Antibody or antigen binding fragment thereof and application thereof

Abstract

The invention provides a target antibody or an antigen binding fragment thereof and application thereof, and belongs to the field of antibodies. Wherein the antibody or the antigen binding fragment thereof comprises a heavy chain complementarity determining region HCDR, and the HCDR comprises at least one selected from SEQ ID NO: 1-18 or an amino acid sequence having at least 80% homology with the HCDR. The antibody or the antigen-binding fragment thereof can bind to TIM-1 protein with high specificity and affinity, increase the blocking of TIM-1 activity so as to enable the TIM-1 to interact with ligands such as phosphatidylserine, TIM-4 and galectin-9, and enhance immune response.

Description

Technical Field

[0001] The present application belongs to the field of antibodies, and specifically, relates to antibodies or antigen-binding fragments thereof and applications thereof. Background Art

[0002] In recent years, immunotherapy has developed rapidly, providing new avenues for the treatment of a variety of diseases such as cancer, autoimmune diseases and chronic inflammation. TIM-1, also known as HAVCR1, T cell immunoglobulin and mucin domain protein 1 (TIM-1), is a receptor protein that plays a key role in immune regulation. It is mainly expressed on activated T cells, B cells and certain epithelial cells, such as those in the kidney and liver. TIM-1 has attracted much attention due to its role in regulating immune responses and its association with pathological conditions such as chronic inflammation, viral infection, autoimmune diseases and cancer.

[0003] TIM-1 interacts with phosphatidylserine, a molecule exposed on the surface of apoptotic cells, thereby promoting the clearance of these apoptotic cells. This process is essential for maintaining immune homeostasis. Abnormal expression or overactivation of TIM-1 is associated with the progression of immune-related diseases, including chronic kidney disease (CKD), asthma, and even certain cancers, making it a potential therapeutic target. Although existing antibody therapies have shown certain efficacy in treating TIM-1-related diseases, they still have obvious defects due to limitations such as their large molecular size, potential immunogenicity, complex production processes, and target diversity. Summary of the invention

[0004] The present application aims to solve at least one of the above technical problems. To this end, one object of the present application is to provide an antibody targeting TIM-1, which has the advantages of small size, low immunogenicity, simple production process and high specificity.

[0005] Specifically, this application provides the following technical solutions:

[0006] 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, it includes: a heavy chain complementary determining region HCDR, the HCDR comprising at least one selected from SEQ ID NO: 1 to 18 or an amino acid sequence having at least 80% homology thereto. In some examples of the present application, the aforementioned antibody or antigen-binding fragment thereof can bind to the TIM-1 protein with high specificity and affinity, increase the blocking of TIM-1 activity so that it interacts with ligands such as phosphatidylserine, TIM-4 and galectin-9, and enhance the immune response.

[0007] In the second 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. In some examples of the present application, based on the aforementioned nucleic acid molecule, the above-mentioned antibody or antigen-binding fragment thereof can be efficiently expressed in vitro to simplify the production process; in addition, the nucleic acid molecule is convenient for genetic engineering modification to obtain an antibody or antigen-binding fragment thereof that is more suitable for practical applications.

[0008] In the third 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 second 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 can be effectively achieved under the mediation of a regulatory system, thereby achieving a large-scale in vitro preparation of the antibody or its antigen-binding fragment.

[0009] In the fourth aspect of the present application, the present application proposes a method for preparing the antibody or antigen-binding fragment thereof described in the first aspect. According to an embodiment of the present application, the method comprises: introducing the expression vector described in the third aspect of the present application into a cell; culturing the cell under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment thereof. In some examples of the present application, based on the above method, antibodies or antigen-binding fragments thereof that can bind to TIM-1 protein with high specificity and affinity can be efficiently prepared in vitro.

[0010] 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 antibody or antigen-binding fragment thereof described in the first aspect of the present application, the nucleic acid molecule described in the second aspect, or the expression vector described in the third aspect. Wherein, the aforementioned recombinant cell is obtained by transfecting or transforming the expression vector. In some examples of the present application, the recombinant cell can efficiently express the above-mentioned antibody or antigen-binding fragment thereof under suitable conditions, and the obtained antibody or antigen-binding fragment thereof can bind to the TIM-1 protein with high specificity and affinity.

[0011] In the sixth 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 aforementioned engineered protein can effectively target the TIM-1 protein, increase the blocking of TIM-1 activity so that it interacts with ligands such as phosphatidylserine, TIM-4 and galectin-9, and enhance the immune response.

[0012] In the seventh aspect of the present application, the present application proposes a composition. According to an embodiment of the present application, the composition includes: the antibody or antigen-binding fragment thereof described in the first aspect of the present application, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the recombinant protein of the sixth aspect. As mentioned above, the antibody or its antigen-binding fragment can bind to the TIM-1 protein with high specificity and affinity. Therefore, a composition comprising the antibody or its antigen-binding fragment, such as a food composition, a pharmaceutical composition, etc., can also effectively target the TIM-1 protein, increase the blocking of TIM-1 activity so that it interacts with ligands such as phosphatidylserine, TIM-4 and galectin-9, and enhance the immune response.

[0013] In the eighth aspect of the present application, the present application proposes the use of the antibody or antigen binding fragment thereof described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, the recombinant protein of the sixth aspect, or the composition described in the seventh aspect in the preparation of a drug, wherein the drug is used to treat or prevent TIM-1-mediated related diseases. In some examples of the present application, the drug prepared based on the antibody or antigen binding fragment thereof, recombinant protein, nucleic acid molecule, expression vector, recombinant cell or pharmaceutical composition can bind to TIM-1 protein with high specificity and affinity, increase the blocking of TIM-1 activity so that it interacts with ligands such as phosphatidylserine, TIM-4 and galectin-9, and enhance the immune response.

