Antibodies or antigen-binding fragments thereof and uses thereof

Abstract

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

Description

Technical Field

[0001] This application belongs to the field of antibodies, specifically relating to antibodies or their antigen-binding fragments and their applications. Background Technology

[0002] In recent years, immunotherapy has developed rapidly, providing new avenues for treating various 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 crucial role in immune regulation. It is primarily expressed on activated T cells, B cells, and certain epithelial cells, such as those in the kidneys and liver. TIM-1 has attracted considerable attention due to its role in regulating immune responses and its association with pathological conditions such as chronic inflammation, viral infections, 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 cells. This process is crucial for maintaining immune homeostasis. Aberrant 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. While existing antibody therapies have shown some efficacy in treating TIM-1-related diseases, significant limitations remain due to their large molecular size, potential immunogenicity, complex manufacturing processes, and target diversity. Summary of the Invention

[0004] This application aims to solve at least one of the aforementioned technical problems. Therefore, one objective of this application is to provide an antibody targeting TIM-1, which has advantages such as small size, low immunogenicity, simple manufacturing process, and high specificity.

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

[0006] In a first aspect of this application, an antibody or its antigen-binding fragment thereof is provided. According to embodiments of this application, it includes a heavy chain complementarity-determining region (HCDR), said HCDR comprising at least one of SEQ ID NO:1-18 or an amino acid sequence having at least 80% homology with it. In some examples of this application, the aforementioned antibody or its antigen-binding fragment can bind to the TIM-1 protein with high specificity and affinity, increasing the blocking of TIM-1 activity to enable it to interact with ligands such as phosphatidylserine, TIM-4, and galactolectin-9, thereby enhancing the immune response.

[0007] In a second aspect of this application, a nucleic acid molecule is proposed. According to embodiments of this application, the nucleic acid molecule encodes the antibody or antigen-binding fragment thereof described in the first aspect of this application. In some examples of this application, based on the aforementioned nucleic acid molecule, the aforementioned antibody or antigen-binding fragment thereof can be efficiently expressed in vitro, simplifying the production process; furthermore, the nucleic acid molecule is easily genetically engineered to obtain antibodies or antigen-binding fragments thereof that are more suitable for practical applications.

[0008] In a third aspect, this application provides an expression vector. According to embodiments of this application, the expression vector carries the nucleic acid molecule described in the second aspect of this application. In some examples of this application, after the expression vector is introduced into suitable recipient cells, the expression of the aforementioned antibody or its antigen-binding fragment can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro preparation of the antibody or its antigen-binding fragment.

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

[0010] In a fifth aspect of this application, a recombinant cell is provided. According to embodiments of this application, the recombinant cell carries the antibody or its antigen-binding fragment described in the first aspect, the nucleic acid molecule described in the second aspect, or the expression vector described in the third aspect. The recombinant cell is obtained by transfecting or transforming the expression vector. In some examples of this application, the recombinant cell can efficiently express the aforementioned antibody or its antigen-binding fragment under suitable conditions, and the obtained antibody or its antigen-binding fragment can bind to the TIM-1 protein with high specificity and affinity.

[0011] In a sixth aspect of this application, a recombinant protein is proposed. According to embodiments of this application, the recombinant protein comprises: the antibody or its antigen-binding fragment described in the first aspect of this application. In some examples of this application, the aforementioned engineered protein can effectively target the TIM-1 protein, increasing the blocking of TIM-1 activity to enable it to interact with ligands such as phosphatidylserine, TIM-4, and galactolectin-9, thereby enhancing the immune response.

[0012] In a seventh aspect of this application, a composition is proposed. According to embodiments of this application, the composition comprises: an antibody or antigen-binding fragment thereof as described in the first aspect of this application, a nucleic acid molecule as described in the second aspect, an expression vector as described in the third aspect, recombinant cells as described in the fifth aspect, or a recombinant protein as described in the sixth aspect. As previously mentioned, the antibody or antigen-binding fragment thereof can bind to the TIM-1 protein with high specificity and affinity. Therefore, compositions containing the antibody or antigen-binding fragment thereof, such as food compositions, pharmaceutical compositions, etc., can also effectively target the TIM-1 protein, increasing the blocking of TIM-1 activity to allow it to interact with ligands such as phosphatidylserine, TIM-4, and galactolectin-9, thereby enhancing the immune response.

[0013] In an eighth aspect of this application, 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 described in the sixth aspect, or the composition described in the seventh aspect in the preparation of a medicament for the treatment or prevention of TIM-1-mediated diseases is disclosed. In some examples of this application, medicaments prepared based on the antibody or antigen-binding fragment thereof, recombinant protein, nucleic acid molecule, expression vector, recombinant cell, or pharmaceutical composition are capable of binding to the TIM-1 protein with high specificity and affinity, increasing the blocking of TIM-1 activity to enable it to interact with ligands such as phosphatidylserine, TIM-4, and galactolectin-9, thereby enhancing the immune response.

