Stable CD3-CD19 resisting mini-type difunctional antibody of disulfide bond and preparation method thereof

Abstract

The invention relates to a stable CD3-CD19 resisting mini-type difunctional antibody of disulfide bond and a preparation method thereof. The preparation method comprises the steps of based on the CD3-CD19 resisting antibody, introducing amino acid Cys mutation in the CD3VL-resisting 100th amino acid Ser and CD3VH-resisting 44th amino acid Gly, wherein the expressed product has the structure of disulfide bond formed by the CD3VL-resisting 100th and the CD3VH-resisting 44th cysteines shown in the sequences 3 and 4. The stable CD3-CD19 resisting mini-type difunctional antibody of disulfide bond introduces cysteine mutation in the bits of VL-100 and VH-44 in the CD3-resisting variable region, and the disulfide bond is formed in a bacterium periplasm cavity between the variable region fragments of the two expressed single chains, i.e., between the CD3VL-CD19VH resisting segment and the CD19VL-CD3VH resisting segment, thereby enhancing the stability of the antibody; besides, a mutational site is located in a conservative framework region and two mutational sites in the space conformation are closer enough without generating tension so that the activity of the transformed difunctional antibody remains unchanged and the stability is enhanced.

Description

technical field [0001] The invention belongs to the field of antibodies, and relates to a miniature bifunctional antibody, especially a disulfide bond-stabilized anti-CD3-anti-CD19 miniature bifunctional antibody and a preparation method thereof. Background technique [0002] Non-Hodgkin's lymphoma (NHL) is a group of malignant tumors originating from lymphoid tissue, and its morbidity and mortality rank the fifth among malignant tumors. Traditional radiotherapy and chemotherapy are satisfactory for early and mid-stage patients, but not effective for late-stage patients, especially those with lower pathological grades. A large number of cases cannot be fully recovered because of the tiny residual lesions, and are the source of future recurrence. For advanced patients, high-dose chemotherapy and whole-body radiation therapy are generally used, combined with bone marrow transplantation (BMT) and peripheral blood stem cell transplantation (PBSCT). High-dose chemotherapy and wh...

AI Technical Summary

Problems solved by technology

The anti-CD3-anti-CD19 mini bifunctional antibody is composed of the expression products of two cistrons CD3VL-CD19VH and CD19VL-CD3VH under the control of the promoter phoA, and the VH and VL in the same cistron pass through the Linker (that is, the short peptide GlyGlyGlyGlySer) Connection, because the G4S short peptide is short, the VH and VL in the same cistron produce steric hindrance, cannot be paired, and can only be reversely paired with the VH and VL on another cistron, and it is expressed in the bacterial periplasm Dimers are formed with the help of folding enzymes and molecular chaperones in the cavity. The two scFv fragments in the dimer are connected by non-covalent bonds, which are easy to dissociate and inactivate, and have poor stability.

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