Specific binding protein targeting tau and use thereof

Abstract

The present invention relates to an antibody targeting Tau or an antigen-binding fragment thereof, which can specifically bind to Tau. The present invention also relates to the use of the specific binding protein in the treatment, prevention and / or diagnosis of neurodegenerative diseases.

Description

Specific binding proteins targeting Tau and their applications Technical Field

[0001] The present disclosure belongs to the field of biotechnology, specifically to the field of antibody therapy, and more specifically to a specific binding protein targeting Tau. Background Art

[0002] Alzheimer's disease (AD), the most common form of dementia, is a neurodegenerative disorder characterized by the accumulation of Aβ protein plaques and tau protein neurofibrillary tangles (NFTs). Clinical manifestations include cognitive impairment and memory loss, accompanied by various neuropsychiatric symptoms and behavioral disturbances. The multifactorial and complex nature of AD significantly complicates its treatment.

[0003] Alzheimer's disease drugs are divided into three categories: disease-modifying therapies, symptomatic cognitive enhancement therapies, and drugs for treating neuropsychiatric symptoms. Disease-modifying therapies account for the largest proportion. Among these drugs, one-third are biologics targeting Aβ and Tau, and the other half are small molecules targeting inflammation and synaptic plasticity.

[0004] Currently available drugs for Alzheimer's disease are all symptomatic cognitive enhancement therapies or drugs that treat neuropsychiatric symptoms and cannot truly slow the progression of Alzheimer's disease. Among disease-modifying therapies, Aβ and Tau proteins are the best targets for Alzheimer's disease treatment. Excessive accumulation of Aβ and Tau leads to the formation of amyloid plaques and neurofibrillary tangles, respectively, and the formation of oligomers further proliferates and exacerbates the pathology.

[0005] Passive immunotherapy (antibodies) has some obvious advantages over active immunotherapy (vaccines). Elderly Alzheimer's patients have low responsiveness to vaccines and cannot achieve the expected effective treatment. On the other hand, antibody therapy is more reliable in titer and intensity, reducing the chance of unpredictable adverse reactions. Even if adverse reactions occur, antibody therapy is easier to terminate than vaccines to avoid further and more pathogenic adverse reactions.

[0006] Aducanumab and Lecanemab are the first two FDA-approved drugs for the treatment of Alzheimer's disease. However, more and more studies have found that Tau plays a crucial role in Alzheimer's disease.

[0007] Therefore, there is a need to develop antibodies against different fragments of Tau for the treatment and diagnosis of neurodegenerative diseases such as Alzheimer's disease. Summary of the Invention

[0008] The present invention provides a novel specific binding protein for Tau, aiming to obtain more effective Tau antibodies and provide new ideas for the diagnosis and treatment of Alzheimer's disease.

[0009] One aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to Tau and / or a fragment thereof, comprising at least one heavy chain variable region and at least one light chain variable region.

[0010] In some embodiments, the heavy chain variable region comprises: a HCDR1 as shown in any one of SEQ ID NOs: 121-145;

[0011] a HCDR2 as shown in any one of SEQ ID NOs: 146-174; and a HCDR3 as shown in any one of SEQ ID NOs: 175-196 or having an amino acid sequence of AR.

[0012] In some embodiments, the light chain variable region includes: LCDR1 as shown in any one of SEQ ID NOs: 197-218; and / or LCDR2 having an amino acid sequence of AAS, DTS, FAT, KLS, KVS, LAS, LVS, RMS, RVS, TVS or WAS; and / or LCDR3 as shown in any one of SEQ ID NOs: 219-242.

[0013] In some embodiments, the heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 121, SEQ ID NO: 146 and SEQ ID NO: 175, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 122, SEQ ID NO: 147 and SEQ ID NO: 176, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 123, SEQ ID NO: 148 and an amino acid sequence of AR, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 124, SEQ ID NO: 149 and SEQ ID NO: 177, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 125, SEQ ID NO: 150 and SEQ ID NO: 178, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 126, SEQ ID NO: 151 and SEQ ID NO: NO: 179; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 126, SEQ ID NO: 152 and SEQ ID NO: 179, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 126, SEQ ID NO: 167 and SEQ ID NO: 185, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 127, SEQ ID NO: 153 and SEQ ID NO: 180, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 128, SEQ ID NO: 154 and SEQ ID NO: 181, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 129, SEQ ID NO: 154 and SEQ ID NO: 181, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 130, SEQ ID NO: 155 and SEQ ID NO: SEQ ID NO: 182; or, HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 130, SEQ ID NO: 156, and SEQ ID NO: 182, respectively; or, HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 130, SEQ ID NO: 157, and SEQ ID NO: 182, respectively;or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 130, SEQ ID NO: 158 and SEQ ID NO: 182, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 131, SEQ ID NO: 157 and SEQ ID NO: 183, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 132, SEQ ID NO: 159 and SEQ ID NO: 184, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 132, SEQ ID NO: 160 and SEQ ID NO: 184, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 133, SEQ ID NO: 161 and SEQ ID NO: 185, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 134, SEQ ID NO: 162 and SEQ ID NO: 186, respectively; or, : 135, SEQ ID NO: 163 and SEQ ID NO: 187; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 136, SEQ ID NO: 164 and SEQ ID NO: 188, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 137, SEQ ID NO: 165 and SEQ ID NO: 189, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 138, SEQ ID NO: 166 and SEQ ID NO: 190, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 139, SEQ ID NO: 168 and SEQ ID NO: 190, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 140, SEQ ID NO: 169 and SEQ ID NO: 191, respectively; or, SEQ ID NO: 141, SEQ ID NO: 170 and SEQ ID NO: 192; or, SEQ ID NO: 142, SEQ ID NO: 171 and SEQ ID NO: 193, respectively;or, the HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO: 143, SEQ ID NO: 172, and SEQ ID NO: 194, respectively; or, the HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO: 144, SEQ ID NO: 173, and SEQ ID NO: 195, respectively; or, the HCDR1, HCDR2, and HCDR3 set forth in SEQ ID NO: 145, SEQ ID NO: 174, and SEQ ID NO: 196, respectively.

[0014] In some embodiments, the light chain variable region comprises: LCDR1, LCDR2, and LCDR3, respectively, as set forth in SEQ ID NO: 197, with an amino acid sequence of AAS and SEQ ID NO: 220; or LCDR1, LCDR2, and LCDR3, respectively, as set forth in SEQ ID NO: 197, with an amino acid sequence of KVS and SEQ ID NO: 219; or LCDR1, LCDR2, and LCDR3, respectively, as set forth in SEQ ID NO: 197, with an amino acid sequence of KVS and SEQ ID NO: 235; or LCDR1, LCDR2, and LCDR3, respectively, as set forth in SEQ ID NO: 197, with an amino acid sequence of KLS and SEQ ID NO: 219; or LCDR1, LCDR2, and LCDR3, respectively, as set forth in SEQ ID NO: 198, with an amino acid sequence of AAS and SEQ ID NO: 220; or LCDR1, LCDR2, and LCDR3, respectively, as set forth in SEQ ID NO: 199, with an amino acid sequence of RVS and SEQ ID NO: 221; or LCDR1, LCDR2, and LCDR3, respectively, as set forth in SEQ ID NO: NO: 200, the amino acid sequence is KVS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 222; or SEQ ID NO: 200, the amino acid sequence is TVS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 225, respectively; or SEQ ID NO: 200, the amino acid sequence is TVS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 240, respectively; or SEQ ID NO: 201, the amino acid sequence is WAS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 223, respectively; or SEQ ID NO: 202, the amino acid sequence is KVS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 219, respectively; or SEQ ID NO: 202, the amino acid sequence is LVS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 224, respectively; or SEQ ID NO: 202, the amino acid sequence is RVS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 224; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 203, the amino acid sequence of KVS and SEQ ID NO: 219, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 203, the amino acid sequence of WAS and SEQ ID NO: 226, respectively;or SEQ ID NO: 203, the amino acid sequence of WAS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 227, respectively; or SEQ ID NO: 204, the amino acid sequence of WAS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 228, respectively; or SEQ ID NO: 205, the amino acid sequence of DTS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 229, respectively; or SEQ ID NO: 206, the amino acid sequence of DTS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 229, respectively; or SEQ ID NO: 206, the amino acid sequence of WAS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 228, respectively; or SEQ ID NO: 207, the amino acid sequence of LVS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 230, respectively; or SEQ ID NO: 208, the amino acid sequence of LVS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 231, respectively. NO: 209, an amino acid sequence of DTS and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 231; or SEQ ID NO: 210, an amino acid sequence of RMS and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 232, respectively; or SEQ ID NO: 210, an amino acid sequence of RMS and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 233, respectively; or SEQ ID NO: 211, an amino acid sequence of LAS and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 234, respectively; or SEQ ID NO: 212, an amino acid sequence of LVS and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 230, respectively; or SEQ ID NO: 213, an amino acid sequence of FAT and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 236, respectively; or SEQ ID NO: 214, an amino acid sequence of DTS and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 237; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 215, whose amino acid sequence is DTS and SEQ ID NO: 238, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 216, whose amino acid sequence is WAS and SEQ ID NO: 239, respectively;or LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 217, whose amino acid sequence is LAS and SEQ ID NO: 241, respectively; or LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 218, whose amino acid sequence is LVS and SEQ ID NO: 242, respectively.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 121, SEQ ID NO: 146 and SEQ ID NO: 175, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 197, the amino acid sequence of KVS and SEQ ID NO: 219, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 121, SEQ ID NO: 146 and SEQ ID NO: 175, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 198, the amino acid sequence of AAS and SEQ ID NO: 220, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 121, SEQ ID NO: 146 and SEQ ID NO: 175, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 197, the amino acid sequence of KVS and SEQ ID NO: 220, respectively. NO: 219; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 122, SEQ ID NO: 147 and SEQ ID NO: 176, respectively, and SEQ ID NO: 199, the amino acid sequence of RVS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 221, respectively; or HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 123, SEQ ID NO: 148 and the amino acid sequence of AR, respectively, and SEQ ID NO: 200, the amino acid sequence of KVS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 222, respectively; or HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 124, SEQ ID NO: 149 and SEQ ID NO: 177, respectively, and SEQ ID NO: 201, the amino acid sequence of WAS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 222, respectively. LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 223; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 125, SEQ ID NO: 150 and SEQ ID NO: 178, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 202, the amino acid sequence of LVS and SEQ ID NO: 224, respectively;or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 126, SEQ ID NO: 151 and SEQ ID NO: 179, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 203, the amino acid sequence of KVS and SEQ ID NO: 219, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 126, SEQ ID NO: 152 and SEQ ID NO: 179, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 197, the amino acid sequence of KVS and SEQ ID NO: 219, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 126, SEQ ID NO: 167 and SEQ ID NO: 185, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 212, the amino acid sequence of LVS and SEQ ID NO: 230, respectively; or, NO: 127, SEQ ID NO: 153 and SEQ ID NO: 180, and the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 200, the amino acid sequence of TVS and SEQ ID NO: 225, respectively; or, the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 128, SEQ ID NO: 154 and SEQ ID NO: 181, and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 200, the amino acid sequence of TVS and SEQ ID NO: 225, respectively; or, the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 129, SEQ ID NO: 154 and SEQ ID NO: 181, and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 200, the amino acid sequence of TVS and SEQ ID NO: 225, respectively; or, the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 155 and SEQ ID NO: 182, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 197, amino acid sequence KVS, and SEQ ID NO: 219, respectively;or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 130, SEQ ID NO: 156 and SEQ ID NO: 182, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 197, the amino acid sequence of KLS and SEQ ID NO: 219, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 130, SEQ ID NO: 157 and SEQ ID NO: 182, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 197, the amino acid sequence of KVS and SEQ ID NO: 219, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 130, SEQ ID NO: 155 and SEQ ID NO: 182, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 202, the amino acid sequence of KVS and SEQ ID NO: 219, respectively; or, NO: 130, SEQ ID NO: 158 and SEQ ID NO: 182, and the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 202, the amino acid sequence of KVS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 219, respectively; or, the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 131, SEQ ID NO: 157 and SEQ ID NO: 183, and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 203, the amino acid sequence of WAS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 226, respectively; or, the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 131, SEQ ID NO: 157 and SEQ ID NO: 183, and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 203, the amino acid sequence of WAS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 227, respectively; or, HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 159 and SEQ ID NO: 184, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 197, the amino acid sequence of which is AAS, and SEQ ID NO: 220, respectively;or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 132, SEQ ID NO: 159 and SEQ ID NO: 184, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 204, the amino acid sequence of which is WAS and SEQ ID NO: 228, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 132, SEQ ID NO: 159 and SEQ ID NO: 184, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 205, the amino acid sequence of which is DTS and SEQ ID NO: 229, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 132, SEQ ID NO: 159 and SEQ ID NO: 184, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 206, the amino acid sequence of which is DTS and SEQ ID NO: 229, respectively; or, NO: 132, SEQ ID NO: 160 and SEQ ID NO: 184, and the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 206, the amino acid sequence of WAS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 228, respectively; or, the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 133, SEQ ID NO: 161 and SEQ ID NO: 185, respectively, and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 207, the amino acid sequence of LVS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 230, respectively; or, the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 134, SEQ ID NO: 162 and SEQ ID NO: 186, respectively, and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 202, the amino acid sequence of RVS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 224, respectively; or, the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 135, SEQ ID NO: HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 163 and SEQ ID NO: 187, and LCDR1, LCDR2 and LCDR3 as set forth in SEQ ID NO: 209, amino acid sequence DTS and SEQ ID NO: 231, respectively;or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 135, SEQ ID NO: 163 and SEQ ID NO: 187, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 210, the amino acid sequence of which is RMS and SEQ ID NO: 232, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 136, SEQ ID NO: 164 and SEQ ID NO: 188, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 210, the amino acid sequence of which is RMS and SEQ ID NO: 233, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 137, SEQ ID NO: 165 and SEQ ID NO: 189, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 211, the amino acid sequence of which is LAS and SEQ ID NO: 234, respectively; or, NO: 138, SEQ ID NO: 166 and SEQ ID NO: 190, and the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 197, the amino acid sequence of KVS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 235, respectively; or, the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 139, SEQ ID NO: 168 and SEQ ID NO: 190, and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 213, the amino acid sequence of FAT and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 236, respectively; or, the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 140, SEQ ID NO: 169 and SEQ ID NO: 191, and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 214, the amino acid sequence of DTS and the LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 237, respectively; or, the HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 141, SEQ ID NO: HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 170 and SEQ ID NO: 192, and LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 215, the amino acid sequence of which is DTS, and SEQ ID NO: 238, respectively;or, HCDR1, HCDR2 and HCDR as shown in SEQ ID NO: 142, SEQ ID NO: 171 and SEQ ID NO: 193, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 209, the amino acid sequence of which is DTS and SEQ ID NO: 231, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 143, SEQ ID NO: 172 and SEQ ID NO: 194, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 216, the amino acid sequence of which is WAS and SEQ ID NO: 239, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 143, SEQ ID NO: 172 and SEQ ID NO: 194, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 200, the amino acid sequence of which is TVS and SEQ ID NO: 240, respectively; or, SEQ ID NO: 144, SEQ ID NO: 173 and SEQ ID NO: 195, and SEQ ID NO: 217, the amino acid sequence of LAS and SEQ ID NO: 241, respectively; or, SEQ ID NO: 145, SEQ ID NO: 174 and SEQ ID NO: 196, and SEQ ID NO: 218, the amino acid sequence of LVS and SEQ ID NO: 242, respectively.

[0016] In some embodiments, these CDRs may contain amino acid mutations while maintaining the antibody's ability to specifically bind to CD40. Preferably, the amino acid mutations are amino acid substitutions, and the number of amino acid substitutions may be 1-3.

[0017] In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-60, or an amino acid sequence that is at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical thereto.