[0014] In a ninth aspect of this application, a medicament is proposed. According to embodiments of this application, the medicament comprises: an antibody or antigen-binding fragment thereof as described in the first aspect, a nucleic acid molecule as described in the second aspect, an expression vector as described in the third aspect, recombinant cells as described in the fifth aspect, a recombinant protein as described in the sixth aspect, or a composition as described in the seventh aspect. The medicament is used to treat or prevent TIM-1-mediated diseases. In some examples of this application, medicaments prepared based on antibodies or antigen-binding fragments thereof, recombinant proteins, nucleic acid molecules, expression vectors, recombinant cells, or medicament compositions can effectively target the TIM-1 protein, increasing the blocking of TIM-1 activity to allow it to interact with ligands such as phosphatidylserine, TIM-4, and galactohemagglutinin-9, thereby enhancing the immune response.

[0015] In a tenth aspect of this application, the use of the antibody or antigen-binding fragment thereof described in the first aspect in the preparation of a kit for detecting TIM-1 is proposed. In some examples of the application, kits prepared based on antibodies or antigen-binding fragments thereof, recombinant proteins, nucleic acid molecules, expression vectors, or recombinant cells can be used for portable detection of TIM-1 protein.

[0016] In the eleventh aspect of this application, a kit is provided. According to an embodiment of this application, the kit comprises the antibody or its antigen-binding fragment described in the first aspect. As previously mentioned, the antibody or its antigen-binding fragment can bind to the TIM-1 protein with high specificity and affinity. Therefore, the kit containing the antibody or its antigen-binding fragment can be used for efficient and portable detection of TIM-1. In some examples of this 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 assess an individual's condition, such as determining whether the individual's TIM-1 level is higher or lower than normal after obtaining the individual's TIM-1 level.

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

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the chromatographic results of TIM-1 protein (antigen) provided in the embodiments of this application;

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

[0021] Figure 3 This is a schematic diagram of the phage display library construction results provided in the embodiments of this application;

[0022] Figure 4 This is a schematic diagram of the biological screening results provided in an embodiment of this application;

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

[0024] Figure 6 This is a schematic diagram of the purification results of nanobody provided in the embodiments of this application;

[0025] Figure 7 This is a schematic diagram showing the binding affinity detection results between the nanobody and TIM-1 provided in the embodiments of this application;

[0026] Figure 8 This is a schematic diagram of the SDS-PAGE gel electrophoresis results of the nanobody provided in the embodiments of this application;

[0027] Figure 9 This is a schematic diagram of the affinity detection results between the nanobody and the TIM-1 protein provided in the embodiments of this application. Detailed Implementation

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

[0029] In this application, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0030] In this application, the term "antibody" refers to an immunoglobulin, which is composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the immunoglobulin heavy chain differ, thus their antigenicity also differs. Immunoglobulins can be classified into five classes: IgM, IgD, IgG, IgA, and IgE. The same class of immunoglobulins can also be divided into different subclasses based on their amino acid composition, such as IgG1, IgG2, IgG3, and IgG4. The immunoglobulin light chain is divided into κ chains or λ chains based on the difference in its constant region. In some examples, the variable region of the antibody heavy chain may further include a heavy chain constant region containing human or mouse IgG1, IgG2, IgG3, IgG4, or variants thereof; the variable region of the antibody light chain may further include a light chain constant region containing human or mouse κ, λ chains, or variants thereof.

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

[0032] In this application, the term "monoclonal antibody (abbreviated as MABS)" refers to an antibody that can recognize only one specific antigenic epitope. Common MABS consist of two lighter light chains and two heavier heavy chains, linked by disulfide bonds to form a tetrapeptide molecule. The amino-terminal (N-terminus) amino acid sequence of this heavy or light chain varies considerably and is called the variable region (V-terminus), while the carboxyl-terminus (C-terminus) of the heavy or light chain is relatively stable and changes very little, and is called the constant region (C-terminus). The V-termini of the L-chain and H-chain are called VL and VH, respectively. MABS can also be small molecule antibodies, which mainly include Fab antibodies, Fv antibodies, single-chain antibodies, single-domain antibodies, and the smallest recognition unit.

[0033] In this application, the term "polyclonal antibody (abbreviated as polyclonal antibody)" refers to an antibody that can recognize multiple antigenic epitopes, such as an antibody that can recognize two antigenic epitopes (abbreviated as biclonal antibody), an antibody that can recognize three antigenic epitopes, or an antibody that can recognize four antigenic epitopes. It is interpreted in a broad sense, and the specific structure is not limited, as long as it can recognize multiple antigenic epitopes.

[0034] In this application, the term "nanobody" refers only to the heavy chain variable region (VHH), which binds specifically to the antigen through the heavy chain variable region.

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

[0036] In this application, the term "exogenous" means a substance produced outside of an organism, cell, or human body, depending on the circumstances.

