CN116355088A

CN116355088A - Antibodies to type IV collagen alpha 5 chain

Info

Publication number: CN116355088A
Application number: CN202211700701.7A
Authority: CN
Inventors: 李彦磊; 刘培; 田入勇; 史敏龙; 钱永洪
Original assignee: Nanjing Jinsirui Science and Technology Biology Corp
Current assignee: Nanjing Jinsirui Science and Technology Biology Corp
Priority date: 2021-12-28
Filing date: 2022-12-28
Publication date: 2023-06-30

Abstract

Provided herein are antibodies or antigen binding fragments thereof that target the type IV collagen alpha 5 chain. Also provided herein is the use of the antibody or antigen binding fragment thereof in the diagnosis of Alport syndrome.

Description

Antibodies to type IV collagen alpha 5 chain

The present application claims priority to the chinese patent application of application number 202111630329.2, filed 12 months 28 of 2021, which is incorporated herein by reference in its entirety.

Technical Field

The invention belongs to the field of antibodies, and relates to a monoclonal antibody specific to type IV collagen, in particular to a monoclonal antibody specific to an alpha 5 chain thereof.

Background

Alport syndrome (Alport syndrome) is also known as hereditary progressive nephritis, and is characterized clinically by hematuria, proteinuria and progressive renal hypofunction, and some patients can combine extra-renal manifestations such as sensorineural deafness, ocular abnormalities, esophageal leiomyoma and the like. The disease is caused by mutation of the gene COL4An (n=3, 4, 5) encoding the glomerular basement membrane type iv collagen α3 to α5 chain. About 85% of Alport syndrome patients are X-linked dominant inherited Alport syndrome (X-linked Alport syndrome, XL Alport syndrome, OMIM 301050) caused by COL4a 5 or COL4a 5 and COL4A6 gene mutations, with male patients having a relatively high severity and 40 years old kidney failure up to 90% and female patients having a relatively low severity. About 15% of Alport syndrome patients are autosomal inherited Alport syndrome caused by COL4A3 or COL4A4 gene mutation, with autosomal recessive inherited Alport syndrome (autosomal recessive Alport syndrome, AR Alport syndrome, OMIM 203780) being the predominant patient, almost all with renal failure occurring before age 30.

Epidemiological data based on Alport syndrome of demographics have not been available yet, but the inheritance of haematuria occurs clinically. Alport syndrome is more common in kidney disease. Alport syndrome is most common if it is characterized by progression to End Stage Renal Disease (ESRD). Data from the united states sector shows: alport syndrome gene frequency is approximately 1:5000 or 1:10000. In foreign kidney biopsy specimens, alport syndrome accounts for 1.6% -4%, and in several groups of relatively large kidney biopsy pathology studies in China (including 13519 cases, 2315 cases and 1100 cases), 0.729%, 1.2% and 0.818% of patients are diagnosed as Alport syndrome respectively. Different data also show that Alport syndrome accounts for 0.2% -5% of patients with end-stage renal disease, 1.8% -3% of patients with chronic renal failure in children, and 0.6% -2.3% of patients receiving renal transplantation at all ages. However, alport syndrome is more common in patients with persistent hematuria, especially in children, accounting for 11% -27%.

The diagnosis of clinical symptom syndromes is currently mainly carried out by the following methods:

1. according to clinical syndrome manifestation diagnosis, a typical Alport syndrome clinical manifestation is 'hematuria + deafness + renal failure family history', the diagnosis method is widely applied and is mastered by most clinical doctors, and is the simplest and most commonly used method for diagnosing Alport syndrome. However, the diagnosis of Alport syndrome is not exact according to the clinical syndrome manifestation, which genetic type cannot be distinguished, and the patients with Alport syndrome without deafness symptoms are easy to miss diagnosis.

According to the kidney tissue electron microscope diagnosis: typical pathological changes of Alport syndrome patients with kidney tissue electron microscopy are uneven glomerular basement membrane thickness, tearing of dense layer, delamination, basket reticulation and worm erosion like changes. However, there is a limitation in diagnosing Alport syndrome according to an electron microscope, and the early study finds that the renal tissue electron microscope of the patient suffering from Alport syndrome of small age is atypical, often appears as diffuse thinning of glomerular basement membrane, and is easy to misdiagnose as thin basement membrane nephropathy. Second, even if the patient's kidney tissue electron microscope is typically altered in Alport syndrome, it is not possible to distinguish which genotype.

2. Diagnosis based on gene mutation: the X-linked dominant inheritance type Alport syndrome pathogenic gene of Alport syndrome is COL4A 5 gene or COL4A 5 gene and COL4A6 gene co-mutation; the autosomal recessive inheritance type Alport syndrome is caused by homozygous mutation or compound heterozygous mutation of the COL4A3 gene or COL4A4 gene; the autosomal dominant inheritance type Alport syndrome is caused by heterozygous mutation of COL4A3 gene or COL4A4 gene. Gene mutation detection can confirm the diagnosis of Alport syndrome and is important in predicting the risk of disease progression, prenatal genetic diagnosis and genetic counseling for re-fertility, and guiding therapy. However, this method has about 10% of missed tests and cannot judge the progress of the disease.

3. Diagnosis based on kidney tissue or skin tissue type IV collagen alpha chain staining: the kidney tissue or skin tissue type IV collagen alpha 5 chain staining method can not only diagnose Alport syndrome, but also diagnose X-linked dominant inheritance type Alport syndrome in men, women and autosomal recessive inheritance type. The staining of kidney tissue type IV collagen alpha 5 chains of male patients with X-linked dominant type Alport syndrome is negative in glomeruli and Bao Manshi capsule, and the staining of skin basement membrane type IV collagen alpha 5 chains is negative; the kidney tissue type IV collagen alpha 5 chain of female patients is discontinuously positive in glomerulus and Bao Manshi capsule, and the skin basement membrane type IV collagen alpha 5 chain is discontinuously positive. Kidney tissue type IV collagen alpha 5 chain staining of autosomal inheritance Alport syndrome patients was negative in glomeruli and positive in Bao Manshi capsule; skin basement membrane type iv collagen alpha 5 chain staining was positive.

Therefore, the detection of the expression of type IV collagen in kidney and skin basement membrane using type IV collagen α5 chain antibodies is the most desirable diagnostic method for Alport syndrome.

Immunofluorescence examination and immunohistochemical detection of antigens are common disease diagnosis methods, have the advantages of high sensitivity and convenient detection, and the method relies on high-specificity antibodies to recognize antigens. Therefore, there is a need to develop an anti- α5 chain mab with high affinity.

Disclosure of Invention

In one aspect, provided herein is an antibody or antigen binding fragment thereof that targets the type IV collagen a 5 chain, comprising a heavy chain variable region, HCDR1 of which comprises the amino acid sequence NYWMH (SEQ ID NO: 1); HCDR2 includes amino acid sequence RIHPSDSAINYSPKFTG (SEQ ID NO: 2); HCDR3 comprises the amino acid sequence GGYDSY (SEQ ID NO: 3).

In some embodiments, the antibody or antigen binding fragment thereof further comprises a light chain variable region, LCDR1 of which comprises amino acid sequence RASQDISNYLN (SEQ ID NO: 4); LCDR2 comprises the amino acid sequence YTS LHS (SEQ ID NO: 5); LCDR3 includes amino acid sequence QQVNTLPWT (SEQ ID NO: 6).

In some embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 7 or a functional variant having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 7.