[0018] In some embodiments, the light chain variable region comprises the amino acid sequence shown in any one of SEQ ID NOs:61-120, or an amino acid sequence that is at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical thereto.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO: 1 and a light chain variable region as set forth in SEQ ID NO: 61; or comprises a heavy chain variable region as set forth in SEQ ID NO: 2 and a light chain variable region as set forth in SEQ ID NO: 62; or comprises a heavy chain variable region as set forth in SEQ ID NO: 3 and a light chain variable region as set forth in SEQ ID NO: 63; or comprises a heavy chain variable region as set forth in SEQ ID NO: 4 and a light chain variable region as set forth in SEQ ID NO: 64; or comprises a heavy chain variable region as set forth in SEQ ID NO: 5 and a light chain variable region as set forth in SEQ ID NO: 65; or comprises a heavy chain variable region as set forth in SEQ ID NO: 6 and a light chain variable region as set forth in SEQ ID NO: 66; or comprises a heavy chain variable region as set forth in SEQ ID NO: 7 and a light chain variable region as set forth in SEQ ID NO: 67; or comprises a heavy chain variable region as set forth in SEQ ID NO: 8 and a light chain variable region as set forth in SEQ ID NO: 68; or comprises a heavy chain variable region as set forth in SEQ ID NO: 9 and a light chain variable region as set forth in SEQ ID NO: NO:69; or comprising the heavy chain variable region of SEQ ID NO:10 and the light chain variable region of SEQ ID NO:70; or comprising the heavy chain variable region of SEQ ID NO:11 and the light chain variable region of SEQ ID NO:71; or comprising the heavy chain variable region of SEQ ID NO:12 and the light chain variable region of SEQ ID NO:72; or comprising the heavy chain variable region of SEQ ID NO:13 and the light chain variable region of SEQ ID NO:73; or comprising the heavy chain variable region of SEQ ID NO:14 and the light chain variable region of SEQ ID NO:74; or comprising the heavy chain variable region of SEQ ID NO:15 and the light chain variable region of SEQ ID NO:75; or comprising the heavy chain variable region of SEQ ID NO:16 and the light chain variable region of SEQ ID NO:76; or comprising the heavy chain variable region of SEQ ID NO:17 and the light chain variable region of SEQ ID NO:77; or comprising the heavy chain variable region of SEQ ID NO:18 and the light chain variable region of SEQ ID NO:19; NO:18 and the light chain variable region shown in SEQ ID NO:78; or the heavy chain variable region shown in SEQ ID NO:19 and the light chain variable region shown in SEQ ID NO:79; or the heavy chain variable region shown in SEQ ID NO:20 and the light chain variable region shown in SEQ ID NO:80; or the heavy chain variable region shown in SEQ ID NO:21 and the light chain variable region shown in SEQ ID NO:81;or comprising a heavy chain variable region as set forth in SEQ ID NO: 22 and a light chain variable region as set forth in SEQ ID NO: 82; or comprising a heavy chain variable region as set forth in SEQ ID NO: 23 and a light chain variable region as set forth in SEQ ID NO: 83; or comprising a heavy chain variable region as set forth in SEQ ID NO: 24 and a light chain variable region as set forth in SEQ ID NO: 84; or comprising a heavy chain variable region as set forth in SEQ ID NO: 25 and a light chain variable region as set forth in SEQ ID NO: 85; or comprising a heavy chain variable region as set forth in SEQ ID NO: 26 and a light chain variable region as set forth in SEQ ID NO: 86; or comprising a heavy chain variable region as set forth in SEQ ID NO: 27 and a light chain variable region as set forth in SEQ ID NO: 87; or comprising a heavy chain variable region as set forth in SEQ ID NO: 28 and a light chain variable region as set forth in SEQ ID NO: 88; or comprising a heavy chain variable region as set forth in SEQ ID NO: 29 and a light chain variable region as set forth in SEQ ID NO: 89; or comprising a heavy chain variable region as set forth in SEQ ID NO: 30 and a light chain variable region as set forth in SEQ ID NO: NO:90; or comprising the heavy chain variable region of SEQ ID NO:31 and the light chain variable region of SEQ ID NO:91; or comprising the heavy chain variable region of SEQ ID NO:32 and the light chain variable region of SEQ ID NO:92; or comprising the heavy chain variable region of SEQ ID NO:33 and the light chain variable region of SEQ ID NO:93; or comprising the heavy chain variable region of SEQ ID NO:34 and the light chain variable region of SEQ ID NO:94; or comprising the heavy chain variable region of SEQ ID NO:35 and the light chain variable region of SEQ ID NO:95; or comprising the heavy chain variable region of SEQ ID NO:36 and the light chain variable region of SEQ ID NO:96; or comprising the heavy chain variable region of SEQ ID NO:37 and the light chain variable region of SEQ ID NO:97; or comprising the heavy chain variable region of SEQ ID NO:38 and the light chain variable region of SEQ ID NO:98; or comprising the heavy chain variable region of SEQ ID NO:39 and the light chain variable region of SEQ ID NO:40; NO:39 and the light chain variable region shown in SEQ ID NO:99; or the heavy chain variable region shown in SEQ ID NO:40 and the light chain variable region shown in SEQ ID NO:100; or the heavy chain variable region shown in SEQ ID NO:41 and the light chain variable region shown in SEQ ID NO:101; or the heavy chain variable region shown in SEQ ID NO:42 and the light chain variable region shown in SEQ ID NO:102;or comprising a heavy chain variable region as set forth in SEQ ID NO:43 and a light chain variable region as set forth in SEQ ID NO:103; or comprising a heavy chain variable region as set forth in SEQ ID NO:44 and a light chain variable region as set forth in SEQ ID NO:104; or comprising a heavy chain variable region as set forth in SEQ ID NO:45 and a light chain variable region as set forth in SEQ ID NO:105; or comprising a heavy chain variable region as set forth in SEQ ID NO:46 and a light chain variable region as set forth in SEQ ID NO:106; or comprising a heavy chain variable region as set forth in SEQ ID NO:47 and a light chain variable region as set forth in SEQ ID NO:107; or comprising a heavy chain variable region as set forth in SEQ ID NO:48 and a light chain variable region as set forth in SEQ ID NO:108; or comprising a heavy chain variable region as set forth in SEQ ID NO:49 and a light chain variable region as set forth in SEQ ID NO:109; or comprising a heavy chain variable region as set forth in SEQ ID NO:50 and a light chain variable region as set forth in SEQ ID NO:110; or comprising a heavy chain variable region as set forth in SEQ ID NO:51 NO: 51 and the light chain variable region shown in SEQ ID NO: 111; or comprising the heavy chain variable region shown in SEQ ID NO: 52 and the light chain variable region shown in SEQ ID NO: 112; or comprising the heavy chain variable region shown in SEQ ID NO: 53 and the light chain variable region shown in SEQ ID NO: 113; or comprising the heavy chain variable region shown in SEQ ID NO: 54 and the light chain variable region shown in SEQ ID NO: 114; or comprising the heavy chain variable region shown in SEQ ID NO: 55 and the light chain variable region shown in SEQ ID NO: 115; or comprising the heavy chain variable region shown in SEQ ID NO: 56 and the light chain variable region shown in SEQ ID NO: 116; or comprising the heavy chain variable region shown in SEQ ID NO: 57 and the light chain variable region shown in SEQ ID NO: 117; or comprising the heavy chain variable region shown in SEQ ID NO: 58 and the light chain variable region shown in SEQ ID NO: 118; or comprising the heavy chain variable region shown in SEQ ID NO: 59 and the light chain variable region shown in SEQ ID NO: NO:119; or comprising the heavy chain variable region shown in SEQ ID NO:60 and the light chain variable region shown in SEQ ID NO:120.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof is a humanized antibody.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region. Preferably, the heavy chain constant region is selected from hIgG1, hIgG2, hIgG3, or hIgG4 heavy chain constant regions. In some embodiments, the light chain constant region may be selected from a kappa chain or a lambda chain. In some embodiments, the above-mentioned specific antibody or antibody or antigen-binding fragment thereof may be a prepared polyclonal antibody or a monoclonal antibody.

[0022] Another aspect of the present invention provides a chimeric antigen receptor, which includes an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises the antibody or antigen-binding fragment thereof described in the present invention.

[0023] Another aspect of the present invention provides a modified immune cell comprising the chimeric antigen receptor of the present invention.

[0024] Another aspect of the present invention provides a multispecific antibody comprising two or more antigen-binding domains, wherein one of the antigen-binding domains comprises the antibody or antigen-binding fragment thereof according to the present invention.

[0025] Another aspect of the present invention provides an antibody composition, comprising a first antibody and a second antibody, wherein the first antibody and / or the second antibody is selected from the antibodies or antigen-binding fragments thereof that specifically bind to Tau and / or its fragments described in the present invention.

[0026] In some embodiments, the first antibody comprises a first heavy chain variable region and a first light chain variable region, and the second antibody comprises a second heavy chain variable region and a second light chain variable region.

[0027] In some embodiments, the first antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 7 and a light chain variable region as set forth in SEQ ID NO: 67, or comprises a heavy chain variable region as set forth in SEQ ID NO: 9 and a light chain variable region as set forth in SEQ ID NO: 69, or comprises a heavy chain variable region as set forth in SEQ ID NO: 18 and a light chain variable region as set forth in SEQ ID NO: 78, or comprises a heavy chain variable region as set forth in SEQ ID NO: 47 and a light chain variable region as set forth in SEQ ID NO: 107, or comprises a heavy chain variable region as set forth in SEQ ID NO: 57 and a light chain variable region as set forth in SEQ ID NO: 117, and the second antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 12 and a light chain variable region as set forth in SEQ ID NO: 72, or comprises a heavy chain variable region as set forth in SEQ ID NO: 13 and a light chain variable region as set forth in SEQ ID NO: 73, or comprises a heavy chain variable region as set forth in SEQ ID NO: 14 and a light chain variable region as set forth in SEQ ID NO: 74, or comprises a heavy chain variable region as set forth in SEQ ID NO: 15 NO:15 and the light chain variable region shown in SEQ ID NO:75, or the heavy chain variable region shown in SEQ ID NO:37 and the light chain variable region shown in SEQ ID NO:97, or the heavy chain variable region shown in SEQ ID NO:44 and the light chain variable region shown in SEQ ID NO:104.

[0028] In some embodiments, the second antibody comprises a heavy chain variable region as set forth in SEQ ID NO:49 and a light chain variable region as set forth in SEQ ID NO:109, and the first antibody comprises a heavy chain variable region as set forth in SEQ ID NO:14 and a light chain variable region as set forth in SEQ ID NO:74, or comprises a heavy chain variable region as set forth in SEQ ID NO:16 and a light chain variable region as set forth in SEQ ID NO:76, or comprises a heavy chain variable region as set forth in SEQ ID NO:18 and a light chain variable region as set forth in SEQ ID NO:78, or comprises a heavy chain variable region as set forth in SEQ ID NO:20 and a light chain variable region as set forth in SEQ ID NO:80, or comprises a heavy chain variable region as set forth in SEQ ID NO:21 and a light chain variable region as set forth in SEQ ID NO:81, or comprises a heavy chain variable region as set forth in SEQ ID NO:22 and a light chain variable region as set forth in SEQ ID NO:82, or comprises a heavy chain variable region as set forth in SEQ ID NO:23 and a light chain variable region as set forth in SEQ ID NO:83, or comprises a heavy chain variable region as set forth in SEQ ID NO:25 and a light chain variable region as set forth in SEQ ID NO: The light chain variable region is shown in ID NO:85.

[0029] In some embodiments, the second antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 55 and a light chain variable region as set forth in SEQ ID NO: 115, and the first antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 13 and a light chain variable region as set forth in SEQ ID NO: 73, or comprises a heavy chain variable region as set forth in SEQ ID NO: 15 and a light chain variable region as set forth in SEQ ID NO: 75, or comprises a heavy chain variable region as set forth in SEQ ID NO: 17 and a light chain variable region as set forth in SEQ ID NO: 77, or comprises a heavy chain variable region as set forth in SEQ ID NO: 18 and a light chain variable region as set forth in SEQ ID NO: 78, or comprises a heavy chain variable region as set forth in SEQ ID NO: 20 and a light chain variable region as set forth in SEQ ID NO: 80, or comprises a heavy chain variable region as set forth in SEQ ID NO: 21 and a light chain variable region as set forth in SEQ ID NO: 81, or comprises a heavy chain variable region as set forth in SEQ ID NO: 22 and a light chain variable region as set forth in SEQ ID NO: 82, or comprises a heavy chain variable region as set forth in SEQ ID NO: 23 and a light chain variable region as set forth in SEQ ID NO: 83. The heavy chain variable region shown in SEQ ID NO: 23 and the light chain variable region shown in SEQ ID NO: 83, or the heavy chain variable region shown in SEQ ID NO: 25 and the light chain variable region shown in SEQ ID NO: 85; or the heavy chain variable region shown in SEQ ID NO: 27 and the light chain variable region shown in SEQ ID NO: 87.

[0030] In some embodiments, the second antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 59 and a light chain variable region as set forth in SEQ ID NO: 119, and the first antibody comprises a heavy chain variable region as set forth in SEQ ID NO: 14 and a light chain variable region as set forth in SEQ ID NO: 74, or comprises a heavy chain variable region as set forth in SEQ ID NO: 16 and a light chain variable region as set forth in SEQ ID NO: 76, or comprises a heavy chain variable region as set forth in SEQ ID NO: 17 and a light chain variable region as set forth in SEQ ID NO: 77, or comprises a heavy chain variable region as set forth in SEQ ID NO: 18 and a light chain variable region as set forth in SEQ ID NO: 78, or comprises a heavy chain variable region as set forth in SEQ ID NO: 20 and a light chain variable region as set forth in SEQ ID NO: 80, or comprises a heavy chain variable region as set forth in SEQ ID NO: 21 and a light chain variable region as set forth in SEQ ID NO: 81, or comprises a heavy chain variable region as set forth in SEQ ID NO: 22 and a light chain variable region as set forth in SEQ ID NO: 82, or comprises a heavy chain variable region as set forth in SEQ ID NO: 23 and a light chain variable region as set forth in SEQ ID NO: 83. The heavy chain variable region is shown in NO:23 and the light chain variable region is shown in SEQ ID NO:83.

[0031] Another aspect of the present invention provides an isolated polypeptide comprising a first domain and a second domain, wherein the first domain comprises at least 20 amino acids from positions 1 to 55 from the N-terminus of the Tau protein, and the second domain comprises at least 15 amino acids from positions 151 to 369 from the N-terminus of the Tau protein.

[0032] In some embodiments, the first domain includes at least 20 amino acids from positions 1 to 25 from the N-terminus of the Tau protein, and the second domain includes at least 17 amino acids from positions 170 to 240 from the N-terminus of the Tau protein.

[0033] In some embodiments, the first domain comprises at least 20 amino acids from positions 1-22 of the Tau protein from the N-terminus, and the second domain comprises at least 17 amino acids from positions 170-200 of the Tau protein from the N-terminus. In some embodiments, the first domain comprises amino acids from positions 1-22 of the Tau protein from the N-terminus, and the second domain comprises amino acids from positions 174-191 of the Tau protein from the N-terminus, wherein amino acid position 181 is phosphorylated. In some embodiments, the first domain comprises at least 20 amino acids from positions 1-22 of the Tau protein from the N-terminus, and the second domain comprises at least 17 amino acids from positions 210-230 of the Tau protein from the N-terminus. In some embodiments, the first domain comprises amino acids from positions 1-22 of the Tau protein from the N-terminus, and the second domain comprises amino acids from positions 210-227 of the Tau protein from the N-terminus, wherein amino acid position 217 is phosphorylated.

[0034] In some embodiments, the first domain comprises at least 20 amino acids from positions 1 to 22 of the Tau protein from the N-terminus, and the second domain comprises at least 17 amino acids from positions 220 to 240 of the Tau protein from the N-terminus. In some embodiments, the first domain comprises amino acids from positions 1 to 22 of the Tau protein from the N-terminus, and the second domain comprises amino acids from positions 224 to 240 of the Tau protein from the N-terminus, wherein amino acid 231 is phosphorylated.

[0035] In some embodiments, the isolated polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NOs: 251-253.

[0036] In some embodiments, the first domain and the second domain are directly connected or connected through a linker.

[0037] Another aspect of the present invention provides a method for detecting Tau in a sample, comprising the step of detecting Tau in the sample using the antibody or antigen-binding fragment thereof.

[0038] In some embodiments, the method further comprises detecting Tau using the isolated polypeptide described herein.

[0039] In some embodiments, the sample is whole blood, red blood cell concentrates, platelet concentrates, white blood cell concentrates, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, stool, urine, cultured cells, saliva, oral secretions, and / or nasal secretions.

[0040] In one embodiment, the method is a method for non-diagnostic purposes.

