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Bispecific antibody and application thereof in cancer treatment

A bispecific antibody and a specific technology, applied in the field of bispecific antibody and its application in the treatment of cancer, can solve problems such as B cell depletion, and achieve the effects of reducing the secretion of immune factors, reducing toxic side effects, and prolonging the half-life in vivo

Pending Publication Date: 2022-07-08
骏蓦(北京)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with CD3-BsAbs, some disadvantages of currently clinically approved CAR-T cells are: (1) patients need to undergo lymphatic dissection before infusion of CAR-T cells, (2) CAR-T cells must be produced individually for each patient. T cells, while CD3-BsAbs can be used as off-the-shelf therapeutic drugs that can be mass-produced, (3) CAR-T cells remain in the patient after the tumor is cleared, resulting in persistent CD19-targeting CAR-T cells B cell depletion while CD3-BsAbs are cleared from the blood over time

Method used

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  • Bispecific antibody and application thereof in cancer treatment
  • Bispecific antibody and application thereof in cancer treatment
  • Bispecific antibody and application thereof in cancer treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of CD3 single domain antibody

[0032] Select healthy adult alpacas, mix the recombinant human CD3 antigen (preserved in our laboratory) and Freund's complete adjuvant at a ratio of 1:1, and subcutaneously inject the alpacas on the back for four times in total. The immunization interval is Two weeks. After successful immunization, 10 mL of alpaca peripheral blood was collected to construct a phage display library.

[0033] Lymphocytes were separated from the collected peripheral blood of alpaca using a lymphocyte separation kit (Sigma-Aldrich); 1 × 10 7 The total RNA was extracted by the Trizol method. The steps were as follows: add 1 mL of Trizol (purchased from Sigma) to the EP tube containing lymphocytes, pipet repeatedly, and place on ice for 5 minutes; add 250 μL of chloroform, vortex for 30 seconds, and continue Place on ice for 5 minutes; centrifuge at 12,000g for 15 minutes at 4°C, transfer the aqueous phase to a new EP tube; add an equal...

Embodiment 2

[0042] Example 2 Design and preparation of bispecific antibodies

[0043] 2.1 Design of bispecific antibodies

[0044] The traditional structural antibodies targeting CD19 and CD3 were preserved by our laboratory, and their nucleic acid sequences were cloned into the PTT5 vector, wherein the antibody targeting CD19 had HCDR1 as shown in SEQ ID NO: 1, and as shown in SEQ ID NO: 2. HCDR2 shown in SEQ ID NO:3, LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, and LCDR3 shown in SEQ ID NO:6; and Has a heavy chain variable region shown in SEQ ID NO:10 and a light chain variable region shown in SEQ ID NO:11.

[0045] In this example, traditional CD3-targeting antibodies and single-domain antibodies were used to construct bispecific antibodies, so as to verify the corresponding anti-tumor effects. like figure 1 As shown in A, CD19-CD3 VH / VL BsAbs have traditional antibody structures and bind to CD19 and CD3 targets, respectively; as shown in figure 1 As shown in B, the CD19...

Embodiment 3

[0048] Example 3 In vitro anti-tumor experiments of bispecific antibodies

[0049] 3.1 Benefit cell isolation and culture

[0050] Human PBMC cells were isolated by Ficoll density gradient centrifugation: 10 mL of fresh human peripheral blood was drawn, mixed with 10 mL of serum-free RPMI1640 medium, and slowly added to the upper layer of 10 mL of density gradient centrifugation fluid Ficoll; centrifuged at 12,000 rpm for 10 min at room temperature; taken out the centrifuge tube, discarded Remove the upper layer of plasma, and then carefully aspirate the white layer PBMC between the plasma and Ficoll, and place it in a 50 mL centrifuge tube; add 15 mL of serum-free RPMI1640 medium, resuspend and centrifuge at 3000 rpm for 10 min, repeat the operation twice; add 15 mL of 10% serum-containing RPMI1640 medium at 37°C 5% CO 2 cultured under conditions.

[0051] 3.2 Tumor cell culture

[0052] In the present invention, human acute myeloid leukemia cell line THP-1, acute lymphocy...

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Abstract

The invention provides a bispecific antibody and application thereof in cancer treatment, the bispecific antibody comprises a first antigen binding domain specifically bound with a first antigen and a second antigen binding domain specifically bound with a second antigen, the first antigen is CD19, the second antigen is CD3, the first antigen binding domain targeting the CD19 can be bound with a target antigen in a high affinity manner, and the second antigen binding domain targeting the CD19 can be bound with the target antigen in a high affinity manner. A CD3-targeting second antigen binding domain selects a single-domain antibody structure with medium affinity, so that T cell immune response can be effectively mediated, and the situation that bispecific antibodies are gathered in T cell-enriched tissues such as spleen and lymph nodes in vivo to affect the anti-tumor effect can be prevented; a traditional antibody structural domain with a light chain and heavy chain structure is selected and combined with a single-domain antibody structural domain, so that the in-vivo half-life period of the bispecific antibody can be prolonged, and the anti-tumor activity is improved; in addition, the bispecific antibody provided by the invention can also reduce excessive immune factor secretion in the treatment process, and is helpful for reducing toxic and side effects.

Description

Technical field: [0001] The invention belongs to the field of tumor immunotherapy, and specifically provides a bispecific antibody and its application for treating cancer. Background technique: [0002] Malignant tumor is one of the intractable diseases that seriously threaten human health, and it also shows an increasing trend year by year in our country. Pay attention to. Hematological tumor cells mostly originate in the bone marrow, which is generally believed to be caused by the uncontrolled rapid clonal proliferation and accumulation of some immature blood cell precursor cells, and can also affect the growth and proliferation of cells in other normal non-hematopoietic tissues and organs. Clinical manifestations include fever, infection, varying degrees of anemia, enlarged liver, spleen, and lymph nodes, bone and joint pain, and complications caused by hematopoietic dysfunction. Hematological tumors can be divided into various types. Taking leukemia as an example, acco...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00A61P35/02
CPCC07K16/2803C07K16/2809C07K16/005A61P35/00A61P35/02C07K2317/31C07K2317/56C07K2317/565C07K2317/569C07K2317/22C07K2317/92A61K2039/505
Inventor 余亚杰窦欣童
Owner 骏蓦(北京)生物科技有限公司
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