A kind of bcma binding protein and its preparation method and application

A protein and amino acid binding technology, applied in the field of BCMA binding protein and its preparation, can solve the problems of weak internalization and weak binding

Active Publication Date: 2020-10-23
NONA BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the anti-BCMA antibodies in the prior art have defects such as weak internalization and weak binding to 293T-huBCMA cells, so there is an urgent need for an anti-BCMA antibody that has both sufficient affinity for drug production and excellent internalization

Method used

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  • A kind of bcma binding protein and its preparation method and application
  • A kind of bcma binding protein and its preparation method and application
  • A kind of bcma binding protein and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1 Antigen preparation, mouse immunization and hybridoma preparation

[0054] 1. Antigen Preparation

[0055] (ACRO cat.no:BCA-C52H7)

[0056] supplier name NCBI ID Cat# Acrobio huBCMA-ECD-Fc Q02223 BCA-H522y Acrobio BCMA-His BCA-C52H7 Acrobio CynoBCMA-Fc G7NPN8 BCA-C5253 Acrobio CynoBCMA-His BCA-C5225 Acrobio BAFF-his-avitag Q9Y275 BAF-H82Q2

[0057] 2. Immunity

[0058] Fully human anti-BCMA antibodies were identified from hybridomas generated from H2L2 mice immunized with BCMA-ECD-Fc protein (Harbor Pharmaceuticals, EP2379727B1). For the first injection of 50 μg of the fusion protein, CFA was used as an immune adjuvant for immunization, and then 25 μg of protein and Ribi adjuvant (Sigma-Aldrich; Sigma Adjuvant System; Catalog Number S6322) and strengthened 7 times. On days 50, 78, and 107, blood was collected for testing, and the binding affinity of mouse serum was detected by FAC...

Embodiment 2

[0062] Example 2 Antibody Screening and Sequencing

[0063] 1. ELISA screening

[0064] Freshly prepared hBCMA ECD-Fc protein or hFc at 1 μg / ml in PBS was added to 96-well plates (Corning 9018), coated overnight at 4°C, then discarded and washed 3 times with PBST. Plates were blocked with 5% milk for 2 hours at room temperature and washed 3 times with PBST. 100 μl / well of hybridoma supernatant was added and incubated at room temperature for 1 hour, then washed 3 times with PBST. 100 μl / well of secondary antibody was added and incubated for 1 hour at room temperature, followed by washing. Add 100 μl / well TMB to the plate and incubate at room temperature for 15 min, then stop and read.

[0065] 2. FACS screening

[0066] For flow cytometry screening, adherent cells were digested with TypLE (CAT#12605010, Gibco) for 3 minutes at 37°C and the digestion was terminated with complete medium containing 10% FBS. The cells were washed and counted with FACS buffer (CAT#14190250, Gib...

Embodiment 3

[0077] Example 3. Antibody Production, Purification and Validation

[0078] 1. Recombinant Antibody Production and Purification

[0079] After obtaining the light and heavy chain variable domain sequences encoding antibody molecules, conventional recombinant DNA technology can be used to combine the light and heavy chain variable domain sequences with the corresponding human antibody light and heavy chain constant domain sequences. Fusion expression to obtain recombinant antibody molecules. In this example, the antibody heavy chain variable domain sequence (VH) was genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human IgG1 antibody heavy chain constant domain sequence to encode the full-length IgG1 antibody. heavy chain. The antibody light chain variable domain sequence (VL) was genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human antibody Ig kappa light chain constant domain seq...

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Abstract

The invention discloses a BCMA binding protein and a preparation method and application thereof. The BCMA binding protein includes a heavy chain variable region and a light chain variable region, the light chain variable region includes LCDR1, LCDR2 and LCDR3; the heavy chain variable region includes HCDR1, HCDR2 and HCDR3. The amino acid sequences of the LCDR1, LCDR2 and LCDR3 and the HCDR1, HCDR2 and HCDR3 are detailed in the present invention. The BCMA-binding protein of the present invention has good specific affinity, it binds better on tumor cell lines than the control antibody, and its internalization effect is better than that of the control, so it can be further developed as an ADC candidate. Antibodies have partial blocking function in ELISA tests, and may gain additional efficacy when used as mAbs or CAR-T therapy.

Description

technical field [0001] The invention belongs to the field of biomacromolecules, and in particular relates to a BCMA-binding protein and its preparation method and application. Background technique [0002] Multiple myeloma (MM) has been characterized by uncontrolled proliferation of bone marrow-derived plasma cells with abnormal secretion of immunoglobulins or free light chains, ranking second among the most common hematological malignancies, accounting for about 10% of. In the United States, there are more than 30,000 new diagnoses and more than 12,000 deaths each year. Despite new targeted therapies such as protease inhibitors (Bortezomb), it is widely considered incurable and more efforts are needed in drug development. [0003] B cell maturation antigen (BCMA), also known as TNFRSF17 or CD269, is a specific antigen expressed only in the B cell lineage, especially terminally differentiated B cells. It is normally absent on naive and memory B cells and is highly express...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
CPCC07K16/2878A61P35/00C07K2317/56C07K2317/565C07K2317/92C07K2317/73C07K2317/76A61K2039/505
Inventor 王正何云戎一平
Owner NONA BIOSCIENCES CO LTD
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