Antibody binding to p-Tau217 phosphorylated protein or antigen binding part thereof and application thereof

Abstract

The invention belongs to the field of antibodies, and particularly relates to a p-Tau217 phosphorylated protein binding antibody or an antigen binding part thereof and application thereof. The invention provides the amino acid sequences of the heavy chain CDR and the light chain CDR of the antibody specifically bound with the p-Tau217 phosphorylated protein, and the antibody can specifically recognize and bind the p-Tau217 phosphorylated protein and has the advantages of high affinity and good specificity to the protein. Based on the advantages, the antibody can be applied to diagnosis of diseases (such as nervous system degenerative diseases) related to abnormal p-Tau217 phosphorylated protein level, and especially can be applied to clinical diagnosis of Alzheimer's disease.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application No. 2024109951311, filed on July 23, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of antibodies, specifically relating to an antibody that specifically binds to p-Tau217 phosphorylated protein and its application in the detection of neurodegenerative diseases. Background Technology

[0004] Alzheimer's disease (AD), also known as senile dementia, is a neurodegenerative disease. One of the typical pathological features of AD is neurofibrillary tangles (NFT). In AD patients, tau protein in neurons undergoes excessive phosphorylation, causing microtubules to aggregate and form neurofibrillary tangles. The stages of Alzheimer's disease include cognitively unimpaired (CU), mild cognitive impairment, and mild cognitive impairment (MCI).

[0005] Microtubule-associated protein (MAPT) Tau is a major neuronal microtubule-associated protein encoded by a single gene on human chromosome 17, which can promote microtubule assembly and stabilize microtubule structure, and promote axonal transport of neurons. Human Tau protein consists of 441 amino acids, which consists of NTR (N-terminal region), PRR (proline-rich region), MBR (microtubule binding region) and CTR (C-terminal region). Tau is a highly phosphorylated protein, with a total of 85 phosphorylatable sites, including 45 serine residues, 35 threonine residues and 5 tyrosine residues. Low-level phosphorylation of Tau protein can function, while high-level abnormal phosphorylation leads to loss of biological activity of Tau protein. In the AD brain, Tau protein hyperphosphorylation induces a vicious cycle, promoting the spread of Tau pathology in the brain and accumulation in human cerebrospinal fluid (CSF). The sites with increased phosphorylation levels in the early stages of AD include Thr181, Ser199, Thr205, Thr212, Thr217, Thr231, Ser262, Ser396 and Ser404, especially Thr-181 and Thr-217 are associated with early diagnosis of AD. Glycogen synthase kinase-3β (GSK-3β) as a serine / threonine kinase is an important tau protein kinase that regulates the phosphorylation of more than 20 sites of tau protein pT181, pS198, pT217 and pS404. The main distribution of anti-Tau protein antibody is the use of antibodies for therapeutic drugs or diagnostic reagents. In 2016, LILLY company AU2016223095 A1 disclosed antibodies against Tau protein and their therapeutic uses, and Johnson & Johnson developed new anti-PHF-tau antibodies in 2012 and 2016, respectively, which are disclosed in patent documents WO2013096380 A3, WO2016112078A2.

[0006] Alzheimer's disease has a long course of 20-30 years, and the researchers found that the level of plasma p-Tau217 increased about 20 years before the occurrence of MCI, and the increase in the level of plasma p-tau217 was higher than that of p-Tau231 and p-Tau181. Compared with p-Tau231, p-Tau181, Aβ42 / 40 and GFAP biomarkers, only plasma p-Tau217 showed Aβ-dependent dynamic changes in the preclinical and MCI stages of AD patients within 4-6 years, and the increase in plasma p-Tau217 was associated with the clinical deterioration and brain atrophy of preclinical AD. Mattsson-Carlgren et al. showed that plasma p-Tau217 is the best marker for predicting cognitive decline in Aβ-positive CU patients, and plasma p-tau217 can predict the decline in cognitive ability of AD. Binette et al. found that plasma p-Tau217 is a predictor of the development of MCI into AD, which is better than plasma biomarkers such as Aβ42 / 40, NfL and GFP.

[0007] Currently, there is no effective treatment for AD, so its early diagnosis and prevention are particularly important. Aβ42 / Aβ40 ratio, p-tau181, Aβ positron emission tomography (Aβ-PET) and tau-PET are commonly used methods for diagnosing AD, but due to their traumatic and expensive shortcomings, their use in clinical practice is limited. The development of ultra-sensitive, high-throughput analysis technologies represented by single molecule array (Simoa) and Meso Scale Discovery (MSD) platforms has promoted the research of AD peripheral biomarkers. p-Tau217 is a peripheral biomarker for early AD screening and clinical trial treatment response monitoring, and has good application prospects. High sensitivity and specificity p-Tau217 phosphorylated protein monoclonal antibodies are important raw materials for the development of p-Tau217 detection methods, and have potential application value.

[0008] However, there are few types of p-Tau217 phosphorylated protein monoclonal antibody diagnostic raw materials on the market, and the antibody affinity is not high, which can only reach the nanomolar (nM) level. For example, the mouse anti-p-Tau217 phosphorylated protein monoclonal antibody (Anti-h p-Tau217 R13323) developed by Medix company has an antibody affinity KD of 6.3x 10 -10 M (0.63 nM). For clinical detection of Alzheimer's disease, more specific antibodies with higher affinity are needed, such as those reaching the picomolar (pM) level, to achieve ultra-high sensitivity detection of p-Tau217 in blood samples, brain tissue and cerebrospinal fluid samples. SUMMARY

[0009] A first object of the present application is to provide an antibody or antigen-binding portion thereof specifically binding to a p-Tau217 phosphorylated protein, comprising (a) heavy chain CDRs comprising: a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3; wherein the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 comprise, respectively: (1) the amino acid sequence set forth in SEQ ID NO: 9, 10, 11; or (2) the amino acid sequence set forth in SEQ ID NO: 19, 20, 21, or a variant thereof comprising up to 3 amino acid mutations in the CDR regions; and (b) light chain CDRs comprising: a light chain CDR1, a light chain CDR2 and a light chain CDR3; wherein the light chain CDR1, the light chain CDR2 and the light chain CDR3 comprise, respectively: (1) the amino acid sequence set forth in SEQ ID NO: 14, 15, 16; or (2) the amino acid sequence set forth in SEQ ID NO: 24, 25, 26, or a variant thereof comprising up to 3 amino acid mutations in the CDR regions.

[0010] As a preferred embodiment, the heavy chain CDRs and light chain CDRs are selected from the following sequences: a. the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2 and the light chain CDR3 are set forth in SEQ ID NO: 9, 10, 11, 14, 15 and 16, respectively; or b. the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2 and the light chain CDR3 are set forth in SEQ ID NO: 19, 20, 21, 24, 25 and 26, respectively.

