Anti-tmem106b antibodies and methods of use thereof
An antibody and ligand technology, applied in anti-TMEM106B antibody and its application field, can solve the problem that the function of TMEM106B has not been fully characterized.
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Embodiment 1
[0342] Example 1: TMEM106B is a risk factor in various neurodegenerative disorders
[0343]To search for genetic risk factors for FTLD, a genome-wide association study (GWAS) was performed in 2509 control subjects and 515 subjects with pathologically confirmed FTLD-TDP, some of whom harbored GRN mutations (vanDeerlin et al. , 2010, Nat Genetics, 42:234-239). This GWAS identified 3 SNPs within a 68 kb region on chromosome 7p21.3 that were associated with the occurrence of FTLD-TDP with genome-wide significance (rs6966915, rs102004 and the upper marker SNP rs1990622; p-value range = 5.00 × 10 -11 to 1.08×10-11). Three significantly associated SNPs were within the same linkage disequilibrium (LD) block as nine other nominally associated SNPs, and all spanned the locus of the TMEM106B gene. In this case, minor alleles for each significant SNP in the control cohort were largely under-represented in FTLD-TDP patients (32.1 vs 43.6% in controls for the rs1990622 minor C allele; 10...
Embodiment 2
[0350] Example 2: Generation of GST and murine Fc-conjugated human TMEM106B
[0351] Various human TMEM106B polypeptides and TMEM106B polypeptide fusion proteins were generated as described below. Mammalian recombinant expression of various TMEM106B polypeptides can be performed by cloning synthetic genes into mammalian expression vectors based on TMEM106B cDNA, followed by transient transfection and expression in HEK293T cells. Each TMEM106B expression construct contains a heterologous signal peptide, as well as a His-tag, human IgG1 Fc, glutathione-S-transferase, or murine IgG1 Fc for fusion constructs. Some TMEM106B expression constructs comprise the C-terminal region of TMEM106B (the putative extracellular domain (ECD) (amino acid residues 122-274 of SEQ ID NO: 1)). Other TMEM106B expression constructs include a truncated version of the ECD (amino acid residues 122-210 of SEQ ID NO: 1) to avoid expression of a small hydrophobic fragment (located at approximately amino aci...
Embodiment 3
[0368] Example 3: Construction of TMEM106B expression plasmids for DNA immunization
[0369] A DNA immunization approach was used to develop antibodies against TMEM106B. The cDNA sequences encoding human TMEM106B, mouse TMEM106B and cynomolgus TMEM106B (SEQ ID NO: 1, 2 and 315, respectively) were cloned into pCAGGS mammalian expression vector (KeraFAST EH1017) for DNA immunization. Transient transfection of expression constructs into HEK293T cells, followed by the use of commercially available anti-TMEM106B antibodies (EMD Millipore MAB-N473, Thermo-Fischer PA5-6338, Abcam ab140185, Abcam ab116023, Protein Tech 20995-1-AP, Expression of each TMEM106B polypeptide was confirmed by Western blot and intracellular and extracellular FACS analysis performed by LifeSpan Biosciences LS-C145601, Abgent A112796, MyBioSource MBS9412982, SigmaSAB2106773, Bethyl Labs A303-439A). The expression constructs were then used for DNA immunization in mice as described below.
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