In some embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 8 or a functional variant having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 8.

In some embodiments, the heavy chain variable region has the amino acid sequence set forth in SEQ ID NO. 7 and the light chain variable region has the amino acid sequence set forth in SEQ ID NO. 8.

In some embodiments, the antibody is a monoclonal antibody, a single chain antibody, or a single domain antibody.

In some embodiments, the antibody is a murine antibody or a humanized antibody.

In some embodiments, the heavy chain of the antibody comprises the signal peptide sequence shown in SEQ ID NO. 9 and/or the light chain of the antibody comprises the signal peptide sequence shown in SEQ ID NO. 10.

In some embodiments, the heavy and/or light chain of the antibody comprises a purification tag.

In some embodiments, the antigen binding fragment is a Fab, fab ', or F (ab') ₂ 。

In some embodiments, the antibody or antigen binding fragment thereof further comprises an Fc fragment.

In some embodiments, the antibody or antigen binding fragment thereof binds to type IV collagen α5 chain EC as determined by ELISA ₅₀ A value of about 22.13ng/mL; or a KD of less than 4nM, preferably about 3.176 ×10, as measured by biological membrane layer interference (BLI) technique for binding to type IV collagen alpha 5 chain ^-9 M。

In another aspect, provided herein are nucleic acid molecules encoding the antibodies or antigen binding fragments thereof described above.

In another aspect, provided herein are expression vectors comprising the above nucleic acid molecules.

In another aspect, provided herein are host cells comprising the above nucleic acid molecules or expression vectors.

In another aspect, provided herein are host cells expressing the antibodies or antigen binding fragments thereof described above.

In some embodiments, the host cell is a hybridoma cell formed by fusion of a mouse cell with a myeloma cell, preferably a SP2/0, YB2/0, NS0 or P3X63Ag8.653 cell.

In some embodiments, the host cell is a mammalian cell, preferably a CHO cell, a CHO-S cell, a HEK-293 cell, a BHK cell or a PER-C6 cell.

In another aspect, provided herein are immunoconjugates comprising the above antibody or antigen binding fragment thereof and a detectable label.

In some embodiments, the detectable label has enzymatic activity.

In some embodiments, the detectable label is a fluorescent group, a fluorescent protein, or a radioisotope.

In another aspect, provided herein are detection kits comprising an antibody or antigen-binding fragment or immunoconjugate thereof described above.

In some embodiments, the detection kit further comprises an antibody targeting the type IV collagen α1 chain and/or an antibody targeting the type IV collagen α3 chain.

In some embodiments, the detection kit further comprises instructions for how to use the antibody, antigen binding fragment, or immunoconjugate to diagnose Alport syndrome.

The antibodies, antigen binding fragments, nucleic acid molecules, host cells, immunoconjugates, etc., provided herein can be used to detect the presence or amount of type IV collagen α5 chain in a sample, and can also be used to identify Alport syndrome in a subject.

In another aspect, provided herein is a method of detecting the presence or amount of type IV collagen α5 chain in a sample, comprising:

1) Contacting the sample with the above antibody or antigen binding fragment or immunoconjugate thereof, an

2) Determining the presence or absence of type IV collagen α5 chain or amount thereof in the sample by detecting the presence or absence of a binding complex of type IV collagen α5 chain with the antibody or antigen binding fragment thereof or amount thereof.

In another aspect, provided herein is a method of identifying Alport syndrome in a subject, comprising:

1) Contacting a biological sample from said subject with an antibody or antigen-binding fragment or immunoconjugate as described above, an

2) Detecting the presence or amount of a binding complex formed by the type IV collagen α5 chain and the antibody or antigen binding fragment thereof to determine the presence or severity of Alport syndrome in the subject.

In some embodiments, the biological sample is from kidney tissue and/or skin tissue.

In some embodiments, the Alport syndrome is an X-linked dominant inherited Alport syndrome or an autosomal recessive inherited Alport syndrome.

In another aspect, provided herein is the use of an antibody or antigen-binding fragment or immunoconjugate as described above for the preparation of a kit for diagnosing Alport syndrome.

The monoclonal antibody provided by the invention specifically binds to human IV type collagen alpha 5 chain antibody, and can be used for diagnosing Alport syndrome in kidney disease diagnosis. The monoclonal antibody provided by the invention is not combined with alpha 3, alpha 1 or other collagen chains, and can diagnose specific types of Alport syndrome, such as X-linked dominant inheritance type Alport syndrome of men, women and autosomal recessive inheritance type.

Drawings

Fig. 1 shows the results of mouse serum titers after immunization of mice with type IV collagen polypeptides.

FIG. 2 shows the results of the antibody purity test in the examples. Sample order: m: a Marker;1:18G6-2 reduction, purity 99%;2: mouse IgG reduction.

FIG. 3 shows the EC of antibodies in the examples ₅₀ And (5) measuring the result.

Figure 4 shows the results of affinity assays for antibodies in the examples.

Figure 5 shows the results of the antibody thermal acceleration experiments in the examples.

Detailed Description

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The term "or" refers to a single element of a list of selectable elements unless the context clearly indicates otherwise. The term "and/or" means any one, any two, any three, any more, or all of the listed selectable elements.

The term "about" as used herein means a value within + -10% of a given numerical value.

The terms "comprising," "including," "having," and similar referents used herein do not exclude the presence of unrecited elements. These terms also include cases consisting of only the recited elements.

The term "antibody" as used herein is used in its broadest sense to include an immunoglobulin or other type of molecule comprising one or more antigen binding domains that specifically bind an antigen, a protein or polypeptide that exhibits binding specificity for a particular antigen. Specific examples of antibodies can include whole antibodies (e.g., classical four-chain antibody molecules), single chain antibodies, single domain antibodies, multispecific antibodies, and the like. Classical antibody molecules are typically tetramers composed of 2 identical heavy chains and 2 identical light chains interconnected by disulfide bonds. Heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxy terminus according to the conservative differences in amino acid sequences. The variable region is used to recognize and bind antigen and the constant region (e.g., fc fragment) is used to initiate downstream effects such as antibody dependent cell-mediated cytotoxicity (ADCC). Within the variable regions of the heavy and light chains, the amino acid composition and arrangement order of the three partial regions, respectively, have a higher degree of variation, which is a critical position for binding of antibodies to antigens and is therefore also referred to as a Complementarity Determining Region (CDR). The amino acid sequence of the CDRs can be readily determined using art-recognized numbering schemes, e.g., kabat, chothia, IMGT, abM or contacts. In a specific embodiment, the CDRs of the antibodies described herein have been determined according to the IMGT numbering scheme. Herein, the three heavy chain complementarity determining regions are referred to as HCDR1, HCDR2 and HCDR3, respectively, and the three light chain complementarity determining regions are referred to as LCDR1, LCDR2 and LCDR3, respectively.

Antibodies can be divided into five main different types based on the amino acid sequence of the heavy chain constant region of the antibody: igA, igD, igE, igG and IgM. These antibody types can be further classified into subclasses according to the size of the hinge region, the position and molecular weight of the inter-chain disulfide bond, for example, igGl, igG2a, igG2b, igG3, and the like. Light chains can be classified into two types, kappa and lambda, depending on the amino acid composition and arrangement of the antibody light chain constant region. Subunit structures and three-dimensional conformations of different classes of immunoglobulins are known in the art.