[0041] Another aspect of the present invention provides an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to the present invention.

[0042] In some embodiments, the nucleic acid molecule is an mRNA molecule.

[0043] Another aspect of the present invention provides an expression vector comprising the isolated nucleic acid of the present invention.

[0044] In some embodiments, the expression vector can be a eukaryotic cell expression vector and / or a prokaryotic cell expression vector, such as a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector or a herpes simplex vector.

[0045] In some embodiments, the expression vector is present in nanoparticles, liposomes, exosomes, microbubbles, or a gene gun.

[0046] Another aspect of the present invention provides a host cell comprising the isolated nucleic acid or the expression vector of the present invention.

[0047] In some embodiments, the host cell is a conventional host cell in the art, as long as the expression vector can stably express the carried nucleic acid molecule as the antibody or antigen-binding fragment thereof of the present invention. Preferably, the host cell is a prokaryotic cell and / or a eukaryotic cell, the prokaryotic cell is preferably an E. coli cell such as TG1, BL21 (expressing single-chain antibodies or Fab antibodies), and the eukaryotic cell is preferably a HEK293 cell or a CHO cell (expressing full-length IgG antibodies). The expression vector is transformed into a host cell to obtain the host cell of the present invention. The transformation method is a conventional transformation method in the art, preferably a chemical transformation method, a heat shock method or an electroporation method.

[0048] Another aspect of the present invention provides an antibody-drug conjugate, comprising: the antibody or antigen-binding fragment thereof according to the present invention; and a drug covalently linked to the antibody or antigen-binding fragment thereof.

[0049] In some embodiments, the drug is selected from an immunosuppressant and a cytotoxic drug.

[0050] Another aspect of the present invention provides a pharmaceutical composition comprising: the antibody or antigen-binding fragment thereof, or the antibody-drug conjugate according to the present invention; and a pharmaceutically acceptable carrier.

[0051] In some embodiments, the pharmaceutical composition further comprises a therapeutic agent selected from an immunosuppressant and a cytotoxic drug.

[0052] In some embodiments, the pharmaceutically acceptable carrier may be a conventional carrier in the art, and the carrier may be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including a pharmaceutically acceptable excipient, filler, or diluent. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the specific binding protein and / or other small molecule drug or antibody or polypeptide, and 0.01 to 99.99% of a pharmaceutical carrier, wherein the percentages are the mass percentages of the pharmaceutical composition.

[0053] In some embodiments, the pharmaceutical composition can be administered parenterally, by injection, or orally. The pharmaceutical composition can be prepared in a form suitable for administration, such as a solid, semisolid, or liquid form, and can be in the form of an aqueous solution, a non-aqueous solution, or a suspension, a powder, a tablet, a capsule, a granule, an injection, or an infusion. It can be administered intravascularly, subcutaneously, intraperitoneally, intramuscularly, by inhalation, intranasally, by airway instillation, or by intrathoracic instillation. The pharmaceutical composition can also be administered in the form of an aerosol or spray, such as nasally; or, it can be administered intrathecally, intramedullary, or intraventricularly, and can also be administered transdermally, percutaneously, topically, enterally, intravaginally, sublingually, or rectally. The pharmaceutical composition can be prepared into various dosage forms as needed, and can be administered by a physician based on factors such as the patient's type, age, weight, general disease condition, and mode of administration to determine the dosage that is beneficial to the patient.

[0054] In some embodiments, the specific binding protein and other active ingredients in the pharmaceutical composition can be administered simultaneously or sequentially.

[0055] Another aspect of the present invention provides use of the antibody or antigen-binding fragment thereof, the isolated nucleic acid, the antibody-drug conjugate or the pharmaceutical composition according to the present invention in the preparation of a medicament for preventing, treating and / or diagnosing Alzheimer's disease.

[0056] In some embodiments, the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, or frontotemporal dementia.

[0057] In some embodiments, the neurodegenerative disease is selected from Alzheimer's disease.

[0058] Another aspect of the present invention provides a kit comprising one or more kits, wherein the kit comprises the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the pharmaceutical composition according to the present invention.

[0059] In some embodiments, the kit comprises a first kit and a second kit, wherein the first kit comprises the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the pharmaceutical composition according to the present invention, and the second kit comprises a therapeutic agent selected from an immunosuppressant and a cytotoxic drug.

[0060] Another aspect of the present invention provides a drug delivery device comprising the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the pharmaceutical composition according to the present invention.

[0061] In some embodiments, the drug delivery device is a pre-filled syringe.

[0062] Another aspect of the present invention provides a method for preventing, treating and / or diagnosing Alzheimer's disease, comprising administering to a subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate or the pharmaceutical composition according to the present invention.

[0063] In some embodiments, the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, or frontotemporal dementia.

[0064] In some embodiments, the neurodegenerative disease is selected from Alzheimer's disease.

[0065] Another aspect of the present invention provides a detection kit comprising the antibody or antigen-binding fragment thereof, the multispecific antibody or the antibody composition of the present invention.

[0066] In some embodiments, the detection kit further comprises the isolated polypeptide described in the present disclosure,

[0067] In some embodiments, the detection kit further comprises a detectable marker that can be linked to the antibody or its antigen-binding fragment, the multispecific antibody or the antibody composition, and the detectable marker is linked to the antibody or the bispecific binding protein, or is separately present in the kit.

[0068] In some embodiments, the detection kit further comprises a substrate corresponding to the detectable label and / or instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] FIG1 shows the titer detection results of the serum of mice immunized with Tau441 and Tau(1-45).

[0070] FIG2 shows the titer detection results of the serum of mice immunized with Tau441 and Tau (187-208).

[0071] FIG3 shows the titer test results of the serum of mice immunized with P181-KLH.

[0072] FIG4 shows the titer test results of the serum of mice immunized with P231-KLH.

[0073] FIG5 shows the titer test results of the serum of mice immunized with P217-KLH.

[0074] FIG6 shows the binding results of Tau(1-45) antibody and different peptides of Tau441.

[0075] Figure 7 shows the binding results of Tau (187-208) antibody with different peptides of Tau441, from left to right: full-length Tau441, Tau (1-45), Tau (1-22), Tau (12-34), Tau (23-44) and blank control.

[0076] FIG8 shows the binding results of Tau (187-208) antibody and different peptides of Tau441.

[0077] FIG9 shows the binding results of the P181 antibody to different peptides of Tau441.

[0078] FIG10 shows the binding results of the P217 antibody to different peptides of Tau441.

[0079] FIG11 shows the binding results of the P231 antibody to different peptides of Tau441.

[0080] FIG12 shows the affinity determination results of Tau(1-45) antibody and human Tau441.

[0081] FIG13 shows the affinity determination results of Tau(187-208) antibody and human Tau441.

[0082] FIG14 shows the results of paired screening of antibodies binding to the middle end of Tau and antibodies binding to Tau (1-22).

[0083] FIG15 shows the results of paired screening of antibodies binding to the middle end of Tau and antibodies binding to Tau (1-22).

[0084] FIG16 shows the results of paired screening of antibodies binding to the middle end of Tau and antibodies binding to Tau (1-22).

[0085] FIG17 shows the results of paired screening of antibodies binding to phosphorylated P181 and antibodies binding to Tau (1-22).

[0086] FIG18 shows the results of paired screening of antibodies binding to phosphorylated P217 and antibodies binding to Tau (1-22).

[0087] FIG19 shows the results of paired screening of antibodies binding to phosphorylated P231 and antibodies binding to Tau (1-22).

[0088] FIG20 shows the results of paired screening of antibodies binding to phosphorylated P231 and antibodies binding to Tau (1-22).

[0089] FIG21 shows the results of paired screening of antibodies binding to phosphorylated P231 and antibodies binding to Tau (1-22).

[0090] FIG22 shows the results of paired screening of antibodies binding to phosphorylated P231 and antibodies binding to Tau (1-22).

[0091] FIG23 shows the results of paired screening of antibodies binding to the middle end of Tau-55# and antibodies binding to Tau(1-22).

[0092] FIG24 shows the results of paired screening of antibodies binding to the middle end of Tau-70# and antibodies binding to Tau(1-22).

[0093] FIG25 shows the results of paired screening of antibodies binding to the middle end P217-19# and antibodies binding to Tau (1-22).

[0094] FIG26 shows the results of paired screening of antibodies binding to the middle end P231-5# and antibodies binding to Tau (1-22).

[0095] FIG27 shows the results of paired screening of antibodies binding to phosphorylated P217-6## and antibodies binding to Tau(1-22).

[0096] FIG28 shows the results of paired screening of antibodies binding to phosphorylated P231-7# and antibodies binding to Tau (1-22).

[0097] FIG29 shows the results of paired screening of antibodies binding to phosphorylated P181-1# and antibodies binding to Tau (1-22).

[0098] Figure 30 shows the antibody pairing screening results of antibodies binding to the middle end P217-19#, Tau(187-208)-70# and phosphorylated P217-6# and antibodies binding to Tau(1-22) on the simoa platform.

[0099] Figure 31 shows the results of antibody pairing screening on the simoa platform for antibodies that bind to phosphorylated P181-1# and P231-7# and antibodies that bind to Tau (1-22).

[0100] FIG32 shows the HPLC chromatograms of three standards: pT181-N22, pT217-N22, and pT231-N22.

[0101] FIG33 shows the MALDI-MSI and MALDI-ISD results of the standard pT181-N22.

[0102] FIG34 shows the MALDI-MSI and MALDI-ISD results of the standard pT217-N22.

[0103] FIG35 shows the MALDI-MSI and MALDI-ISD results of the standard pT231-N22.

[0104] FIG36 shows the binding results of Tau antibodies to Tau441GSK 3beta-phosphorylated, pT231-N22, pT217-N22, pT181-N22 and BSA (Control).

[0105] FIG37 shows the results of ELISA for determining the affinity of Tau antibodies. DETAILED DESCRIPTION

[0106] The following are preferred embodiments of the present disclosure, and the present disclosure is not limited to the following preferred embodiments. It should be noted that, for those skilled in the art, any modifications and improvements made based on this invention and creative concept fall within the scope of protection of the present disclosure. The reagents used, for which the manufacturer is not indicated, are all commercially available conventional products.

[0107] Alzheimer's disease (AD), the most common form of dementia, is a neurodegenerative disease characterized by the deposition of Aβ protein plaques and tau protein neurofibrillary tangles (NFTs). Clinical manifestations include cognitive impairment and memory loss, accompanied by various neuropsychiatric symptoms and behavioral disorders.

[0108] Tau is a microtubule-associated protein. Under physiological conditions, its primary function is to regulate microtubule stability. The tau hyperphosphorylation hypothesis posits that tau becomes hyperphosphorylated in the brains of AD patients, gradually losing its ability to stabilize the microtubule system. Tau then accumulates and aggregates in the brain, forming paired helical fibers. These fibers then transform into NFTs, destabilizing microtubules and disrupting the microtubule system, leading to neuronal death and ultimately the development of AD. Tau spread appears to follow a prion-like transmission mechanism: the first step is inducing misfolding, and the second step allows for the transport of misfolded and intact prions, known as seeding and propagation, respectively. Following these two steps, tau species spread between neurons through the extracellular space or along axonal pathways, inducing tau aggregation in surrounding neurons and further accelerating the pathological process. Two anti-tau strategies exist: promoting the clearance of tau aggregates and inhibiting abnormal tau phosphorylation and aggregation.

[0109] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0110] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0111] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0112] The term "specific binding protein" generally refers to a molecule that specifically binds to an antigenic determinant. Antigen binding molecules or specific binding proteins include, for example, antibodies, antibody fragments, and scaffold antibodies or antigen-binding fragments thereof.

[0113] The term "antibody" of the present invention encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies or trispecific antibodies), single-chain molecules and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0114] The term "monoclonal antibody" of the present invention refers to an antibody obtained from a substantially homogeneous antibody population, i.e., except for possible trace amounts of variant antibodies (e.g., containing naturally occurring mutations or generated during the production of monoclonal antibody preparations, typically present in small amounts), the individual antibodies comprised by the antibody population are identical and / or bind to the same epitope. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.

[0115] The term "multispecific antibody" of the present invention is used in its broadest sense to encompass antibodies with multiple epitope specificities. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has multiple epitope specificities; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable domains, each single variable domain binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies (diabodies), and trispecific antibodies (triabodies), antibody fragments linked together covalently or non-covalently, etc.

[0116] The term "valence" of the present invention represents that an antigen binding molecule has a specified number of binding domains. Therefore, the terms "bivalent," "tetravalent," and "hexavalent" represent that there are two binding domains, four binding domains, and six binding domains in an antigen binding molecule, respectively. The bispecific antibody is at least "bivalent," and can be "trivalent," "tetravalent," or "multivalent." In some cases, the antibody has two or more binding sites and is bispecific. That is, even in the presence of more than two binding sites (i.e., the antibody is trivalent or multivalent), the antibody can also be bispecific.

[0117] The terms "antibody or antigen-binding fragment thereof" and "antibody" of the present invention are used interchangeably herein to refer to antibodies that are substantially similar to natural antibody structures. "Native antibody" refers to naturally occurring immunoglobulin molecules. For example, natural IgG class antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two light chains and two heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH) (also referred to as a variable heavy chain domain or a heavy chain variable domain) and three constant domains (CH1, CH2, and CH3) (also referred to as a heavy chain constant region). From the N-terminus to the C-terminus, each light chain has a variable region (VL) (also referred to as a variable light chain domain or a light chain variable domain) and a light chain constant domain (CL) (also referred to as a light chain constant region). The heavy chains of antibodies can be one of five types: α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), which can be further divided into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of antibodies can be one of two types, kappa and lambda, based on the amino acid sequence of their constant domains.

[0118] Within the light chain and heavy chain, the variable region and the constant region are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0119] The term "variable region" or "variable domain" of the present invention refers to the domain of the antibody heavy chain or light chain that participates in the binding of an antigen-binding molecule to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0120] The term "variable" of the present invention refers to that certain segments of the variable domain are generally different in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed throughout the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) within the light and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions, most of which adopt a β-sheet configuration and are connected by three HVRs, which form loops connected and, in some cases, form part of the β-sheet structure. The HVRs in each chain are tightly held together by the FR region and, together with the HVRs of the other chains, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, 5th edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not directly involved in the binding of the antibody to the antigen, but have other effector functions, such as participating in the antibody-dependent cellular toxicity of the antibody.

[0121] The term "hypervariable region" or "HVR" of the present invention refers to a region in an antibody variable domain region that is highly variable in sequence and / or forms structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs: three present in VH (H1, H2, H3) and three present in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and / or from "complementarity determining regions (CDRs)", where amino acid residues have the highest sequence variability and / or are involved in antigen recognition. Exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia et al., J. Mol. Biol. 196:901-917 (1987). Exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3) occur at amino acid residues 24-34 (L1), amino acid residues 50-56 (L2), amino acid residues 89-97 (L3), amino acid residues 31-35 (H1), amino acid residues 50-65 (H2), and amino acid residues 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). For comparison, the following table lists the corresponding amino acid residues of the CDRs defined in the references cited above. In the present application, the amino acid sequences of the CDRs listed above are all shown according to the IMGT definition rules (the sequences shown in the claims of the present application are also shown according to the IMGT definition rules). However, it is well known in the art that the CDRs of antibodies can be defined in the art by various methods, such as the Kabat definition rules based on sequence variability and the Chothia definition rules based on the position of the structural loop regions (see J Mol Biol 273:927-48, 1997). It should be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or its region (e.g., variable region) should be understood to cover the complementary determining region defined by any of the above-mentioned known schemes described in the present invention. Although the scope of protection claimed in the present invention is based on the sequences shown in the IMGT definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.

[0122] "Framework" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. The FR of a variable domain is typically composed of the following four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0123] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0124] A "humanized antibody" comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises at least one, typically two, variable domains in which all or substantially all HVRs (e.g., CDRs) correspond to HVRs of a non-human antibody, and all or substantially all FRs correspond to FRs of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0125] A "humanized antibody" has an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source utilizing human antibody libraries or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues.

[0126] The present invention also relates to amino acid sequence variants, which can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions, insertions, and / or substitutions of residues in the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be performed to obtain a final construct having the desired properties, such as antigen-binding activity. Sites for substitution generally include HVRs and framework (FR). See the table below for possible amino acid substitutions.