[0011] As a preferred embodiment, the antibody or antigen-binding portion thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is selected from the amino acid sequence set forth in SEQ ID NO: 8 or 18, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 or 18; and the light chain variable region is selected from the amino acid sequence set forth in SEQ ID NO: 13 or 23, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13 or 23. As a preferred embodiment, the heavy chain variable region and the light chain variable region are selected from the following sequences: A. the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13; or B. the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0012] As a preferred solution, the antibody is a rabbit-derived antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0013] As a preferred solution, the antigen-binding portion is a Fab, a Fab', a F(ab')2, a Fd, a Fv, a scFv, or a SdAb.

[0014] As a preferred solution, the antibody or the antigen-binding portion thereof further comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is any one of an IgG type, an IgA type, an IgM type, an IgE type, and an IgD type; and the light chain constant region is a κ chain or a λ chain.

[0015] As a preferred solution, the antibody or the antigen-binding portion thereof comprises a heavy chain and a light chain.

[0016] The heavy chain comprises an amino acid sequence shown in SEQ ID NO: 7, and the light chain comprises an amino acid sequence shown in SEQ ID NO: 12; or the heavy chain comprises an amino acid sequence shown in SEQ ID NO: 17, and the light chain comprises an amino acid sequence shown in SEQ ID NO: 22.

[0017] A second object of the present application is to provide a nucleic acid molecule encoding the antibody or the antigen-binding portion thereof that specifically binds to the p-Tau217 phosphorylated protein.

[0018] A third object of the present application is to provide an expression vector comprising the nucleic acid molecule.

[0019] A fourth object of the present application is to provide a host cell comprising the expression vector or having the nucleic acid molecule integrated in its genome.

[0020] A fifth object of the present application is to provide a cell-free expression system comprising i) the expression vector or the nucleic acid molecule; and ii) other substances, energy, and environmental conditions required for expression of the antibody. A sixth object of the present application is to provide a complex comprising i) the antibody or the antigen-binding portion thereof that specifically binds to the p-Tau217 phosphorylated protein; and ii) a detectable label.

[0021] A seventh object of the present application is to provide a conjugate comprising i) the antibody or the functional fragment thereof that specifically binds to the p-Tau217 phosphorylated protein, or the complex; and ii) a solid support.

[0022] The eighth object of the present application is to provide the use of the antibody or antigen-binding portion thereof specifically binding to the p-Tau217 phosphorylated protein in any one of the following aspects: 1) detecting the p-Tau217 phosphorylated protein; 2) preparing a product for detecting the p-Tau217 phosphorylated protein.

[0023] The ninth object of the present application is to provide the use of the antibody or antigen-binding portion thereof specifically binding to the p-Tau217 phosphorylated protein in any one of the following aspects: (1) diagnosing or aiding in the diagnosis of a disease associated with abnormal level of the p-Tau217 phosphorylated protein; (2) preparing a product for diagnosing or aiding in the diagnosis of a disease associated with abnormal level of the p-Tau217 phosphorylated protein.

[0024] As a preferred aspect, the disease is a neurodegenerative disease.

[0025] As a more preferred aspect, the disease comprises Alzheimer's disease.

[0026] The tenth object of the present application is to provide a method for preparing the antibody or antigen-binding portion thereof specifically binding to the p-Tau217 phosphorylated protein, comprising: expressing the nucleic acid molecule by using a host cell or a cell-free expression system, and then recovering the produced antibody or antigen-binding portion thereof from the culture medium and / or the cultured host cell.

[0027] The antibody provided by the present application can specifically recognize and bind to the p-Tau217 phosphorylated protein, and has the advantages of high affinity and good specificity. Based on the above advantages, the antibody can be applied to the diagnosis of a disease associated with abnormal level of the p-Tau217 phosphorylated protein, and in particular, can be applied to the clinical diagnosis of Alzheimer's disease. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the specific embodiments or prior art description will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0029] Figure 1 Figure 1 shows the reactivity of the anti-p-Tau217 phosphorylated protein antibody in the rabbit antiserum after the third immunization of rabbit No. 1 with different human p-Tau217 polypeptides in the ELISA detection of an embodiment of the present application.

[0030] Figure 2Figure 4 shows the reactivity of the rabbit anti-serum against p-Tau217 phosphorylated protein after the 4th immunization of rabbits with the p-Tau217 phosphorylated protein in ELISA assay.

[0031] Figure 3 Figure 5 shows the purity of the rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibody (10E4) after purification in SDS-PAGE assay. In this figure, lane M: Protein Marker (Bio-rad, CAT#: 1610394); lane 1: purified rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibody (10E4).

[0032] Figure 4 Figure 6 shows the purity of the rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibody (19A4) after purification in SDS-PAGE assay. In this figure, lane M: Protein Marker (Bio-rad, CAT#: 1610394); lane 1: purified rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibody (19A4).

[0033] Figure 5 Figure 7 shows the binding curve of the rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibody (10E4) to 3 different human p-Tau217 peptides in ELISA assay.

[0034] Figure 6 Figure 8 shows the binding curve of the rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibody (19A4) to 3 different human p-Tau217 peptides in ELISA assay.

[0035] Figure 7 Figure 9 shows the binding curve of the rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibodies (10E4 and 19A4) to recombinant human p-tau217 phosphorylated protein. DETAILED DESCRIPTION

[0036] The specific embodiments of the present application are described in detail below. It should be appreciated that the detailed description and specific examples given are intended for purposes of illustration only and are not intended to limit the scope of the present application. Those skilled in the art will recognize that changes can be made to the specific embodiments described which will be "substantially equivalent," while still falling within the scope of the present application. For example, features illustrated or described as part of one embodiment can be used with another embodiment to yield still a further embodiment.

[0037] Terminology

[0038] Unless otherwise defined, all terms (including technical and scientific terms) used in the disclosure of the present application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. By further guidance, the following definitions are used for better understanding the teachings of the present application. The terms used in the specification of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0039] The selection scope of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, including any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, B, C, and / or D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0040] The terms "containing", "including" and "comprising" used in the present application are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.

[0041] The numerical ranges expressed in endpoints in the present application include all values and fractions within the range and the recited endpoints.

[0042] In the present application, the concentration values involved include fluctuations within a certain range. For example, it can fluctuate within a corresponding accuracy range. For example, 2%, it can be allowed to fluctuate within ±0.1%. For larger values or values that do not need to be controlled too finely, it is also allowed that the meaning includes larger fluctuations. For example, 100mM, it can be allowed to fluctuate within ±1%, ±2%, ±5%, etc. For molecular weight, it is allowed that the meaning includes a fluctuation of ±10%.

[0043] In the present application, the descriptions such as "a plurality of", "a plurality of" and the like are used without special limitation, which means greater than or equal to 2 in quantity.

[0044] In the present application, the technical features described in an open way include both the closed technical solution consisting of the listed features and the open technical solution containing the listed features.

[0045] In the present application, "preferably", "more preferably", "more preferably", "suitably" only describe the better effect of the embodiments or examples, and it should be understood that it does not constitute a limitation on the protection scope of the present application.

[0046] In the present application, "optionally", "optional", "optional", "optionally", "optional", "optional" means optional, that is, selected from "have" or "no" two parallel schemes. If there are multiple "optional" or "optional" in a technical solution, unless otherwise specified, and there is no contradiction or mutual restriction, each "optional" or "optional" is independent.