"antigen-binding fragment" of an antibody refers to a fragment of an amino acid in an antibody molecule that is involved in antigen-specific binding, e.g., fab 'and F (ab') ₂ Etc. Those skilled in the art know how to obtain such antigen binding fragments. For example, classical antibody molecules can be digested with papain to give Fab fragments, which are digested with pepsin to give F (ab') ₂ Disconnection of F (ab') by treatment with a reducing agent ₂ Disulfide bonds between the hinge regions form Fab' fragments.

Single chain antibodies (single chain fragment variable, scFv) are composed of an antibody heavy chain variable region and a light chain variable region linked by a short peptide into one peptide chain. By correct folding, the variable regions from the heavy and light chains interact through non-covalent bonds to form Fv fragments, so that scfvs can better retain their affinity for antigen.

"Single domain antibody (single domain antibody, sdAb)", or also referred to as "V" _H H antibody ", refers to an antibody molecule having antigen binding capacity, including a heavy chain variable region without a light chain. Structurally, single domain antibodies can also be considered fragments of classical four-chain antibody molecules. Single domain antibodies were first discovered in camelids and subsequently developedThe study personnel found more single domain antibodies with antigen binding capacity by screening with an antibody library (e.g., phage display library). Single domain antibodies have several advantages over common antibody molecules (e.g., classical antibody molecules), including, for example, but not limited to: the molecular weight is smaller, so that the antibody can easily reach tissues or parts which are difficult to reach by common antibody molecules when being used for a human body, or can contact antigen epitopes which are difficult to reach by the common antibody molecules in proteins or polypeptides; more stable and able to withstand, for example, changes in temperature and pH, and the action of denaturants and proteases.

As used herein, "Fc fragment" refers to the handle region of a Y-shaped classical antibody molecule, i.e., the crystallizable fragment (fragment crystallizable, fc), comprising the second and third constant domains (CH 2 and CH3 domains) of the heavy chain. The antibody Fc region can be obtained by hydrolyzing an antibody molecule with a proteolytic enzyme such as papain. In some examples, the Fc region may comprise a hinge, CH2, and CH3. Dimerization between two Fc-containing polypeptides may be mediated when the Fc region comprises a hinge. The Fc fragment may be from IgG, igM, igD, igE or IgA. In some examples, the Fc region is from IgG1, igG2, igG3, or IgG4."Fc fragment" also includes variant Fc fragments derived from natural Fc fragments, which have been altered but still retain their effector function. A "variant Fc fragment" comprises an amino acid sequence having at least one amino acid change in the amino acid sequence of the native Fc fragment. In some examples, the variant Fc-fragment has at least one amino acid substitution compared to the parent Fc-fragment (native Fc-fragment), e.g., about 1 to about 10 amino acids are substituted, and preferably about 1 to about 5 amino acid substitutions, in the parent Fc-fragment. In some examples, the variant Fc-fragment Fc-region has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the parent Fc-fragment. The effector functions of an "Fc fragment" may include binding to Fc receptors, clq binding and Complement Dependent Cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), mediated phagocytosis, and the like.

"murine antibody" refers to an antibody in which the variable and constant regions, if any, are derived from mouse or rat immunoglobulin sequences. The murine antibody can be conveniently obtained by immunizing a mouse or rat with the corresponding antigen and isolating the antibody of interest therefrom. Alternatively, it can be obtained by isolating and culturing cells expressing the antibody of interest (e.g., B cells) after immunization of mice or rats with the corresponding antigen. Alternatively, after a mouse or a rat is immunized with the corresponding antigen, cells expressing the antibody of interest are isolated and cultured, and fused with immortalized cells such as myeloma cells to obtain hybridoma cells, and the hybridoma cells are cultured to obtain the antibody of interest (e.g., monoclonal antibody) in a long term and in a large amount. In some embodiments, the "murine antibody" is a mouse antibody. "humanized antibody" refers to a chimeric antibody obtained by artificial engineering a non-human antibody, i.e., an antibody whose variable and constant regions (if any) are not derived from human immunoglobulin, so as to contain the amino acid sequence of a human antibody. The humanized antibody may comprise constant and/or framework regions of a human antibody. Humanized antibodies can be obtained by genetic engineering means, for example, replacing the constant region of a murine antibody with that of a human antibody and/or replacing the framework region of a murine antibody with that of a human antibody. Such humanized modifications generally do not affect the binding specificity of the original antibody to the corresponding antigen and thus such antigens are also included within the scope of the present invention.

The term "monoclonal antibody" as used herein refers to a homogeneous antibody directed against only one specific epitope. In contrast to polyclonal antibodies, which typically include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope on the antigen. The modifier "monoclonal" refers to a homogeneous characteristic of the antibody and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies of the invention may be produced by hybridoma methods or recombinant DNA methods well known in the art, or may be obtained by screening methods described elsewhere herein.

The term "immunoconjugate" as used herein refers to an antibody or antigen-binding fragment thereof attached to other chemical groups or peptide fragments. For the purposes of the present invention, the chemical groups or peptide fragments may facilitate detection of the antibody or antigen-binding fragment thereof, or the antibody or antigen-binding fragment thereof with a corresponding antigen (e.g., IVDetection of immune complexes (i.e., antigen-antibody complexes) formed by the type collagen α5 chain may thus include a "detectable label". The "detectable label" may be used to indicate the presence or amount of the corresponding antigen in a sample, or to track the location of the corresponding antigen in a cell or in vivo. Examples of detectable labels include various enzymes that can be used in immunoassays, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP); a fluorescent group (e.g., FAM, FITC) or a fluorescent protein (e.g., GFP); radioisotopes (e.g ³ H、 ¹⁴ C、 ³⁵ S). When the detectable label is an enzyme, the presence or amount of the antibody or antigen binding fragment thereof linked to the enzyme can be determined by the enzymatic activity of the enzyme.

The term "purification tag" as used herein refers to an amino acid sequence that facilitates isolation of a polypeptide or protein of interest from a cell culture or supernatant that expresses the polypeptide or protein. Examples include, but are not limited to, his6 tags, flag tags, MBP tags, GST tags, SUMO tags, and the like.

For an antibody or antigen-binding fragment thereof, "targeting," "directed against," or "specifically binding" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) having a higher binding affinity for another molecule (e.g., an antigen) relative to other molecules that are concurrently present in the environment. One molecule may target, target or specifically bind more than one molecule, e.g., a bispecific antibody may have a higher binding affinity for two different antigens relative to the other molecule. The binding affinity of an antibody for an antigen can be measured by measurement of some parameter, such as the EC of the antibody binding to the antigen ₅₀ Values or KD values.

EC ₅₀ (concentration for 50%of maximal effect) means a concentration causing 50% of the maximum effect. When used in an enzyme-linked immunosorbent assay (ELISA) to indicate the binding capacity of an antibody molecule to a corresponding antigen, it may refer to the concentration of the antibody molecule that produces half of the maximum detection signal (e.g., colorimetric or fluorescent intensity). EC (EC) ₅₀ The lower the value, the greater the binding affinity to the antigen.

KD values can also be used to measure the binding affinity of an antibody to its antigen. KD is the equilibrium dissociation constant between an antibody and its antigen, i.e., k _off /k _on Is a ratio of (2). Thus the lower the KD value (lower concentration), the higher the affinity of the antibody.