[0037] In this application, the amino acid sequences of the CDRs listed above are all as shown in the Kabat definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as the IMGT rule, the Chothia rule, etc. Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined as described in any of the known schemes above. Although the scope of protection claimed in this application is based on the sequences shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also be included in the scope of protection of this application.

[0038] In this application, the term "amino acid" is represented by a single-letter or three-letter code, with the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Aspartic acid (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: Histidine; I: Ile (isoleucine); L: Leu (leucine); K: Lysine (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Proline (proline); S: Serine (serine); T: Threonine (threonine); W: Tryptophan (tryptophan); Y: Tyrosine (tyrosine); V: Valine (valine).

[0039] For nucleotides, the terms "homology," "identity," or "similarity" are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percentage of "sequence homology" between a first and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. This is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first sequence—is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using standard settings and known computer algorithms for sequence alignment, such as NCBI Blast v2.0. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those 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 peptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more peptides or their designated sequences at optimal alignment and comparison (where appropriate insertions or deletions of nucleotides are made). The percentage of homology between two sequences varies with the number of identical positions shared by these sequences at optimal alignment (i.e., homology % = number of identical positions / total number of positions × 100), where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and identity percentage determination between two sequences can be performed using mathematical algorithms, as described in the non-limiting examples below.

[0041] In this application, without substantially affecting antibody activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain sequences of the antibody or its functional fragments. These are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences described in this application can have at least 80% identity (or homology) with the reference sequences, meaning at least 80%, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity with each reference sequence. The sequence consistency described in this application can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this invention are shown from the N-terminus to the C-terminus.

[0042] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0043] In this application, the term "composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining an active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely pulverized solid carrier, or both.

[0044] In this application, the term "pharmaceuticalally acceptable" means a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0045] In this application, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. The use of any conventional excipients that are incompatible with the compounds of this invention, such as any adverse biological effects they produce or interactions that occur harmfully with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0046] In this application, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, conjugates, or pharmaceutical compositions of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

[0047] In this application, the term "treatment" is used to refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in individuals susceptible to disease but not yet diagnosed with it; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing symptoms associated with the disease. As used herein, "treatment" covers any administration of a drug or compound to an individual to treat, cure, alleviate, improve, reduce, or suppress the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0048] In this application, the terms "effective amount" or "effective dose" refer to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0049] The amino acid sequences involved in this application are shown in Table 1.

[0050] This application discloses an antibody or antigen-binding fragment thereof targeting the TIM-1 protein, a method for its preparation, a nucleic acid molecule, an expression vector, a recombinant cell, a recombinant protein, a composition, a pharmaceutical use, a drug, a use in a preparation kit, a kit, a method for treating a disease, and a use for treating a disease, which will be described in detail below.

[0051] Antibody or its antigen-binding fragment

[0052] In one aspect of this application, an antibody or antigen-binding fragment thereof is provided, comprising a heavy chain complementarity-determining region (HCDR), said HCDR containing an amino acid sequence selected from at least one of SEQ ID NO: 1 to 18 or having at least 80% homology with it. In some examples of this application, antibodies or antigen-binding fragments thereof having the aforementioned HCDR amino acid sequence are capable of binding to the TIM-1 protein with high affinity and specificity.

[0053] In some examples of this application, the aforementioned HCDRs include: HCDR1, HCDR2, and HCDR3, wherein HCDR1 contains amino acid sequences as shown in SEQ ID NO:1-6 or having at least 80% homology with SEQ ID NO:1-6, HCDR2 contains amino acid sequences as shown in SEQ ID NO:7-12 or having at least 80% homology with SEQ ID NO:7-12, and HCDR3 contains amino acid sequences 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 based on the aforementioned HCDR sequences exhibit high binding affinity and specificity to the TIM-1 protein.

[0054] In some preferred embodiments of this application, the aforementioned HCDR includes HCDR1, HCDR2, and HCDR3, respectively, with amino acid sequences as shown in SEQ ID NO:1–18. Experimental verification has shown that antibodies or their antigen-binding fragments having amino acid sequences as shown in SEQ ID NO:1–18 exhibit high binding affinity and specificity to the TIM-1 protein.

[0055] In some examples of this application, the aforementioned antibody or its antigen-binding fragment further includes a light chain complementarity-determining region (LCDR), wherein the LCDR includes LCDR1, LCDR2, and LCDR3.

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

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

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

[0059] In some examples of this application, at least a portion of the aforementioned heavy chain framework region (HFR) is independently derived from at least one of alpaca-derived antibodies, mouse-derived antibodies, rabbit-derived antibodies, primate-derived antibodies, or mutants thereof. The aforementioned primate-derived antibodies include human antibodies and monkey-derived antibodies. In some preferred examples of this application, at least a portion of the aforementioned heavy chain framework region (HFR) is independently derived from alpaca-derived antibodies.