[0047] In the present application, the term "p-Tau217" refers to the form of phosphorylation of the serine residue at position 217 of the protein Tau, which can include phosphorylation modification or other modification of other sites in addition to the serine at position 217.

[0048] In the present application, the term "antibody" refers to an immunoglobulin molecule, which is usually a tetramer composed of two identical heavy chains and two identical light chains connected to each other by disulfide bonds. According to the differences in the conservation of amino acid sequences, the heavy and light chains are divided into variable regions (V) at the amino terminal and constant regions (C) at the carboxyl terminal. Within the variable regions of the heavy and light chains, the amino acid composition and arrangement order of three local regions have a higher degree of variation, which are the key positions for antibody binding to antigen, and are therefore also called complementarity determining regions (CDRs). In the present application, the three heavy chain complementarity determining regions are referred to as VH CDR1, VH CDR2 and VH CDR3, respectively, and the three light chain complementarity determining regions are referred to as VL CDR1, VL CDR2 and VL CDR3, respectively. The variable regions of a heavy chain and a light chain interact to form an antigen binding site (Fv). According to the amino acid sequence of their heavy chain constant region, antibodies can be divided into different classes. There are five main types of intact antibodies: IgA, IgD, IgE, IgG and IgM, and some of these can be further divided into subclasses, for example, IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The subunit structure and three-dimensional conformation of immunoglobulins of different classes are known in the art. The present application is intended to include antibodies of any of the aforementioned classes or subclasses.

[0049] In the present invention, the term "antigen binding portion" of an antibody means an antigen binding fragment and antibody analogs of an antibody, encompassing a digested fragment or a functional variant of the antibody, which generally includes at least a portion of the antigen binding region or variable region (e.g., one or more CDRs) of the parental antibody, for example, an antibody fragment capable of binding to a p-Tau217 phosphorylated protein or a portion thereof comprising the phosphorylated Ser217 site, including but not limited to Fab, Fab', F(ab')2, Fd, Fv, scFv, or SdAb.

[0050] The term "monoclonal antibody" as used herein refers to an antibody that is uniform, i.e., directed against only one specific epitope of an antigen. In contrast to ordinary polyclonal antibody preparations, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant on an antigen. The modifier "monoclonal" indicates the uniform character of the antibody, not the method by which it is produced. Monoclonal antibodies of the present invention are preferably produced by recombinant DNA methods, or are obtained by screening methods described elsewhere in the present invention.

[0051] In the present invention, "percent (%) identity" with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity, and not considering any conservative substitutions as part of the identity. Sequence comparisons for the determination of percent amino acid identity can be performed in a number of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0052] In the present invention, the term "mutation" includes deletion, substitution and / or insertion.

[0053] In the present invention, the term "nucleic acid molecule" refers to a polynucleotide that is not in its natural state as found in nature, including polynucleotides isolated by biological techniques from nature (including from organisms), and also including polynucleotides that are artificially synthesized.

[0054] In the present invention, the term "cell-free expression system", also known as cell-free protein synthesis (CFPS), is a technology for synthesizing proteins in vitro. It extracts necessary biomolecules (such as ribosomes, tRNA, enzymes, etc.) from cells, and uses these molecules to synthesize proteins without living cells.

[0055] Antibody or antigen-binding portion thereof

[0056] The present disclosure relates to an antibody or antigen binding portion thereof that specifically binds to a p-Tau217 phosphorylated protein, the antibody or antigen binding portion thereof comprising (a) heavy chain CDRs comprising: a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3; wherein the heavy chain CDR1, the heavy chain CDR2, and the heavy chain CDR3 each comprises: (1) the amino acid sequence set forth in SEQ ID NO: 9, 10, 11; or (2) the amino acid sequence set forth in SEQ ID NO: 19, 20, 21, or a variant thereof comprising up to 3 amino acid mutations in the CDR regions; and (b) light chain CDRs comprising: a light chain CDR1, a light chain CDR2, and a light chain CDR3; wherein the light chain CDR1, the light chain CDR2, and the light chain CDR3 each comprises: (1) the amino acid sequence set forth in SEQ ID NO: 14, 15, 16; or (2) the amino acid sequence set forth in SEQ ID NO: 24, 25, 26, or a variant thereof comprising up to 3 amino acid mutations in the CDR regions. In some embodiments, the heavy chain CDRs and light chain CDRs are selected from the following sequences: a. the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 are set forth in SEQ ID NO: 9, 10, 11, 14, 15, and 16, respectively; or b. the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2, and the light chain CDR3 are set forth in SEQ ID NO: 19, 20, 21, 24, 25, and 26, respectively.

[0057] In some embodiments, the antibody or antigen binding portion thereof comprises a heavy chain variable region selected from the amino acid sequence set forth in SEQ ID NO: 8 or 18, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 or 18; and a light chain variable region selected from the amino acid sequence set forth in SEQ ID NO: 13 or 23, or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13 or 23. In some embodiments, the heavy chain variable region and the light chain variable region are selected from the following sequences:

[0058] A. the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13; or

[0059] B. the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0060] In some embodiments, the antibody is a rabbit-derived antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0061] In some embodiments, the antigen binding moiety is a Fab, Fab', F(ab')2, Fd, Fv, scFv, or SdAb.

[0062] In some embodiments, the antigen binding moiety is a Fab, Fab', F(ab')2, Fd, Fv, scFv, or SdAb.

[0063] Fab'(Fragment antigen binding prime) is usually obtained by further enzymatic digestion of the Fab fragment. Pepsin can be used to cleave the hinge region of the antibody, resulting in an antibody fragment of approximately Fab size.

[0064] F(ab')2(Fragment antigen binding 2) is usually obtained by using an enzyme digestion method called Panning separation on the intact antibody. This method involves exposing the antibody to specific conditions that cause the Fc fragment to be digested by enzymes, resulting in two Fab fragments bound together to form a F(ab')2 fragment.

[0065] Fd(Fragment antigen binding) is usually obtained by enzymatic digestion or PCR amplification of the intact antibody. The enzymatic method is similar to the preparation of Fab, but it is directed at the heavy chain.

[0066] Fv(Variable fragment) is usually obtained by synthesizing the variable regions of the two chains and connecting them together, or by enzymatic digestion from existing antibodies.

[0067] scFv(Single-chain variable fragment) is usually obtained by connecting the Fv of the heavy chain and the Fv of the light chain of the antibody to form a single-chain variable fragment.

[0068] SdAb (Single-domain antibody) can be obtained by immunizing an animal or screening an in vitro display library.

[0069] In some embodiments, the antibody or antigen-binding portion thereof further comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is any one of IgG type, IgA type, IgM type, IgE type, IgD type; the light chain constant region is kappa chain or lambda chain.

[0070] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain; the heavy chain comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7, and the light chain comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12; or the heavy chain comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17, and the light chain comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22.