The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues. Such polymers of amino acid residues may contain natural or unnatural amino acid residues and include, but are not limited to, peptides, oligopeptides, dimers, trimers and multimers of amino acid residues. Both full-length proteins and fragments thereof are encompassed within this definition. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for the purposes of the present invention, a "polypeptide" refers to a protein that includes modifications to the native sequence, such as deletions, additions and substitutions (which are typically conservative in nature), so long as the protein retains the desired activity. These modifications may be purposeful, such as induced via site-directed mutagenesis; or may be occasional, such as through mutation of the host producing the protein or errors due to PCR amplification.

The term "functional variant" as used herein refers to a variant molecule obtained after the introduction of one or more amino acid insertions, deletions or substitutions on the basis of the parent protein molecule (e.g. the native protein molecule), which still retains at least part of the function of the parent protein molecule (in particular the function of interest, such as the binding capacity to the corresponding antigen). For example, a functional variant of an antibody molecule may retain at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of its parent molecule's binding capacity for an antigen, or even have a higher binding capacity than the parent molecule. In some embodiments, a functional variant of an antibody molecule may retain at least 80%, 85%, 90%, 95% or even 100% or more of its parent molecule's binding affinity for antigen. For antibody molecules or antigen binding fragments thereof, functional variants typically include amino acid changes in the variable region framework sequences and/or constant regions, although it is not excluded that one or a few amino acid changes may be made to the CDR region sequences.

The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to a polymer of nucleotides. Such nucleotide polymers may contain natural and/or unnatural nucleotides and include, but are not limited to, DNA, RNA, and PNA. "nucleic acid sequence" refers to a linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

The term "vector" refers to a nucleic acid molecule (e.g., a nucleic acid, plasmid, virus, or the like) that can be engineered to contain a polynucleotide of interest (e.g., a coding sequence for a polypeptide of interest) or that can replicate in a host cell. The carrier may include one or more of the following components: an origin of replication, one or more regulatory sequences (such as promoters and/or enhancers) that regulate the expression of the polynucleotide of interest, and/or one or more selectable marker genes (such as an antibiotic resistance gene and a gene useful in colorimetric assays, e.g., β -galactose). The term "expression vector" refers to a vector used to express a polypeptide of interest in a host cell.

"host cell" refers to a cell that may be or have been a vector or recipient of an isolated polynucleotide. The host cell may be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells such as yeast; a plant cell; insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, CHO cells, HEK-293 cells, BHK cells or PER-C6 cells, as well as derived cells thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO-S and CHO-DS cells. In some embodiments, the anti- α5 single chain antibodies provided herein are secreted by mammalian cells. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells may be isolated cells or cell lines, and also include cells transfected in vivo with the nucleic acid molecules or expression vectors provided herein.

"subject" includes animals, such as mammals, including, but not limited to, primates, rodents, apes, felines, canines, equines, bovines, porcines, sheep, goats, mammalian laboratory animals, mammalian farm animals, mammalian sports animals, and mammalian pets. The subject may be male or female and may be any suitable-aged subject, including infant, young, adult, and geriatric subjects. In some examples, a subject refers to an individual in need of diagnosis or treatment of a disease or disorder. In some examples, the subject undergoing diagnosis or treatment may be a patient suffering from, or at risk of suffering from, a disorder associated with the diagnosis or treatment. In a particular example, the subject is a human, such as a human patient. The term is generally used interchangeably with "patient," "test subject," "treatment subject," and the like.

As used herein, "sample" or "biological sample" refers to a substance from a subject. Such substances include, but are not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, white blood cells, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

When referring to amino acid or nucleotide sequences, the term "sequence identity (sequence identity)" (also referred to as "sequence identity") refers to the amount of degree of identity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Typically, sequence alignment (alignment) is performed and gaps (gaps), if any, introduced prior to calculating the percent identity between two amino acid or nucleotide sequences. If at a certain alignment the amino acid residues or bases in the two sequences are identical, then the two sequences are considered to be identical or matched at that position; amino acid residues or bases in the two sequences differ, and are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence identity. In other algorithms, the number of gaps and/or the gap length are also considered. Common sequence alignment algorithms or software include DANMAN, CLUSTALW, MAFFT, BLAST, MUSCLE, etc. For the purposes of the present invention, the disclosed alignment software BLAST (available from https:// www.ncbi.nlm.nih.gov /), is used to obtain optimal sequence alignments by using default settings and to calculate sequence identity between two amino acid or nucleotide sequences.

Provided herein are monoclonal antibodies, or functional fragments thereof, directed against type IV collagen a 5 chain comprising a heavy chain variable region comprising an amino acid sequence that replaces, inserts or deletes 1, 2 or 3 amino acid residues in an HCDR1, HCDR2, HCDR3 sequence, respectively, and a light chain variable region comprising an amino acid sequence that replaces, inserts or deletes 1, 2 or 3 amino acid residues in an LCDR1, LCDR2 and LCDR3 sequence, respectively:

the amino acid sequence of HCDR1 is NYWMH (SEQ ID NO: 1);

the amino acid sequence of HCDR2 is RIHPSDSAINYSPKFTG (SEQ ID NO: 2);

the amino acid sequence of HCDR3 is GGYDSY (SEQ ID NO: 3);

the amino acid sequence of LCDR1 is RASQDISNYLN (SEQ ID NO: 4);

the amino acid sequence of LCDR2 is YTS LHS (SEQ ID NO: 5);

the amino acid sequence of LCDR3 is QQVNTLPWT (SEQ ID NO: 6).

The present invention provides a monoclonal antibody, or a functional fragment thereof, directed against type IV collagen a 5 chain comprising a heavy chain variable region comprising an amino acid sequence having 80%, 85%, 90% or 95% identity to an HCDR1, HCDR2, HCDR3 sequence, respectively, and a light chain variable region comprising an amino acid sequence having 80%, 85%, 90% or 95% identity to an LCDR1, LCDR2 and LCDR3 sequence, respectively:

The amino acid sequence of HCDR1 is NYWMH (SEQ ID NO: 1);

the amino acid sequence of HCDR2 is RIHPSDSAINYSPKFTG (SEQ ID NO: 2);

the amino acid sequence of HCDR3 is GGYDSY (SEQ ID NO: 3);

the amino acid sequence of LCDR1 is RASQDISNYLN (SEQ ID NO: 4);

the amino acid sequence of LCDR2 is YTS LHS (SEQ ID NO: 5);

the amino acid sequence of LCDR3 is QQVNTLPWT (SEQ ID NO: 6).

In some embodiments, the heavy chain variable region of the anti- α5 chain antibody comprises the amino acid sequences set forth in the following HCDR1, HCDR2, and HCDR3 sequences, and the light chain variable region comprises the amino acid sequences set forth in LCDR1, LCDR2, and LCDR 3:

the amino acid sequence of HCDR1 is NYWMH (SEQ ID NO: 1);

the amino acid sequence of HCDR2 is RIHPSDSAINYSPKFTG (SEQ ID NO: 2);

the amino acid sequence of HCDR3 is GGYDSY (SEQ ID NO: 3);

the amino acid sequence of LCDR1 is RASQDISNYLN (SEQ ID NO: 4);

the amino acid sequence of LCDR2 is YTS LHS (SEQ ID NO: 5);

the amino acid sequence of LCDR3 is QQVNTLPWT (SEQ ID NO: 6).

In some embodiments, the anti-human type IV collagen α5 chain antibodies provided herein further comprise an antibody framework region.

In some embodiments, the heavy chain sequence of the anti-human type IV collagen a 5 chain antibody provided herein comprises the amino acid sequence shown below: QVQLQQPGAELVKPGASVKVSCKASGYTFTNYWMHWVKQRPGQGLEWIGRIHPSDS AINYSPKFTGKATLTVDKSSSTAYMQLTSLTSEDSAVYYCAIGGYDSYWGQGTLVTVSA (SEQ ID NO: 7).