[0014] In the ninth aspect of the present application, the present application proposes a drug. According to an embodiment of the present application, the drug includes: the antibody or antigen binding fragment thereof described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, the recombinant protein in the sixth aspect, or the composition described in the seventh aspect, and the drug is used to treat or prevent TIM-1-mediated related diseases. 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 effectively target TIM-1 protein, increase the blocking of TIM-1 activity so that it interacts with ligands such as phosphatidylserine, TIM-4 and galectin-9, and enhance immune response.

[0015] In the tenth aspect of the present application, the present application proposes the use of the antibody or antigen-binding fragment thereof described in the first aspect in preparing a kit for detecting TIM-1. In some examples of the application, the kit 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 TIM-1 protein.

[0016] In the eleventh aspect of the present application, the present application proposes a kit. According to an embodiment of the present application, the kit comprises the antibody or antigen-binding fragment thereof described in the first aspect. As previously mentioned, the antibody or antigen-binding fragment thereof can bind to the TIM-1 protein with high specificity and affinity, and therefore, the kit comprising the antibody or antigen-binding fragment thereof can be used for efficient and portable detection of TIM-1. In some examples of the present application, the kit can be used for scientific research, such as qualitative or quantitative detection of TIM-1 protein in biological samples, and can also be used to judge the status of an individual, such as after obtaining the TIM-1 level of the individual, judging whether the TIM-1 level is higher or lower than the normal level.

[0017] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 It is a schematic diagram of the chromatography results of TIM-1 protein (antigen) provided in the examples of the present application;

[0020] Figure 2 This is a schematic diagram of the TIM-1 protein purification results provided in the examples of the present application;

[0021] Figure 3 Schematic diagram of the construction results of the phage display library provided in the examples of the present application;

[0022] Figure 4 is a schematic diagram of the bio-panning results provided in the examples of the present application;

[0023] Figure 5 This is a schematic diagram of the results of ELISA screening of positive clones provided in the examples of the present application;

[0024] Figure 6 This is a schematic diagram of the purification results of Nanobodies provided in the Examples of the present application;

[0025] Figure 7 Schematic diagram of the results of the binding affinity test between the Nanobody and TIM-1 provided in the Examples of the present application;

[0026] Figure 8 This is a schematic diagram of the SDS-PAGE gel electrophoresis results of the Nanobody provided in the Examples of the present application;

[0027] Fig. 9 It is a schematic diagram of the affinity test results of the Nanobody and TIM-1 protein provided in the Examples of the present application. DETAILED DESCRIPTION

[0028] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, and should not be understood as limiting the present application.

[0029] In the present application, 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 technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present application, "plurality" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0030] In the present application, the term "antibody" refers to an immunoglobulin, which is composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so its antigenicity is also different. Immunoglobulins can be divided into five categories, namely IgM, IgD, IgG, IgA, and IgE. The same class of immunoglobulins can also be divided into different subclasses according to the amino acid composition, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin light chains are divided into κ chains or λ chains according to different constant regions. In some examples, the antibody heavy chain variable region may further include a heavy chain constant region, which includes human or mouse IgG1, IgG2, IgG3, IgG4 or variants thereof; the antibody light chain variable region may further include a light chain constant region, which includes human or mouse κ, λ chains or variants thereof.

[0031] The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, which is the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, which is the constant region. The variable region includes three hypervariable regions (HVRs) and four relatively conserved framework regions (FRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0032] 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 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.

[0033] 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.

[0034] In the present application, the term "Nanobody" comprises only the heavy chain variable region (VHH), which specifically binds to an antigen via the heavy chain variable region.

[0035] In the present application, the term "antigen binding fragment" refers to a Fab fragment, a Fab' fragment, a F(ab')2 fragment, and a Fv fragment ScFv fragment that binds to human TIM-1, which contains one or more CDR regions of the antibody of the present invention selected from SEQ ID NO: 1 to SEQ ID NO: 18. The Fv fragment contains the variable region of the heavy chain and the variable region of the light chain of the antibody, but has no constant region, and is the smallest antibody fragment with all antigen binding sites. Generally, the Fv antibody also contains a polypeptide linker between the VH and VL domains, and is capable of forming the structure required for antigen binding. Two antibody variable regions can also be connected into a polypeptide chain using different connectors, which is called a single-chain antibody or single-chain Fv.

[0036] In this application, the term "exogenous" refers to a substance produced outside the body of an organism, cell or human body, as the case may be.

[0037] In the present application, the amino acid sequences of the above-listed CDRs are all shown according to the Kabat 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 IMGT 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 its region (such as a variable region) should be understood to cover the complementary determining region defined by any of the above-mentioned known schemes described by the present invention. Although the scope of protection claimed in the present application is based on the sequence shown in the Kabat definition rules, the amino acid sequences corresponding to the definition rules of other CDRs should also be included in the protection scope of the present application.

[0038] In the present application, the term "amino acid" is represented by a single-letter or three-letter code and has the following meaning: 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).

[0039] With respect to nucleotides, the terms "homology", "identity" or "similarity" are used to describe or compare the degree of nucleotide similarity of two or more nucleotide sequences. The percentage of "sequence homology" between a first sequence and a second sequence can be calculated by dividing [the number of nucleotides in the first sequence that are identical to the nucleotides at corresponding positions in the second sequence] minus [the total number of nucleotides in the first sequence] and then multiplying by [100%], where each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence - relative to the first nucleotide sequence - is considered to be a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0, using standard settings. Some other techniques, computer algorithms and arrangements for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0 967 284, EP 1 085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185 and GB 2357768-A.

