved in PBS) to a 96-well culture plate, set a blank, and set up no coating antigen as a control. Place the plate at 4°C overnight. The next day, wash 3 times with PBST (0.1% tween20), and then block with MPBS (5% skim milk) at room temperature for 3h. After washing 3 times, add the primary antibody (monoclonal phage prepared in the previous step) and incubate at room temperature at 100rpm for 1h. Wash 5 times, then add the secondary antibody HRP-M13 (diluted 1:8000 with 5% milk). Add 100μl of the secondary antibody to each well and incubate at room temperature for 1h. Wash four times. Then add 100μl of TMB color substrate to each well under light-proof conditions, incubate at room temperature for 10 minutes (until blue appears), and then add 50μl 1M H ₂ SO ₄ /well to terminate the reaction. Use an enzyme reader to measure the OD450 value, and the results are shown in Figure 5. The positive clones were sent for sequencing, and six nanobodies were identified based on the CDR regions (especially CDR3).

Example 5: Purification of Nanobodies

The nanobody sequence was cloned into pTT5 and then purified using the same method as described in Example 1 for purifying Siglec-7. The chromatogram of the harvest is shown in Figure 6, and the SDS-PAGE result is shown in Figure 7, indicating that the nanobody protein was obtained.

Example 6: ELISA characterization of the binding affinity of nanobodies to Siglec-7

0.2 μg Siglec-7/well (total volume 100 μl, dissolved in PBS) was added to a 96-well culture plate, and a blank was set up without coating antigen as a control. The plate was placed at 4°C overnight. The next day, it was washed 3 times with PBST (0.1% tween20) and then blocked with MPBS (5% skim milk) at room temperature for 2 hours. Wash 3 times, then add the primary antibody (using each nano antibody at a concentration of 10 ³ nM to 10 ^-4 nM) and incubate at room temperature at 80 rpm for 1 hour. Then wash 5 times with PBST (0.1%), and then incubate with the secondary antibody (mouse anti-human IgG-Fc, sinobiological, 10702-MM01T) at a dilution of 1:8000 at room temperature and 80 rpm for 1 hour. Then, the wells were washed five times with 0.1% PBST, and then 100 μl of TMB colorimetric substrate was added to each well in the dark and incubated at room temperature for 10 minutes (until blue color appeared), and then 50 μl of 1M H ₂ SO ₄ /well was added to terminate the reaction. The OD450 value was measured using an ELISA instrument, and the results are shown in Figure 8, indicating that the six nanobodies prepared above have a high binding affinity to the Siglec-7 protein.

Example 7: Single Cycle Kinetics and Affinity SPR

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 fixed on the Cytiva CM5 sensor chip. Once the sensor graph became standard, protein concentrations ranging from 50 nM to 3.12 nM were injected into each channel. Using single-cycle kinetics, the binding parameters were determined, as shown in Figure 9, and the corresponding values are shown in Table 3. The results show that the six nanobodies prepared above have a high binding affinity to the Siglec-7 protein.

Table 3

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

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (12)

1. An antibody or antigen binding fragment thereof comprising a heavy chain variable region CDR sequence or an amino acid sequence having at least 80% identity thereto selected from at least one of the following: SEQ ID NO. 1-18.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

The heavy chain variable region CDR1 sequences are respectively shown as the amino acid sequences of SEQ ID NO. 1-6 or the amino acid sequences with at least 80% of the identity with SEQ ID NO. 1-6;

a heavy chain variable region CDR2 sequence shown as the amino acid sequences of SEQ ID NO 7-12 or the amino acid sequence with at least 80% identity with SEQ ID NO 7-12;

a heavy chain variable region CDR3 sequence shown as the amino acid sequences of SEQ ID NO. 13-18 or the amino acid sequence with at least 80% identity with SEQ ID NO. 13-18;

Preferably, the antibody or antigen binding fragment thereof comprises: heavy chain variable region CDR1 sequences shown as SEQ ID NO. 1-6 amino acid sequences respectively;

