Targeted functional molecule modified antibody complex
An antibody complex and functional molecule technology, applied in the field of pharmacy, can solve the problems of low response rate, ineffectiveness, and large molecular scale of brain tumor treatment, and achieve the effect of improving treatment effect, good application prospect, and expanding the scope of clinical application.
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Embodiment 1
[0056] Example 1 Synthesis and Characterization of Targeted Molecules
[0057] Synthesis and characterization of pHA targeting molecules: Polypeptides were prepared by solid-phase synthesis technology. First, weigh MBHA resin (substitution degree: 0.54mmol / g), add N,N-dimethylformamide for swelling, and then add 20% piperidine -DMF solution to remove the Fmoc protecting group on the resin, after washing, add 8.8 times the amount of Fmoc-amino acid (dissolved in the DMF solution of HBTU / HOBT / DIEA), place in an air shaker (37 ° C, 180rpm ) Shake for 45 minutes. After the reaction, wash with fresh DMF and drain the resin, add 20% piperidine-DMF solution to remove the Fmoc protecting group on the α-amino group. Repeat the above operation according to the sequence (pHA sequence: p-Hydroxybenzoic acid-SSC). After all the amino acids are condensed, the resin is washed and dried, and the peptide cleavage reagent (TFA / TIPS / H 2 O=95 / 2.5 / 2.5, v / v) After stirring for 2 h, the TFA was r...
Embodiment 2
[0060] Example 2 Preparation and Characterization of Targeting Functional Molecule-PDL1 Antibody Complex
[0061] Preparation of target functional molecule-PDL1 antibody complex:
[0062] Add Sulfo-SMCC (1 mg / mL) to the αPDL1 solution, react at room temperature for 30 min, and remove excess Sulfo-SMCC through a desalting column. Add 108 μL of PBS solution (2 mg / mL) of pHA, mix well, place at 4°C for 12 hours, then dialyze twice in a 14K Da dialysis bag with pure water (30 min each time), collect the liquid in the dialysis bag to obtain pHA modification PDL1 antibody complex (pHA-αPDL1). Prepare mRAP-αPDL1 and mCDX-αPDL1 in the same way.
[0063] MMP enzymatic hydrolysis verification:
[0064] Add 1 μL APMA (1M) to MMP-2 and dilute to 100 μL with the reaction solution, and incubate at 37°C for 1 hour. After activation, mix 50 μL of enzyme with 50 μL of mRAP-αPDL1 and mCDX-αPDL1, incubate at 37°C for 2 hours, and then dialyze.
[0065]Characterization of Targeting Functiona...
Embodiment 3
[0067] Example 3 Characterization of Functional Binding Activity of Targeting Functional Molecule-PDL1 Antibody Complex
[0068] ELISA binding activity:
[0069] Coat the 96-well ELISA plate with 100 μL / well of mouse PD-L1 protein, place it at room temperature for 2 hours, wash the plate twice, add BSA at room temperature to block for 1 hour, add pHA-αPDL1, mRAP-αPDL1, mCDX- αPDL1 or unmodified αPDL1 (600ng / mL, 400ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 25ng / mL, 10ng / mL, 5ng / mL, 2ng / mL), shake at 37°C After incubating on the bed for 1 h, after washing the plate, add anti-rat Ig(H+L) / HRP and incubate for 1 h, add 100 μL of chromogenic solution and leave at room temperature for 10 min, add 100 μL of 1M sulfuric acid to terminate the reaction, and measure the absorbance value with a microplate reader at a detection wavelength of 450 nm. see attached results Image 6 (A).
[0070] SPR binding activity:
[0071] Using surface plasmon resonance (SPR) technology, the binding activity...
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