[0040] For polypeptides, the terms "(substantial) homology", "identity" or "similarity" are used to describe or compare the degree of amino acid similarity between two or more polypeptides or their designated sequences when optimally aligned and compared (where nucleotides are appropriately inserted or deleted). The % homology between two sequences varies with the number of identical positions shared by these sequences when the sequences are optimally aligned (i.e., % homology = number of identical positions / total number of positions × 100), wherein the optimal alignment is determined by taking into account the number of gaps that need to be introduced to achieve optimal alignment of the two sequences and the length of each gap. Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0041] 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.

[0042] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for the expression of transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell that contains the vector.

[0043] In this application, the term "composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with a carrier that constitutes one or more accessory ingredients. Generally, the composition is prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely divided solid carrier, or both.

[0044] In the present application, the term "pharmaceutically acceptable" ingredients are substances that are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic response), ie, substances with a reasonable benefit / risk ratio.

[0045] In the present application, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients incompatible with the compound of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of the present invention.

[0046] In the present application, the term "administration" refers to introducing a predetermined amount of material into a patient by a suitable method. The antibody or antigen binding fragment, recombinant protein, multispecific antibody, conjugate or pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the expected tissue. Various modes of administration are expected, including peritoneal, intravenous, intramuscular, subcutaneous injection, etc., but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous or subcutaneous injection.

[0047] In the present application, the term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, especially humans, including: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit an individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0048] In the present application, 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.

[0049] In the present application, the amino acid sequences involved are shown in Table 1.

[0050] The present application proposes an antibody or antigen-binding fragment thereof targeting TIM-1 protein and a preparation method thereof, a nucleic acid molecule, an expression vector, a recombinant cell, a recombinant protein, a composition, a pharmaceutical use, a drug, a use in preparing a kit, a kit, a disease treatment method, and a disease treatment use, which will be described in detail below.

[0051] Antibodies or antigen-binding fragments thereof

[0052] In one aspect of the present application, the present application proposes an antibody or an antigen-binding fragment thereof, comprising: a heavy chain complementary determining region HCDR, wherein the HCDR comprises at least one selected from SEQ ID NO: 1 to 18 or an amino acid sequence having at least 80% homology thereto. In some examples of the present application, an antibody or an antigen-binding fragment thereof having the aforementioned HCDR amino acid sequence can bind to TIM-1 protein with high affinity and specificity.

[0053] In some examples of the present application, the aforementioned HCDR includes: HCDR1, HCDR2 and HCDR3, wherein the aforementioned HCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 1-6 or having at least 80% homology with SEQ ID NO: 1-6, the aforementioned HCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 7-12 or having at least 80% homology with SEQ ID NO: 7-12, and the aforementioned HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 13-18 or having at least 80% homology with SEQ ID NO: 13-18. Antibodies or antigen-binding fragments thereof based on the aforementioned HCDR sequences have high binding affinity and specificity to the TIM-1 protein.

[0054] In some preferred examples of the present application, the aforementioned HCDR includes: HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 1 to 18, respectively. Experimental verification shows that antibodies or antigen-binding fragments thereof having amino acid sequences as shown in SEQ ID NOs: 1 to 18 have high binding affinity and specificity to TIM-1 protein.

[0055] In some examples of the present application, the aforementioned antibody or antigen-binding fragment thereof further includes: a light chain complementary determining region LCDR, and the aforementioned LCDR includes: LCDR1, LCDR2 and LCDR3.

[0056] It should be noted that one or more amino acid residues in the aforementioned HCDR targeting TIM-1 protein can be replaced by other amino acid residues from the same side chain family, and the retained function of the altered antibody can be tested using the functional assay method described herein.

[0057] It should be noted that one or more amino acid residues in the aforementioned HCDR targeting TIM-1 protein can also be replaced by amino acids having the same or similar amino acid type or physicochemical properties, and the retained function of the altered antibody can be tested using the functional assay method described herein.

[0058] In some examples of the present application, the aforementioned antibody or antigen-binding fragment thereof further includes: a heavy chain framework region HFR, wherein the HFR includes: HFR1, HFR2, HFR3 and HFR4.

[0059] In some examples of the present application, at least a portion of the aforementioned heavy chain framework region HFR is independently derived from at least one of an alpaca antibody, a mouse antibody, a rabbit antibody, a primate antibody, or a mutant thereof. Among them, the aforementioned primate antibody includes a human antibody and a monkey antibody. In some preferred examples of the present application, at least a portion of the aforementioned heavy chain framework region HFR is independently derived from an alpaca antibody.

[0060] In some examples of the present application, the HFR1 comprises an amino acid sequence as shown in SEQ ID NO: 19-24 or having at least 80% homology to SEQ ID NO: 19-24, the HFR2 comprises an amino acid sequence as shown in SEQ ID NO: 25-30 or having at least 80% homology to SEQ ID NO: 25-30, the HFR3 comprises an amino acid sequence as shown in SEQ ID NO: 31-36 or having at least 80% homology to SEQ ID NO: 31-36, and the HFR4 comprises an amino acid sequence as shown in SEQ ID NO: 37-42 or having at least 80% homology to SEQ ID NO: 37-42. The aforementioned framework region sequences help maintain the stability of the antibody structure.

[0061] In some preferred examples of the present application, the HFR includes: HFR1, HFR2, HFR3 and HFR4 having amino acid sequences shown in SEQ ID NOs: 19 to 42, respectively. The antibody structure having the aforementioned framework region sequence is more stable.

[0062] In some examples of the present application, the antibody or antigen-binding fragment thereof further includes: a light chain framework region LFR, and the LFR includes: LFR1, LFR2, LFR3 and LFR4.