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

[0061] In some preferred embodiments of this application, the HFR includes HFR1, HFR2, HFR3, and HFR4, respectively, as shown in SEQ ID NO:19-42. Antibody structures having the aforementioned framework region sequences are more stable.

[0062] In some examples of this application, the antibody or its antigen-binding fragment further includes a light chain framework region (LFR), wherein the LFR includes LFR1, LFR2, LFR3, and LFR4.

[0063] In some examples of this application, at least a portion of the aforementioned light chain framework region (LFR) is independently derived from at least one of mouse antibodies, rabbit antibodies, primate antibodies, or mutants thereof. The aforementioned primate antibodies include human antibodies and monkey antibodies.

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

[0065] In some examples of this application, at least a portion of at least one of the aforementioned heavy chain constant region and the light chain constant region is derived from at least one of primate-derived antibodies and murine antibodies or mutants thereof. The aforementioned primate-derived antibodies include human antibodies and monkey antibodies.

[0066] In some examples of this application, the heavy chain constant region and the light chain constant region are derived from mouse IgG antibodies or mutants thereof, or human IgG antibodies or mutants thereof, preferably human IgG antibodies or mutants thereof.

[0067] In some examples of this application, the light chain constant region comprises human or mouse κ, λ chains or variants thereof.

[0068] In some examples of this application, the aforementioned antibodies include polyclonal antibodies, monoclonal antibodies, or nanobodies; the aforementioned antigen-binding fragments include those selected from F(ab')2 fragments, Fab' fragments, Fab fragments, Fv fragments, scFv fragments, scFv-Fc fusion proteins, or minimal recognition units.

[0069] Nanobodies offer advantages such as small size, stability, and solubility. In some preferred embodiments of this application, the aforementioned antibodies are selected from nanobodies. Based on the small size of nanobodies, tissue penetration is increased, thereby enhancing infiltration into the tumor microenvironment.

[0070] In some preferred embodiments of this application, the amino acid sequence of the aforementioned nanobody is as shown in any of SEQ ID NO:43-48. Nanobodies with the aforementioned amino acid sequence have advantages such as small size, low immunogenicity, simple manufacturing process, and high specificity.

[0071] Nucleic acid molecules

[0072] In another aspect of this application, a nucleic acid molecule is proposed that encodes an antibody or an antigen-binding fragment thereof as described above. In some examples of this application, antibodies or antigen-binding fragments thereof can be efficiently expressed in vitro based on the aforementioned nucleic acid molecule.

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

[0074] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned in this specification and claims actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is actually disclosed as well. In addition, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0075] expression carrier

[0076] In another aspect of this application, an expression vector is proposed that carries the nucleic acid molecule as described above. In some examples of this application, the aforementioned expression vector enables the efficient in vitro preparation of antibodies or antigen-binding fragments.

[0077] It should be noted that when linking the aforementioned nucleic acid molecules to the vector, the nucleic acid molecules can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. These control elements can originate directly from the vector itself or be exogenous, i.e., not derived from the vector itself. Of course, the connection between the nucleic acid molecules and the control elements must be operably established.

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

[0079] In this document, the term "operably ligated" refers to ligating a foreign gene to a vector such that the control elements within the vector, such as amino acid sequences controlling transcription and amino acid sequences controlling translation, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include viral vectors, plasmids, bacteriophages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into suitable recipient cells, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of the proteins encoded by the nucleic acid molecules.

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

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

[0082] Preparation method of antibody or its antigen-binding fragment

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

[0084] In some examples of this application, the cells are eukaryotic cells.

[0085] In some specific examples of this application, the eukaryotic cells are mammalian cells. When the cells are mammalian cells, the expression efficiency of the antibody or its antigen-binding fragment is higher.

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

[0087] Recombinant cells

[0088] In another aspect of this application, a recombinant cell is provided, carrying the aforementioned antibody or its antigen-binding fragment, the aforementioned nucleic acid molecule, or the aforementioned expression vector. In some examples of this 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 suitable conditions.

[0089] It should be noted that the recombinant cells described in this application are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells can be fungi including Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, Trichoderma, etc.; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described in this application are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0090] The aforementioned “transformation” or “transfection” refers to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this application and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0091] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for antibody expression. Those skilled in the art will readily understand that suitable conditions for antibody expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, 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 optimal conditions for antibody expression based on the specific environment of their laboratory.

[0092] Recombinant protein

[0093] In another aspect of this application, a recombinant protein is proposed, comprising: the aforementioned antibody or its antigen-binding fragment. In some examples of this application, the recombinant protein is capable of binding to the TIM-1 protein with high affinity and high specificity.

[0094] In some examples of this application, the aforementioned recombinant protein may further include at least one selected from bioactive proteins or fragments thereof, bioactive peptides or fragments thereof.