In some embodiments, the light chain sequence of the anti-human type IV collagen a 5 chain antibody provided herein comprises the amino acid sequence shown below: DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSTLHSGVP SRFSGSGSGTDYSLTINSLAQEDIATYFCQQVNTLPWTFGGGTKLAIK (SEQ ID NO: 8).

In some embodiments, the heavy and/or light chain of the anti- α5 chain mab further comprises a signal peptide sequence.

In some embodiments, the heavy chain signal peptide sequence is MRWSCIILFLVATATGVRS (SEQ ID NO: 9).

In some embodiments, the light chain signal peptide sequence is MSSAQFLGLLLLCFQGTRC (SEQ ID NO: 10).

In some embodiments, the invention provides antibodies against human type IV collagen α5 chain, further comprising a constant region sequence.

In some embodiments, the antibodies provided herein against human type IV collagen anti- α5 chains are of the IgG subtype, in some embodiments, of the IgG1 subtype.

In some embodiments, the invention provides an anti-human type IV collagen α5 chain antibody having a dissociation constant KD of less than 4nM between the human type IV collagen α5 chain.

In some embodiments, the monoclonal antibodies provided herein are produced by immunizing a mouse with a polypeptide of type IV collagen as an immunogen, and secreting hybridoma cells formed by fusing spleen cells of the mouse with myeloma cells, including but not limited to SP2/0, YB2/0, NS0, or p3x63ag8.653 cells, by immunizing the mouse.

In some embodiments, the anti- α5 chain antibodies provided herein are produced from ascites in mice.

In some embodiments, the anti- α5 chain antibodies provided herein can be used to prepare Alport syndrome detection kits.

In some embodiments, the detection kit of the present invention may further comprise an anti- α3 antibody and/or an anti- α1 antibody.

In some embodiments, the detection kit of the present invention is a variety of detection kits for detecting type IV collagen within the common general knowledge in the art, such as immunofluorescence detection kit, immunohistochemical detection kit, and the like.

Antibodies or antigen binding fragments thereof targeting type IV collagen alpha 5 chain

Provided herein are antibodies or antigen-binding fragments thereof that specifically bind to type IV collagen alpha 5 chains. The antibody or antigen binding fragment thereof binds to collagen type IV alpha 5 chain with relatively high binding affinity. The ability of an antibody or antigen binding fragment thereof to bind to type IV collagen α5 chain can be measured by an assay method such as an enzyme-linked immunosorbent assay (ELISA), as set forth in the examples below. In addition, it can also be determined by other protein interaction assay methods known in the art, such as Surface Plasmon Resonance (SPR) and Biological Layer Interference (BLI) techniques.

In some embodiments, the antibody or antigen binding fragment thereof that targets the type IV collagen α5 chain is obtained by immunizing a mouse with the type IV collagen α5 chain. In some embodiments, the antibody or antigen-binding fragment thereof that targets the type IV collagen α5 chain is obtained by genetic engineering techniques, such as by introducing into a host cell an expression vector that expresses the antibody or antigen-binding fragment thereof and culturing the host cell.

In some embodiments, the antigen binding fragment is a single chain antibody (scFv). In addition, the inventors expect that, based on the CDR sequences provided herein, the corresponding single domain antibodies (sdabs) can be obtained by one of skill in the art.

Provided herein are heavy and light chain CDR sequences of antibodies or antigen binding fragments thereof that target type IV collagen α5 chain, as shown in SEQ ID NOs 1-3 and 4-6, respectively.

Based on the CDR sequences provided herein, one of skill in the art can construct a variety of polypeptide constructs (including antibodies or antigen binding fragments thereof) having type IV collagen α5 chain binding capacity, including the use of Framework Regions (FR) and/or constant regions from different antibody molecules in combination with these CDR sequences. These framework regions include native framework region sequences from human antibodies or animal (e.g., mouse, rat, sheep, camel, etc.) antibodies. These framework regions may also include variants of the sequence of the framework regions resulting from alterations to the sequence of the natural framework regions. Polypeptide constructs that specifically bind type IV collagen a 5 chains can be readily obtained by combining CDR sequences provided herein with different framework region sequences to form heavy chain variable regions and testing their ability to bind to type IV collagen a 5 chains.

In some embodiments, the heavy chain variable region of an antibody or antigen binding fragment thereof that targets the type IV collagen α5 chain provided herein comprises an amino acid sequence that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID No. 7.

In some embodiments, the light chain variable region of an antibody or antigen binding fragment thereof that targets the type IV collagen α5 chain provided herein comprises an amino acid sequence that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID No. 8.

It will be appreciated by those skilled in the art that, based on the specific sequences provided herein, the corresponding variants of the type IV collagen α5 chain-targeting antibody molecules provided herein can be obtained by substitution, deletion, addition, and validation or screening of a few amino acids for the binding capacity or biological activity of the resulting product to the type IV collagen α5 chain, and such variants are intended to be included within the scope of the present invention. For example, an antibody molecule provided herein may have at least 1 and no more than 10, e.g., no more than 5, 4, 3, 2, or 1 amino acid changes over its full length or variable region sequence or CDR sequence. For example, there may be at least 1 and NO more than 10, such as NO more than 5, 4, 3, 2 or 1 amino acid changes in the heavy chain variable region sequence shown in SEQ ID NO. 7, or there may be at least 1 and NO more than 10, such as NO more than 5, 4, 3, 2 or 1 amino acid changes in the light chain variable region sequence shown in SEQ ID NO. 8, or there may be NO more than 5, 4, 3, 2 or 1 amino acid changes in total in the heavy chain CDRs of SEQ ID NO. 1-3, or there may be NO more than 5, 4, 3, 2 or 1 amino acid changes in total in the light chain CDRs of SEQ ID NO. 4-6, or there may be NO more than 3, 2 or 1 amino acid changes in any one of the HCDRs shown in SEQ ID NO. 1-3, or there may be NO more than 3, 2 or 1 amino acid changes in any one of the LCDRs shown in SEQ ID NO. 4-6, or any combination of these.

It is contemplated that the antibodies or antigen-binding fragments thereof described herein may comprise conservative amino acid substitutions. Conservative amino acid substitutions can generally be described as the substitution of one amino acid residue for another amino acid residue of similar chemical structure, with little or no effect on the function, activity, or other biological properties of the polypeptide. Conservative amino acid substitutions are well known in the art. Conservative substitutions may be, for example, the substitution of one amino acid in the following groups (a) - (e) with another amino acid within the same group: (a) small aliphatic nonpolar or low polar residues: ala, ser, thr, pro and Gly; (b) Polar negatively charged residues and (uncharged) amides: asp, asn, glu and Gln; (c) a polar positively charged residue: his, arg and Lys; (d) large aliphatic nonpolar residues: met, leu, ile, val and Cys; and (e) an aromatic residue: phe, tyr and Trp.

Immunoconjugates

Provided herein are conjugates comprising at least one antibody or antigen-binding fragment thereof provided herein that specifically binds to type IV collagen alpha 5 chain and at least one other functional moiety.

The other moiety may be a chemical group, for example a therapeutic agent, such as a cytotoxic agent, or may be a tracer. In some examples, the functional moiety may be a targeting moiety, a small molecule drug (e.g., a non-polypeptide drug of less than 500 Da), a toxin, a cytostatic agent, a cytotoxic agent, an immunosuppressive agent, a radioactive agent suitable for diagnostic purposes, a radioactive metal ion for therapeutic purposes, or the like.