[0071] In some embodiments, the variants (including variants with less than 100% identity and variants with at least one mutation) have the same or similar (e.g., performance difference less than or equal to 10%, 8%, 5%, 3%, 2%, 1%, or 0.5%) performance in specifically binding to the phosphorylated protein or polypeptide of p-Tau217 as the parent (i.e., the antibody or antigen-binding portion thereof before “deletion, substitution, and / or insertion”), including specificity and affinity.

[0072] In some embodiments, the substitution of an amino acid is a "conservative substitution", also known as a substitution to a "sibling" amino acid residue, which refers to the substitution of an amino acid residue by another amino acid residue having a side chain of similar chemical properties, e.g., charge or hydrophobicity. Examples of conservative substitutions are the substitution of a basic side chain amino acid (e.g., lysine, arginine, histidine) for another; a hydrophilic polar side chain (e.g., aspartic acid, glutamic acid, cysteine, serine, threonine, tyrosine, tryptophan) for another; a nonpolar side chain (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) for another; a beta-branched side chain (e.g., threonine, valine, isoleucine) for another; and a hydrophobic side chain (e.g., methionine, leucine, valine, isoleucine, alanine) for another. Conservative substitutions typically have little effect on the properties of the resulting protein.

[0073] Nucleic acid molecule

[0074] The present application also provides a nucleic acid molecule encoding the antibody or antigen binding portion thereof specifically binding to the p-Tau217 phosphorylated protein.

[0075] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding the heavy chain variable region of the antibody or antigen binding portion thereof, and a nucleotide sequence encoding the light chain variable region of the antibody or antigen binding portion thereof.

[0076] In some embodiments, the nucleic acid molecule can be genomic DNA, cDNA, mRNA or synthetic other RNA, or a combination thereof.

[0077] The skilled person can design the corresponding nucleotide sequence according to the amino acid sequence of the antibody provided by the present application, and prepare it in an isolated state by using standard genetic engineering methods, molecular biology methods, biochemical methods, etc. Based on codon degeneracy, the skilled person can design multiple not exactly identical nucleotide sequences, but all of which encode the same amino acid sequence. These modified nucleotide sequences are also included in the scope of the present application.

[0078] Biological material

[0079] The present application also provides a biological material comprising the nucleic acid molecule, which is selected from one of an expression cassette, a vector, a transposon, a host cell.

[0080] The present application also provides an expression vector comprising the nucleic acid molecule.

[0081] The present application also provides a host cell comprising the expression vector, or having the nucleic acid molecule integrated in its genome.

[0082] The term "vector" as used herein refers to any molecule (e.g., nucleic acid, plasmid, or virus, etc.) used to transfer nucleotide coding information into a host cell.

[0083] The term "expression cassette" or "expression vector" as used herein refers to a vector suitable for expressing a gene of interest (a nucleotide sequence to be expressed) in a host cell, usually including the gene of interest, a promoter, a terminator.

[0084] The term "transposon" as used herein is a DNA sequence that is capable of moving position in a genome, also known as a jumping gene or a jumping element. They exist in a variety of organisms, including bacteria, plants, animals, and fungi. Transposons can move across the genome, inserting into new locations and causing changes in the structure of the genome. With the use of transposon systems, foreign genes can be introduced into a specific site in the host genome, enabling genome editing, gene therapy, and transgenic applications.

[0085] The term "host cell" as used herein refers to a cell that has been or is capable of being transformed with a nucleic acid sequence and thereby expresses a selected gene of interest. The term includes the progeny of the parent cell, whether or not the progeny is identical to the original parent cell in morphology or in genomic make-up, so long as the selected gene of interest is present. Commonly used host cells include bacteria, yeast, mammalian cells, etc. A commonly used method for introducing foreign genes into host cells is "transfection", which allows the uptake of foreign or exogenous DNA by the host cell, which can be induced by physical and chemical methods (e.g., by calcium chloride treatment) to make the cell in the most suitable physiological state for uptake and accommodation of foreign DNA, i.e., "competent state".

[0086] A cell-free expression system

[0087] The present application also provides a cell-free protein synthesis (CFPS) system, which comprises: i) the expression vector or the nucleic acid molecule described above; and ii) other substances, energy and environmental conditions required for the expression of the antibody.

[0088] The cell-free protein synthesis system extracts a crude extract from cultured cells, removes endogenous DNA and mRNA, and then supplements ribosomes, tRNA, aminoacyl tRNA synthetases, and other macromolecular components such as initiation, elongation, and termination factors to start the translation program.

[0089] In some embodiments, the cell-free protein synthesis system is a system derived from prokaryotic cells, such as an E. coli system, which is one of the most popular protein expression systems at present due to its easy large-scale fermentation in low-cost medium and high protein yield.

[0090] In some embodiments, the cell-free expression system is a system derived from eukaryotic cells, such as wheat germ extract (WGE), rabbit reticulocyte lysate (RRL), and insect cell extract (ICE), which are more advantageous in producing more complex proteins, but are also more complex and costly.

[0091] In the field of antibody production, various cell-free expression systems have been developed and applied. Here are some specific cell-free expression systems and their application methods:

[0092] (1) E. coli cell-free expression system: This system uses cell extracts from E. coli for protein synthesis. Studies have shown that by optimizing energy supply and metabolic pathways, protein production can be efficiently achieved in the E. coli cell-free system. For example, using phosphocreatine and glucose as dual energy sources can extend the time of cell-free protein synthesis and improve the efficiency of ATP supply.

[0093] (2) Streptomyces cell-free expression system: Studies have shown that by optimizing the CFPS system of Streptomyces, the protein expression capacity has been significantly improved. By adding translation initiation factors, elongation factors, and ribosome recycling factors, the protein production of high GC content genes has been further improved.

[0094] (3) Pichia pastoris cell-free expression system: The CFPS system based on Pichia pastoris is developed for expressing complex proteins that require post-translational modifications. This system has the advantages of easy preparation and high protein yield, and is suitable for expressing glycosylated proteins and membrane proteins, etc.

[0095] (4) Recombinant expression system: The recombinant expression system provides a flexible protein production method by using a protein synthesis system with recombinant elements. This system can simulate the entire cell transcription and translation process and allows detailed and in-depth study of individual components and reaction networks. The application of cell-free expression systems in antibody production has shown its great potential in rapid, efficient, and high-throughput protein production.

[0096] Complex

[0097] The present application also provides a complex comprising: i) the antibody or antigen binding portion thereof that specifically binds to p-Tau217 phosphorylated protein; and ii) a detectable label.

[0098] In some embodiments, the detectable label can be selected from any one or more of a chromophore, a digoxin-labeled probe, an electron-dense substance, colloidal gold, or an enzyme. The following non-limiting section lists these labels:

[0099] i) an enzyme that generates a detectable signal, such as by colorimetry, fluorescence and luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase and glucose-6-phosphate dehydrogenase.

[0100] ii) a fluorophore, such as a fluorescent, quantum dot, fluorescent microsphere, luminescent compound and dye, such as an organic dye like fluorescein, phenanthroline, rhodamine, or a fluorescein-based dye like FITC (fluorescein isothiocyanate), TRITC (tetramethylrhodamine), Cy3 (cyanine 3), Cy5 (cyanine 5).