[0063] In some examples of the present application, at least a portion of the aforementioned light chain framework region LFR is independently derived from at least one of a mouse antibody, a rabbit antibody, a primate antibody, or a mutant thereof, wherein the aforementioned primate antibody includes a human antibody and a monkey antibody.

[0064] In some examples of the present application, the aforementioned antibody or antigen-binding fragment thereof further includes: at least one of a heavy chain constant region and a light chain constant region.

[0065] In some examples of the present application, at least a portion of at least one of the aforementioned heavy chain constant region and the aforementioned light chain constant region is from at least one of a primate antibody and a mouse antibody or a mutant thereof, wherein the aforementioned primate antibody includes a human antibody and a monkey antibody.

[0066] In some examples of the present application, the heavy chain constant region and the light chain constant region are derived from a murine IgG antibody or a mutant thereof or a human IgG antibody or a mutant thereof, preferably a human IgG antibody or a mutant thereof.

[0067] In some examples of the present application, the light chain constant region comprises a human or murine κ, λ chain or a variant thereof.

[0068] In some examples of the present application, the aforementioned antibodies include polyclonal antibodies, monoclonal antibodies or nanoantibodies; the aforementioned antigen-binding fragment includes a fragment selected from F(ab')2 fragment, Fab' fragment, Fab fragment, Fv fragment, scFv fragment, scFv-Fc fusion protein or minimum recognition unit.

[0069] Based on the advantages of nano antibodies such as small size, stability and solubility. In some preferred examples of the present application, the aforementioned antibody is selected from nano antibodies. Based on the tiny size of nano antibodies, tissue penetration is increased, thereby enhancing the infiltration into the tumor microenvironment.

[0070] In some preferred examples of the present application, the amino acid sequence of the aforementioned nanobody is shown in any one of SEQ ID NOs: 43 to 48. The nanobody having the aforementioned amino acid sequence has the advantages of small size, low immunogenicity, simple production process and high specificity.

[0071] Nucleic acid molecules

[0072] In another aspect of the present application, the present application proposes a nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described above. In some examples of the present application, the antibody or antigen-binding fragment thereof can be efficiently expressed in vitro based on the aforementioned nucleic acid molecule.

[0073] In some examples of the present application, the nucleic acid molecule is DNA.

[0074] It should be noted that, for the nucleic acid molecules mentioned in the specification and claims of this application, those skilled in the art should 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 DNA form or RNA form, and disclosing one of them means that the other is also disclosed.

[0075] Expression vector

[0076] In another aspect of the present application, the present application proposes an expression vector, wherein the expression vector carries the nucleic acid molecule as described above. In some examples of the present application, the antibody or antigen-binding fragment can be efficiently prepared in vitro based on the aforementioned expression vector.

[0077] It should be noted that when the above nucleic acid molecule is connected to a vector, the nucleic acid molecule and the control element on the vector can be directly or indirectly connected, as long as these control elements can control the translation and expression of the nucleic acid molecule. 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 molecule and the control element can be operably connected.

[0078] According to the embodiments of the present application, the vector may refer to a cloning vector or an expression vector, which can be obtained by operably connecting the nucleic acid to a commercially available vector (such as a plasmid or a viral vector). The vector in the present invention is not particularly limited, and commonly used plasmids can be used, such as pSeTag2, PEE14, pMH3, etc.

[0079] In this article, the term "operably linked" refers to connecting the exogenous gene to the vector so that the control elements in the vector, such as the amino acid sequence of transcription control such as and the amino acid sequence of translation control such as, can play their expected functions of regulating the transcription and translation of the exogenous gene. Commonly used vectors can be, for example, viral vectors, plasmids, phages, etc. After the expression vectors according to some specific embodiments of the present invention are introduced into suitable recipient cells, the expression of the nucleic acid molecules described above can be effectively achieved under the mediation of the regulatory system, thereby achieving the in vitro acquisition of a large amount of protein encoded by the nucleic acid molecules.

[0080] In some examples of the present application, the vector is a eukaryotic vector or a prokaryotic vector.

[0081] In some examples of the present application, the vector includes at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector, and an adeno-associated virus vector.

[0082] Method for preparing antibody or antigen-binding fragment thereof

[0083] In another aspect of the present application, the present application proposes a method for preparing the aforementioned antibody or its antigen-binding fragment, comprising: introducing the aforementioned expression vector into cells; culturing the cells under conditions suitable for protein expression and secretion, so as to obtain the aforementioned antibody or its antigen-binding fragment.

[0084] In some examples of the present application, the cell is a eukaryotic cell.

[0085] In some specific examples of the present application, the eukaryotic cell is a mammalian cell. When the cell is a mammalian cell, the expression efficiency of the antibody or antigen-binding fragment thereof is higher.

[0086] It should be noted that the eukaryotic cells do not include animal germ cells, fertilized eggs or embryonic stem cells.

[0087] Recombinant cells

[0088] In another aspect of the present application, the present application proposes a recombinant cell carrying the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule or the aforementioned expression vector. In some examples of the present application, the recombinant cell is obtained by transfecting or transforming the expression vector, and the aforementioned recombinant cell can efficiently express the aforementioned antibody or antigen-binding fragment under appropriate conditions.

[0089] It should be noted that the recombinant cells described in the present 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, Schizosaccharomyces, 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.

[0090] The aforementioned "transformation" or "transfection" refers to the introduction of nucleic acid (e.g., vector) into cells by various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of the present application, and can be used for the expression and / or secretion of the target protein. Suitable host cells can be transformed or transfected with the DNA sequence of the present invention, and can be used for the expression and / or secretion of the target protein. Examples of suitable host cells that can be used for 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.

[0091] It should be noted that the "suitable conditions" described in the specification of this application refer to conditions suitable for the expression of antibodies in this application. It is easy for those skilled in the art to understand that conditions suitable for antibody expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell states, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions for optimal antibody expression according to the specific environment of the laboratory.