In some embodiments, the immunoconjugate is an Antibody Drug Conjugate (ADC) comprising one or more antibodies or antigen-binding fragments thereof provided herein and a therapeutic agent, which is cytotoxic, inhibits cell growth, or provides some therapeutic benefit. In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (i.e., a radioactive conjugate). In some examples, the antibody drug conjugates provided herein allow for targeted delivery of drug moieties to tumors. In some cases, this may cause targeted killing of tumor cells.

In some examples, therapeutic agents include, for example, daunomycin (daunomycin), doxorubicin (doxorubicin), methotrexate (methotrexa), vindesine (vindeline), maytansinoids (maytansinoid), and the like. In some examples, the therapeutic agent has intracellular activity. In some examples, immunoconjugates that bind the type IV collagen α5 chain are internalized and the therapeutic agent has the activity of blocking cellular protein synthesis, blocking nucleic acid synthesis, and causing cell growth arrest or death.

In some embodiments, the immunoconjugate comprises one or more antibodies or antigen-binding fragments thereof provided herein and a tracer. The immunoconjugates can be used for research or diagnostic purposes, e.g., for in vivo detection of cancer. The tracer may directly or indirectly generate a detectable signal. For example, the tracer may be a radioisotope, e.g ³ H、 ¹⁴ C、 ³² P、 ³⁵ S、 ¹²³ I, a step of I; fluorescent (fluorophore) or chemiluminescent (chromophore) compounds, such as fluorescent isothiocyanates, rhodamines, or luciferins; a developer; or metal ions. In some embodiments, the tracer is a radioactive atom for scintillation photography studies, e.g ⁹⁹ Tc or ¹²³ I, or spin labeling for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), e.g ⁸⁹ Zr、 ¹²³ I、 ¹⁹ F、 ¹³ C、 ¹⁵ N and ¹⁷ O。 ⁸⁹ zr can chelate with a variety of metal chelators and bind to antibodies, for example for PET imaging.

The attachment of the antibodies or antigen binding fragments thereof provided herein to other functional moieties may be covalent or non-covalent. Examples of non-covalent attachment may include via a Biotin-Avidin System (Biotin-Avidin System). Examples of covalent linkages may include various chemical linkers, including peptide linkers, cleavable linkers, or non-cleavable linkers. Exemplary linker components include 6-Maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4- (2-pyridylthio) pentanoate (SPP), N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC), and N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB).

In some embodiments, the linker may comprise an amino acid residue. Exemplary amino acid linker components include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Exemplary dipeptides include: valine-citrulline (vc or val-cit), alanine-phenylalanine (afa-phe). Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit) and glycine-glycine (gly-gly-gly). Amino acid residues comprising an amino acid linker component include naturally occurring residues as well as non-naturally occurring amino acid analogs, such as citrulline. The amino acid linker component may be designed and optimized in terms of its selectivity for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C and D, cytoplasmic proteases.

The antibodies or antigen binding fragments thereof provided herein can be made with a variety of bifunctional protein coupling reagents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as the dibutylimido matrix), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene).

In some embodiments, provided herein are multi-specific antibody molecules, e.g., bispecific antibody molecules, comprising at least one binding domain that binds to type IV collagen a 5 chain (e.g., an antibody or antigen binding fragment of the invention, or a heavy chain variable region or a light chain variable region) and one or more additional binding domains. The one or more additional binding domains may bind to a second antigen or protein other than the type IV collagen α5 chain.

In some embodiments, the multispecific antibody, or antigen-binding fragment thereof, further comprises an Fc fragment. The presence of the Fc fragment facilitates multimerization of the binding domain and may provide a related effector function.

Detection or diagnostic kit comprising antibodies or antigen binding fragments thereof targeting type IV collagen alpha 5 chain

The antibodies or antigen-binding fragments thereof provided herein that target type IV collagen α5 chains, and other forms of molecules (e.g., immunoconjugates or bispecific antibody molecules) comprising the antibodies or antigen-binding fragments thereof, can specifically bind to type IV collagen α5 chains in a sample. The amount of type IV collagen α5 chain (or the presence or absence) in a sample can be conveniently determined by detecting the amount of a complex of type IV collagen α5 chain-antibody or antigen binding fragment thereof with its antigen (formed by type IV collagen α5 chain), or detecting the amount of antibody or antigen binding fragment thereof in the complex.

As described above, for this purpose, the antibodies or antigen binding fragments thereof provided herein may be conjugated to various detection tags for detection by various means, including, but not limited to, bioluminescence, fluorescence, radiolabel, yield of enzymatic reaction products, and the like. Comparing the amount of type IV collagen α5 chain detected in the sample with the amount of normal type IV collagen α5 chain in a normal population can be used to determine the disease condition or severity of the subject providing the sample. The change of the alpha 5 chain content of the IV type collagen can be repeatedly detected with time in the treatment process of the subjects, and can be used for determining whether a treatment means is effective or not, so that a basis is provided for adjusting a treatment scheme.

The antibodies or antigen-binding fragments thereof provided herein, and other forms of molecules (e.g., immunoconjugates or bispecific antibody molecules) comprising the antibodies or antigen-binding fragments thereof, can be placed in a container to form a detection or diagnostic kit. These containers may be in the form of boxes, ampoules, vials, tubes, bags or suitable containers known in the art. These containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for preserving protein preservation. If desired, instructions for use are provided with the container. Instructions may generally include information about how to use the antibody or antigen-binding fragment thereof for an immunoassay, e.g., may include instructions for how to quantify the sample type IV collagen α5 chain; a description of how Alport syndrome is diagnosed based on the detection results. The instructions may be printed directly on the container (if present) or as a label affixed to the container or as a separate paper, book, card or folded print provided in or with the container.

The technical scheme of the invention is further described in detail below through examples and with reference to the accompanying drawings. Unless otherwise indicated, the methods and materials of the examples described below are all conventional products available commercially. Those skilled in the art will appreciate that the methods and materials described below are exemplary only and should not be construed as limiting the scope of the invention.

The embodiments of the present invention are not limited to the examples described above, and those skilled in the art can make various changes and modifications in form and detail without departing from the spirit and scope of the present invention, which are considered to fall within the scope of the present invention.

Example 1: immunization of animals

After emulsification of 25. Mu.g of human type IV collagen. Alpha.5 chain polypeptide (GenScript synthesis) conjugated with hemocyanin (KLH) in PBS and Freund's adjuvant (antigen: adjuvant=1:1) with stirring, subcutaneous immunization was performed at 200. Mu.l/3 Balb/C and 3C 57BL/6 mice for 6-8 weeks, followed by repeated immunization every 2 weeks for boosting, for a total of 3 times. The second immunization was followed by a week of first blood collection, the third immunization was followed by a week of second blood collection, and ELISA was performed on serum. One Balb/C and one C57BL/6 with the highest serum titers were selected for the last immunization at the third week after the third immunization, and fusion was performed 4 days after the last immunization.