[0095] In some examples of this application, the bioactive protein or fragment thereof includes at least one selected from protein tags, protein toxins or fragments thereof, tumor necrosis factor or fragments thereof, interferon or fragments thereof, biological response regulators or fragments thereof, and Fc fragments.

[0096] The term "protein tag" typically refers to a polypeptide or protein fused together with a target protein (antibody or antigen-binding fragment) for expression. It can be used for the expression, detection, detection, or purification of the target protein. Examples include, but are not limited to, His tags (also known as His-Tag, sequence HHHHHH), Flag tags (also known as Flag-Tag, sequence DYKDDDDK), GST tags (also known as GST-Tag, glutathione thiotransferase tag), SUMO tags, and C-Myc tags.

[0097] The term "toxin" generally refers to substances toxic to the host, including protein toxins and non-protein toxins. Protein toxins include, but are not limited to, abrin, ricin A, Pseudomonas exotoxin, and diphtheria toxin. In this application, the protein toxin is preferably an enzymatic protein toxin.

[0098] The term "tumor necrosis factor" usually refers to substances that can cause hemorrhagic necrosis in various tumors, including but not limited to TNF-α and TNF-β.

[0099] The term "interferon" typically refers to a glycoprotein that can directly kill or inhibit viruses. This includes, but is not limited to, IFN-α, IFN-β, and IFN-γ.

[0100] The term "biological response modulators" generally refers to a class of protein substances that enhance the body's anti-tumor effects directly or indirectly through the immune system. These include, but are not limited to, lymphokines, IL-2, IL-6, IL-10, and GM-CSF.

[0101] The term "Fc fragment" typically refers to the Fc region of IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM, including CH2, CH3 regions and optionally a hinge region. Preferably, the IgG, IgA1, IgA2, IgD, IgE, or IgM are derived from alpaca, mouse, or primate sources. More preferably, the IgG, IgA1, IgA2, IgD, IgE, or IgM are derived from alpaca sources.

[0102] Composition

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

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

[0105] Pharmaceutical uses

[0106] In another aspect of this application, the application proposes the use of 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 in the preparation of a medicament for the treatment or prevention of TIM-1-mediated diseases.

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

[0108] drug

[0109] In another aspect of this application, a medicament is provided, 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, said medicament for treating or preventing TIM-1-mediated diseases.

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

[0111] In some examples of this application, pharmaceutically acceptable excipients are further included.

[0112] In some examples of this application, the excipients include one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers.

[0113] In some examples of this application, the pharmaceutical composition is an injectable formulation.

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

[0115] The drug of this application contains a safe and effective amount of the active ingredient (antibody) of this application and pharmaceutically acceptable excipients. Such excipients include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration; the dosage form of the drug of this application is an injection, oral formulation (tablets, capsules, oral liquid), transdermal formulation, or sustained-release formulation. For example, it is prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The drug is preferably manufactured under aseptic conditions.

[0116] The effective amount of the active ingredient described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0117] Pharmaceutically acceptable excipients described in this application include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

[0118] Uses in the preparation kit

[0119] In another aspect of this application, the use of the aforementioned antibody or its antigen-binding fragment in the preparation of a kit for detecting TIM-1 is proposed.

[0120] As previously described, the antibody or its antigen-binding fragment of this application can specifically bind to TIM-1. Therefore, the antibody or its antigen-binding fragment can be used to detect TIM-1 protein. Furthermore, it can be used to prepare TIM-1-related kits for scientific research, such as qualitative or quantitative detection of TIM-1 protein molecules in biological samples. More specifically, it can be used in kits involving the specific binding properties of TIM-1 and antibodies, such as immunoblotting and immunoprecipitation. These kits may contain any one or more of the following: antagonists, the antibody of this application, or pharmaceutical reference materials; protein purification columns; immunoglobulin affinity purification buffers; and cell assay diluents. The antibody of this application can be used for different types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, it can be used to test for TIM-1-mediated diseases (such as chronic kidney disease and asthma) by detecting the serum or blood of a subject.

[0121] Reagent test kit

[0122] In another aspect of this application, a kit is provided comprising the antibody or its antigen-binding fragment described in the first aspect. The antibody or its antigen-binding fragment in the kit provided by this application can effectively bind to the TIM-1 protein. Furthermore, under suitable conditions, the nucleic acid molecule, expression vector, or recombinant cell can express the antibody or its antigen-binding fragment. Further, the kit containing the above substances can bind to TIM-1 with high specificity and can be used for effective detection of TIM-1. The kit can be used for scientific research, such as qualitative or quantitative detection of TIM-1 in biological samples, and can also be used to assess individual status, such as determining whether an individual's TIM-1 level is too high or too low than normal after obtaining the individual's TIM-1 level. The biological sample can be cells, tissues, etc.

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

[0124] Disease treatment methods

[0125] In another aspect of this application, a method for preventing and / or treating TIM-1-mediated diseases is provided. According to embodiments of this application, the method includes administering to a subject a pharmaceutically acceptable amount of an antibody or its antigen-binding fragment, expression vector, recombinant cells, recombinant protein, composition, or drug.