[0101] iii) a group with an electron density that can be detected by electron microscopy or by its electrical properties, such as conductivity, amperometry, voltammetry and resistance.

[0102] iv) a detectable group, such as a group whose molecular size is sufficient to induce a detectable modification in its physical and / or chemical properties; such detection can be achieved by optical methods (such as diffraction, surface plasmon resonance, surface variation and contact variation angle) or physical methods (such as atomic force spectroscopy and tunnel effect).

[0103] v) an electron-dense substance, such as a radioactive molecule (such as 32 P, 35 S or 125 I).

[0104] In some embodiments, the detectable label is selected from one or more of alkaline phosphatase, acridinium ester, horseradish peroxidase, trispyridyl ruthenium, isoluminol or a rare earth element, preferably alkaline phosphatase, acridinium ester or horseradish peroxidase.

[0105] In some embodiments, the detectable label is selected from fluorescein, phenanthroline, rhodamine, FITC (fluorescein isothiocyanate), TRITC (tetramethylrhodamine), Cy3 (cyanine 3) or Cy5 (cyanine 5).

[0106] Conjugate

[0107] The present application also provides a conjugate comprising: I) the antibody or functional fragment thereof that specifically binds to the p-Tau217 phosphorylated protein, or the complex described; and II) a solid support.

[0108] The term "solid support" as used in the present application refers to a medium in which the antibody is immobilized by physical or chemical means.

[0109] In some embodiments, the solid support can comprise polystyrene, polypropylene, polycarbonate, cyclic olefin, glass, or quartz. In some embodiments, the solid support can be a microtiter (or "multiwell") plate, a microarray plate, a micro well plate, or a chip. In some embodiments, the solid support can be a bead, such as a magnetic bead.

[0110] In some embodiments, the conjugate is made based on the Single Molecule Array (Simoa) or Meso Scale Discovery (MSD) technology.

[0111] Use

[0112] The present application also provides use of the antibody or antigen binding portion thereof specifically binding to the p-Tau217 phosphorylated protein in detecting p-Tau217. That is, a method for detecting p-Tau217 phosphorylated protein, comprising: using the antibody or antigen binding portion thereof specifically binding to the p-Tau217 phosphorylated protein to detect a sample to be tested.

[0113] In practical applications, the detection of the p-Tau217 phosphorylated protein can be based on diagnostic or auxiliary diagnostic purposes, or can be based on non-diagnostic purposes, such as for scientific research, drug development, and therapeutic monitoring, etc.

[0114] The present application also provides use of the antibody or antigen binding portion thereof specifically binding to the p-Tau217 phosphorylated protein in the preparation of a product for detecting p-Tau217 phosphorylated protein.

[0115] The present application also provides use of the antibody or antigen binding portion thereof specifically binding to the p-Tau217 phosphorylated protein in the diagnosis or auxiliary diagnosis of a disease associated with abnormal level of p-Tau217 phosphorylated protein. That is, a method for diagnosing or aiding in the diagnosis of a disease associated with abnormal level of p-Tau217 phosphorylated protein, comprising: using the antibody or antigen binding portion thereof specifically binding to the p-Tau217 phosphorylated protein to detect a sample to be tested.

[0116] The present application also provides use of the antibody or antigen binding portion thereof specifically binding to the p-Tau217 phosphorylated protein in the preparation of a product for diagnosing or aiding in the diagnosis of a disease associated with abnormal level of p-Tau217 phosphorylated protein.

[0117] In some embodiments, the disease is a neurodegenerative disease.

[0118] In some preferred embodiments, the disease comprises Alzheimer's disease.

[0119] In some embodiments, the sample to be detected in the application is blood plasma, serum, cerebrospinal fluid (CSF), tissue, cells or other related biological fluids, etc. Among them, the blood-based p-Tau217 detection has the advantages of non-invasiveness and high efficiency, and is more advantageous in clinical application.

[0120] Detection product

[0121] The application also provides a detection product comprising the antibody or antigen-binding portion thereof specifically binding to the p-Tau217 phosphorylated protein, or comprising the complex, or comprising the conjugate; the detection product includes a reagent, a test strip or a kit.

[0122] In some specific embodiments, the detection product includes but is not limited to an ELISA (enzyme-linked immunosorbent assay) kit, an immunohistochemical reagent, a Western blot kit, an immunoblotting kit, an immunofluorescence staining kit, an immunoprecipitation kit, an immunochromatography reagent, an immunohistochemical tissue staining kit, an immunoprecipitation-mass spectrometry kit, an immunohistochemical flow cytometry analysis kit, a magnetic bead immunoprecipitation kit, etc.

[0123] Preparation method

[0124] The application also provides a method for preparing the antibody or antigen-binding portion thereof specifically binding to the p-Tau217 phosphorylated protein, which comprises: expressing the nucleic acid molecule by using a host cell or a cell-free expression system, and then recovering the produced antibody or antigen-binding portion thereof from the culture medium and / or the cultured host cell.

[0125] In the present invention, the method of culturing host cells is typically a serum-free culture method, and the cells are typically cultured in serum-free suspension. After the antibody of the present invention is produced in culture, it can be purified from the cell culture contents according to standard procedures in the art, including ammonium sulfate precipitation, affinity column, column chromatography, gel electrophoresis, etc. Such techniques are within the skill in the art and are not limited to the several mentioned in the present invention. Another method of expressing antibodies can utilize expression in animals, particularly transgenic animals or nude mice. This involves the use of an expression system utilizing the animal casein promoter, which when transgenically incorporated into a mammal, allows the female animal to produce the desired recombinant protein in its milk. The culture fluid in which the antibody is secreted can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column with a modified buffer. Non-specifically bound components are washed away. The bound antibody is eluted using a pH gradient method, and the antibody fragments are detected using SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and multimers can also be removed using conventional methods, such as molecular sieving, ion exchange. The resulting product is immediately frozen, such as at -70°C, or lyophilized.

[0126] Examples

[0127] Embodiments of the present invention will be described in detail with reference to the following Examples. It is to be understood that the Examples are only by way of illustration and are not intended to limit the scope of the present invention. In the following Examples, the experimental methods are not specifically indicated, and the guidelines given in the present invention are preferred, and the experimental methods in the art or conventional conditions can also be used, or other experimental methods known in the art can be used, or the conditions recommended by the manufacturer can be used.

[0128] In the following specific examples, the measurement parameters of the raw material components may, unless otherwise specified, have slight deviations within the weighing accuracy range. With respect to temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0129] Example 1: Preparation of rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibody single B cells

[0130] 1. The amino acid sequence of human Tau (SEQ ID NO: 1) was obtained from GenBank (Accession No. P10636-8);

[0131] 2. Human p-Tau217 phosphorylated polypeptide immunogen-I (SEQ ID NO: 2) and human p-Tau217 phosphorylated polypeptide immunogen-II (SEQ ID NO: 3) were designed using Optimum Antigen Design Tool (https: / / www.genscript.com / antigen-design.html) from Genscript Nanjing Biological Technology Co., Ltd., and synthesized by Genscript Nanjing Biological Technology Co., Ltd.