Preferably, the antibody or antigen binding fragment thereof comprises: the heavy chain variable region CDR2 sequences are shown as the amino acid sequences of SEQ ID NO 7-12 respectively;

preferably, the antibody or antigen binding fragment thereof comprises: the heavy chain variable region CDR3 sequences are shown as SEQ ID NO. 13-18 amino acid sequences respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, further comprising a heavy chain framework region sequence, at least a portion of which is derived from at least one of an alpaca-derived antibody, a shark-derived antibody, a murine antibody, a rabbit-derived antibody, a primates-derived antibody or a mutant thereof, preferably an alpaca-derived antibody;

optionally, the antibody or antigen binding fragment thereof further comprises 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-derived antibody, or a mutant thereof;

optionally, the antibody or antigen binding fragment thereof further comprises a light chain variable region comprising a light chain complementarity determining region LCDR and a light chain framework region;

Optionally, 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-origin antibody, or a mutant thereof;

Optionally, the antibody or antigen binding fragment thereof further comprises a light chain constant region, at least a portion of which is derived from at least one of a murine antibody, a rabbit antibody, a primate-derived antibody, or a mutant thereof.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody comprises at least one selected from the group consisting of a polyclonal antibody and a monoclonal antibody; or (b)

The antigen binding fragment comprises at least one selected from the group consisting of a F (ab ') 2 fragment, a Fab' fragment, a Fab fragment, a F (ab) 2 fragment, an Fv fragment, an scFv-Fc fusion protein, an scFv-Fv fusion protein, and a minimal recognition unit;

preferably, the antibody is a nanobody;

optionally, the nanobody has an amino acid sequence as shown in SEQ ID NO 19-24.

5. A recombinant protein comprising: the antibody or antigen-binding fragment thereof of any one of claims 1-4;

optionally, further comprising at least one member selected from the group consisting of a biologically active protein or fragment thereof, a biologically active polypeptide or fragment thereof;

Optionally, the biologically active protein or fragment thereof comprises at least one selected from the group consisting of a protein tag, a protein toxin or fragment thereof, a tumor necrosis factor or fragment thereof, an interferon or fragment thereof, a biological response modifier or fragment thereof, and an Fc fragment.

6. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-4 or the recombinant protein of claim 5;

Optionally, the nucleic acid molecule is DNA.

7. An expression vector carrying the nucleic acid molecule of claim 6;

Optionally, the expression vector is a eukaryotic expression vector or a prokaryotic expression vector;

Preferably, the expression vector is a plasmid expression vector.

8. A recombinant cell comprising:

carrying the nucleic acid molecule of claim 6 or the expression vector of claim 7; or (b)

Expressing the antibody or antigen-binding fragment thereof of any one of claims 1-4 or the recombinant protein of claim 5;

Optionally, the recombinant cell is obtained by introducing the expression vector of claim 7 into a host cell;

optionally, the recombinant cell is a mammalian cell.

9. A pharmaceutical composition comprising:

the antibody or antigen-binding fragment thereof of any one of claims 1-4, the recombinant protein of claim 5, the nucleic acid molecule of claim 6, the expression vector of claim 7, or the recombinant cell of claim 8.

10. Use of the antibody or antigen binding fragment thereof of any one of claims 1-4, the recombinant protein of claim 5, the nucleic acid molecule of claim 6, the expression vector of claim 7, the recombinant cell of claim 8, or the pharmaceutical composition of claim 9 in the manufacture of a medicament for treating or preventing a tumor;

Optionally, the tumor includes 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 hematological tumors.

11. Use of the antibody or antigen binding fragment thereof of any one of claims 1-4, the recombinant protein of claim 5, the nucleic acid molecule of claim 6, the expression vector of claim 7, or the recombinant cell of claim 8 in the preparation of a kit for detecting Siglec-7.

12. A kit, comprising:

the antibody or antigen-binding fragment thereof of any one of claims 1-4, the recombinant protein of claim 5, the nucleic acid molecule of claim 6, the expression vector of claim 7, or the recombinant cell of claim 8.