Example 2: preparation of anti-human IV type collagen alpha 5 chain mouse monoclonal antibody

1) Hybridoma fusion and screening

The spleens of the mice of example 1 were extracted and homogenized to produce a single cell suspension, and the single cell suspensions of the two mice were mixed. A single cell suspension of myeloma cells (SP 2/0) was also prepared. Spleen cells were removed using an electrofusion apparatus: fusion was performed with SP 2/0=2:1. The fused cells were resuspended in 300ml containingHybridoma cell selection agents thymine pyrimidine, hypoxanthine and aminopterin in DMEM/10% FBS medium, and using a pipette in 200 u l volume into 15X 96 well plate. The plates were incubated at 37℃with 5% CO ₂ Is cultured. After 7 days of culture, hybridomas secreting the polypeptide directed against α5 were initially screened using an indirect ELISA as described below.

2) Indirect ELISA detection method

An indirect ELISA was used to assess the binding capacity of antibodies to polypeptides in the supernatant. ELISA plates were coated with 1. Mu.g/ml of type IV collagen. Alpha.5 chain polypeptide in 100. Mu.l/well PBS at 4℃overnight. Plates were washed with PBS-T (0.05% Tween) and blocked with 200. Mu.l/well of 1% BSA in PBST for 0.5 hours at 37 ℃. The blocking solution was then discarded, 100 μl of hybridoma cell culture supernatant was added to each well, and then incubated for 1 hour at room temperature. Plates were washed three times with PBST and incubated with 100 μl/well of horseradish peroxidase conjugated goat anti-mouse IgG (GenScript) ℃ for 0.5 hours. Plates were washed five times with PBST, then TMB chromogenic solution (GenScript) was added and incubated in the dark for 15 minutes at room temperature. The reaction was stopped by adding 50. Mu.l of 1M HCl stop solution (Sigma). Reading the plate at 450nm by using an enzyme-labeled instrument to obtain positive clone 22 holes and OD ₄₅₀ Greater than 0.5.

3) Mouse hybridoma subcloning

All clones above were prepared for hybridoma cell suspensions to be cloned, and peritoneal macrophages (as feeder cells) were diluted to 5X 10 per ml with 20% serum in HT medium (20% FBS+2% HT+2ml IL-6+78% DMEM) ⁴ Cells were pre-plated at 0.1ml per well, the cells to be subcloned were diluted with DMEM to 5, 30 or 100 cells per ml, mixed at 0.1ml per well and plated into pre-plated feeder cell plates at a total volume of 200 μl per well, hybridoma cell numbers per well of 0.5, 3 and 10, 32 wells per dilution; 37 ℃ and 7.5% CO ₂ Wetting and culturing for 7-10 days, and detecting antibodies after macroscopic cloning appears; and (3) observing under an inverted microscope, marking a positive hole in which only a single clone grows, and repeating the steps for 2-3 times again by cloning until the positive hole rate is 100%. Antibody detection, cell expansion culture and freezingAnd (5) storing.

Example 3: variable region sequencing of mouse monoclonal antibodies and antibody production

1) The cell clone obtained in example 2 was used to extract RNA using the RNeasy Plus Micro Kit kit (QIAGEN, cat: 74034). RNA was then reverse transcribed into cDNA, with the addition of random and Oligo dT primers, using SMART script reverse transcriptase (TaKaRa, cat# 639536). PCR amplification (Genscript) to obtain antibody heavy chain and light chain variable region sequences, inserting pcDNA3.1 expression plasmid, transforming into DH5 alpha competent cells, shaking for 3-4 hours, directly sucking 100 mu L of bacterial liquid into a plate, covering the plate, and taking the plate onto a desk oscillator for plating for 3min. The coated plate is taken back to the ultra clean bench, and then the plate is placed in a 37 ℃ water-proof constant temperature incubator for overnight culture for 12-16h. Single colony shaking was picked and sequenced. Clone 18G6-2 was finally obtained. The antibody amino acid sequence is as follows, the bolded sequence is the signal peptide, CDR sequences (determined according to IMGT numbering scheme) are underlined:

Heavy chain variable region (containing signal peptide) (134 aa)

Light chain variable region (containing signal peptide) (126 aa)

/>

Antibodies can be produced in a variety of ways, such as in CHO-S cells, in mouse ascites, or in hybridomas. The following hybridoma production mode is described as an example:

2) Hybridoma production, hybridoma cells were cultured in shake flasks at 37℃for 6 days, and supernatants were harvested for antibody purification. The column was re-equilibrated with a buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0). The harvested cell culture supernatant is then diluted 1:1 with 2 Xthe above buffer and sterilized by filtration. The filtered supernatant and protein A column were incubated for 2 hours at room temperature, after washing the column with 1 Xthe above buffer, igG was eluted using sterile 0.1M sodium citrate (pH 3.5), and the eluate was collected and neutralized with one-ninth volume of sterile 1M Tris-HCl (pH 9.0). Under sterile conditions, the product buffer was exchanged for PBS (ph 7.4) to remove any elution buffer and concentrate the sample. After concentration, the antibodies were quantified by OD280nm using an extinction coefficient Ec of 1.43 (0.1%).

3) Purified antibodies were analyzed by SDS-PAGE using 10% pre-gel (GenScript) using a BioRad electrophoresis system. The gel was stained with Estain2.0 (GenScript) and molecular size was estimated to be about 150KD and purity to be about 99% by comparing the stained bands with protein Ladder (GenScript), as shown in FIG. 2.

Example 4: determination of monoclonal antibody subtype

The capture antibody (goat anti-mouse Ig) in the kit (SBA Clonotyping System-HRP 1 kit) was diluted to the target concentration with PBS (phosphate buffer, PH 7.4), 100 μl/well, coated overnight at 4 degrees; the coating solution was thrown out, 150. Mu.l of PBST wash (PBS+Tween 20) was added to each well to wash the plate once, and blocking solution (1% BSA,1% sucrose, 0.5% Tween 20) was added at 37℃for 2 hours; the hybridoma was used to enumerate 9 wells, 100 μl wells, and incubated at 37deg.C; removing hybridoma supernatant, adding 250 μl of washing solution into each well for three times, adding corresponding 100 μl of HRP-labeled goat anti-mouse kappa, lambda, ig, igG1, igG2a, igG2b and IgG3 enzyme-labeled secondary antibodies into the well, and incubating at 37deg.C for 30min; removing enzyme-labeled secondary antibody, washing the plate for 4-5 times in 250 mu l of PBST each well, adding 100 mu l of TMB color development liquid (GeneScript) into the well, and developing at 25 ℃ in an incubator; mu.l of stop solution was added to each well. Read immediately on a microplate reader at 450 nm. The antibody was determined to be of the IgG1.K subtype.

₅₀ Example 5: determination of monoclonal antibodies EC

Indirect ELISA was used to assess the binding capacity of purified antibodies to polypeptides. ELISA plates were coated with 0.5. Mu.g/ml of polypeptide in 100. Mu.l/well PBS at 4℃overnight. Plates were washed with PBS-T (0.05% Tween) and blocked with 250. Mu.l/well of 1% BSA in PBST for 2 hours at 37 ℃. The blocking solution was then discarded and 1. Mu.g/ml of hybridoma production purification was added to the first well Antibodies were 100 μl and diluted in 3-fold gradients for a total of 11 test concentration gradients and 1 zero well. Then incubated at room temperature for 1 hour. Plates were washed three times with PBST and incubated with 100 μl/well horseradish peroxidase conjugated goat anti-mouse IgG (Fc specific) (GenScript) for 0.5 hours at 37 ℃. Plates were washed 4 times with PBST, then TMB chromogenic solution (GenScript) was added and incubated in the dark for 15 minutes at room temperature. The reaction was quenched by the addition of 50. Mu.l of 1M HCl stop solution. Plates were read at 450nm using an microplate reader. EC as in FIG. 3, 18G6-2 ₅₀ 22.13ng/ml;

example 6: monoclonal antibody affinity assay

The affinity of the antibodies was detected using a Fortebio Octet detection instrument and Streptavidin (SA) biosensors were equilibrated with 200 μl PBST. The biotin-labeled antigen polypeptide (Genscript synthesis) was diluted to a concentration of 10. Mu.g/ml in a volume of 200. Mu.l, and the antigen was immobilized on the SA biosensor. The SA biosensor after curing was equilibrated with 200 μl PBS. Antibody 18G6-2 was diluted at 7 concentration points (nM): 100. 50, 25, 12.5, 6.25, 3.13, 1.56, 200 μl in volume, bind to immobilized antigen on the SA biosensor. Antigen and antibody were dissociated in 200 μl PBST. The dissociation equilibrium constant KD (M) of the antibody was calculated as 3.176E-09 from the experimental results, as shown in FIG. 4.