[0127] Conservative amino acid substitutions

[0128] The term "polynucleotide" or "nucleic acid" or "nucleotide sequence" herein refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), virally derived RNA, or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0129] "Antibody fragments" comprise a portion of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv; diabodies, triabodies, tetrabodies, crossover Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibodies formed from antibody fragments and single-domain antibodies (single-domain antibodies).

[0130] The term "antigen binding domain" or "antigen binding site" of the present invention refers to the part of an antigen binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen binding domain" refers to a part of an antibody that comprises a region that specifically binds to and is complementary to a part or all of an antigen. In the case where an antigen molecule is very large, an antigen binding molecule can only bind to a specific portion of an antigen, which is referred to as an epitope. Antigen binding domains can be provided by, for example, one or more variable domains (also referred to as variable regions). Preferably, antigen binding domains comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one aspect, antigen binding domains can bind to its antigen and block or partially block the function of the antigen.

[0131] The term "antigenic determinant" of the present invention is synonymous with "antigen" and "epitope", and refers to a site on a polypeptide macromolecule (e.g., a stretch of continuous amino acids or a conformational configuration consisting of different regions of non-continuous amino acids) to which an antigen-binding portion binds, thereby forming an antigen-binding portion-antigen complex. Antigenic determinants can be present, for example, on the surface of tumor cells, on the surface of microbially infected cells, on the surface of other diseased cells, on the surface of immune cells, free in serum, and / or in the extracellular matrix (ECM). Unless otherwise indicated, the protein used as an antigen in the present invention can be any natural form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The antigen can also be a human protein, or the antigen can be a "full-length", unprocessed protein, as well as any form of protein produced by intracellular processing, or a naturally occurring protein variant, such as a splice variant or allelic variant.

[0132] "Specific binding" refers to binding selectivity for an antigen and can be distinguished from unwanted or non-specific binding. The ability of an antigen binding molecule to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., surface plasmon resonance (SPR) technology and traditional binding assays. In one embodiment, for example, as measured by SPR, the degree of binding of an antigen binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen binding molecule to the antigen. In certain embodiments, the dissociation constant (Kd) of the molecule bound to the antigen is ≤1M, ≤100nM, ≤10nM, ≤1nM, ≤0.1nM, ≤0.01nM or ≤0.001nM (e.g., 10-7M or lower, e.g., 10-7M to 10-13M, e.g., 10-9M to 10-13M).

[0133] "Affinity" or "binding affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Binding affinity can typically be expressed in terms of the dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (Koff and Kon, respectively). Thus, equivalent affinities can include different rate constants as long as the ratio of the rate constants remains the same. Affinity can be measured by conventional methods known in the art, such as surface plasmon resonance (SPR).

[0134] The term "isolated" nucleic acid molecule or polynucleotide of the present invention refers to a nucleic acid molecule, DNA or RNA, that has been separated from its natural environment. In the present invention, a recombinant polynucleotide encoding a polypeptide contained in a vector is also isolated. Other examples of isolated polynucleotides include recombinant polynucleotides in heterologous host cells or polynucleotides purified in solution. Isolated polynucleotides include polynucleotide molecules that are normally contained in cells containing the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or in a chromosomal location different from its natural chromosomal location. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, in positive and negative strand forms, and in double-stranded form. The isolated polynucleotides or nucleic acids of the present invention further include such molecules that are synthetically generated. In addition, the polynucleotides or nucleic acids can be or can include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0135] As used herein, the term "connector" refers to a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) or a non-polypeptide, such as an alkyl chain. In some embodiments, two or more connection sons can be connected in series. When multiple connection sons are present, each connection son can be the same or different. In general, the connection son provides flexibility or prevents / improves steric hindrance. Connection sons are generally not cut; however, in some aspects, such cutting may be required. Therefore, in some embodiments, the connection son may include one or more protease cleavable sites, which may be located within the connection son sequence or on the connection son flanks at either end of the connection son sequence. In some embodiments, the connection son is a peptide connection son. In some embodiments, the peptide linker can comprise at least about two, at least about three, at least about four, at least about five, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 amino acids. In some embodiments, the linker comprises one or more amino acids. In some embodiments, the linker comprises a Gly-Ser (GS) linker. In some embodiments, the GS linker comprises (GS), wherein n is an integer between 1 and 10. In some embodiments, the GS linker comprises (GS), wherein n is an integer between 1 and 10. In some embodiments, the peptide linker is synthetic, i.e., non-naturally occurring. In one aspect, the peptide linker comprises a peptide (or polypeptide) (e.g., a naturally or non-naturally occurring peptide) comprising an amino acid sequence that links or genetically fuses a first linear amino acid sequence to a second linear amino acid sequence, wherein the first linear amino acid sequence is not naturally linked or genetically fused to the second linear amino acid sequence in nature. For example, in one aspect, the peptide linker can comprise a non-naturally occurring polypeptide that is a modified form of a naturally occurring polypeptide (e.g., comprising a mutation such as an addition, substitution, or deletion).

[0136] The terms "vector" or "expression vector" and "expression construct" of the present invention are used interchangeably to refer to DNA molecules that introduce a specific gene operably linked thereto into a target cell and direct its expression. The vectors include vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. The expression vectors of the present invention comprise an expression cassette. The expression vectors can transcribe large amounts of stable mRNA. Once the expression vector is within the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cell's transcription and / or translation machinery. The term "expression cassette" of the present invention refers to a recombinantly or synthetically produced polynucleotide having a series of nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. The recombinant expression cassette can be introduced into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.

[0137] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, and also include the progeny of such cells. Host cells include "transformants / transformants" and "transformed cells," including primary transformed cells and progeny derived therefrom. The nucleic acid of the progeny may not be completely identical to that of the parent cell and may contain mutations. Host cells are any type of cells that can be used to produce the antibodies or antigen-binding fragments thereof of the present invention. Host cells include cultured cells, for example, cultured mammalian cells, such as CHO cells, HEK293 cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, and also include transgenic animals, transgenic plants, or cells contained within cultured plant or animal tissues.

[0138] The term "antibody drug conjugate" or "ADC" refers to a binding protein (such as an antibody or an antibody or antigen-binding fragment thereof) chemically linked to one or more chemical drugs. In a preferred embodiment, the ADC comprises a binding protein, a drug, and a linker connecting the binding protein and the drug.

[0139] The term "chimeric antigen receptor" or "CAR" refers to a receptor with desired antigen specificity and signaling domains to propagate intracellular signals when antigens bind. For example, T lymphocytes recognize specific antigens via the interaction of T cell receptors (TCRs) with short peptides presented by class I or class II major histocompatibility complex (MHC) molecules. For initial activation and clonal expansion, initial T cells rely on antigen presenting cells (APCs) that provide additional costimulatory signals. In some embodiments, monocytes and macrophages can be engineered to, for example, express chimeric antigen receptors (CARs). Modified cells can be recruited to the tumor microenvironment, where they are used as powerful immune effectors by infiltrating tumors and killing target cancer cells. CAR may include an antigen binding domain, a transmembrane domain, and an intracellular domain. The antigen binding domain is bound to an antigen on a target cell. Examples of cell surface markers that can be used as antigens bound to the antigen binding domains of CAR include those associated with viruses, bacteria, parasitic infections, autoimmune diseases, and cancer cells (e.g., tumor antigens).

[0140] The term "modified immune cell" refers to an immune cell that has been genetically modified to express CAR. In some embodiments, the immune cell is a T cell, or a cell derived therefrom. In some embodiments, the immune cell is a natural killer (NK) cell, or a cell derived therefrom. In some embodiments, the immune cell is a B cell, or a cell derived therefrom. In some embodiments, the immune cell is a B cell, or a cell derived therefrom. In some embodiments, the immune cell is a B cell, or a cell derived therefrom. In some embodiments, the immune cell is a monocyte or a macrophage, or a cell derived therefrom.

[0141] The term "kit of parts" refers to a combination of one or more active ingredients in one unit. When there are multiple active ingredients in the kit of parts, the active ingredients can be administered simultaneously or sequentially.

[0142] The term "drug delivery device" refers to any means for administering a drug to a subject.

[0143] The term "prefilled syringe" refers to a syringe that has been loaded with medication before the operator of the syringe approaches or uses it. The prefilled syringe can be of any material (e.g., glass, plastic, or metal). In some embodiments, the prefilled syringe is a glass syringe.

[0144] An "effective amount" of a drug refers to the amount necessary to cause a physiological change in a cell or tissue to which it is administered. An "effective amount" encompasses an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0145] A "therapeutically effective amount" of a drug (e.g., a pharmaceutical composition) refers to an amount effective to achieve the desired therapeutic or preventive effect at a dosage and dosing interval and time. For example, a therapeutically effective amount of a drug eliminates, alleviates / reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0146] The term "individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, the individual or subject is a human.

[0147] The term "pharmaceutical composition" refers to a mixture containing one or more antibodies or antigen-binding fragments thereof disclosed herein, together with other chemical components, such as physiologically / pharmaceutically acceptable carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, thereby facilitating the absorption of the active ingredient and thereby exerting its biological activity.

[0148] The term "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers and / or preservatives.

[0149] The term "treatment" refers to administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or their antigen-binding fragments disclosed herein, or a nucleic acid molecule encoding the antibody or its antigen-binding fragment, to a patient suffering from one or more diseases or symptoms for which the therapeutic agent has a therapeutic effect. Typically, the therapeutic agent is administered to the patient or population being treated in an amount effective to alleviate the one or more diseases or symptoms, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable degree.

[0150] The term "detectable label" encompasses a label that is directly or indirectly detectable and is attached to the antibody or bispecific binding protein or present separately in the kit. Suitable labels include, but are not limited to, molecules detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Suitable labels include, but are not limited to, fluorescent dyes (e.g., GFT and its variants, FITC, TRITC, fluorescein, and rhodamine), electron-dense reagents (e.g., gold), enzymes (e.g., horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase), molecules containing radionuclides (i.e., radioisotopes), chemiluminescent molecules, electrochemiluminescent molecules, biotin, digoxigenin / digoxigenin, or haptens and other entities that are or can be made detectable. The antibodies, antigen-binding fragments thereof, or bispecific binding proteins of the present disclosure are detectably labeled by being attached to a "detectable label."

[0151] Example

[0152] Example 1. Preparation of Tau Antibodies

[0153] 1. Mouse Immunization

[0154] (1) Immunization of mice with immunogen Tau (1-45)

[0155] Immunogens Tau441 (purchased from Sigma) and Tau(1-45) (purchased from Sigma) were used to immunize 6-8 week old female Balb / C and C57BL / 6 mice housed under SPF conditions. For the primary immunization, Tau441 protein and Freund's complete adjuvant (purchased from Sigma) were mixed in a 1:1 ratio and emulsified. 0.3 mL of the mixture was then injected subcutaneously and intraperitoneally, yielding 100 μg of Tau441 per mouse. For the primary booster immunization, Tau(1-45) and Freund's incomplete adjuvant (purchased from Sigma) were mixed in a 1:1 ratio and emulsified. 0.3 mL of the mixture was then injected intraperitoneally, yielding 100 μg of Tau(1-45) per mouse. A two-week interval separated the primary and first booster immunizations, and a two-week interval separated each subsequent booster immunization. Tau441 and Tau(1-45) were mixed in a 1:1 ratio. Blood was collected one week after each booster immunization, and the antibody titers of the immunogens Tau441 and Tau (1-45) in the serum were detected by ELISA.

[0156] (2) Immunization of mice with immunogen Tau (187-208)

[0157] Immunizations with Tau441 (purchased from Sigma) and Tau(187-208)-KLH (hemocyanin) were performed on 6-8 week-old female Balb / C and C57BL / 6 mice housed under SPF conditions. For the primary immunization, 0.3 mL of a 1:1 mixture of Tau441 and Freund's complete adjuvant (purchased from Sigma) was injected subcutaneously at multiple sites and intraperitoneally, giving 100 μg of Tau441 per mouse. For the primary booster immunization, 0.3 mL of a 1:1 mixture of Tau(187-208)-KLH and Freund's incomplete adjuvant (purchased from Sigma) was injected intraperitoneally, giving 100 μg of Tau(187-208)-KLH per mouse. A two-week interval separated the primary immunization and the first booster immunization, and a two-week interval separated each subsequent booster immunization. Tau441 and Tau(187-208)-KLH were mixed in a 1:1 ratio. Blood was collected one week after each booster immunization, and serum antibody titers against Tau441 and Tau(187-208)-KLH were measured by ELISA.

[0158] (3) Immunization of mice with immunogen P181

[0159] Immunization with the immunogen P181-KLH (KLH-CKTPPAPK(pT)PPSSGEPPKS, SEQ ID NO:243) was performed on 6-8 week old female Balb / C and C57BL / 6 mice housed under SPF conditions. For the primary immunization, P181-KLH protein was emulsified in a 1:1 ratio with Freund's complete adjuvant (Sigma), and 0.3 mL of the mixture was injected subcutaneously at multiple sites and intraperitoneally, giving 100 μg of P181-KLH per mouse. For the primary booster immunization, P181-KLH was emulsified in a 1:1 ratio with Freund's incomplete adjuvant (Sigma), and 0.3 mL of the mixture was injected intraperitoneally, giving 100 μg of P181-KLH per mouse. A two-week interval was allowed between the primary and first booster immunizations, and a two-week interval was allowed between each subsequent booster immunization. Blood was collected one week after each booster immunization, and the antibody titers of P181-KLH and P181-BSA in the serum were detected by ELISA.

[0160] (4) Immunization of mice with immunogen P231

[0161] Immunization with the immunogen P231-KLH (KLH-KKVAVVR(pT)PPKSPSSAKC, SEQ ID NO:244) was performed on 6-8 week old female Balb / C and C57BL / 6 mice housed under SPF conditions. For the primary immunization, P231-KLH protein was emulsified in a 1:1 ratio with Freund's complete adjuvant (Sigma), and 0.3 mL of the mixture was injected subcutaneously at multiple sites and intraperitoneally, giving 100 μg of P231-KLH per mouse. For the primary booster immunization, P231-KLH was emulsified in a 1:1 ratio with Freund's incomplete adjuvant (Sigma), and 0.3 mL of the mixture was injected intraperitoneally, giving 100 μg of P231-KLH per mouse. A two-week interval was allowed between the primary and first booster immunizations, and a two-week interval was allowed between each subsequent booster immunization. Blood was collected one week after each booster immunization, and the antibody titers of P231-KLH and P231-BSA in the serum were detected by ELISA.

[0162] (5) Immunization of mice with immunogen P217

[0163] Immunization with the immunogen P217-KLH (KLH-CSRTPSLP(pT)PPTREPKKVA, SEQ ID NO:245) was performed on 6-8 week old female Balb / C and C57BL / 6 mice housed under SPF conditions. For the primary immunization, P217-KLH protein was emulsified in a 1:1 ratio with Freund's complete adjuvant (Sigma) and injected subcutaneously and intraperitoneally (0.3 mL, 100 μg per mouse). For the primary booster immunization, P217-KLH was emulsified in a 1:1 ratio with Freund's incomplete adjuvant (Sigma) and injected intraperitoneally (0.3 mL, 100 μg per mouse). A two-week interval was allowed between the primary and first booster immunizations, and a two-week interval was allowed between each subsequent booster immunization. Blood was collected one week after each booster immunization, and the antibody titers of P217-KLH and P217-BSA in the serum were detected by ELISA.

[0164] 2. Mouse titer detection

[0165] (1) Tau(1-45) titer detection in mice immunized

[0166] The ELISA method was used to detect the titer of the serum of immunized mice. The results in Figure 1 show that the sera of mice immunized with Tau441 and Tau (1-45) bound to the immunogens to varying degrees, exhibiting antigen-antibody reactions. The highest dilution gradient of the immunogen Tau441 was greater than 64K, and the highest dilution gradient of the immunogen Tau (1-45) was around 64K.

[0167] (2) Tau (187-208) titer detection in mice immunized

[0168] The ELISA method was used to detect the titer of the serum of immunized mice. The results in Figure 2 show that the sera of mice immunized with Tau441 and Tau (187-208) bound to the immunogens to varying degrees, exhibiting antigen-antibody reactions. The highest dilution gradient of the immunogen Tau441 was around 16K.