[0126] It should be noted that the terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one implementation, 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 infections, etc. Subjects can be humans, but also include 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 its antigen-binding fragment, nucleic acid molecule, expression vector, recombinant protein, composition, or drug described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

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

[0129] Uses in disease treatment

[0130] In another aspect of this application, the use of the aforementioned antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, recombinant protein, composition, or drug in the treatment or prevention of TIM-1-mediated diseases is proposed. In some examples of this application, administration of an effective dose of the antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, recombinant protein, composition, or drug to a subject can effectively treat TIM-1-mediated diseases.

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

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

[0133]

[0134]

[0135]

[0136]

[0137] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified 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. The pTT5-TIM-1 plasmid was constructed using Gibson assembly.

[0141] The extracted plasmids were transfected into HEK293F cells using PEI (polyvinylimide) transfection reagent. Parameters and dosages are shown in Table 2. The mammalian expression system was transfected using the pTT5-TIM-1 plasmid.

[0142] Table 2. Parameters and dosage of the transfection system

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

[0144] Before transfection, preheat DMEM in a 37°C water bath. Fill two 50ml tubes with 15ml of DMEM each, then add the plasmid to one tube and the PEI to the other. Let both tubes stand at room temperature for 12 minutes. Then transfer the PEI (in DMEM) to the plasmid tube and incubate at room temperature for 15-16 minutes. HEK293F cells were grown at a density of 2 million / ml, for a total volume of 400ml. After plasmid-PEI incubation, it was dropwise transferred to 400ml of cells and then incubated at 37°C for 20 minutes without shaking. The cells were then cultured for four days in a shaker at 120 rpm and 5% CO2. The cells were then centrifuged at 4347rcf for 30 minutes, and the supernatant was passed through 0.45μm and 0.22μm filters, respectively. The filtered samples were 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 using an AKTA Primeplus purification system. 0.2 M 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. PBS was used as liquid A, and 0.1 M acetic acid solution was used as liquid B to elute bound proteins. The peak chromatogram of the harvested protein is shown below. Figure 1 As shown, this indicates that the TIM-1 protein was obtained.

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

[0147] To immunize alpacas and generate specific nanobodies, the antigen needed to be as pure as possible. Therefore, the purified protein (TIM-1-TEV-Fc) was digested using Tobacco Etch Virus protease (TEV protease). The TEV protease was added to the collected protein and stored overnight at 4°C. Subsequently, the digested protein was passed through a protein A column and a nickel (Ni-NTA) column to completely remove undigested protein, Fc, and TEV protease, respectively. Afterward, the protein A and nickel columns were 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] Adult male alpacas were immunized four times with the prepared TIM-1 protein. The alpacas received 0.3 mg for the first immunization, followed by 0.4 mg for the next three immunizations, with a two-week interval between immunizations. Lymphocytes were collected after the fourth immunization. Total RNA was extracted using the Omega Biotek kit, and genomic DNA was removed using DNaSe. Total RNA was reverse transcribed into cDNA using the PrimeScript™ First-Strand cDNA Synthesis Kit (Takara, 6110A). A specific gene fragment of VHH was amplified using VHH primers, and then VHH was cloned into the phage plasmid pR2 using the Beyotime Seamless Cloning Kit (D7010M) via Gibson assembly. The VHH amplification primers and pR2 plasmid sequence amplification primers are shown in Table 1.

[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 the cloning master mixture (Beyotime). The reaction was carried out in a 50°C water bath for 1 hour. The ligation product was recovered using a PCR product recovery kit and then stored on ice. The GA product was mixed with 500 μl of TG1 electrocompetent cells (Biosearch 60502-1) and transferred to an ice-cold electroporation cuvette with a 0.2 cm gap (the cuvette was pre-soaked in 75% ethanol for half an hour and then placed in a fume hood under sterile conditions). The voltage was set to 2.5 kV / cm and held for 5 ms in a BTX ECM 299 system. The electroporated TG1 cells were then recovered in 2 ml of recovery medium at 37°C and 180 rpm for 1 hour. 0.2 μl and 1 μl of the 2 ml (preparative dilution) were plated on 10 cm LB / Amp / 2% Glucose plates and incubated at 37°C for 14 hours. Then, colony counting was performed to determine the library size. The results are as follows: Figure 3 As shown, the library size is approximately 2.4 x 10^6. 6 CFU.

[0152] Phage library amplification: Remaining TG1 cells were seeded onto 150 mm LB / Amp / 2% G plates and incubated for 13 h. Then, a mixture of 2TY and glycerol (20%) was added, and colonies were scraped and collected in centrifuge tubes. The tubes were vortexed and rapidly frozen in liquid nitrogen, then stored at -80°C for future use. 100 μl of the library culture and 10 μl of glycerol were added to 100 mL of 2TY medium (containing 2% glucose and 100 μl ampicillin). 12 PFU helper phage KM13 (purchased from MRC Molecular Biology Laboratory). The culture was shaken at 37°C until the logarithmic growth phase of the bacteria (OD600, 0.5 to 0.6), and then incubated for 45 minutes for infection.

