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Bispecific egfr/cd16 antigen-binding protein

A protein-binding, multi-specific technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-animal/human immunoglobulin, immunoglobulin, etc.

Pending Publication Date: 2020-11-20
AFFIMED GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this approach demonstrated that simply changing the mode of action from receptor blockade to elimination of target cells by recruiting immune cells to kill target cells did not succeed in avoiding serious side effects, as at μg / kg / d Relatively low doses in the range tested in cynomolgus monkeys had to be discontinued due to severe hepatic and renal toxicity observed

Method used

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  • Bispecific egfr/cd16 antigen-binding protein
  • Bispecific egfr/cd16 antigen-binding protein
  • Bispecific egfr/cd16 antigen-binding protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1 : Generation and Production of EGFR / CD16A Antigen Binding Proteins

[0122] Material

[0123]

[0124] Production of GFR / CD16A antigen-binding protein

[0125] tandem diabody

[0126] Tandem diabodies were constructed as described in Reusch et al., 2014, mAbs 6:3, 728-739 ( image 3 ). To construct tandem diabodies, anti-EGFR Fv domains (SEQ ID NO: 1, 2) were combined with anti-CD16A Fv domains (SEQ ID NO: 12, 13). The expression module of the tandem diabody was cloned such that the anti-EGFR domain and the anti-CD16A domain were arranged in the order VH_EGFR-L1-VL_CD16A-L2-VH_CD16A-L3-VL_EGFR. 9 amino acid linker (G 2 S) 3 (SEQ ID NO: 36) for linker L1 and L3, linker of 6 amino acids (G 2 S) 2 (SEQ ID NO: 35) is used for the linker L2 to obtain the EGFR / CD16A tandem diabody consisting of two polypeptides having the amino acid sequence shown in SEQ ID NO: 27.

[0127] aTriFlex

[0128] aTriFlex was constructed as described in WO 2017 / 064221 ...

Embodiment 2

[0171] Bispecific EGFR / CD16A antigen-binding protein in the presence or absence of 10 mg / mL polyclonal human IgG Combination of white and primary human NK cells

[0172] method:

[0173] Isolation of PBMCs from Buffy Coats and Enrichment of Human NK Cells

[0174] PBMCs were isolated from buffy coats (German Red Cross, Mannheim, Germany) by density gradient centrifugation. The buffy coat sample was diluted with 2-3 times the volume of PBS (Invitrogen, product number: 14190-169), and spread on the lymphocyte separation agent (Lymphoprep) (Stem Cell Technologies, product number :07861) and centrifuge at 800×g for 25 minutes at room temperature (without brake). PBMCs located in the interface were collected, washed 3 times with PBS, and then cultured overnight in complete RPMI 1640 medium supplemented with 10% FCS without stimulation. To enrich for NK cells, harvest PBMCs from overnight cultures and use EasySep for immunomagnetic separation of untouched human NK cells accor...

Embodiment 3

[0183] Binding of EGFR / CD16A tandem diabody to CHO cells expressing recombinant human EGFR or EGFRvIII

[0184] Cell binding assay and flow cytometry analysis

[0185] Aliquots of the indicated cells were incubated with 100 μL of serial dilutions of His-tagged tandem diabodies serially diluted in FACS buffer (Invitrogen, product number: 14190-169) for 45 minutes at 37°C. FACS buffer contained 2% heat-inactivated FCS (Invitrogen, product number: 10270-106), 0.1% sodium azide (Roth, Karlsruhe, Germany, product number: A1430.0100). After repeated washing with FACS buffer, first detect the cell-bound antibody with 10 μg / mL anti-His mAb13 / 45 / 31-2 (DIANOVA, Hamburg, Germany, product number: DIA910-1MG), and then use 15 μg / mL FITC-conjugated Goat anti-mouse IgG (DIANOVA, product number: 115-095-062) was used to detect cell-bound antibodies. After the final staining step, cells were washed again and resuspended in 0.2 mL FACS buffer containing 2 μg / mL propidium iodide (PI) (Sigma,...

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Abstract

Described are tetravalent, bispecific EGFR / CD16A antigen-binding proteins for engaging NK-cells towards EGFR-positive cells. EGFR / CD16A antigen-binding proteins with different pharmacokinetic (PK) properties are described. Further described is the use of bispecific EGFR / CD16A antigen-binding proteins for the treatment of an EGFR-positive malignancy, such as EGFR-positive tumors.

Description

technical field [0001] The present invention relates to tetravalent, bispecific EGFR / CD16A antigen-binding proteins for binding NK cells to EGFR-positive cells, and their use in the treatment of EGFR-positive malignancies, such as EGFR-positive tumors. Background technique [0002] Epidermal growth factor receptor (EGFR) is a validated target for the treatment of several solid tumors. Current monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) targeting EGFR work primarily by blocking signaling. Furthermore, the therapeutic efficacy of these drugs depends on the mutational status of the receptor or signaling pathway, such as the T790M Gatekeeper mutation in the tyrosine kinase domain or mutations downstream of signal transduction cascades such as RAS or RAF, This may lead to treatment-intrinsic or acquired resistance in many patients. In addition, therapies targeting EGFR are associated with side effects that are thought to affect prescription rates. [000...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P35/00C07K16/18C07K16/28
CPCC07K16/18C07K16/283C07K16/2863C07K2317/31C07K2317/33C07K2317/62C07K2317/622C07K2317/64C07K2317/73C07K2317/76C07K2317/92C07K2317/94C07K2319/00A61P35/00C07K2317/565C07K2317/624C07K2317/55C07K2317/52A61K2039/505A61K47/65A61K39/001129A61K39/001104
Inventor 迈克尔·克卢格迈克尔·特萨伊维卡·法斯克克里斯蒂娜·埃尔万格尔乌维·罗伊施迈克尔·达姆拉特埃里希·拉伊科维奇马丁·特雷德
Owner AFFIMED GMBH
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