[0169] (3) P181-KLH titer detection in mice immunized with P181

[0170] The titer of the serum of immunized mice was detected by ELISA. The results in Figure 3 showed that the serum of mice immunized with P181-KLH had varying degrees of binding to the immunogen, showing antigen-antibody reactions. The highest dilution gradient of the immunogen P181-KLH was around 64K.

[0171] (4) P231-KLH titer detection in mice immunized with P231

[0172] The titer of the serum of immunized mice was detected by ELISA. The results in Figure 4 showed that the serum of mice immunized with P231-KLH had varying degrees of binding to the immunogen, exhibiting antigen-antibody reactions. The highest dilution gradient of the immunogen P231-KLH was around 64K.

[0173] (5) P217-KLH titer detection in mice immunized with P217-KLH

[0174] The titer of the serum of immunized mice was detected by ELISA. The results in Figure 5 showed that the serum of mice immunized with P217-KLH had varying degrees of binding to the immunogen, showing antigen-antibody reactions. The highest dilution gradient of the immunogen P217-KLH was around 64K.

[0175] 3. Fusion, Screening, and Subcloning

[0176] The selected mice were given two consecutive booster immunizations. Three to four days after the second booster immunization, the mice were sacrificed, spleen cells were collected, counted, and centrifuged at 400 g / min for 5 minutes. Appropriate amount of DMEM medium was added to resuspend the spleen cells and the density of spleen cells was adjusted to 1 × 10 8cells / mL. Plasma cells were screened out using a CD138 kit (purchased from STEMCELL). The screened plasma cells were mixed with mouse myeloma cells SP2 / 0 at a ratio of 1:5 in terms of the number of live cells, and then washed twice with DMEM medium at 400G / min for 5 minutes, and cell fusion was performed using the PEG fusion method. After fusion, centrifuge at 1000rpm / min for 7 minutes. The centrifuged cells were diluted into 400mL DMEM medium (purchased from Sigma) containing 20% ​​Australian fetal bovine serum (purchased from Gibco) and 1×HAT (purchased from Gibco). The percentages are by mass. Then 200μL per well was added to a 96-well cell culture plate and placed in a 5% CO2, 37°C incubator. The percentages are by volume. After 7-10 days, the supernatant of the cell fusion plate was screened using ELISA (coated with Tau441 protein), and the OD value in the ELISA was calculated. 450nm The positive clones with a p-value > 0.4 were supplemented with 100 μL of 400 mL of DMEM medium containing 20% ​​Australian fetal bovine serum and 1×HT (purchased from Gibco) and cultured overnight in a 5% CO2, 37°C incubator. The next day, the positive clones were rescreened by ELISA.

[0177] Based on the results of the secondary screening, positive hybridoma cells were selected for subcloning using a gradient dilution method in 96-well plates. Culture was performed in DMEM supplemented with 15% Australian fetal bovine serum in a 5% CO2, 37°C incubator. Seven to ten days after subcloning, screening was performed using ELISA (coated with Tau441 and Tau(1-45) proteins, respectively) to identify a single double-positive clone. Subcloning was continued for one to two more rounds.

[0178] After the third round of subcloning, the best monoclone was selected and expanded in DMEM medium containing 15% Australian fetal bovine serum in a 5% CO2, 37°C incubator. The hybridoma cells of the present invention were obtained by freezing in liquid nitrogen and can be used for subsequent antibody production and purification.

[0179] Example 2. Production and purification of Tau antibodies

[0180] Balb / C mice aged 6-8 weeks were purchased and housed under SPF conditions. Mice were pretreated with liquid paraffin (purchased from Xilong Science) one week in advance. Two mice pretreated with sensitizers were injected with each cell line, and each mouse was injected with (0.5-1)×10 6Hybridoma cells. Closely observe the growth of ascites and the health status of the mice starting around the 6th day after injection. When there is obvious mobile ascites in the mouse's abdominal cavity, start collecting the ascites. Use an alcohol cotton ball to wipe the needle insertion site (approximately 0.5-1 cm away from the right hind limb), and then insert a No. 12 syringe needle (purchased from KDL) into the mouse's abdominal cavity. Due to the pressure in the mouse's abdominal cavity, the ascites will flow out along the needle. The ascites that flows out is collected in a 50mL centrifuge tube (purchased from NEST). The collected ascites is immediately centrifuged at 4500 rpm for 5 minutes, the supernatant is taken, and stored in a -20°C refrigerator. It is collected about once every two days, and under normal circumstances, about 5ml can be collected from each mouse.

[0181] Hybridoma ascites was purified using a 1 mL protein A gravity column. The protein A column was equilibrated with 5 column volumes of equilibration buffer (PBS phosphate buffer, pH 7.2, purchased from Solebro). The mouse ascites fluid to be purified was thawed and 2 mL was removed. The fluid was centrifuged at 10,000 rpm / min for 15 min, and the supernatant was then filtered through a 0.22 μm filter. After the column was equilibrated, the ascites supernatant was loaded onto the protein A column. After loading, the protein A column was washed with equilibration buffer, which was 4 column volumes of protein A. The Tau antibody bound to the protein A column was eluted with eluent (0.1 M citric acid buffer, pH 3.0). The eluted antibody was collected and the pH was neutralized by adding 15% 2 M Tris-HCl buffer (pH 8.0). The percentages are by volume. The eluate was then ultrafiltered using a 30 kD ultrafiltration tube to replace the liquid for about 5 times. The replaced antibody was collected and stored at -80°C.

[0182] The purified antibodies were tested for protein concentration (Nanodrop), purity, and subtype. The results are shown in Table 1.

[0183] Table 1

[0184] Example 3. Tau antibody amino acid sequence determination

[0185] After the subclone culture supernatant in Example 1 was tested for antigen-antibody binding, 1×10 5 -1x10 6 Add 1 ml of Trizol to each hybridoma cell and mix well. Freeze the sample at -80°C or proceed directly to the following sequencing steps.

[0186] RNA extraction: Take 0.5ml of Trizol sample and transfer it to a 1.5ml centrifuge tube, add 100μL chloroform to each tube, shake vigorously on a shaker for 15 seconds, and let it stand at room temperature for 5 minutes; precool the centrifuge to 4℃, centrifuge the above sample at 12000g for 15 minutes, and after centrifugation, stratify. Use a pipette to carefully transfer the top transparent layer to a new 1.5ml centrifuge tube free of RNase and DNase, then add 250μL of isopropanol to each tube, mix it by inverting it upside down several times, and let it stand at room temperature for 10 minutes; centrifuge it at 12000g in a centrifuge precooled to 4℃ for 10 minutes, remove the supernatant and retain the precipitate; add 1ml 75% ethanol to each tube, invert it upside down several times to suspend the precipitate; centrifuge it at 7500g in a centrifuge precooled to 4℃ for 5 minutes, remove the supernatant and retain the precipitate; keep the centrifuge tube mouth open, let it dry at room temperature for 5 minutes, and add 10μL to each tube. Dissolve in DEPC-treated water and incubate at 55°C for 10 minutes to ensure complete dissolution of the RNA; centrifuge at 7500g for 5 minutes in a centrifuge precooled to 4°C, and carefully aspirate the supernatant into the same centrifuge tube to obtain total RNA; perform 1 μL of the above total RNA on 1% agarose gel electrophoresis to detect the integrity of the total RNA, and take 2 μL to measure the RNA concentration using a nucleic acid concentration meter.

[0187] Reverse transcription and PCR: Use immediately after total RNA extraction Reverse transcription was performed using a 1st strand cDNA kit (purchased from Takara) to minimize RNA degradation. Total RNA was primed with the Oligo dT Primer included in the kit and the reaction mixture was prepared in a 1.5 ml centrifuge tube according to the ratios shown in Table 2.

[0188] Table 2

[0189] Place the centrifuge tube containing the above reaction solution in a metal heater and react at 65°C for 5 minutes. After the reaction is completed, place it on ice for rapid cooling. Prepare the reaction solution in the above 1.5 ml centrifuge tube according to the ratio in Table 3 below.

[0190] Table 3

[0191] After slowly mixing, the centrifuge tube containing the reaction solution was placed in a metal heater, reacted at 45°C for 60 minutes, then at 75°C for 15 minutes, and cooled on ice to obtain cDNA after total RNA reverse transcription. The obtained cDNA was used as a template for two PCR reactions to amplify the light chain and heavy chain respectively. For the heavy chain, MH1 (ctt ccg gaa ttc SAR GTN MAG CTG SAG SAG TC, SEQ ID NO: 246) and IgG1 (gga aga tct ATA GAC AGATGG GGG TGT CGT TTT GGC, SEQ ID NO: 247) were used as primers. For the light chain, Kc (ggt gca tgc GGA TAC AGT TGG TGC AGC ATC, SEQ ID NO: 248) and Mk (gg gag ctc GAY ATT GTG MTS ACM CAR WCT MCA, SEQ ID NO: 249) were used as primers. The rest of the PCR reaction system was configured according to Table 4 below. Among them, in the primer, S is c or g; R is a or g; N is a, c, g or t; M is a or c; Y is c or t; W is a or t.

[0192] Table 4

[0193] After configuration, perform PCR reaction according to Table 5 below:

[0194] Table 5

[0195] After the reaction, 2 μL of PCR product was subjected to 1% agarose gel electrophoresis to determine whether the amplification was successful. The PCR reaction solution with obvious amplification bands was recovered using DNA recovery reagent (purchased from Tiangen) to obtain the amplified fragments.

[0196] Cloning and sequencing: The purified light and heavy chain amplified bands were subjected to TA cloning using the pMDTM19-T Vector Cloning Kit (purchased from Takara). 2.5 μL of Solution I in the kit was mixed with 0.5 μL of the purified fragment, 0.5 μL of pMD T vector, and 1.5 μL of sterile water, and incubated at 4°C for 30 min. A DH5a competent cell (purchased from Kangti Life) was taken and allowed to stand on ice for 10 minutes before adding the above mixture. After further standing for 30 minutes, heat shock was performed in a 42°C water bath for 1 minute, and then quickly placed on ice. 1 ml of LB liquid medium was added and cultured with shaking at 37°C and 220 rpm for 1 hour. The cell was then plated onto a solid LB plate containing 50 μg / ml carbenicillin and cultured overnight until monoclonal bacteria grew. Three monoclonal colonies were picked and sent for sequencing. Sequencing was performed using the pMD19T sequencing primer GAGCGGATAACAATTTCACACAGG (SEQ ID NO: 250) to obtain the light and heavy chain DNA sequences, and thus the antibody amino acid sequence.

[0197] Recombinant expression: 1) Cell preparation: Thaw and resuscitate the 293F cells used and culture them in suspension in a 37°C 5% CO2 incubator. Adjust the shaker speed according to the shaker model (about 85-120 rpm). Monitor the cell growth at the same time. When the cell density reaches 4-6×10 6 Cells / mL, with a viability of more than 95%, were subcultured; generally, newly recovered cells needed to be subcultured 2-3 times before being used for expression, with a subculture density of 0.6×10 6 Cultured for 3 days, 0.3×10 6 Culture for 4 days. The specific situation can be adjusted according to the cells and needs. The expression density is generally 2×10 6 2) Expression: Using a 25 mL transfection system as an example, prepare two 15 mL sterile centrifuge tubes. Add 1 mL of transfection buffer and 25 μg of plasmid to tube 1 (culture medium or a dedicated transfection buffer can be used as the transfection buffer). Add 1 mL of transfection buffer and 125 μL of transfection reagent (PEI) to tube 2. Mix the solutions in the two centrifuge tubes and let them stand at room temperature for 10 minutes. Then, slowly add the mixture to the prepared cells (2 × 10 6cells / mL, viability >95%); and place the cells in an incubator for culture (incubator parameters are the same as above). After 24 hours of culture, add feed, 0.25mL of 100× feed. (Note: the final concentration of the plasmid is 1μg / mL, and the mass ratio of plasmid to transfection reagent is 1:3-1:6). 3) Expression level detection: After 5-7 days of culture, SDS-PAGE can be used to run the gel to check the expression level. 4) After the expression is completed, centrifuge it at 10,000 rpm / min and filter it with a 0.22μm filter membrane; 5) Perform subsequent purification work. If not purified, freeze it in a -80 refrigerator.

[0198] Antibody purification: Purify using a 1ml protein A gravity column. The protein A column is equilibrated with 5 column volumes of equilibration buffer (PBS phosphate buffer, pH 7.2, purchased from Solebro). Centrifuge the expression supernatant to be purified at 12,000 rpm for 30 minutes, carefully aspirate the supernatant and filter it with a 0.22 μm filter membrane. After the column is equilibrated, load the ascites supernatant onto the protein A column. After loading, wash the protein A column with equilibration buffer, which is 4 column volumes of the protein A column. Elute the antibody bound to the protein A column with eluent (0.1 M citric acid buffer, pH 3.0). Collect the eluted antibody and add 15% 2M Tris-HCl buffer (pH 8.0) to neutralize the pH. The percentages are volume percentages. Then, use a 30KD ultrafiltration tube to ultrafilter the eluate to replace the liquid, replacing it about 5 times in total. Collect the replaced antibodies and store them at -80°C. The purified antibodies are tested for protein concentration (Nanodrop) and purity.

[0199] The variable region sequencing results are shown in Table 6 below.

[0200] Example 4. Identification of Antibodies

[0201] Tau (1-22) and Tau phosphorylated peptides containing the following sequences were synthesized as antibody detection antigens:

[0202] pT181-N22:MAEPRQEFEVMEDHAGTYGLGDKTPPAPK(pT)PPSSGEPPKS(SEQ ID NO:251);

[0203] pT217-N22:MAEPRQEFEVMEDHAGTYGLGDSRTPSLP(pT)PPTREPKKVA(SEQ ID NO:252);

[0204] pT231-N22:MAEPRQEFEVMEDHAGTYGLGDKKVAVVR(pT)PPKSPSSAK (SEQ ID NO:253).

[0205] 1. Binding experiments between antibodies and antigens with different peptides

[0206] (1) Binding experiments of Tau (1-45) antibodies and different peptides

[0207] The purified Tau (1-45) antibody from Example 3 was tested for binding to Tau441 (UniProt: #P10636-8), Tau441 (1-45), Tau441 (1-22), Tau441 (12-34), and Tau441 (23-44) using ELISA. First, 100 μL of 0.1 μg / ml Tau441 peptide fragments were coated onto an ELISA plate (purchased from NEST) and incubated overnight at 4°C. The next day, 150 μL of blocking solution was added to each well and blocked in a 37°C incubator for 1.5 hours. Subsequently, 100 μL of 5 μg / ml Tau (1-45) antibody was added to each well and incubated at 37°C for 1 hour. After 1 hour, 100 μL of anti-mouse enzyme-labeled secondary antibody (purchased from Jackson) was added to each well and incubated at 37°C for 1 hour. After incubation, wash four times with PBST (Solebol) and pat dry. Add 100 μL of one-component TMB colorimetric solution (Solebol) to each well and develop at room temperature for 5-10 minutes. Finally, add 50 μL of stop solution (Solebol) to each well and measure the OD40 on a microplate reader.

[0208] The binding results of each Tau (1-45) antibody with different peptides are shown in Figure 6, showing that antibodies 4#, 5#, 6#, 8#, 9#, 10#, 11#, 12#, 14#, 16#, 17#, 18#, 19#, 22#, 24#, 28#, 30#, 38#, 41#, and 42# bind to amino acids 1-22 of N-Tau, antibodies 19#, 22#, 26#, 27#, 28#, 29#, 31#, 32#, 37#, 38#, 39#, 40#, 41#, 42#, 43#, 44#, 47#, 48#, and 49# bind to amino acids 12-34 of N-Tau, and antibodies 7#, 13#, 27#, 28#, and 29# bind to amino acids 23-44 of N-Tau.

[0209] (2) Binding experiments of Tau (187-208) antibodies with different peptides

[0210] Purified Tau (187-208) antibodies were tested for binding to Tau441, Tau (1-45), Tau441 (1-22), Tau441 (12-34), and Tau441 (23-44) using ELISA. First, 100 μL of 0.1 μg / mL Tau441 peptide fragments were coated onto an ELISA plate (purchased from NEST) and incubated overnight at 4°C. The next day, 150 μL of blocking solution was added to each well and blocked in a 37°C incubator for 1.5 hours. Subsequently, 100 μL of 5 μg / mL Tau (187-208) antibody was added to each well and incubated at 37°C for 1 hour. After 1 hour, 100 μL of anti-mouse enzyme-linked secondary antibody (purchased from Jackson) was added to each well and incubated at 37°C for 1 hour. After incubation, the plates were washed four times with PBST (purchased from Solebro) and patted dry. Add 100 μL of TMB colorimetric solution (purchased from Solebro) to each well and develop at room temperature for 5-10 minutes. Finally, add 50 μL of stop solution (purchased from Solebro) to each well to stop the reaction and measure OD40 on a flow cytometer.

