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BCMA binding protein and preparation method and application thereof

A technology for binding proteins and amino acids, applied in the field of BCMA binding proteins and their preparation, which can solve the problems of weak internalization and weak binding

Active Publication Date: 2020-06-05
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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  • BCMA binding protein and preparation method and application thereof
  • BCMA binding protein and preparation method and application thereof
  • BCMA binding protein and preparation method and application thereof

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, 50 μg of the above fusion protein was immunized with CFA as an immune adjuvant, and then 25 μg protein and Ribi adjuvant (Sigma-Aldrich; Sigma AdjuvantSystem; 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 FACS using ...

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 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, wherein the light chain variable region includes LCDR1, LCDR2 and LCDR3, the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 and aminoacid sequences of the HCDR1, HCDR2 and HCDR3 are shown in detail in the specification. The BCMA binding protein has good specific affinity, and has better binding on a tumor cell line and better internalization effects when compared with a control antibody, and therefore the BCMA binding protein can be further developed as an ADC candidate; and after ELISA tests are performed, the BCMA binding protein antibody has a function of partial blocking, and additional curative effects can be achieved when the BCMA binding protein is adopted as mAbs or CAR-T during treatment.

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 Applications(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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