[0132] 3. The above two human p-Tau217 phosphorylated polypeptides were coupled with carrier protein KLH (Sigma-Aldrich, CAT#: 374805) to prepare immunogen-I and immunogen-II, respectively. One New Zealand white rabbit (No. #1 rabbit) was immunized with immunogen-I, and one New Zealand white rabbit (No. #4 rabbit) was immunized with immunogen-II.

[0133] 4. 200 μL of each of the immunogen-I and immunogen-II prepared in step 3 above were mixed with 200 μL of Freund's complete adjuvant (Sigma-Aldrich, CAT#: F5881) at a volume ratio of 1:1 to form an emulsion for primary immunization. Each New Zealand white rabbit was subcutaneously injected with 80 μg of immunogen.

[0134] 5. The immunization was boosted every two weeks for a total of four times. 200 μL of each of the immunogen-I and immunogen-II prepared in step 3 above were mixed with 200 μL of Freund's incomplete adjuvant (Sigma-Aldrich, CAT#: F5506) at a volume ratio of 1:1 to form an emulsion for boosting immunization. Each New Zealand white rabbit was subcutaneously injected with 80 μg of immunogen.

[0135] 6. Blood was taken 7 days after the third antigen immunization, and the level of anti-p-Tau217 phosphorylated polypeptide antibody in the rabbit serum was detected by ELISA:

[0136] Biotin-conjugated human p-Tau217 monophosphorylated polypeptide (threonine 217 phosphorylated, SEQ ID NO: 4), biotin-conjugated human p-Tau217 non-phosphorylated polypeptide (without phosphorylation site, SEQ ID NO: 5), and biotin-conjugated human p-Tau217 diphosphorylated polypeptide (both serine 214 and threonine 217 sites phosphorylated, SEQ ID NO: 6) were used to coat Elisa plates, respectively, to evaluate the presence of rabbit anti-human p-Tau217 antibodies in the serum of immunized animals and cell culture supernatant by ELISA. The above three p-Tau217 polypeptides were synthesized by Genscript Nanjing Biotechnological Co., Ltd.

[0137] 1) Add 100 μL of PBS buffer containing 1 μg / mL streptavidin (GenScript, Z02043) to each well of the ELISA plate and coat overnight at 4°C;

[0138] 2) Discard the coating solution and wash the wells once with 100 μL of PBS-T buffer (0.05% Tween 20);

[0139] 3) Add 200 μL of PBS-T buffer containing 1% BSA to each well of the ELISA plate and block at 37°C for 0.5 hours;

[0140] 4) Discard the blocking solution and wash the wells once with 100 μL of PBS-T buffer (0.05% Tween 20);

[0141] 5) Prepare peptide buffer solutions with a concentration of 1 μg / mL using PBS buffer for biotin-conjugated human p-Tau217 monophosphorylated peptide, biotin-conjugated human p-Tau217 nonphosphorylated peptide, and biotin-conjugated human p-Tau217 diphosphorylated peptide.

[0142] 6) Add 100 μL of the above-mentioned peptide buffer to each well of the 96-well plate from step 4, and then block at room temperature for 1 hour;

[0143] 7) Discard the blocking solution; dilute the collected rabbit serum with PBS buffer at a ratio of 1:1000, and add 100 μL of the diluted rabbit serum to the first row of wells in a 96-well plate to perform a 2-fold serial dilution, for a total of 10 concentrations. Add PBS buffer to the last well as a negative control and incubate at room temperature for 1 hour.

[0144] 8) After incubation, discard the supernatant, wash the plate three times with PBS-T buffer, and add 100 μL / well of mouse anti-rabbit IgG Fc secondary antibody containing horseradish peroxidase (GenScript, CAT#:A01856). Incubate at 37°C for 0.5 hours.

[0145] 9) Wash the 96-well plate five times with PBS-T buffer, then add TMB chromogenic solution and incubate in the dark at room temperature for 15 minutes. Terminate the reaction by adding 50 μL of 1M HCl stop solution and read the plate at 450 nm using a microplate reader.

[0146] ELISA detection of rabbit serum after three immunization and three kinds of p-Tau217 polypeptide reactivity, the results show that the rabbit serum after immunization with human p-Tau217 phosphorylated polypeptide produces antibodies against human p-Tau217 phosphorylated polypeptide, the antibodies in rabbit serum have high reactivity with human p-Tau217 monophosphorylated and diphosphorylated polypeptide, and have less reactivity with non-phosphorylated polypeptide. Among them, the immunogen used by No. 1 rabbit is human p-Tau217 phosphorylated polypeptide immunogen-I, and the immunogen used by No. 4 rabbit is human p-Tau217 phosphorylated polypeptide immunogen-II. The antibody titers of No. 1 rabbit and No. 4 rabbit serum against human p-Tau217 phosphorylated polypeptide are high, and the detection results are shown in Figure 1 and Figure 2 .

[0147] Example 2: ELISA analysis of cell supernatant anti-p-Tau217 phosphorylated protein antibody and human p-tau217 polypeptide reactivity:

[0148] 1. Select No. 1 rabbit and No. 4 rabbit, take 15 mL of blood to separate PBMC cells, and separate positive B cells from PBMC.

[0149] 2. The enriched B cells are plated into 96-well cell culture plates at a density of 10 cells per well, and the 96-well cell culture plates are placed in a 5% CO2, 37°C incubator for 7 days.

[0150] 3. On the 7th day, collect the supernatant of the 96-well cell culture plate, and analyze the presence of anti-p-Tau217 phosphorylated protein antibodies in the supernatant by ELISA. The detection sample is 100 μL / well of 96-well plate cell culture supernatant. The antigen coating and the rest of the experimental methods are consistent with the ELISA detection of p-Tau217 phosphorylated protein antibody level in rabbit serum in Example 1.

[0151] The ELISA detection results of human anti-p-Tau217 phosphorylated protein antibodies in cell culture supernatant are shown in Table 1, and the results show that the anti-human p-Tau217 phosphorylated protein antibodies in the cell supernatant of the 96-well plate can specifically recognize human p-Tau217 monophosphorylated polypeptide and biotin-conjugated human p-Tau217 diphosphorylated polypeptide, and have no cross reaction with p-Tau217 non-phosphorylated polypeptide.

[0152] Table 1 ELISA detection of cell culture supernatant

[0153]

[0154]

[0155] In Table 1, antigen A: biotin-conjugated human p-Tau217 monophosphorylated polypeptide; antigen B: biotin-conjugated human p-Tau217 unphosphorylated polypeptide; antigen C: biotin-conjugated human p-Tau217 diphosphorylated polypeptide.

[0156] Example 3: Sequencing of variable regions of rabbit anti-human p-Tau217 phosphorylated monoclonal antibodies and antibody production

[0157] 1. Antibody sequencing was performed on rabbit B cells (9C11, 19A4, 20D9, 34F11, 10E4, 11E12, 14A9 and 41F5) that produced anti-human p-Tau217 phosphorylated protein antibodies. Total RNA was extracted from 3 x 10 6 -5 x 10 6 single B cells using TRIzol (Ambion, CAT#: 15596-026) and reverse-transcribed into cDNA using antibody subtype-specific primers and universal primers (Takara, CAT#: 6110A).