[0211] The results are shown in FIG7 , which show that all Tau (187-208) antibodies bind to amino acids 1-22 of N-Tau.

[0212] (3) Binding experiments of Tau (187-208) antibodies with different peptides

[0213] Purified Tau (187-208) antibodies were tested for binding to Tau441, Tau (1-45), Tau441 (1-22), Tau441 (151-441), and Tau441 (187-208) using ELISA. First, 100 μL of 0.1 μg / mL Tau441 peptide fragments were coated onto an ELISA plate (purchased from NEST) and incubated overnight at 4°C. The next day, 150 μL of blocking solution was added to each well and blocked in a 37°C incubator for 1.5 hours. Subsequently, 100 μL of 5 μg / mL Tau (187-208) antibody was added to each well and incubated at 37°C for 1 hour. After 1 hour, 100 μL of anti-mouse enzyme-linked secondary antibody (purchased from Jackson) was added to each well and incubated at 37°C for 1 hour. After incubation, the plates were washed four times with PBST (purchased from Solebro) and patted dry. Add 100 μL of TMB colorimetric solution (purchased from Solebro) to each well and develop at room temperature for 5-10 minutes. Finally, add 50 μL of stop solution (purchased from Solebro) to each well to stop the reaction and measure OD40 on a flow cytometer.

[0214] The results are shown in FIG8 . Antibodies 70# and 75# specifically bind to the Tau (187-208) site.

[0215] (4) Binding experiments of P181 antibody with different peptides

[0216] The purified P181 antibody was tested for binding to Tau441, P181-KLH, pT181-N22, and BSA using ELISA. First, 100 μL of 0.1 μg / mL Tau441 peptide fragments were coated onto an ELISA plate (purchased from NEST) and refrigerated overnight at 4°C. The next day, 150 μL of blocking buffer was added to each well and blocked in a 37°C incubator for 1.5 hours. Subsequently, 100 μL of 5 μg / mL P181 antibody was added to each well and incubated at 37°C for 1 hour. After 1 hour, 100 μL of anti-mouse enzyme-linked secondary antibody (purchased from Jackson) was added to each well and incubated at 37°C for 1 hour. After incubation, the plates were washed four times with PBST (purchased from Solebro) and patted dry. 100 μL of one-component TMB colorimetric solution (purchased from Solebro) was added to each well and developed at room temperature for 5-10 minutes. Finally, 50 μL of stop solution (purchased from Solebro) was added to each well to stop the cell culture, and the OD40 was detected by flow cytometer.

[0217] The results are shown in FIG9 , and both antibodies 1# and 2# specifically bind to the P181 phosphorylation site.

[0218] (5) Binding experiments of P217 antibody with different peptides

[0219] The purified P217 antibody was tested for binding to Tau441, P217-KLH, pT217-N22, and BSA using ELISA. First, 100 μL of 0.1 μg / mL Tau441 peptide fragments were coated onto an ELISA plate (purchased from NEST) and refrigerated overnight at 4°C. The next day, 150 μL of blocking buffer was added to each well and blocked in a 37°C incubator for 1.5 hours. Subsequently, 100 μL of 5 μg / mL P217 antibody was added to each well and incubated at 37°C for 1 hour. After 1 hour, 100 μL of anti-mouse enzyme-linked secondary antibody (purchased from Jackson) was added to each well and incubated at 37°C for 1 hour. After incubation, the plates were washed four times with PBST (purchased from Solebro) and patted dry. 100 μL of one-component TMB colorimetric solution (purchased from Solebro) was added to each well and developed at room temperature for 5-10 minutes. Finally, 50 μL of stop solution (purchased from Solebro) was added to each well to stop the cell culture, and the OD40 was detected by flow cytometer.

[0220] The results are shown in Figure 10. Only antibody #6 specifically binds to the P217 phosphorylation site, while antibodies #1, #11, #19, and #24 bind to the full length of Tau441.

[0221] (6) Binding experiments of P231 antibody with different peptides

[0222] The purified P231 antibody was tested for binding to Tau441, P231-KLH, pT231-N22, and BSA using ELISA. First, 100 μL of 0.1 μg / mL Tau441 peptide fragments were coated onto an ELISA plate (purchased from NEST) and refrigerated overnight at 4°C. The next day, 150 μL of blocking buffer was added to each well and blocked in a 37°C incubator for 1.5 hours. Subsequently, 100 μL of 5 μg / mL P231 antibody was added to each well and incubated at 37°C for 1 hour. After 1 hour, 100 μL of anti-mouse enzyme-linked secondary antibody (purchased from Jackson) was added to each well and incubated at 37°C for 1 hour. After incubation, the plates were washed four times with PBST (purchased from Solebro) and patted dry. 100 μL of one-component TMB colorimetric solution (purchased from Solebro) was added to each well and developed at room temperature for 5-10 minutes. Finally, 50 μL of stop solution (purchased from Solebro) was added to each well to stop the cell culture, and the OD40 was detected by flow cytometer.

[0223] The results are shown in Figure 11. Antibodies 2#, 3#, 6#, 7#, 8#, 9#, 11#, 12# and 14# specifically bind to the P231 phosphorylation site, and 5# binds to the full length of Tau441.

[0224] 2. Affinity Determination of Tau Antibodies and Human Tau441

[0225] Purified tau antibodies were tested for binding affinity to human tau441 using ELISA. First, 100 μL of 0.1 μg / mL tau441 peptide fragments were coated onto an ELISA plate (NEST) and refrigerated overnight at 4°C. The next day, 150 μL of blocking buffer was added to each well and blocked in a 37°C incubator for 1.5 hours. Tau antibodies were serially diluted in PBS buffer, and 100 μL of antibody was added to each well and incubated at 37°C for 1 hour. After 1 hour, 100 μL of anti-mouse enzyme-linked secondary antibody (Jackson) was added to each well and incubated at 37°C for 1 hour. After incubation, the plate was washed four times with PBST (Solebol) and patted dry. 100 μL of one-component TMB colorimetric solution (Solebol) was added to each well and developed at room temperature for 5-10 minutes. Finally, 50 μL of stop solution (Solebol) was added to each well, and the OD40 value was measured on an analyzer.

[0226] The result is as follows:

[0227] (1) The affinity determination results of Tau(1-45) antibody and human Tau441 are shown in Figure 12. Except for antibodies 22# and 49#, the affinities of other antibodies reached the nM level.

[0228] (2) The affinity determination results of Tau (187-208) antibody and human Tau441 are shown in FIG13 . The affinity of the antibodies reaches the nM level.

[0229] 3. Determination of Tau Antibody Affinity Constants

[0230] Affinity constants were determined using a high-throughput molecular interaction instrument (purchased from GE). Specific operations and methods were based on the instrument manual and the detailed methods provided by the manufacturer. Specifically, affinity determination was performed using a Protein A chip (purchased from Cytiva). First, the purified antibody was diluted to 10 μg / ml with 1× HBS-EP Buffer (purchased from Cytiva). The antigen Tau441 solution was diluted with 1× HBS-EP Buffer to the following concentrations: 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM.

[0231] The surface plasmon resonance principle of a high-throughput molecular interaction instrument is used to detect the binding and dissociation of antibodies and immunogens. After the run is completed, the curve is drawn and fitted using the accompanying software to obtain the ka, kd, and KD values.

[0232] The results are shown in Table 7 below, indicating that the affinity of the antibodies reached the nM level.

[0233] Table 7

[0234] Example 5. Antibody pair screening

[0235] 1. Antibody pair screening using ELISA

[0236] 100 μL of 2 μg / ml capture antibody (binding to Tau (1-22)) was coated onto an ELISA plate (purchased from NEST) and refrigerated overnight at 4°C. The next day, 150 μL of blocking solution was added to each well and blocked in a 37°C incubator for 1.5 hours. Subsequently, 100 μL of 25 ng / ml Tau441, pT181-N22, pT231-N22, or 1 μg / ml pT217-N22 antigen was added to each well and incubated at 37°C for 1 hour. After 1 hour, 100 μL of 2 μg / ml detection antibody (binding to Tau (187-208), p231, p217, or p181) was added and incubated at 37°C for 1 hour. After 1 hour, 100 μL of anti-biotin enzyme-labeled secondary antibody (purchased from Jackson) was added to each well and incubated at 37°C for 1 hour. After incubation, wash four times with PBST (Solebol) and pat dry. Add 100 μL of one-component TMB colorimetric solution (Solebol) to each well and develop at room temperature for 5-10 minutes. Finally, add 50 μL of stop solution (Solebol) to each well and measure the OD450 value on a microplate reader.

[0237] Antigen preparation:

[0238] pT181-N22:MAEPRQEFEVMEDHAGTYGLGDKTPPAPKTPPSSGEPPKS (SEQ ID NO: 251);

[0239] pT217-N22:MAEPRQEFEVMEDHAGTYGLGDSRTPSLPTPPTREPKKVA (SEQ ID NO: 252);

[0240] pT231-N22: MAEPRQEFEVMEDHAGTYGLGDKKVAVVRTPPKSPSSAK (SEQ ID NO: 253).

[0241] (1) Screening of paired antibodies that bind to the middle end of Tau and antibodies that bind to Tau (1-22)

[0242] The ELISA sandwich method was used to pair the Tau antibodies binding to the middle end (Tau-70# / Tau-59# / Tau-75# / Tau-88#; P217-1# / P217-11# / P217-19# / P217-24#; P231-5# / P231-20#) with the antibodies binding to N-Tau (1-22) for screening.

[0243] The results are shown in Figures 14-16, which show that when the concentration of Tau441 antigen is 25 ng / ml, the pairing results of most antibody pairs are good.

[0244] (2) Screening of paired antibodies binding to phosphorylated P181 and antibodies binding to Tau (1-22)

[0245] The antibody binding to phosphorylated P181 was paired with the antibody binding to N-Tau (1-22) for screening using the sandwich ELISA method.

[0246] The results are shown in FIG17 , which show that when the concentration of pT181-N22 antigen is 25 ng / ml, the pairing effect of the antibody binding to phosphorylated P181-1# and the antibody binding to N-Tau (1-22) is better than that of the antibody binding to P181-2#.

[0247] (3) Screening of paired antibodies binding to phosphorylated P217 and antibodies binding to Tau (1-22)

[0248] ELISA sandwich method was used to screen the antibodies binding to phosphorylated P217 and N-Tau (1-22).

[0249] The results are shown in FIG18 , which show that when the concentration of pT217-N22 antigen was 1 μg / ml, the pairing results of most antibody pairs were good.

[0250] (4) Screening of paired antibodies binding to phosphorylated P231 and antibodies binding to Tau (1-22)

[0251] The ELISA sandwich method was used to screen the antibodies binding to phosphorylated P231 and those binding to N-Tau (1-22).

[0252] The results are shown in Figures 19-22, which show that when the concentration of pT231-N22 antigen is 25 ng / ml, the pairing effect of the antibody binding to phosphorylated P231-7# and the antibody binding to N-Tau (1-22) is better than the pairing effect of the other antibodies.

[0253] 2. Detection limit of antibody pair

[0254] Based on the antibody pairing results from the previous step, select the optimal antibody pair and determine the detection limit of the antibody pair using ELISA. Coat 100 μL of 0.2 μg / mL antibody onto an ELISA plate (NEST) and refrigerate overnight at 4°C. The next day, add 150 μL of blocking solution to each well and block in a 37°C incubator for 1.5 hours. Tau antigen is serially diluted in PBS buffer, followed by 100 μL of antibody per well and incubation at 37°C for 1 hour. After 1 hour, add 100 μL of anti-mouse enzyme-linked secondary antibody (Jackson) per well and incubate at 37°C for 1 hour. After incubation, wash four times with PBST (Solebol) and pat dry. Add 100 μL of one-component TMB colorimetric solution (Solebol) per well and develop at 37°C for 5-10 minutes. Finally, add 50 μL of stop solution (Solebol) per well and measure the OD40 on an analyzer.

[0255] The result is as follows:

[0256] (1) The results of paired screening of antibodies binding to the middle end of Tau-55# and antibodies binding to Tau (1-22) are shown in Figure 23, which shows that the detection limits of the six antibody pairs tested (Tau-55# and Tau5#, Tau-55# and Tau6#, Tau-55# and Tau11#, Tau-55# and Tau14#, Tau-55# and Tau32#, Tau-55# and Tau48#) are between 25 and 100 pg / ml.

[0257] (2) The results of paired screening of antibodies binding to the middle end of Tau-70# and antibodies binding to Tau (1-22) are shown in Figure 24, which shows that the detection limits of the six antibody pairs tested (Tau-70# and Tau5#, Tau-70# and Tau6#, Tau-70# and Tau11#, Tau-70# and Tau14#, Tau-70# and Tau32#, Tau-70# and Tau48#) are between 6.25 and 3.125 pg / ml.

[0258] (3) The results of paired screening of antibodies binding to the middle end P217-19# and antibodies binding to Tau (1-22) are shown in Figure 25, which shows that the detection limits of the six antibody pairs tested (P217-19# and Tau5#, P217-19# and Tau6#, P217-19# and Tau11#, P217-19# and Tau14#, P217-19# and Tau32#, P217-19# and Tau48#) are all less than 3.125 pg / ml.

[0259] (4) The results of paired screening of antibodies binding to the middle end P231-5# and antibodies binding to Tau (1-22) are shown in FIG26 , which shows that the detection limits of the six antibody pairs tested (P231-5# and Tau5#, P231-5# and Tau6#, P231-5# and Tau11#, P231-5# and Tau14#, P231-5# and Tau32#, P231-5# and Tau48#) are between 100 and 200 pg / ml.

[0260] (5) The results of paired screening of antibodies binding to phosphorylated P217-6# and antibodies binding to Tau (1-22) are shown in FIG27 , which shows that the detection limits of the 10 antibody pairs tested (P217-6# and Tau6#, P217-6# and Tau14#, P217-6# and Tau50#, P217-6# and Tau52#, P217-6# and Tau57#, P217-6# and Tau61#, P217-6# and Tau62#, P217-6# and Tau63#, P217-6# and Tau65#, P217-6# and Tau78#) are between 0.44 and 59.6 pg / ml.

[0261] (6) The results of paired screening of antibodies binding to phosphorylated P231-7# and antibodies binding to Tau (1-22) are shown in FIG28 , which shows that the detection limits of the eight antibody pairs tested (P231-7# and Tau11#, P231-7# and Tau24#, P231-7# and Tau57#, P231-7# and Tau61#, P231-7# and Tau62#, P231-7# and Tau63#, P231-7# and Tau65#, P231-7# and Tau78#) are between 0.15 and 4.35 pg / ml.

[0262] (7) The results of paired screening of antibodies binding to phosphorylated P181-1# and antibodies binding to Tau (1-22) are shown in FIG29 , which shows that the detection limits of the eight antibody pairs tested (P181-1# and Tau11#, P181-1# and Tau24#, P181-1# and Tau50#, P181-1# and Tau57#, P181-1# and Tau61#, P181-1# and Tau62#, P181-1# and Tau65#) are between 0.44 and 59.6 pg / ml.