Example 7: antibody thermal acceleration experiments

The thermal acceleration stabilization experiment is used for determining the preservation time of the antibody at a certain temperature, and determining the validity period of the antibody according to an Arrhenius equation. Carrying out heat acceleration for one day in a constant temperature incubator at 37 ℃ which is equivalent to preserving for 1.5 months at 4 ℃; the 18G6-2 purified antibody was dispensed at 20. Mu.l/tube and then stored at-20℃for further use.

Taking out samples from the first 21 days, the first 17 days, the first 14 days, the first 11 days, the first 7 days and the first 3 days, respectively placing the samples at the temperature of 4 ℃ and the temperature of 37 ℃ for preservation, finally taking out the antibodies to be measured which are stored under different conditions (including always at the temperature of-20 ℃) on the same day of detection, uniformly measuring the binding capacity of the purified antibodies after heat acceleration on the polypeptides by using an indirect ELISA method, and evaluating the stability of the antibodies. ELISA plates were coated with 1. Mu.g/ml of polypeptide in 100. Mu.l/well PBS at 4℃overnight. Plates were washed 1 time with PBS-T (0.05% Tween) and blocked with 250. Mu.l/well of 1% BSA in PBST for 2 hours at 37 ℃. The blocking solution was then discarded, and 100. Mu.l of purified antibody was added to the first well at 30. Mu.g/ml, and diluted according to a 3-fold gradient for a total of 11 test concentration gradients and 1 zero well. Then incubated at 37℃for 1 hour. Plates were washed three times with PBST and incubated with 100. Mu.l/well goat anti-mouse IgG antibody (Jackson, 115-035-071) for 0.5 hours at 37 ℃. Plates were washed 4 times with PBST, then TMB chromogenic solution (GenScript) was added and incubated in the dark for 15 minutes at 25 ℃. The reaction was quenched by the addition of 50. Mu.l of 1M HCl stop solution. Plates were read at 450nm using an microplate reader. According to Arrhenius equation, the antibody can be stably stored at 37deg.C for 21 days, and according to Arrhenius equation conversion, the antibody can be stably stored at 4deg.C for 31.5 months.

Claims

1. An antibody or antigen-binding fragment thereof that targets the type IV collagen a 5 chain, comprising a heavy chain variable region, HCDR1 of which comprises the amino acid sequence NYWMH (SEQ ID NO: 1); HCDR2 includes amino acid sequence RIHPSDSAINYSPKFTG (SEQ ID NO: 2); HCDR3 comprises the amino acid sequence GGYDSY (SEQ ID NO: 3).

2. The antibody or antigen-binding fragment thereof of claim 1, further comprising a light chain variable region, LCDR1 of which comprises amino acid sequence RASQDISNYLN (SEQ ID NO: 4); LCDR2 comprises the amino acid sequence YTS LHS (SEQ ID NO: 5); LCDR3 includes amino acid sequence QQVNTLPWT (SEQ ID NO: 6).

3. The antibody or antigen binding fragment thereof of claim 1 or 2, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID No. 7 or a functional variant having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID No. 7.

4. The antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID No. 8 or a sequence complementary to SEQ ID NO:8, a functional variant having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in seq id no.

5. The antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein the heavy chain variable region has the amino acid sequence set forth in SEQ ID No. 7 and the light chain variable region has the amino acid sequence set forth in SEQ ID No. 8.

6. The antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein the antibody is a monoclonal antibody, a single chain antibody, or a single domain antibody.

7. The antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the antibody is a murine antibody or a humanized antibody.

8. The antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the heavy chain of the antibody comprises the signal peptide sequence shown in SEQ ID No. 9 and/or the light chain of the antibody comprises the signal peptide sequence shown in SEQ ID No. 10; or the heavy and/or light chain of the antibody comprises a purification tag.

9. The antibody or antigen-binding fragment thereof of any one of claims 1-8, wherein the antigen-binding fragment is Fab, fab ', or F (ab') ₂ 。

10. The antibody or antigen-binding fragment thereof of any one of claims 1-9, further comprising an Fc fragment.

11. The antibody or antigen-binding fragment thereof of any one of claims 1-10, which binds to type IV collagen α5 chain EC as determined by ELISA ₅₀ A value of about 22.13ng/mL; or has a KD of less than 4nM, preferably about 3.176 ×10, as measured by biological membrane layer interference (BLI) technique for binding to type IV collagen alpha 5 chain ^-9 M。

12. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1-11.

13. An expression vector comprising the nucleic acid molecule of claim 12.

14. A host cell comprising the nucleic acid molecule of claim 12 or the expression vector of claim 13.

15. A host cell expressing the antibody or antigen-binding fragment thereof of any one of claims 1-11.

16. The host cell of claim 15, which is a hybridoma cell formed by fusion of a mouse cell with a myeloma cell, preferably a SP2/0, YB2/0, NS0 or p3x63ag8.653 cell.

17. The host cell of claim 15, wherein the host cell is a mammalian cell, preferably a CHO cell, CHO-S cell, HEK-293 cell, BHK cell or PER-C6 cell.

18. An immunoconjugate comprising the antibody or antigen binding fragment thereof of any one of claims 1-11 and a detectable label.

19. The immunoconjugate of claim 18, wherein the detectable label is enzymatically active, or the detectable label is a fluorescent group, a fluorescent protein, or a radioisotope.

20. A test kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1-11 or the immunoconjugate of claim 18 or 19.

21. The test kit of claim 20, further comprising an antibody targeting type IV collagen α1 chain and/or an antibody targeting type IV collagen α3 chain.

22. The test kit of claim 20 or 21, further comprising instructions for how to use the antibody, antigen binding fragment, or immunoconjugate to diagnose Alport syndrome.

23. A method of detecting the presence or amount of type IV collagen α5 chain in a sample, comprising:

1) Contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1-11 or the immunoconjugate of claim 18 or 19, and

24. A method of identifying Alport syndrome in a subject, comprising:

1) Contacting a biological sample from the subject with the antibody or antigen-binding fragment thereof of any one of claims 1-11 or the immunoconjugate of claim 18 or 19, and

25. The method of claim 24, wherein the biological sample is from kidney tissue and/or skin tissue.

26. The method of claim 24 or 25, wherein the Alport syndrome is X-linked dominant inherited Alport syndrome or autosomal recessive inherited Alport syndrome.

27. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-11 or an immunoconjugate according to claim 18 or 19 for the preparation of a reagent for diagnosing Alport syndrome.