[0092] Recombinant protein

[0093] In another aspect of the present application, the present application proposes a recombinant protein, including: the aforementioned antibody or antigen-binding fragment thereof. In some examples of the present application, the recombinant protein can bind to TIM-1 protein with high affinity and high specificity.

[0094] In some examples of the present application, the aforementioned recombinant protein may further include at least one selected from a biologically active protein or a fragment thereof, a biologically active polypeptide or a fragment thereof.

[0095] In some examples 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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 are derived from alpaca, mouse or primate. More preferably, IgG, IgA1, IgA2, IgD, IgE or IgM are derived from alpaca.

[0102] Composition

[0103] In another aspect of the present application, the present application proposes a composition, including: the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell or the aforementioned recombinant protein. In some examples of the present application, the aforementioned composition can be used to prepare related products targeting TIM-1 protein, such as drugs, kits, etc.

[0104] It should be noted that the composition includes combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present application. For example, the components contained in the composition can be administered to the subject as a whole, or separately. When the components contained in the composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0105] Pharmaceutical use

[0106] In another aspect of the present application, the present application proposes the use of the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned recombinant protein or the aforementioned composition in the preparation of a drug, which is used to treat or prevent TIM-1-mediated related diseases.

[0107] In some examples of the present application, the TIM-1-mediated related diseases include: chronic kidney disease and asthma, etc.

[0108] drug

[0109] In another aspect of the present application, the present application proposes a drug, comprising: the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, the aforementioned recombinant protein or the aforementioned composition, wherein the drug is used to treat or prevent TIM-1-mediated related diseases.

[0110] In some examples of the present application, the aforementioned TIM-1-mediated related diseases include: chronic kidney disease and asthma.

[0111] In some examples of the present application, a pharmaceutically acceptable excipient is further included.

[0112] In some examples of the present application, the auxiliary material includes: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0113] In some examples of the present application, the pharmaceutical composition is an injection.

[0114] It should be noted that the pharmaceutical composition includes combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present invention. For example, the components contained in the pharmaceutical composition can be administered to the subject as a whole, or separately. When the components contained in the pharmaceutical composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0115] The drug of the present application contains a safe and effective amount of the active ingredient (antibody) of the present application and a pharmaceutically acceptable adjuvant. Such adjuvants 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 drug 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 drug is preferably manufactured under sterile conditions.

[0116] 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 aforementioned 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 administered daily, or the dose may be reduced proportionally.

[0117] The pharmaceutically acceptable excipients 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.

[0118] Use in the preparation of kits

[0119] In another aspect of the present application, the present application proposes the use of the aforementioned antibody or antigen-binding fragment thereof in preparing a kit for detecting TIM-1.

[0120] As mentioned above, the antibody or antigen-binding fragment thereof of the embodiment of the present application can specifically bind to TIM-1, and therefore, the antibody or antigen-binding fragment thereof can be used to detect TIM-1 protein. Further, it can be used to prepare TIM-1 related kits and used in scientific research, such as qualitatively or quantitatively detecting TIM-1 protein molecules in biological samples. More specifically, it can be used for immunoblotting, immunoprecipitation, etc., which involve using the specific binding properties of TIM-1 and antibodies for detection. These kits may contain any one or more of the following: antagonists, antibodies or drug reference materials of the present application; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents. The antibodies of the present application 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 TIM-1-mediated related diseases (such as chronic kidney disease and asthma, etc.).

[0121] Reagent test kit

[0122] In another aspect of the present application, the present application proposes a kit comprising the antibody or antigen-binding fragment thereof described in the first aspect. The antibody or antigen-binding fragment thereof in the kit provided by the present application can effectively bind to the TIM-1 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 comprising the above substances can bind to TIM-1 with high specificity and can be used to effectively detect TIM-1. The kit can be used for scientific research, such as qualitatively or quantitatively detecting TIM-1 in biological samples, and can also be used to judge the status of an individual, such as after obtaining the TIM-1 level of the individual, judging whether the TIM-1 level is too high or lower than the normal level, and the biological sample can be a cell, tissue, etc.

[0123] In some examples of the present application, the kit is used to detect TIM-1.

[0124] Disease treatment methods

[0125] In another aspect of the present application, the present application provides a method for preventing and / or treating a TIM-1-mediated disease. According to an embodiment of the present application, the method comprises: administering to a subject a pharmaceutically acceptable amount of an antibody or an antigen-binding fragment thereof, an expression vector, a recombinant cell, a recombinant protein, a composition or a drug.

[0126] It should be noted that the terms "subject", "individual" and "patient" are used interchangeably herein and refer to a mammal that is evaluated for treatment and / or treated. In one embodiment, the mammal is a human. The terms "subject", "individual" and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infection, etc. The subject can be a human, but also includes other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0127] The effective amount of the antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, recombinant protein, composition or drug 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 for 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 administered daily, or the dose may be reduced proportionally.

[0128] In some examples of the present application, the TIM-1-mediated related diseases include chronic kidney disease and asthma.

[0129] Disease treatment uses

[0130] In another aspect of the present application, the present application proposes the use of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, recombinant proteins, compositions or drugs in the treatment or prevention of TIM-1-mediated related diseases. In some examples of the present application, administering an effective dose of an antibody or antigen-binding fragment thereof, nucleic acid molecules, expression vectors, recombinant proteins, compositions or drugs to a subject can effectively treat a TIM-1-mediated disease.

[0131] In some examples of the present application, the TIM-1-mediated related diseases include chronic kidney disease and asthma.