[0153] Take 50 ml, centrifuge, remove the supernatant, resuspend the precipitate in 100 mL of 2TY, add 0.1% glucose, 100 μl ampicillin and 100 μl kanamycin, and incubate at 180 rpm and 25 °C for about 16 h. Concentrate the phage by PEG precipitation, resuspend in PBS, and store on ice.

[0154] Example 4: Biological panning

[0155] First round of biological selection

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

[0157] Phage inoculation: Wash the plate three times with PBST (0.1% Tween 20 + PBS). Inoculate with 1×10⁻⁶ phages. 11个 Add 100 μl of the bacteriophage library (per well) to MPBS (5% skim milk + PBS) and then incubate the immunoassay plate on a shaker at 60 rpm 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 for 1 h at room temperature on a shaker (60 rpm) to elute the phages bound in the wells.

[0159] Phage infection of 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 for 45 minutes, and then spread 100 μl and 10 μl of the diluted solution onto 100 mm LB / 2% G / Amp plates respectively. Figure 4 The remaining phage solution was used to infect 4 mL of logarithmic growth phase TG1 bacteria, incubated at 37°C for 45 minutes, spread on 150 mm LB / 2% G / Amp plates, and incubated overnight at 37°C.

[0160] Scraping: Add 4 ml of 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, flash freeze in liquid nitrogen, and store at -80°C.

[0161] Second round of biological screening

[0162] The above library was amplified using the same method as in the first round and then 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 1×10⁻⁶. 8 After adding PFU and phage, the number of washes was increased to 20-30. 10 μl of the phage obtained in the second round was used to infect 1 mL of logarithmic-phase TG1 bacteria, and incubated at 37°C for 45 minutes. 100 μl and 10 μl of the diluted solution were then spread onto 100 mm LB / 2% G / Amp plates. Figure 4 After two rounds of biological screening, phages targeting TIM-1 were significantly enriched.

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

[0164] Preparation of monoclonal bacteriophages:

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

[0166] Coating antigen:

[0167] 0.2 μg TIM-1 / well (total 100 μl, dissolved in PBS) was added to a 96-well plate. A blank control (uncoated antigen) was included. The plate was incubated overnight at 4°C. The next day, the plate was washed three times with PBST (0.1% Tween 20) and then blocked with MPBS (5% skim milk powder) at room temperature for 3 hours. After three washes, the primary antibody (the monoclonal phage prepared in the previous step) was added and incubated at 100 rpm for 1 hour at room temperature. After five washes, the secondary antibody HRP-M13 (diluted 1:8000 with 5% milk powder) was added to each well. The plate was incubated at room temperature for 1 hour with 100 μl of secondary antibody per well. The plate was washed four times. Then, under dark conditions, 100 μl of TMB substrate was added to each well and incubated at room temperature for 10 minutes (until a blue color appeared). The reaction was terminated by adding 50 μl of 1M H2SO4 to each well. The OD450 value was measured using a microplate reader. The results are shown in the table below. 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 for purifying TIM-1. The chromatogram of the protein is shown below. Figure 6 As shown, nanobodies (1A1, 1B9, 1C6, 1D4, 2E11 and 1H6) were obtained.

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

[0171] 0.2 μg TIM-1 / well (total 100 μl, dissolved in PBS) was added to a 96-well plate. A blank control (uncoated antigen) was included. The plate was incubated overnight at 4°C. The next day, the plate was washed three times with PBST (0.1% Tween 20) and then blocked with MPBS (5% skim milk powder) at room temperature for 2 hours. After three washes, primary antibody (concentration of each nanobody ranging from 10...) was added. 3 nM to 10 -4 Incubate with nM at room temperature and 80 rpm for 1 h. Wash five times with PBST (0.1%), then incubate with secondary antibody (mouse anti-human IgG-Fc, Sanofi Beijing, 10702-MM01T) at a dilution of 1:8000 at room temperature and 80 rpm for 1 h. Wash five times with 0.1% PBST, then add 100 μl of TMB substrate to each well under dark conditions and incubate at room temperature for 10 minutes (until blue color develops). Finally, add 50 μl of 1M H2SO4 per well to terminate the reaction. Measure the OD450 value using a microplate reader. The results are as follows: Figure 7 As shown, the nanobodies (1A1, 1B9, 1C6, 1D4, 2E11 and 1H6) have a high affinity for TIM-1.

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

[0173] The band size of the TIM-1 nanobodies was verified by SDS-PAGE electrophoresis using a one-step PAGE gel (12.5%). Each nanobodies were loaded with 7 μl, resulting in a concentration of approximately 1 mg / mL. Band sizes were as follows: Figure 8 As shown, the molecular weights of the obtained samples all meet the requirements.