[0158] 2. Rabbit immunoglobulin heavy and light chain variable region fragments were amplified by RACE PCR and the resulting PCR fragments were subcloned into a pMD18-T vector system (Takara, CAT#: 6011) and the inserts were sequenced using vector-specific primers. The unique V region amino acid sequences of two rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibodies (19A4 and 10E4) were obtained (see Table 2).

[0159] Table 2 CDR region sequences of rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibodies

[0160] Antibody number CDR1 / sequence number CDR2 / sequence number CDR3 / sequence number 19A4 V H ]]> (SEQ ID NO: 9) (SEQ ID NO: 10) (SEQ ID NO: 11) 19A4 V L ]] (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO: 16) 10E4 V H ]] (SEQ ID NO: 19) (SEQ ID NO: 20) (SEQ ID NO: 21) 10E4 V L ]]> (SEQ ID NO: 24) (SEQ ID NO: 25) (SEQ ID NO: 26)

[0161] 3. DNA fragments of the light chains (19A4 light chain: SEQ ID NO: 12; 10E4 light chain: SEQ ID NO: 22) and heavy chains (19A4 heavy chain: SEQ ID NO: 7; 10E4 heavy chain: SEQ ID NO: 17) of the two clones (19A4 and 10E4) were synthesized according to the sequences in Table 2 and inserted into a pTT5 expression vector (NRC Biotechnology Research Institute, National Research Council of Canada), respectively, to construct light and heavy chain expression plasmids of the two rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibodies (19A4 and 10E4).

[0162] 4. The constructed light and heavy chain plasmids of the two clones (19A4 and 10E4) were co-transfected into CHO-3E7 cells respectively, and the cells were cultured at 37°C, 5% CO2 for 6 days in a shaker flask. The supernatant was collected for antibody purification.

[0163] 5. The purification pipeline and protein A purification column were depyrogenated with 0.2M NaOH before purification. The protein A purification column was re-equilibrated with a buffer containing 0.05M Tris and 1.5M NaCl (pH 8.0). Then the harvested cell culture supernatant was diluted 1:1 with 2x the above buffer and filtered to remove bacteria.

[0164] 6. The filtered supernatant was added to the protein A purification column respectively, and incubated at room temperature for 2 hours. The protein A purification column was washed with 1x the above buffer, and the antibody was eluted with sterile 0.1M sodium citrate (pH 3.5). The eluate was collected and neutralized with one-ninth volume of sterile 1M Tris-HCl (pH 9).

[0165] 6. Under sterile conditions, the eluted sample buffer was exchanged to PBS (pH 7.4) to remove all elution buffers and concentrate the sample. After concentration, the antibody was quantified by measuring the absorbance of OD280nm using an extinction coefficient Ec (0.1%) of 1.43.

[0166] 7. The purified antibody was analyzed by SDS-PAGE gel electrophoresis using a 10% precast gel (GenScript, CAT#: M00656) through a BioRad electrophoresis system. The gel after electrophoresis was stained with eStain 2.0 (GenScript), and the antibody molecular size and purity were estimated by comparing the stained bands with the protein Marker (Bio-rad, CAT#: 1610394). The results are shown in Figure 3 (10E4) and Figure 4 (19A4). Thus, two purified rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibodies (19A4 and 10E4) were obtained.

[0167] Example 4: ELISA detection of the specificity of rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibody:

[0168] The binding specificity of the two purified rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibodies 19A4 and 10E4 to different human p-Tau217 polypeptides was detected by ELISA. Among them, the detection sample was the purified anti-p-Tau217 phosphorylated protein monoclonal antibody of clone 10E4 and clone 19A4, and the initial concentration of the antibody was 10 ug / ml, which was diluted according to a 3-fold concentration gradient, a total of 11 test concentrations. The coating antigen and other experimental part methods were consistent with the detection of the level of anti-p-Tau217 phosphorylated protein antibody in rabbit serum by ELISA in Example 1.

[0169] The binding specificity of the antibodies of clone 10E4 and clone 19A4 to different human p-Tau217 polypeptides is shown in Figure 5. Figure 5 (10E4) and Figure 6 (19A4). The results showed that the clone 10E4 antibody specifically bound to human p-Tau217 monophosphorylated polypeptide, and had no reactivity with p-Tau217 non-phosphorylated polypeptide and p-Tau217 diphosphorylated polypeptide; the clone 19A4 antibody specifically bound to human p-Tau217 monophosphorylated and p-Tau217 diphosphorylated polypeptides, and had no reactivity with p-Tau217 non-phosphorylated polypeptide.

[0170] Example 5: ELISA detection of the reactivity of rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibody to p-Tau217 phosphorylated protein

[0171] The binding ability of the purified anti-p-Tau217 phosphorylated protein monoclonal antibodies of clone 19A4 and clone 10E4 to recombinant human p-Tau217 phosphorylated protein (absea CAT#: P00003CB) was evaluated by indirect ELISA.

[0172] 1. Prepare one ELISA plate, add 100 uL of PBS containing 0.5 ug / mL of recombinant human p-Tau217 phosphorylated protein to each well, and coat overnight at 4°C;

[0173] 2. Wash the plate with PBS-T buffer (0.05% Tween), add 200 uL of PBS-T buffer containing 1% BSA to each well, and block at 37°C for 2 hours;

[0174] 3. Discard the blocking solution and dry at 37°C for 2 hours;

[0175] 4. Discard the blocking solution, and add 100 uL of purified anti-p-Tau217 monoclonal antibody of clone 10E4 and clone 19A4 at 10 ug / mL to wells A1 and B1, respectively, and dilute according to a 3-fold concentration gradient, a total of 11 test concentrations.

[0176] 5. Incubate at room temperature for 1 hour. Wash the plate four times with PBS-T buffer and add 100 μL / well of conjugated horse radish peroxidase mouse anti-rabbit IgG Fc (GenScript, CAT#: A01856), incubate at 37°C for 0.5 hours. Wash the plate four times with PBS-T buffer, then add TMB developing solution (FcMACS) and incubate at room temperature in the dark for 15 minutes.

[0177] 6. Add 50 μL of 1M HCl stop solution (Sigma) to stop the reaction. Read the plate using a microplate reader at 450 nm.

[0178] The binding ability of purified rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibodies (19A4 and 10E4) to recombinant human p-Tau217 phosphorylated protein is shown in Figure 7 The results show that both rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibodies (19A4 and 10E4) can specifically bind to p-tau217 phosphorylated protein.

[0179] Example 6: Determination of monoclonal antibody affinity

[0180] 1. The probe (PROBELIFE, CAT#: 160001) with Protein A protein coupled on the surface is soaked in 250 μL of buffer K (PBS + 0.002% Tween 20 + 0.02% BSA) for 10 minutes;

[0181] 2. Two rabbit anti-human p-Tau217 phosphorylated protein monoclonal antibodies (19A4 and 10E4) are respectively configured into working solutions of 5 μg / mL with buffer K;

[0182] 3. Recombinant human p-Tau217 phosphorylated protein is configured into working solutions of four concentrations, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL and 0 μg / mL, with buffer K;

[0183] 4. Add reagents according to the instructions of Gator non-labeled analyzer (Gator Bio) and perform antibody affinity determination according to the instructions. The affinity data is shown in Table 3.

