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研究活化誘導脫氨酶體突變的最佳方法和Globo H抗體原位類型轉換

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Monoclonal antibodies have become a mainstream treatment option in oncology. Most therapeutic targets have been limited to surface receptors overexpressed on cancer cells. Globo H (GH) is a newly appreciated glycolipid for cancer targeting, which is overexpressed on several cancer types including b

reast, colon, lung, ovarian, gastric, pancreatic and prostate cancers as well as on breast cancer stem cells. GH was discovered more than 30 years ago, but only two specific antibodies have been reported; MBr1 and VK9.No antibodies against GH have yet been approved for cancer treatment. A general ob

stacle in the glycobiology field is the difficulty in generating high affinity and specific anti-carbohydrate antibodies. Here, we propose an in situ affinity maturation platform using ectopic expression of activation-induced cytidine deaminase (AID) to increase the affinity of MBr1 antibody; the mo

st famous and well-studied antibody against Globo H.Affinity maturation requires repeated cycles of mutagenesis, selection and amplification. To generate a general platform for affinity maturation of antibodies, we optimized in situ somatic hypermutagenesis in HEK293FT cells. We found that transgene

s integrated into HEK293FT could efficiently accumulate somatic hypermutations (SHM) after repeated transient expression of a hyperactive AID mutant (AID m7.3). Stable expression of AID m7.3 can accumulate SHM in transgenes, but induce genotoxicity and suppress cells replication.Initially, we planne

d to do affinity maturation directly in hybridoma; we infected MBr1 hybridoma cells with mouse AID and repeated several cycles of sorting. We were able to isolate cells with improved binding to GH probes, but unfortunately, we could not prove that those cells produce antibodies with higher affinity

because they did not have any mutation in VL and VH genes. We proposed that improved binding to GH probes is probably attributed to surface molecules other than antibodies.Since hybridoma in situ affinity maturation is hindered by difficulty of introducing AID and nonspecific binding to GH probes, w

e alternatively decided to use HEK293FT to increase the affinity of GH antibodies. We first generated stable cell lines expressing surface GH antibodies in HEK293FT cells (293FT/MBr1). Diversity can be generated by repeated cycles of AID m7.3 expression. Antibodies with improved binding can be isola

ted by high throughput fluorescence-activated cell sorting (FACS). Specificity of improved antibodies can be maintained by sorting out cells that bind to closely related carbohydrate structures like Gb5, type 1 and type 2 H antigens; this assures that only cells that maintain binding to GH specifica

lly will be collected. This approach may offer a general method to improve the affinity and utility of a wide range of anti-glycan antibodies.AID can also induce class switch recombination (CSR). IgM antibodies cannot diffuse to tissues and are not easy to handle and purify. Using ectopic expressio

n of AID directly in MBr1 and other hybridomas, we succeeded to switch hybridomas from IgM to IgG. IgG hybridomas produce IgG antibodies, which can be easily purified and used for applications occasionally not applicable with IgM antibodies.