Novel humanized anti-CD22 antibody-monomethylaristatin e conjugate and preparation method thereof

A humanized and monomethylated technology, which is applied in the field of new humanized anti-CD22 antibody-monomethylaristatin E conjugate and its preparation, can solve the problems of complex and difficult to distinguish mass spectra, and achieve good solubility , the reaction conditions are mild, and the effect of ensuring effectiveness

Active Publication Date: 2016-02-24
BEIJING DONGFANG BIOTECH +1
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Problems solved by technology

In this type of coupling, the thiol reducing agent dithiothreitol (DTT) is used to partially reduce the antibody. Although this reducing agent can effectively reduce the disulfide bond, it cannot be carried out under acidic conditions and is often easily combined with the peptide. Segments form adducts, complicating mass spectra

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  • Novel humanized anti-CD22 antibody-monomethylaristatin e conjugate and preparation method thereof
  • Novel humanized anti-CD22 antibody-monomethylaristatin e conjugate and preparation method thereof
  • Novel humanized anti-CD22 antibody-monomethylaristatin e conjugate and preparation method thereof

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Embodiment Construction

[0046] The present invention is described in detail below in conjunction with accompanying drawing and specific embodiment:

[0047] 1. Coupling process

[0048] 1) Antibody partial reduction

[0049] Add 2-4 times the molar amount of trichloroethyl phosphate (TCEP), preferably 3 times the molar amount of trichloroethyl phosphate (TCEP) to the preparation buffer containing hRFB4, and put it in a 37°C water bath under the protection of argon or nitrogen Stir the reaction for 1-2 hours, preferably 2 hours, and stop the reaction of the sample in an ice-water bath; the preparation buffer is 10mmol / L histidine hydrochloride, 5% trehalose dihydrate, 0.01% Tween 20, pH5.5 .

[0050] The reduced hRFB4 protein solution was exchanged into PBS / D (pH6.5) with a G-25 desalting column or a 30kD membrane-packed ultrafiltration exchange system, where D is diethylenetriaminepentaacetic acid (DTPA). The protein concentration (ε = 1.4 mg / mL·cm) was detected at an absorption wavelength of 280 ...

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Abstract

The invention relates to the technical field of biological pharmacy, and specifically discloses a novel humanized anti-CD22 antibody-monomethyl auristatin-E conjugate. According to the invention, the novel humanized anti-CD22 antibody hRFB4 is adopted. The joints and drug composition of vcMMAE is selected; in a conjugation process, antibody semi-reduction is carried out with a phosphine-type reducing agent tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP); conjugation is carried out with vcMMAE; and conjugate purification is carried out. Through the optimization upon the process, the novel anti-CD22 antibody drug conjugate is prepared. The conjugate has good performances in the respects of crucial physical and chemical analysis indicators and biological activity detection indicators. In both in-vitro and in-vivo pharmacodynamic detections, the conjugate performs a highly efficient and specific target cell-killing effector function.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a novel humanized anti-CD22 antibody-monomethylaristatin E conjugate and a preparation method thereof. Background technique [0002] Studies have found that there are many determinant molecules (cluster of differentiation) specifically expressed on the surface of B lymphocytes, that is, CD molecules, such as: CD19, CD20, CD22, CD72 and CD80; among these antigen molecules, they are used as drug targets. There are mainly two molecules, CD20 and CD22, that have been extensively studied and applied clinically (CancerRes.2012Nov1; 72(21):5556-65.). CD22, also known as sialic acid-binding immunoglobulin-like lectin, is an antigen molecule specifically expressed on the surface of B cells, because this molecule is only expressed on the membranes of B cells and pre-B cells, but not on the membranes of progenitor B cells and plasma cells , so it is considered to be an ideal dru...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00A61P35/00
Inventor 梁爽汪瑞李春菊周建华白义白先宏
Owner BEIJING DONGFANG BIOTECH
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