[0132] Table 1. Amino acid sequences involved in this application

[0133]

[0134]

[0135]

[0136]

[0137] The scheme of the present application 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 application and should not be considered as limiting the scope of the present application. If no specific technology or conditions are indicated in the embodiments, the technology or conditions described in the literature in this area or the product specification are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0138] Example 1 Expression of TIM-1 protein

[0139] The specific steps for expressing, purifying and eluting TIM-1 protein are as follows:

[0140] Primers were designed to amplify the TIM-1 gene fragment using human cDNA as a template, and the vector was selected from pTT5. Gibson assembly was used to construct the pTT5-TIM-1 plasmid.

[0141] 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-TIM-1 plasmid.

[0142] Table 2 Parameters and dosage of transfection system

[0143] Reagents Dosage Polyethyleneimine 1.2 ml (1 mg / ml) Plasmids 0.4 mg Culture medium (DMEM) 30 ml

[0144] Before transfection, DMEM was preheated in a 37°C water bath. Two 50ml test tubes were filled with 15ml DMEM each, and then the plasmid was added to one test tube and PEI was added to the other test tube. The two test tubes were left to stand at room temperature for 12 minutes. The PEI (in DMEM) was then transferred to the plasmid test tube and incubated at room temperature for 15-16 minutes. HEK293F cells were grown at a density of 2 million / ml in a total volume of 400ml. After the plasmid-PEI incubation, it was transferred dropwise to 400ml cells and then left to stand in an incubator at 37°C for 20 minutes without shaking. After that, the cells were cultured in a shaking incubator at 120rpm and 5% CO2 for four days. The cells were then centrifuged at 4347rcf for 30 minutes, and the supernatant was then passed through 0.45μm and 0.22μm 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.

[0145] After loading, the protein column was eluted on the AKTAprimeplus purification system. 0.2M Tris-HCl (pH 7) was added to the protein collection tube, and the program was run from 0% B solution to 100% B solution to elute the protein. The liquid A used was PBS and the liquid B was 0.1M acetic acid solution to elute the bound protein. The peak of the harvested protein is shown in Figure 1 As shown, it is shown that TIM-1 protein was obtained.

[0146] Example 2 SDS-PAGE purification of TIM-1 protein

[0147] In order to immunize alpacas and produce specific nanobodies, the antigen needs to be as pure as possible. To this end, the purified protein (TIM-1-TEV-Fc) is digested using tobacco etch virus protease (Tobacco Etch Virus protease, also known as TEV protease). TEV protease is added to the collected protein and stored at 4°C overnight. Subsequently, the digested protein is passed through a protein column (Protein A) and a nickel (Ni-NTA) column to completely remove undigested protein, Fc, and TEV enzyme, respectively. Afterwards, the protein A and nickel columns are eluted to verify the removal of the FC tag and TEV protease.

[0148] The results are as follows Figure 2 As shown, the FC tag and TEV protease were successfully removed from the mixture, and purified digested TIM-1 protein was obtained.

[0149] Example 3 Construction of phage display library

[0150] The prepared TIM-1 protein was immunized into adult male alpacas 4 times. The alpaca was immunized with 0.3 mg for the first time and 0.4 mg for the subsequent 3 times. The interval between immunizations 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;

[0151] Using Gibson assembly (GA), 1.5 pmol of pR2 and 6 pmol of amplified VHH were mixed with an appropriate volume of ddH2O 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 TG1 electrocompetent cells (Biosearch 60502-1) and transferred to a 0.2 cm gap ice pre-cooled electrocupant (the cuvette was pre-soaked in 75% ethanol for half an hour, 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 recovery medium at 37°C and 180 rpm for 1 hour. 0.2 μl and 1 μl (prepared dilution) in 2 ml were applied to a 10 cm LB / Amp / 2% Glucose plate and stored in a 37°C incubator for 14 hours. The colonies were then counted to determine the library size. Figure 3 As shown, the library size is approximately 2.4 x 10 6 CFU.

[0152] 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 to scrape the colonies, collected in centrifuge tubes, vortexed and quickly frozen in liquid nitrogen, and then stored at -80°C for future use. 100 μl of library solution and 10 μl of 2TY medium (containing 2% glucose, 100 μl of ampicillin) were added to the 100 mL 2TY medium. 12 pfu helper phage KM13 (purchased from MRC Laboratory of Molecular Biology). 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.

[0153] Take 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.

[0154] Example 4 Biopanning

[0155] First round of bio-panning

[0156] Antigen coating: Prepare antigen (TIM-1) at a concentration of 0.1 mg / ml, add 100 μl antigen to each well (96-well immunoplate), add only PBS as a control, and place overnight at 4°C. Then wash the plate three times with PBST, add 280 μl MPBS (PBS containing 5% skim milk) to each well, and block at room temperature for 2 hours.

[0157] Phage inoculation: Wash the plate three times with PBST (0.1% Tween 20 + PBS). 11个 The library phage (per well) was added to MPBS (5% skim milk + PBS), 100 μl was added to each well, and then the immunoplate was placed on a shaker at 60 rpm and incubated for 1 h.

[0158] Digestion: After washing 10 times with PBST, add 100 μl of 0.5 mg / mL trypsin to each well and digest on a shaker (60 rpm) at room temperature for 1 h to elute the bound phages in the wells.

[0159] Phage infection TG1: Take 10 μl of the phage obtained in the previous step to infect 1 mL of TG1 bacteria in the logarithmic growth phase, incubate at 37°C in a water bath or for 45 minutes, and spread 100 μl and 10 μl of the dilutions on 100 mm LB / 2% G / Amp plates ( Figure 4 ). The remaining phage solution was used to infect 4 mL of TG1 bacteria in the logarithmic growth phase, incubated 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.