[0263] 3. Antibody Pair Screening - Simoa Platform

[0264] Antibody pair screening was performed on a fully automated Simoa HD-X analyzer (Quanterix): (1) The capture antibody was coupled to homemade carboxylated paramagnetic beads. The capture antibody buffer was exchanged into bead coupling buffer (50 mM 2-(N-morpholino)ethanesulfonic acid) using a 0.5 ml centrifugal filter device. The magnetic beads were activated with freshly prepared EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) in cold bead coupling buffer for 30 min, and the activated magnetic beads were then coupled to the capture antibody for 2 h, followed by blocking and washing. (2) Biotinylation of the detection antibody. The detection antibody was exchanged into biotinylation reaction buffer using a 0.5 ml centrifugal filter device, and NHS-PEG 4-biotin (Quanterix) was added at a 40-fold molar ratio of biotin to the antibody for 30 min, followed by washing. The magnetic bead-coupled capture antibody and biotinylated detection antibody were then stored at 4°C until use. (3) Appropriate dilutions of capture and detection antibodies and streptavidin-β-galactosidase (SBG) were prepared and transferred to plastic bottles. (4) Calibrators were prepared by serial dilution to the required concentration using detector / sample diluent and then loaded onto a 96-well plate. These reagents were then loaded onto the Simoa HD-X analyzer. The operating procedures were developed according to the experimental design. (5) After the experiment was completed, the data were extracted and the signal was quantified as average enzyme per bead (AEB) units. The standard curve was plotted using GraphPad Prism 9.5.1 software using a 4-parameter logistic (4PL) function with a weighting factor of 1 / y2. Tau levels in the samples were then inferred from the calibration curve. Coefficient of variation (CV) values ​​below 20% were considered acceptable.

[0265] The antibody pairs described in Table 8 were used for detection on the Simoa platform, and the results are shown in Figures 30 and 31. The detection limits of the five antibody pairs tested were between 0.5 and 50 pg / ml.

[0266] Table 8

[0267] Example 6 pT231, pT217, pT181 Standards

[0268] 1. Synthesis of pT231, pT217, and pT181 Standards

[0269] Tau (1-22) and Tau phosphorylated peptides were synthesized as standards for ELISA assays of Tau antibodies.

[0270] pT181-N22:MAEPRQEFEVMEDHAGTYGLGDKTPPAPK(pT)PPSSGEPPKS(SEQ ID NO:251);

[0271] pT217-N22:MAEPRQEFEVMEDHAGTYGLGDSRTPSLP(pT)PPTREPKKVA(SEQ ID NO:252);

[0272] pT231-N22:MAEPRQEFEVMEDHAGTYGLGDKKVAVVR(pT)PPKSPSSAK (SEQ ID NO:253).

[0273] The HPLC chromatograms of the three standards, pT181-N22, pT217-N22, and pT231-N22, are shown in Figure 32 , demonstrating that the three synthesized standards are of high purity. The MALDI-TOF-MS mass spectra of the three standards, pT181-N22, pT217-N22, and pT231-N22, are shown in Figures 33-35 , demonstrating that the three synthesized standards have the correct molecular weight and sequence.

[0274] 100 μL of 0.1 μg / mL Tau441GSK 3beta-phosphorylated, pT231-N22, pT217-N22, and pT181-N22 were coated onto ELISA plates (purchased from NEST) and refrigerated overnight at 4°C. The next day, 150 μL of blocking solution was added to each well and blocked in a 37°C incubator for 1.5 hours. Subsequently, 100 μL of 5 μg / mL antibody was added to each well and incubated at 37°C for 1 hour. After 1 hour, 100 μL of anti-mouse enzyme-linked secondary antibody (purchased from Jackson) was added to each well and incubated at 37°C for 1 hour. After incubation, the plates were washed four times with PBST (purchased from Solebro) and patted dry. 100 μL of one-component TMB colorimetric solution (purchased from Solebro) was added to each well and developed at room temperature for 5-10 minutes. Finally, 50 μL of stop solution (purchased from Solebro) was added to each well to stop the cell culture, and the OD450 was detected by flow cytometer.

[0275] The results are shown in FIG36 , which show that the binding effects of the antibodies P231, P217, and P181 to the pT231-N22, pT217-N22, and pT181-N22 antigens were significantly higher than those to the synthetic Tau441GSK 3beta-phosphorylated antigen.

[0276] 2. ELISA to determine the affinity of Tau antibodies

[0277] 100 μL of 0.1 μg / mL Tau441GSK 3beta-phosphorylated, pT231-N22, pT217-N22, and pT181-N22 were coated onto ELISA plates (purchased from NEST) and refrigerated overnight at 4°C. The next day, 150 μL of blocking buffer was added to each well and blocked in a 37°C incubator for 1.5 hours. Subsequently, 100 μL of antibody (10 μg / mL, 10-fold dilution, 7 dilutions total) was added to each well and incubated at 37°C for 1 hour. After 1 hour, 100 μL of anti-mouse enzyme-linked secondary antibody (purchased from Jackson) was added to each well and incubated at 37°C for 1 hour. After incubation, the plates were washed four times with PBST (purchased from Solebro) and patted dry. 100 μL of one-component TMB colorimetric solution (purchased from Solebro) was added to each well and developed at room temperature for 5-10 minutes. Finally, 50 μL of stop solution (purchased from Solebro) was added to each well to stop the cell culture, and the OD450 was detected by flow cytometer.

[0278] The results are shown in FIG37 , which are similar to those in FIG36 , and also indicate that the binding effects of the antibodies P231, P217, and P181 to the pT231-N22, pT217-N22, and pT181-N22 antigens were significantly higher than those to the synthetic Tau441GSK 3beta-phosphorylated antigen.

[0279] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to Tau and / or a fragment thereof, comprising at least one heavy chain variable region and at least one light chain variable region, The heavy chain variable region comprises: HCDR1 as shown in any one of SEQ ID NOs: 121-145; HCDR2 as shown in any one of SEQ ID NOs: 146-174; and / or As shown in any one of SEQ ID NOs: 175-196 or the amino acid sequence is HCDR3 of AR; and / or The light chain variable region comprises: LCDR1 as shown in any one of SEQ ID NOs: 197-218; and / or LCDR2 having an amino acid sequence of AAS, DTS, FAT, KLS, KVS, LAS, LVS, RMS, RVS, TVS or WAS; and / or The LCDR3 as shown in any one of SEQ ID NOs: 219-242.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region comprises: HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 121, SEQ ID NO: 146 and SEQ ID NO: 175, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 122, SEQ ID NO: 147 and SEQ ID NO: 176, respectively; or, SEQ ID NO: 123, SEQ ID NO: 148 and HCDR1, HCDR2 and HCDR3 of the amino acid sequence AR, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 124, SEQ ID NO: 149 and SEQ ID NO: 177, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 125, SEQ ID NO: 150 and SEQ ID NO: 178, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 126, SEQ ID NO: 151 and SEQ ID NO: 179, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 126, SEQ ID NO: 152 and SEQ ID NO: 179, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 126, SEQ ID NO: 167 and SEQ ID NO: 185, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 127, SEQ ID NO: 153 and SEQ ID NO: 180, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 128, SEQ ID NO: 154 and SEQ ID NO: 181, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 129, SEQ ID NO: 154 and SEQ ID NO: 181, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 130, SEQ ID NO: 155 and SEQ ID NO: 182, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 130, SEQ ID NO: 156 and SEQ ID NO: 182, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 130, SEQ ID NO: 157 and SEQ ID NO: 182, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 130, SEQ ID NO: 158 and SEQ ID NO: 182, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 131, SEQ ID NO: 157 and SEQ ID NO: 183, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 132, SEQ ID NO: 159 and SEQ ID NO: 184, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 132, SEQ ID NO: 160 and SEQ ID NO: 184, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 133, SEQ ID NO: 161 and SEQ ID NO: 185, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 134, SEQ ID NO: 162 and SEQ ID NO: 186, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 135, SEQ ID NO: 163 and SEQ ID NO: 187, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 136, SEQ ID NO: 164 and SEQ ID NO: 188, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 137, SEQ ID NO: 165 and SEQ ID NO: 189, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 138, SEQ ID NO: 166 and SEQ ID NO: 190, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 139, SEQ ID NO: 168 and SEQ ID NO: 190, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 140, SEQ ID NO: 169 and SEQ ID NO: 191, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 141, SEQ ID NO: 170 and SEQ ID NO: 192, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 142, SEQ ID NO: 171 and SEQ ID NO: 193, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 143, SEQ ID NO: 172 and SEQ ID NO: 194, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 144, SEQ ID NO: 173 and SEQ ID NO: 195, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 145, SEQ ID NO: 174 and SEQ ID NO: 196, respectively; and / or The light chain variable region comprises: LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 197, the amino acid sequence is AAS and as shown in SEQ ID NO: 220, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 197, the amino acid sequence of KVS and SEQ ID NO: 219, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 197, the amino acid sequence of KVS and SEQ ID NO: 235, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 197, the amino acid sequence of KLS and SEQ ID NO: 219, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 198, the amino acid sequence is AAS and as shown in SEQ ID NO: 220, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 199, the amino acid sequence of RVS and SEQ ID NO: 221, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 200, the amino acid sequence of KVS and SEQ ID NO: 222, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 200, the amino acid sequence of TVS and SEQ ID NO: 225, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 200, the amino acid sequence of TVS and SEQ ID NO: 240, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 201, the amino acid sequence of WAS and SEQ ID NO: 223, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 202, the amino acid sequence of KVS and SEQ ID NO: 219, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 202, the amino acid sequence of LVS and SEQ ID NO: 224, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 202, the amino acid sequence of RVS and SEQ ID NO: 224, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 203, the amino acid sequence of KVS and SEQ ID NO: 219, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 203, the amino acid sequence of WAS and SEQ ID NO: 226, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 203, the amino acid sequence of WAS and SEQ ID NO: 227, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 204, the amino acid sequence of WAS and SEQ ID NO: 228, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 205, the amino acid sequence as DTS and SEQ ID NO: 229, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 206, the amino acid sequence as DTS and SEQ ID NO: 229, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 206, the amino acid sequence of WAS and SEQ ID NO: 228, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 207, the amino acid sequence of LVS and SEQ ID NO: 230, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 209, the amino acid sequence as DTS and SEQ ID NO: 231, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 210, the amino acid sequence of RMS and SEQ ID NO: 232, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 210, the amino acid sequence of RMS and SEQ ID NO: 233, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 211, the amino acid sequence of LAS and SEQ ID NO: 234, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 212, the amino acid sequence of LVS and SEQ ID NO: 230, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 213, the amino acid sequence of FAT and SEQ ID NO: 236, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 214, the amino acid sequence as DTS and SEQ ID NO: 237, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 215, the amino acid sequence as DTS and SEQ ID NO: 238, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 216, the amino acid sequence of WAS and SEQ ID NO: 239, respectively; or LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 217, the amino acid sequence of LAS and SEQ ID NO: 241, respectively; or The LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 218, the amino acid sequence is LVS and the LCDR2 is shown in SEQ ID NO: 242 respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:121, SEQ ID NO:146 and SEQ ID NO:175, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:197, the amino acid sequence of KVS and SEQ ID NO:219, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:121, SEQ ID NO:146 and SEQ ID NO:175, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:198, the amino acid sequence is AAS and SEQ ID NO:220, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:121, SEQ ID NO:146 and SEQ ID NO:175, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:197, the amino acid sequence of KVS and SEQ ID NO:219, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:122, SEQ ID NO:147 and SEQ ID NO:176, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:199, the amino acid sequence of RVS and SEQ ID NO:221, respectively; or HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 123, SEQ ID NO: 148 and the amino acid sequence of AR, respectively, and LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 200, the amino acid sequence of KVS and SEQ ID NO: 222, respectively; or HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:124, SEQ ID NO:149 and SEQ ID NO:177, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:201, amino acid sequence WAS and SEQ ID NO:223, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:125, SEQ ID NO:150 and SEQ ID NO:178, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:202, the amino acid sequence of LVS and SEQ ID NO:224, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:126, SEQ ID NO:151 and SEQ ID NO:179, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:203, the amino acid sequence of KVS and SEQ ID NO:219, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:126, SEQ ID NO:152 and SEQ ID NO:179, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:197, the amino acid sequence of KVS and SEQ ID NO:219, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:126, SEQ ID NO:167 and SEQ ID NO:185, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:212, the amino acid sequence of LVS and SEQ ID NO:230, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:127, SEQ ID NO:153 and SEQ ID NO:180, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:200, the amino acid sequence of TVS and SEQ ID NO:225, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:128, SEQ ID NO:154 and SEQ ID NO:181, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:200, the amino acid sequence of TVS and SEQ ID NO:225, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:129, SEQ ID NO:154 and SEQ ID NO:181, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:200, the amino acid sequence of TVS and SEQ ID NO:225, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:130, SEQ ID NO:155 and SEQ ID NO:182, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:197, the amino acid sequence of KVS and SEQ ID NO:219, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 130, SEQ ID NO: 156 and SEQ ID NO: 182, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 197, the amino acid sequence of KLS and SEQ ID NO: 219, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 130, SEQ ID NO: 157 and SEQ ID NO: 182, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 197, the amino acid sequence of KVS and SEQ ID NO: 219, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 130, SEQ ID NO: 155 and SEQ ID NO: 182, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 202, the amino acid sequence of KVS and SEQ ID NO: 219, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:130, SEQ ID NO:158 and SEQ ID NO:182, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:202, the amino acid sequence of KVS and SEQ ID NO:219, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:131, SEQ ID NO:157 and SEQ ID NO:183, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:203, the amino acid sequence of WAS and SEQ ID NO:226, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 131, SEQ ID NO: 157 and SEQ ID NO: 183, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 203, the amino acid sequence of WAS and SEQ ID NO: 227, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:132, SEQ ID NO:159 and SEQ ID NO:184, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:197, the amino acid sequence is AAS and as shown in SEQ ID NO:220, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 132, SEQ ID NO: 159 and SEQ ID NO: 184, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 204, the amino acid sequence of WAS and SEQ ID NO: 228, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:132, SEQ ID NO:159 and SEQ ID NO:184, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:205, the amino acid sequence of DTS and SEQ ID NO:229, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:132, SEQ ID NO:159 and SEQ ID NO:184, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:206, the amino acid sequence of DTS and SEQ ID NO:229, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 132, SEQ ID NO: 160 and SEQ ID NO: 184, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 206, the amino acid sequence of WAS and SEQ ID NO: 228, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:133, SEQ ID NO:161 and SEQ ID NO:185, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:207, the amino acid sequence of LVS and SEQ ID NO:230, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 134, SEQ ID NO: 162 and SEQ ID NO: 186, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 202, the amino acid sequence of RVS and SEQ ID NO: 224, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:135, SEQ ID NO:163 and SEQ ID NO:187, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:209, the amino acid sequence is DTS and as shown in SEQ ID NO:231, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 135, SEQ ID NO: 163 and SEQ ID NO: 187, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 210, the amino acid sequence of RMS and SEQ ID NO: 232, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 136, SEQ ID NO: 164 and SEQ ID NO: 188, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 210, the amino acid sequence of RMS and SEQ ID NO: 233, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:137, SEQ ID NO:165 and SEQ ID NO:189, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:211, the amino acid sequence of LAS and SEQ ID NO:234, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:138, SEQ ID NO:166 and SEQ ID NO:190, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:197, the amino acid sequence of KVS and SEQ ID NO:235, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:139, SEQ ID NO:168 and SEQ ID NO:190, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:213, the amino acid sequence of FAT and SEQ ID NO:236, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 140, SEQ ID NO: 169 and SEQ ID NO: 191, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 214, the amino acid sequence is DTS and as shown in SEQ ID NO: 237, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:141, SEQ ID NO:170 and SEQ ID NO:192, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:215, the amino acid sequence of DTS and SEQ ID NO:238, respectively; or, HCDR1, HCDR2 and HCDR as shown in SEQ ID NO:142, SEQ ID NO:171 and SEQ ID NO:193, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:209, the amino acid sequence is DTS and as shown in SEQ ID NO:231, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 143, SEQ ID NO: 172 and SEQ ID NO: 194, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 216, the amino acid sequence of WAS and SEQ ID NO: 239, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 143, SEQ ID NO: 172 and SEQ ID NO: 194, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 200, the amino acid sequence of TVS and SEQ ID NO: 240, respectively; or, HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:144, SEQ ID NO:173 and SEQ ID NO:195, respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:217, the amino acid sequence of LAS and SEQ ID NO:241, respectively; or, HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO:145, SEQ ID NO:174 and SEQ ID NO:196 respectively, and LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO:218, the amino acid sequence is LVS and SEQ ID NO:242 respectively.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-60, or an amino acid sequence that is at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical thereto; and / or The light chain variable region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 61-120, or an amino acid sequence comprising at least 80%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identity thereto; Preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 61; or comprises a heavy chain variable region as shown in SEQ ID NO: 2 and a light chain variable region as shown in SEQ ID NO: 62; or comprises a heavy chain variable region as shown in SEQ ID NO: 3 and a light chain variable region as shown in SEQ ID NO: 63; or comprises a heavy chain variable region as shown in SEQ ID NO: 4 and a light chain variable region as shown in SEQ ID NO: 64; or comprises a heavy chain variable region as shown in SEQ ID NO: 5 and a light chain variable region as shown in SEQ ID NO: 65; or comprises a heavy chain variable region as shown in SEQ ID NO: 6 and a light chain variable region as shown in SEQ ID NO: 66; or comprises a heavy chain variable region as shown in SEQ ID NO: 7 and a light chain variable region as shown in SEQ ID NO: 67; or comprises a heavy chain variable region as shown in SEQ ID NO: 8 and a light chain variable region as shown in SEQ ID NO: 68; or comprises a heavy chain variable region as shown in SEQ ID NO: 9 and a light chain variable region as shown in SEQ ID NO: NO:69; or comprising a heavy chain variable region as shown in SEQ ID NO:10 and a light chain variable region as shown in SEQ ID NO:70; or comprising a heavy chain variable region as shown in SEQ ID NO:11 and a light chain variable region as shown in SEQ ID NO:71; or comprising a heavy chain variable region as shown in SEQ ID NO:12 and a light chain variable region as shown in SEQ ID NO:72; or comprising a heavy chain variable region as shown in SEQ ID NO:13 and a light chain variable region as shown in SEQ ID NO:73; or comprising a heavy chain variable region as shown in SEQ ID NO:14 and a light chain variable region as shown in SEQ ID NO:74; or comprising a heavy chain variable region as shown in SEQ ID NO:15 and a light chain variable region as shown in SEQ ID NO:75; or comprising a heavy chain variable region as shown in SEQ ID NO:16 and a light chain variable region as shown in SEQ ID NO:76; or comprising a heavy chain variable region as shown in SEQ ID NO:17 and a light chain variable region as shown in SEQ ID NO:77; or comprising a heavy chain variable region as shown in SEQ ID NO:18 NO:18 and the light chain variable region as shown in SEQ ID NO:78; or comprising the heavy chain variable region as shown in SEQ ID NO:19 and the light chain variable region as shown in SEQ ID NO:79; or comprising the heavy chain variable region as shown in SEQ ID NO:20 and the light chain variable region as shown in SEQ ID NO:80; or comprising the heavy chain variable region as shown in SEQ ID NO:21 and the light chain variable region as shown in SEQ ID NO:81;or comprising a heavy chain variable region as shown in SEQ ID NO:22 and a light chain variable region as shown in SEQ ID NO:82; or comprising a heavy chain variable region as shown in SEQ ID NO:23 and a light chain variable region as shown in SEQ ID NO:83; or comprising a heavy chain variable region as shown in SEQ ID NO:24 and a light chain variable region as shown in SEQ ID NO:84; or comprising a heavy chain variable region as shown in SEQ ID NO:25 and a light chain variable region as shown in SEQ ID NO:85; or comprising a heavy chain variable region as shown in SEQ ID NO:26 and a light chain variable region as shown in SEQ ID NO:86; or comprising a heavy chain variable region as shown in SEQ ID NO:27 and a light chain variable region as shown in SEQ ID NO:87; or comprising a heavy chain variable region as shown in SEQ ID NO:28 and a light chain variable region as shown in SEQ ID NO:88; or comprising a heavy chain variable region as shown in SEQ ID NO:29 and a light chain variable region as shown in SEQ ID NO:89; or comprising a heavy chain variable region as shown in SEQ ID NO:30 and a light chain variable region as shown in SEQ ID NO:

31. NO:90; or comprising a heavy chain variable region as shown in SEQ ID NO:31 and a light chain variable region as shown in SEQ ID NO:91; or comprising a heavy chain variable region as shown in SEQ ID NO:32 and a light chain variable region as shown in SEQ ID NO:92; or comprising a heavy chain variable region as shown in SEQ ID NO:33 and a light chain variable region as shown in SEQ ID NO:93; or comprising a heavy chain variable region as shown in SEQ ID NO:34 and a light chain variable region as shown in SEQ ID NO:94; or comprising a heavy chain variable region as shown in SEQ ID NO:35 and a light chain variable region as shown in SEQ ID NO:95; or comprising a heavy chain variable region as shown in SEQ ID NO:36 and a light chain variable region as shown in SEQ ID NO:96; or comprising a heavy chain variable region as shown in SEQ ID NO:37 and a light chain variable region as shown in SEQ ID NO:97; or comprising a heavy chain variable region as shown in SEQ ID NO:38 and a light chain variable region as shown in SEQ ID NO:98; or comprising a heavy chain variable region as shown in SEQ ID NO:39 and a light chain variable region as shown in SEQ ID NO:40; NO:39 and the light chain variable region as shown in SEQ ID NO:99; or comprising the heavy chain variable region as shown in SEQ ID NO:40 and the light chain variable region as shown in SEQ ID NO:100; or comprising the heavy chain variable region as shown in SEQ ID NO:41 and the light chain variable region as shown in SEQ ID NO:101; or comprising the heavy chain variable region as shown in SEQ ID NO:42 and the light chain variable region as shown in SEQ ID NO:102;or comprising a heavy chain variable region as shown in SEQ ID NO:43 and a light chain variable region as shown in SEQ ID NO:103; or comprising a heavy chain variable region as shown in SEQ ID NO:44 and a light chain variable region as shown in SEQ ID NO:104; or comprising a heavy chain variable region as shown in SEQ ID NO:45 and a light chain variable region as shown in SEQ ID NO:105; or comprising a heavy chain variable region as shown in SEQ ID NO:46 and a light chain variable region as shown in SEQ ID NO:106; or comprising a heavy chain variable region as shown in SEQ ID NO:47 and a light chain variable region as shown in SEQ ID NO:107; or comprising a heavy chain variable region as shown in SEQ ID NO:48 and a light chain variable region as shown in SEQ ID NO:108; or comprising a heavy chain variable region as shown in SEQ ID NO:49 and a light chain variable region as shown in SEQ ID NO:109; or comprising a heavy chain variable region as shown in SEQ ID NO:50 and a light chain variable region as shown in SEQ ID NO:110; or comprising a heavy chain variable region as shown in SEQ ID NO:51 NO:51 and the light chain variable region as shown in SEQ ID NO:111; or comprising the heavy chain variable region as shown in SEQ ID NO:52 and the light chain variable region as shown in SEQ ID NO:112; or comprising the heavy chain variable region as shown in SEQ ID NO:53 and the light chain variable region as shown in SEQ ID NO:113; or comprising the heavy chain variable region as shown in SEQ ID NO:54 and the light chain variable region as shown in SEQ ID NO:114; or comprising the heavy chain variable region as shown in SEQ ID NO:55 and the light chain variable region as shown in SEQ ID NO:115; or comprising the heavy chain variable region as shown in SEQ ID NO:56 and the light chain variable region as shown in SEQ ID NO:116; or comprising the heavy chain variable region as shown in SEQ ID NO:57 and the light chain variable region as shown in SEQ ID NO:117; or comprising the heavy chain variable region as shown in SEQ ID NO:58 and the light chain variable region as shown in SEQ ID NO:118; or comprising the heavy chain variable region as shown in SEQ ID NO:59 and the light chain variable region as shown in SEQ ID NO: NO:119; or comprising a heavy chain variable region as shown in SEQ ID NO:60 and a light chain variable region as shown in SEQ ID NO:

120. ; 5. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof is a humanized antibody, Preferably, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region; More preferably, the heavy chain constant region is selected from hIgG1, hIgG2, hIgG3 or hIgG4 heavy chain constant region.

6. A chimeric antigen receptor comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises the antibody or antigen binding fragment thereof according to any one of claims 1 to 5.

7. A modified immune cell comprising the chimeric antigen receptor according to claim 6.

8. A multispecific antibody comprising two or more antigen-binding domains, wherein one antigen-binding domain comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

9. An antibody composition comprising a first antibody and a second antibody, wherein the first antibody and / or the second antibody is selected from the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

10. The antibody composition according to claim 9, wherein The first antibody comprises a heavy chain variable region as shown in SEQ ID NO:7 and a light chain variable region as shown in SEQ ID NO:67, or comprises a heavy chain variable region as shown in SEQ ID NO:9 and a light chain variable region as shown in SEQ ID NO:69, or comprises a heavy chain variable region as shown in SEQ ID NO:18 and a light chain variable region as shown in SEQ ID NO:78, or comprises a heavy chain variable region as shown in SEQ ID NO:47 and a light chain variable region as shown in SEQ ID NO:107, or comprises a heavy chain variable region as shown in SEQ ID NO:57 and a light chain variable region as shown in SEQ ID NO:117, and the second antibody comprises a heavy chain variable region as shown in SEQ ID NO:12 and a light chain variable region as shown in SEQ ID NO:72, or comprises a heavy chain variable region as shown in SEQ ID NO:13 and a light chain variable region as shown in SEQ ID NO:73, or comprises a heavy chain variable region as shown in SEQ ID NO:14 and a light chain variable region as shown in SEQ ID NO:74, or comprises a heavy chain variable region as shown in SEQ ID NO:15 and a light chain variable region as shown in SEQ ID NO: NO:75, or comprising the heavy chain variable region as shown in SEQ ID NO:37 and the light chain variable region as shown in SEQ ID NO:97, or the heavy chain variable region as shown in SEQ ID NO:44 and the light chain variable region as shown in SEQ ID NO:104; or The second antibody comprises a heavy chain variable region as shown in SEQ ID NO:49 and a light chain variable region as shown in SEQ ID NO:109, and the first antibody comprises a heavy chain variable region as shown in SEQ ID NO:14 and a light chain variable region as shown in SEQ ID NO:74, or comprises a heavy chain variable region as shown in SEQ ID NO:16 and a light chain variable region as shown in SEQ ID NO:76, or comprises a heavy chain variable region as shown in SEQ ID NO:18 and a light chain variable region as shown in SEQ ID NO:78, or comprises a heavy chain variable region as shown in SEQ ID NO:20 and a light chain variable region as shown in SEQ ID NO:80, or comprises a heavy chain variable region as shown in SEQ ID NO:21 and a light chain variable region as shown in SEQ ID NO:81, or comprises a heavy chain variable region as shown in SEQ ID NO:22 and a light chain variable region as shown in SEQ ID NO:82, or comprises a heavy chain variable region as shown in SEQ ID NO:23 and a light chain variable region as shown in SEQ ID NO:83, or comprises a heavy chain variable region as shown in SEQ ID NO:25 and a light chain variable region as shown in SEQ ID NO: The light chain variable region shown in NO:85; or The second antibody comprises a heavy chain variable region as shown in SEQ ID NO: 55 and a light chain variable region as shown in SEQ ID NO: 115, and the first antibody comprises a heavy chain variable region as shown in SEQ ID NO: 13 and a light chain variable region as shown in SEQ ID NO: 73, or comprises a heavy chain variable region as shown in SEQ ID NO: 15 and a light chain variable region as shown in SEQ ID NO: 75, or comprises a heavy chain variable region as shown in SEQ ID NO: 17 and a light chain variable region as shown in SEQ ID NO: 77, or comprises a heavy chain variable region as shown in SEQ ID NO: 18 and a light chain variable region as shown in SEQ ID NO: 78, or comprises a heavy chain variable region as shown in SEQ ID NO: 20 and a light chain variable region as shown in SEQ ID NO: 80, or comprises a heavy chain variable region as shown in SEQ ID NO: 21 and a light chain variable region as shown in SEQ ID NO: 81, or comprises a heavy chain variable region as shown in SEQ ID NO: 22 and a light chain variable region as shown in SEQ ID NO: 82, or comprises a heavy chain variable region as shown in SEQ ID NO: 23 and a light chain variable region as shown in SEQ ID NO: NO:83, or the heavy chain variable region as shown in SEQ ID NO:25 and the light chain variable region as shown in SEQ ID NO:85; or the heavy chain variable region as shown in SEQ ID NO:27 and the light chain variable region as shown in SEQ ID NO:87; or The second antibody comprises a heavy chain variable region as shown in SEQ ID NO:59 and a light chain variable region as shown in SEQ ID NO:119, and the first antibody comprises a heavy chain variable region as shown in SEQ ID NO:14 and a light chain variable region as shown in SEQ ID NO:74, or comprises a heavy chain variable region as shown in SEQ ID NO:16 and a light chain variable region as shown in SEQ ID NO:76, or comprises a heavy chain variable region as shown in SEQ ID NO:17 and a light chain variable region as shown in SEQ ID NO:77, or comprises a heavy chain variable region as shown in SEQ ID NO:18 and a light chain variable region as shown in SEQ ID NO:78, or comprises a heavy chain variable region as shown in SEQ ID NO:20 and a light chain variable region as shown in SEQ ID NO:80, or comprises a heavy chain variable region as shown in SEQ ID NO:21 and a light chain variable region as shown in SEQ ID NO:81, or comprises a heavy chain variable region as shown in SEQ ID NO:22 and a light chain variable region as shown in SEQ ID NO:82, or comprises a heavy chain variable region as shown in SEQ ID NO:23 and a light chain variable region as shown in SEQ ID NO: The light chain variable region is shown in ID NO:

83.

11. An isolated polypeptide comprising a first domain and a second domain, wherein the first domain comprises at least 20 amino acids from positions 1 to 55 from the N-terminus of the Tau protein, and the second domain comprises at least 15 amino acids from positions 151 to 369 from the N-terminus of the Tau protein.

12. The isolated polypeptide according to claim 11, wherein The first domain includes at least 20 amino acids from positions 1 to 25 from the N-terminus of the Tau protein, and the second domain includes at least 17 amino acids from positions 170 to 240 from the N-terminus of the Tau protein; Preferably, the first domain and the second domain are directly connected or connected via a linker.

13. The isolated polypeptide according to claim 12, wherein The isolated polypeptide comprises an amino acid sequence as shown in any one of SEQ ID NOs: 251-253.

14. A method for detecting Tau in a sample, the method comprising the step of detecting Tau in the sample using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, Preferably, the method further comprises detecting Tau using the isolated polypeptide described in any one of claims 11-13, Preferably, the sample is whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, feces, urine, cultured cells, saliva, oral secretions and / or nasal secretions.

15. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

16. An expression vector comprising the isolated nucleic acid according to claim 15.

17. A host cell comprising the isolated nucleic acid according to claim 15, or the expression vector according to claim 16.

18. An antibody-drug conjugate comprising: an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5; and a drug covalently linked to the antibody or the antigen-binding fragment thereof, Preferably, the drug is selected from immunosuppressants and cytotoxic drugs.

19. A pharmaceutical composition comprising: the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the antibody-drug conjugate according to claim 18; and a pharmaceutically acceptable carrier, Preferably, the pharmaceutical composition further comprises a therapeutic agent selected from immunosuppressants and cytotoxic drugs.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the isolated nucleic acid according to claim 15, the antibody-drug conjugate according to claim 18 or the pharmaceutical composition according to claim 19 in the preparation of a medicament for preventing, treating and / or diagnosing a neurodegenerative disease, Preferably, the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease or frontotemporal dementia, more preferably selected from Alzheimer's disease.

21. A kit comprising one or more kits, wherein the kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody-drug conjugate according to claim 18, or the pharmaceutical composition according to claim 19; Preferably, the medicine kit comprises a first medicine box and a second medicine box, wherein: The first kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody-drug conjugate according to claim 18, or the pharmaceutical composition according to claim 19, and the second kit comprises a therapeutic agent selected from an immunosuppressant and a cytotoxic drug.

22. A drug delivery device comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody-drug conjugate according to claim 18, or the pharmaceutical composition according to claim 19, Preferably, the drug delivery device is a pre-filled syringe.

23. A method for preventing, treating and / or diagnosing a neurodegenerative disease, comprising administering to a subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the antibody-drug conjugate according to claim 18, or the pharmaceutical composition according to claim 19, Preferably, the neurodegenerative disease includes Alzheimer's disease, Parkinson's disease or frontotemporal dementia, more preferably selected from Alzheimer's disease.

24. A detection kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the multispecific antibody according to claim 8, or the antibody composition according to claim 9 or 10; Preferably, the detection kit further comprises the isolated polypeptide according to any one of claims 11 to 13, Preferably, the detection kit further comprises a detectable marker that can be linked to the antibody or antigen-binding fragment thereof, the multispecific antibody or the antibody composition, and optionally a substrate corresponding to the detectable marker and / or instructions for use.

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