[0174] Example 9: Single-cycle kinetics and affinity SPR

[0175] Each nanobody was concentrated at 0.1 μg / ml. Up to 75-80 μl of each nanobody was injected into the Biacore 8K system, which was then immobilized on a Cytiva CM5 sensor chip. After the sensor pattern was standardized, antigen protein at concentrations of 25 nM-1.56 nM was injected into each channel. Binding parameters, such as..., were determined using single-cycle kinetics. Figure 9 As shown in Table 3, the corresponding values ​​indicate that the six nanobodies prepared above have high binding affinity to the TIM-1 protein.

[0176] Table 3. Results of Nanobody Affinity Detection

[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 this application have been described in detail above; however, this application is not limited thereto. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in this application and are all within the protection scope of this application.

[0179] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0180] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. An antibody or antigen-binding fragment thereof targeting TIM-1, characterized in that, include: Heavy chain complementarity determination regions (HCDRs), HCDR1 as shown in SEQ ID NO: 6, HCDR2 as shown in SEQ ID NO: 12, and HCDR3 as shown in SEQ ID NO:

18.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain framework region (HFR), wherein the HFR includes HFR1, HFR2, HFR3 and HFR4.

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

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The HFR1 contains an amino acid sequence with at least 80% homology to SEQ ID NO: 19-24. The HFR2 contains an amino acid sequence with at least 80% homology to SEQ ID NO: 25-30. The HFR3 contains an amino acid sequence with at least 80% homology to SEQ ID NO: 31-36, and The HFR4 contains an amino acid sequence that is at least 80% homologous to SEQ ID NO: 37-42.

5. The antibody or its antigen-binding fragment according to claim 4, characterized in that, The HFR1 is any amino acid sequence shown in SEQ ID NO:19-24; The HFR2 is any amino acid sequence shown in SEQ ID NO: 25-30; The HFR3 is any amino acid sequence shown in SEQ ID NO: 31-36; The HFR4 is any amino acid sequence shown in SEQ ID NO: 37-42.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1-5, characterized in that, The antibody or its antigen-binding fragment further includes a heavy chain constant region.

7. The antibody or its antigen-binding fragment according to claim 6, characterized in that, At least a portion of the heavy chain constant region is derived from at least one of primate-derived antibodies and murine antibodies or their mutants.

8. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibodies include monoclonal antibodies.

9. The antibody or its antigen-binding fragment according to claim 8, characterized in that, The antibody is a nanobody.

10. The antibody or its antigen-binding fragment according to claim 9, characterized in that, The amino acid sequence of the nanobody is shown in SEQ ID NO:

48.

11. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody or its antigen-binding fragment as described in any one of claims 1 to 10.

12. The nucleic acid molecule according to claim 11, characterized in that, The nucleic acid molecule in question is DNA.

13. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 11 or 12.

14. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, characterized in that, include: The expression vector according to claim 13 is introduced into cells; The cells are cultured under conditions suitable for protein expression and secretion in order to obtain the antibody or its antigen-binding fragment.

15. The method according to claim 14, characterized in that, The cells in question are eukaryotic cells.

16. A recombinant cell, characterized in that, The recombinant cells carry the nucleic acid molecule of claim 11 or 12 or the expression vector of claim 13.

17. A recombinant protein, characterized in that, include: The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10.

18. The recombinant protein according to claim 17, characterized in that, The recombinant protein includes a bioactive protein.

19. The recombinant protein according to claim 17, characterized in that, The bioactive protein includes at least one selected from protein tags, protein toxins, biological response regulators, and Fc fragments.

20. A composition, characterized in that, include: The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10, the nucleic acid molecule carrying the nucleic acid molecule as described in claim 11 or 12, the expression vector as described in claim 13, the recombinant cell as described in claim 16, or the recombinant protein as described in any one of claims 17 to 19.

21. A drug, characterized in that, include: The drug is used to treat or prevent TIM-1-mediated diseases and comprises an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, a nucleic acid molecule carrying the nucleic acid molecule according to claim 11 or 12, an expression vector according to claim 13, a recombinant cell according to claim 16, a recombinant protein according to any one of claims 17 to 19, or a composition according to claim 20.

22. The medicament according to claim 21, characterized in that, The TIM-1-mediated diseases include chronic kidney disease and asthma.

23. The medicament according to claim 22, characterized in that, The drug further includes pharmaceutically acceptable excipients.

24. The medicament according to claim 23, characterized in that, The excipients include one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

25. The medicament according to claim 21, characterized in that, The drug is an injectable form.

26. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 in the preparation of a kit for detecting TIM-1.

27. A reagent kit, characterized in that, The kit contains: The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10.

28. The reagent kit according to claim 27, characterized in that, The kit is used to detect TIM-1.

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