[0160] Scrape: Add 4 ml 2TY (20% glycerol) to scrape the colonies on the 150 mm LB plate obtained in the previous step, collect the library into a centrifuge tube, freeze it in liquid nitrogen, and store it at -80°C.

[0161] Second round of bio-panning

[0162] The above library was amplified in the same way as the first round and infected with KM13. The difference between the two rounds was only the amount of phage added and the number of plate washes. The amount of library phage added was 1×10 8 pfu, and the number of plate washes was changed to 20-30 after adding the library phage. Take 10μl of the phage obtained in the second round to infect 1mL of TG1 bacteria in the logarithmic growth phase, and place in a 37℃ water bath or 45 minutes. Take 100μl and 10μl of the dilutions respectively and spread them on 100mm LB / 2%G / Amp plates ( Figure 4 ). After two rounds of bio-panning, phages targeting TIM-1 were significantly enriched.

[0163] Example 5: Screening of positive clones by monoclonal phage ELISA

[0164] Preparation of monoclonal phage:

[0165] Add 100μl 2TY / 2%G / Amp to each well of a 96-well cell culture plate, inoculate a single clone in each well, and culture at 37℃250rpm for 6 hours. Take another 96-well culture plate, add 200μl 2TY / 2%G / Amp to each well, take 10μl of the bacterial solution from the first plate and inoculate it into the corresponding well of the second plate, and culture at 37℃250rpm for 1.5 hours. Prepare KM13 solution: mix 8μl KM13 with 1ml2TY, then add 5μl KM13 dilution to each well, and culture at 37℃ for 45 minutes. After centrifugation, discard the supernatant and invert to dry. Add 200μl 2TY / 0.1%G / Amp / Kan to each well, incubate at 25℃, 250rpm for 16h, centrifuge the next day, take 150μl from each well (used as the primary antibody for antigen during screening) and transfer to a new plate, and store at 4℃.

[0166] Coating Antigen:

[0167] 0.2μg TIM-1 / well (total volume 100μl, dissolved in PBS) was placed in a 96-well culture plate, and a blank without antigen coating was set as a control. Incubate at 4℃ overnight. The next day, wash three times with PBST (0.1% tween20) and block with MPBS (5% skim milk powder) at room temperature for 3h. After washing three times, add the primary antibody (monoclonal phage prepared in the previous step) and incubate at room temperature at 100rpm for 1h. After washing five times, add the secondary antibody HRP-M13 (diluted 1:8000 with 5% milk powder). Add 100μl of secondary antibody to each well and incubate at room temperature for 1h. Wash four times. Then add 100μl of TMB colorimetric substrate to each well in the dark, incubate at room temperature for 10 minutes (until blue appears), add 50μl1MH2SO4 to each well to terminate the reaction, and measure the OD450 value with an enzyme reader. The results are shown in the table. Figure 5 The positive clones were sent for sequencing, and six nanobodies were identified based on the CDR region (especially CDR3).

[0168] Example 6: Purification of Nanobodies

[0169] The nanobody sequence was cloned into the pTT5 backbone plasmid, and then purified using the same method as in Example 2 to purify TIM-1. The protein chromatogram is shown in Figure 6 As shown, it is shown that Nanobodies (1A1, 1B9, 1C6, 1D4, 2E11 and 1H6) were obtained.

[0170] Example 7: Detection of the binding affinity of nanobodies to TIM-1 by ELISA

[0171] 0.2 μg TIM-1 / well (total volume 100 μl, dissolved in PBS) was placed in a 96-well culture plate, and a blank without antigen coating was set as a control. The plate was placed at 4°C overnight. The next day, the plate was washed three times with PBST (0.1% Tween20) and then blocked with MPBS (5% skim milk powder) at room temperature for 2 hours. After washing three times, the primary antibody (the concentration of each nano antibody ranged from 10 3 nM to 10 -4 nM) at room temperature, 80 rpm for 1 h. After washing five times with PBST (0.1%), incubate with secondary antibody (mouse anti-human IgG-Fc, Beijing Sanofi, 10702-MM01T) at a dilution of 1:8000 at room temperature for 1 h, 80 rpm. After washing five times with 0.1% PBST, add 100 μl TMB color substrate to each well in dark conditions, incubate at room temperature for 10 minutes (until blue appears), then add 50 μl 1MH2SO4 / well to terminate the reaction, and measure the OD450 value with an enzyme reader. The results are as follows Figure 7 As shown, it is shown that nanobodies (1A1, 1B9, 1C6, 1D4, 2E11 and 1H6) have a high binding affinity to TIM-1.

[0172] Example 8: Nanobody TIM-1 SDS-PAGE

[0173] SDS-PAGE electrophoresis was performed using a one-step PAGE gel (12.5%) to verify the band size of the TIM-1 nanobody. The loading volume of each nanobody was 7 μl, and the concentration was about 1 mg / ml. The band size was as follows Figure 8 As shown, it indicates that the molecular weights of the samples obtained meet the requirements.

[0174] Example 9: Single Cycle Kinetics and Affinity SPR

[0175] The concentration of each nanobody was 0.1 μg / ml. A maximum of 75-80 μl of each nanobody was injected into the Biacore 8K system and then fixed on the Cytiva CM5 sensor chip. After the sensor spectrum was standardized, the antigen protein with a concentration of 25nM-1.56nM was injected into each channel. The binding parameters were determined using single-cycle kinetics, such as Fig. 9 As shown, the corresponding values ​​are shown in Table 3. The results show that the 6 nanobodies prepared above have high binding affinity to TIM-1 protein.

