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Bispecific antibodies and uses thereof

A technology of antibody and recombinant antibody, which is applied in the field of biomedicine, can solve the problems of cytokine release syndrome toxicity, etc., and achieve the effect of avoiding systemic activation, high safety, and small side effects

Pending Publication Date: 2022-04-26
HEFEI TG IMMUNOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The activity of bispecific antibodies is affected by the affinity of CD3. CD3 high-affinity bispecific antibodies have a better killing effect in vitro, but there is fatal cytokine release syndrome toxicity in vivo, which is affected by chimeric antigens. Somatic (CAR) T cell toxicity is very similar

Method used

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  • Bispecific antibodies and uses thereof
  • Bispecific antibodies and uses thereof
  • Bispecific antibodies and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0179] Example 1 Design and Construction of CD3×B7H6 Bispecific Antibody Molecule

[0180] In this example, by screening various configurations, the inventors determined a bispecific antibody in the scFv-Fc configuration, and this bispecific antibody was named CD3×B7H6. The antibody contains two monovalent units, one of which is the anti-CD3 ScFv-Fc form, and the other monovalent unit is the anti-B7H6 ScFv-Fc form, and the light chain variable region of the CD3 antibody is linked by a connecting peptide It is connected with the variable region of the heavy chain, and the variable region of the light chain of the B7H6 antibody is connected with the variable region of the heavy chain by using the same connecting peptide as described above, and the connected peptide has the structure shown in SEQ ID NO: 46 The amino acid sequence of the CD3 antibody has a light chain variable region as shown in SEQ ID NO: 13, and a heavy chain variable region as shown in SEQ ID NO: 14, and the B7...

Embodiment 2

[0225] Example 2 Expression and purification of CD3×B7H6 bispecific antibody

[0226] 2.1 Molecular expression of CD3×B7H6 bispecific antibody

[0227] Bispecific molecules that bind CD3 and B7H6 were prepared by transiently transfecting ExpiCHO-S cells (Gibco, Cat. No. A29127) with the pTT5 vector carrying genes encoding CD3×B7H6 chains, in which 18 genes encoding CD3×B7H6 chains have Anti-CD3 ScFv-Fc as shown in SEQ ID NO:49, and anti-B7H6 ScFv-Fc as shown in SEQ ID NO:50-67. The specific experimental operation is as follows: one day before transfection, adjust the cell density of ExpiCHO-S cells to (3-4)×10 6 / mL, 37°C, 8% CO 2 , 120rpm shaking culture overnight. On the day of transfection, cells were grown to 7 × 10 6 – 1×10 7 / mL, ready for transfection when the viability rate is greater than 95%, use fresh pre-warmed ExpiCHO medium (Gibco, Cat. No. A2910002) to dilute the cells to 6×10 6 / mL, and then take the plasmid containing the CD3 polypeptide chain and the B7...

Embodiment 3

[0268] Example 3 Detection of Binding Activity of CD3×B7H6 Bispecific Antibody to Antigen (ELISA)

[0269] In this example, the binding activity of the CD3×B7H6 bispecific antibody to a single antigen (B7H6 antigen or CD3 antigen) and the simultaneous binding of the CD3×B7H6 bispecific antibody to dual antigens (B7H6 antigen and CD3 antigen) were detected respectively. Activity, the specific experimental operation is as follows:

[0270] 3.1 Determination of binding activity of CD3×B7H6 bispecific antibody to single antigen

[0271] Human B7H6 (ACRO biosystems, Cat. No. B76-H52H8) and human CD3 antigen (ACRO biosystems, Cat. 3 , 15mM Na 2 CO 3, pH 9.6) to 2 μg / mL, add 100 μL to each well of the enzyme-linked plate, and leave overnight at 4 °C. Then wash 3 times with PBST (0.05% Tween 20-PBS, pH7.2). Add 300 μL of blocking buffer (1% BSA, 0.05% Tween20-PBS, pH 7.2) to the plate and let stand at room temperature for 2 h. Then wash 3 times with PBST. Add the corresponding ...

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Abstract

The invention provides a bispecific antibody and application thereof. The recombinant antibody contains at least one of the following CDR sequences or amino acid sequences with at least 95% identity with the CDR sequences: CD3 antibody variable region CDR sequences: SEQ ID NO: 1-6; and the variable region CDR sequences of the B7H6 antibody are as shown in SEQ ID NO: 7-12. The recombinant antibody prepared by the invention can target CD3 and B7H6 at the same time, the half-life period is obviously prolonged, and the recombinant antibody shows stronger tumor inhibition capability compared with a single-target antibody.

Description

technical field [0001] The present invention belongs to the field of biomedicine, specifically relates to bispecific antibodies and applications thereof, and more specifically relates to recombinant antibodies, immune cells, nucleic acids, expression vectors, recombinant cells, compositions, the use of the above substances in the preparation of medicines, Reagent test kit. Background technique [0002] Bispecific antibodies are antibodies that can specifically bind two antigenic sites at the same time. Bispecific antibodies for tumor therapy can be divided into three categories according to their mechanism of action: redirecting effector cells; immune regulation; targeting dual binding of tumor cell receptors. Among them, antibodies used for redirection function account for the majority of them, and the two antibodies currently on the market for tumor treatment are also based on T cell redirection. According to the characteristics of bispecific antibodies, it can also be w...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13G01N33/68G01N33/574A61K39/395A61P35/00
CPCC07K16/2809C07K16/2827G01N33/57446G01N33/57423G01N33/57415G01N33/57438G01N33/57484G01N33/6872A61P35/00C07K2317/565C07K2317/56C07K2317/31C07K2317/622C07K2319/30A61K2039/505G01N2333/7051G01N2333/70532G01N33/57492G01N2333/70596C07K2317/73C07K2317/64G01N33/57419C07K16/468C07K2317/52C12N15/63G01N33/574
Inventor 田志刚成赢肖卫华曹国帅孙昊昱孙汭
Owner HEFEI TG IMMUNOPHARMA CO LTD
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