[0184] Table 3 Antibody affinity determination results

[0185] Clone number koff(1 / s) kon(1 / Ms) KD(M) 19A4 <1.00E-006 4.22E+005 <1.00E-012 10E4 <1.00E-006 2.78E+005 <1.00E-012

[0186] The results show that the two rabbit anti-p-Tau217 phosphorylated protein monoclonal antibodies (19A4 and 10E4) have good binding performance to recombinant human p-Tau217 phosphorylated protein, with a binding constant KD in the low nanomolar range (10-9 ) are high affinity antibodies, the KD of the two antibodies obtained (19A4 and 10E4) being in the picomolar range (10 -12 ) are high affinity antibodies.

[0187] The above described embodiments only express several embodiments of the present application, which are described in a more specific and detailed manner, but cannot be understood as limiting the scope of the patent. It should be noted that for ordinary skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application.

Claims

1. An antibody or antigen-binding portion thereof that specifically binds to a p-Tau217 phosphorylated protein, the antibody or antigen-binding portion thereof comprising (a) a heavy chain CDR, the heavy chain CDR comprising: a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3; wherein the heavy chain CDR1, the heavy chain CDR2 and the heavy chain CDR3 respectively comprise: (1) the amino acid sequences set forth in SEQ ID NOs: 9, 10, 11; or (2) the amino acid sequences set forth in SEQ ID NOs: 19, 20, 21, or a variant thereof comprising up to 3 amino acid mutations in the CDR regions; and (b) a light chain CDR, the light chain CDR comprising: a light chain CDR1, a light chain CDR2 and a light chain CDR3; wherein the light chain CDR1, the light chain CDR2 and the light chain CDR3 respectively comprise: (1) the amino acid sequences set forth in SEQ ID NOs: 14, 15, 16; or (2) the amino acid sequences set forth in SEQ ID NOs: 24, 25, 26, or a variant thereof comprising up to 3 amino acid mutations in the CDR regions.

2. The antibody or antigen binding portion thereof that specifically binds a p-Tau217 phosphorylated protein of claim 1, wherein, the heavy chain CDRs and the light chain CDRs are selected from the following sequences: a. the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2 and the light chain CDR3 are set forth in SEQ ID NOs: 9, 10, 11, 14, 15 and 16, respectively; or b. the amino acid sequences of the heavy chain CDR1, the heavy chain CDR2, the heavy chain CDR3, the light chain CDR1, the light chain CDR2 and the light chain CDR3 are set forth in SEQ ID NOs: 19, 20, 21, 24, 25 and 26, respectively.

3. The antibody or antigen-binding portion thereof that specifically binds to a p-Tau217 phosphorylated protein of claim 1, comprising a heavy chain variable region and a light chain variable region, wherein, the heavy chain variable region is selected from the amino acid sequence set forth in SEQ ID NO: 8 or 18, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8 or 18; the light chain variable region is selected from the amino acid sequence set forth in SEQ ID NO: 13 or 23, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13 or 23.

4. The antibody or antigen binding portion thereof specifically binding to a p-Tau217 phosphorylated protein according to claim 3, wherein, the heavy chain variable region and the light chain variable region are selected from the following sequences: A. the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13; or B. the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 18 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

23.

5. The antibody or antigen binding portion thereof specifically binding to a p-Tau217 phosphorylated protein according to any one of claims 1 to 4, wherein, the antibody is a rabbit-derived antibody, a chimeric antibody, a humanized antibody or a human antibody.

6. The antibody or antigen binding portion thereof that specifically binds a p-Tau217 phosphorylated protein of claim 1, wherein, the antigen-binding portion is a Fab, a Fab', a F(ab')2, a Fd, a Fv, a scFv or a SdAb.

7. The antibody or antigen binding portion thereof specifically binding to a p-Tau217 phosphorylated protein according to claim 1, wherein, the antibody or antigen-binding portion thereof further comprises a heavy chain constant region and a light chain constant region; the heavy chain constant region is any one of IgG type, IgA type, IgM type, IgE type, IgD type; the light chain constant region is Kappa chain or Lambda chain.

8. The antibody or antigen binding portion thereof that specifically binds a p-Tau217 phosphorylated protein of claim 1, wherein, the antibody or antigen-binding portion thereof comprises a heavy chain and a light chain; the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 7, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO: 12; or the heavy chain comprises an amino acid sequence as set forth in SEQ ID NO: 17, and the light chain comprises an amino acid sequence as set forth in SEQ ID NO:

22.

9. A nucleic acid molecule encoding the antibody or antigen-binding portion thereof that specifically binds to a p-Tau217 phosphorylated protein according to any one of claims 1-8.

10. An expression vector comprising the nucleic acid molecule according to claim 9.

11. A host cell comprising the expression vector according to claim 10, or having integrated into its genome the nucleic acid molecule according to claim 9.

12. A cell-free expression system comprising: i) the expression vector according to claim 10, or the nucleic acid molecule according to claim 9; and ii) other substances, energy and environmental conditions required for antibody expression.

13. A complex comprising: i) the antibody or antigen-binding portion thereof that specifically binds to a p-Tau217 phosphorylated protein according to any one of claims 1-8; and ii) a detectable label.

14. A conjugate comprising: i) the antibody or functional fragment thereof that specifically binds to a p-Tau217 phosphorylated protein according to any one of claims 1-8, or the complex according to claim 13; and ii) a solid support.

15. Use of the antibody or antigen-binding portion thereof that specifically binds to a p-Tau217 phosphorylated protein according to any one of claims 1-8 in any one of: 1) detecting a p-Tau217 phosphorylated protein; 2) preparing a product for detecting a p-Tau217 phosphorylated protein.

16. Use of the antibody or antigen-binding portion thereof that specifically binds to a p-Tau217 phosphorylated protein according to any one of claims 1-8 in any one of: (1) diagnosing or aiding in the diagnosis of a disease associated with abnormal levels of p-Tau217 phosphorylated protein; (2) preparing a product for diagnosing or aiding in the diagnosis of a disease associated with abnormal levels of p-Tau217 phosphorylated protein.

17. Use according to claim 16, wherein, The disease is a neurodegenerative disease, preferably the disease comprises Alzheimer's disease.

18. A method of making an antibody, or antigen binding portion thereof, that specifically binds to a p-Tau217 phosphorylated protein, comprising: The nucleic acid molecule according to claim 9 is expressed using a host cell, or a cell-free expression system, and the produced antibody or antigen-binding portion thereof is then recovered from the culture medium and / or the cultured host cell. The nucleic acid molecule according to claim 9 is expressed using a host cell, or a cell-free expression system, and the produced antibody or antigen-binding portion thereof is then recovered from the culture medium and / or the cultured host cell.

Images