[0176] Table 3 Nanobody affinity test results

[0177] Nanobody ID Ka(1 / Ms) Kd(1 / s) KD(M) 1A1 <![CDATA[1.10X 10 6 ]]> <![CDATA[1.60X10 -4 ]]> <![CDATA[1.45X 10 -10 ]]> 1B9 <![CDATA[1.40X 10 6 ]]> <![CDATA[1.50X 10 -3 ]]> <![CDATA[1.07X 10 -9 ]]> 1C6 <![CDATA[1.24X 10 6 ]]> <![CDATA[3.78X 10 -4 ]]> <![CDATA[3.05X 10 -10 ]]> 1D4 <![CDATA[1.40X10 6 ]]> <![CDATA[1.67X 10 -3 ]]> <![CDATA[1.19X10 -9 ]]> 2E11 <![CDATA[1.56X10 7 ]]> <![CDATA[4.96X 10 -2 ]]> <![CDATA[3.19x 10 -9 ]]> 1H6 <![CDATA[2.02X 10 6 ]]> <![CDATA[3.63X 10 -3 ]]> <![CDATA[1.80X 10 -9 ]]>

[0178] The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, the technical solution of the present application can be subjected to a variety of simple modifications, including combining various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed in the present application and belong to the protection scope of the present application.

[0179] 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 application. 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.

[0180] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. An antibody or an antigen-binding fragment thereof, characterized in that: include: A heavy chain complementarity determining region HCDR, wherein the HCDR comprises at least one selected from SEQ ID NOs: 1 to 18 or an amino acid sequence having at least 80% homology thereto.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The HCDR comprises: HCDR1, HCDR2 and HCDR3, wherein: The HCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 1-6 or having at least 80% homology to SEQ ID NO: 11-6, The HCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 7-12 or having at least 80% homology to SEQ ID NO: 2, and the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13-18 or having at least 80% homology to SEQ ID NO: 13-18; Preferably, the HCDR comprises: HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NOs: 1 to 18, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: Further including: The light chain complementarity determining region LCDR comprises LCDR1, LCDR2 and LCDR3.

4. The antibody or antigen-binding fragment thereof according to claim 2, characterized in that: The antibody or antigen-binding fragment thereof further comprises: a heavy chain framework region HFR, wherein the HFR comprises: HFR1, HFR2, HFR3 and HFR4; Optionally, at least a portion of the heavy chain framework region HFR is independently derived from at least one of an alpaca antibody, a mouse antibody, a rabbit antibody, a primate antibody or a mutant thereof, preferably an alpaca antibody; Preferably, the HFR1 comprises an amino acid sequence as shown in SEQ ID NO: 19-24 or having at least 80% homology to SEQ ID NO: 19-24, The HFR2 comprises an amino acid sequence as shown in SEQ ID NO: 25-30 or having at least 80% homology to SEQ ID NO: 25-30, The HFR3 comprises an amino acid sequence as shown in SEQ ID NO:31-36 or having at least 80% homology to SEQ ID NO:31-36, and The HFR4 comprises an amino acid sequence as shown in SEQ ID NO:37-42 or having at least 80% homology to SEQ ID NO:37-42; More preferably, the HFR comprises: HFR1, HFR2, HFR3 and HFR4 having amino acid sequences as shown in SEQ ID NOs: 19-42, respectively; Optionally, the antibody or antigen-binding fragment thereof further comprises: a light chain framework region LFR, wherein the LFR comprises: LFR1, LFR2, LFR3 and LFR4; Optionally, at least a portion of the light chain framework region LFR is independently derived from at least one of a murine antibody, a rabbit antibody, a primate antibody or a mutant thereof.

5. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that: The antibody or antigen-binding fragment thereof further comprises: at least one of a heavy chain constant region and a light chain constant region; Optionally, at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a primate antibody and a murine antibody or a mutant thereof.

6. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody comprises a polyclonal antibody, a monoclonal antibody or a nanobody; The antigen binding fragment includes a fragment selected from F(ab')2 fragment, Fab' fragment, Fab fragment, Fv fragment and scFv fragment, scFv-Fc fusion protein or minimum recognition unit; Preferably, the antibody is a nanobody; Preferably, the amino acid sequence of the Nanobody is as shown in any one of SEQ ID NO: 43-48.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; Optionally, the nucleic acid molecule is DNA.

8. An expression vector, characterized in that Carrying the nucleic acid molecule according to claim 7.

9. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, characterized in that: include: Introducing the expression vector of claim 8 into a cell; Cultivating the cells under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment thereof; Optionally, the cell is a eukaryotic cell.

10. A recombinant cell, characterized in that The recombinant cell carries the nucleic acid molecule of claim 7 or the expression vector of claim 8.

11. A recombinant protein, characterized in that include: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; Optionally, further comprising at least one selected from a biologically active protein or a fragment thereof, a biologically active polypeptide or a fragment thereof; Optionally, the biologically active protein or fragment thereof comprises 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.

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

13. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the recombinant cell according to claim 10, the recombinant protein according to claim 11 or the composition according to claim 12 in the preparation of a medicament for treating or preventing TIM-1-mediated related diseases.

14. The use according to claim 13, characterized in that The TIM-1-mediated related diseases include chronic kidney disease and asthma.

15. A drug, characterized in that include: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, the expression vector according to claim 8, the recombinant cell according to claim 10, the recombinant protein according to claim 11 or the composition according to claim 12, wherein the drug is used to treat or prevent TIM-1-mediated related diseases.

16. The drug according to claim 15, characterized in that The TIM-1-mediated related diseases include chronic kidney disease and asthma; Optionally, the drug further comprises a pharmaceutically acceptable excipient; Optionally, the auxiliary materials include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers; Optionally, the drug is an injection.

17. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 in the preparation of a kit for detecting TIM-1.

18. A kit, characterized in that: The kit comprises: The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6.

19. The kit according to claim 18, characterized in that The kit is used for detecting TIM-1.

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