Novel bifunctional antibody conjugate, preparation method and application thereof
A technology of bifunctional antibodies and conjugates, applied in the direction of antibodies, drug combinations, anti-tumor drugs, etc., can solve the problems of unsatisfactory chemical coupling efficiency of bispecific antibodies
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[0102] A typical coupling method such as figure 2 shown. A preferred preparation method of anti-CD3×anti-HER2 bispecific antibody comprises the following steps:
[0103] (1) Antibody dissolution: Dissolve commercial anti-CD3 monoclonal antibody in reaction buffer A (150mM NaCl, 2mM EDTA˙Na 2 , 100mM phosphate buffer, pH8.0); dissolve the anti-medical HER2 monoclonal antibody in reaction buffer B (150mM NaCl, 2mM EDTA˙Na 2 , 100mM phosphate buffer, pH 7.2).
[0104] (2) Dissolution of coupling agent: Quickly weigh Traut’s reagent and Sulfo-SMCC and dissolve them in ultrapure water respectively to prepare a mother liquor.
[0105] (3) Coupling of anti-CD3 monoclonal antibody and Traut's reagent: Mix the prepared Traut's reagent mother solution and anti-CD3 monoclonal antibody at a molar ratio of 5:1 to 10:1; place the well-mixed mixture in On a constant temperature reaction shaker, keep the temperature at 15-28°C and react for 60 minutes.
[0106] (4) Coupling of anti-HER2...
Embodiment 1
[0127] Preparation of anti-CD3×anti-HER2 bispecific antibody
[0128] Experimental steps:
[0129] Take 132 μg of mouse anti-human CD3 monoclonal antibody and dissolve it in reaction buffer A (150 mM NaCl, 2 mM EDTA·Na 2 , 100mM phosphate buffer, pH8.0), weighed 1.2mg of Traut's reagent and dissolved it in 120μL of reaction buffer A, and prepared it as a 10mg / mL stock solution. Anti-CD3 monoclonal antibody was mixed at a molar ratio of 10:1 and incubated at room temperature for 1 hour;
[0130] Take 30 μg of mouse anti-HER2 monoclonal antibody and dissolve it in reaction buffer B (150 mM NaCl, 2 mM EDTA·Na 2 , 100mM phosphate buffer, pH7.2), weighed 1.5mg of Sulfo-SMCC and dissolved it in 300μL of reaction buffer B to prepare a mother solution, diluted 10 times to 0.5mg / mL when used, and mixed Sulfo-SMCC with anti-HER2 Monoclonal antibodies were mixed at a molar ratio of 20:1, 40:1, and 60:1 and incubated at room temperature for 1 hour;
[0131] The Zeba desalting column was...
Embodiment 2
[0137] Preparation of anti-CD3×anti-HER2 bispecific antibody
[0138] Different from Example 1, the specific steps are as follows:
[0139] Take mouse anti-human CD3 monoclonal antibody OKT3 antibody 133μg dissolved in reaction buffer A (150mM NaCl, 2mM EDTA·Na 2 , 100mM phosphate buffer, pH 8.0), weighed 1.3mg of Traut's reagent and dissolved it in 130μL of reaction buffer A to make 10mg / mL stock solution, diluted 50 times to 0.2mg / mL when used, and mixed Traut's reagent with anti CD3 monoclonal antibody was mixed at a molar ratio of 5:1 or 10:1 and incubated at room temperature for 1 hour;
[0140] Take 31 μg of mouse anti-HER2 monoclonal antibody and dissolve in reaction buffer B (150mM NaCl, 2mM EDTA·Na 2 , 100mM phosphate buffer, pH7.2), weighed 1.0mg of Sulfo-SMCC dissolved in 100μL of reaction buffer B, prepared as a mother solution, diluted 20 times to 0.5mg / mL when used, mixed Sulfo-SMCC with anti-HER2 Monoclonal antibodies were mixed at a molar ratio of 4:1 or 20:...
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