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Bispecific t-cell engager with cleavable cytokines for targeted immunotherapy

A non-immunogenic, compound technology that can be used in drug combinations, medical preparations with non-active ingredients, anti-tumor drugs, etc., and can solve the problems of side effects such as T cell depletion and depletion

Pending Publication Date: 2021-12-10
SHENZHEN ENDURING BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Unfortunately, since human healthy tissues and activated T cells may also express PDL1, anti-CD3 / anti-PDL1 BiTEs may kill some of these cells, leading to severe side effects and depletion of the T cell reservoir

Method used

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  • Bispecific t-cell engager with cleavable cytokines for targeted immunotherapy
  • Bispecific t-cell engager with cleavable cytokines for targeted immunotherapy
  • Bispecific t-cell engager with cleavable cytokines for targeted immunotherapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0263] Embodiment 1: the preparation of 30kmPEG-Lys (maleimide)-DBCO ( figure 1 )

[0264] Preparation of 30kmSC-PEG (Compound 2):

[0265] 25 g of 30 kmPEG-OH (MW=30000, 1 eq.) was azeotroped with 360 mL of reagent toluene for 2 hours to remove 75 mL of toluene / water. After constant boiling, the solution was cooled to 45 to 50°C. 166 mg of triphosgene (0.67 eq.) was added to PEG, followed by 131.8 mg of anhydrous pyridine (2 eq.). The reaction was stirred at 50°C for 3 hours. Then 239.8 mg of N-hydroxysuccinimide (2.5 eq.) were added, followed by 164.8 g of anhydrous pyridine (2.5 eq.). The reaction mixture was stirred overnight at 50 °C under nitrogen. Pyridinium salts were filtered. The solvent was removed using a Rotavapor and the residue was recrystallized from 2-propanol. The isolated product was dried in a vacuum oven at 40 °C to obtain 23 g of 30 kmSC-PEG.

[0266] Preparation of 30kmPEG-Lys(Boc)-OH (Compound 3):

[0267] 369 mg of H-lys(boc)-OH (3 eq.), 646.5...

Embodiment 2

[0274] Example 2: Preparation of SCACD3IL2 and SCAPDL1IL10

[0275] The two cytokine-capped single-chain antibody fragment proteins in the present invention are prepared according to the highlighted marks in formula Ib. A 1 -L 3 -C 1 (A 1 -L 3 -C 1 ) of the first protein consists of IL2V+uPA substrate+MMP14 substrate+anti-CD3 (SCACD3IL2), the second protein-A 2 -L 4 -C 2 (A 2 -L 4 -C 2 ) is IL10+uPA+MMP14 substrate+anti-PDL1 (SCAPDL1IL10). Both proteins were produced by recombinant DNA technology in Chinese hamster ovary (CHO) cells knocked out of GS using the pD2531nt-HDP expression vector containing the GS gene (both cell line and vector licensed from Horizon Discovery, Inc) . DNA encoding the first protein (SCACD3IL2) and the second protein (SCAPDL1IL10) were synthesized and cloned into pD2531nt-HDP expression vector, and transfected into CHO-GS(- / -) cells. Stable cell lines with high productivity were obtained by culturing cells in medium containing the GS in...

Embodiment 3

[0280] Example 3: Preparation of 30kmPEG-(SCAPDL1IL10)SCACD3IL2( figure 2 )

[0281] Preparation of compound 9:

[0282] N-Succinimidyl 4-maleimidobutyrate (1 eq.) was reacted with azido-dPEG10-amine (1.5 eq.) in DMSO at room temperature for 45 minutes. The obtained compound 9 azide-PEG10-maleimide was directly used in the next step without further purification.

[0283] Preparation of Compound 10:

[0284] TCEP-HCl (Product No. 580560, Sigma-Aldrich) was added to SCAPDL1IL10 (5-10 mg / mL) at a final concentration of 2-10 mM in 200 mM phosphate buffer (pH 6.8). The reaction was mixed well and left at room temperature for 30 minutes. Reduced SCAPDL1IL10 (1 eq.) was reacted with compound 9 (100 eq.) at room temperature for 1 hour. The reaction was quenched with 10 mM cystine for 10 min at room temperature. Excess compound 9 was removed by desalting column in PBS buffer. Fractions of desired compound 10 azide-SCAPDL1IL10 were pooled and concentrated to 5-10 mg / ml for the n...

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Abstract

A long acting modified T-cell engager bispecific antibody with cytokine caps that provides reduced toxicity and boosted anti-tumor activity is disclosed. Method of making the modified and cytokine capped Bi-specific T-cell engager antibody is also disclosed.

Description

[0001] field of invention [0002] The present invention relates to bispecific antibodies with cleavable immunocytokine caps aimed at reducing toxicity and improving efficacy. In particular, the invention relates to long-acting modified bispecific T cell engager (BiTE) antibodies conjugated to releasable cytokines. [0003] Background of the invention [0004] Advances in cancer biology and oncogenesis over the past two decades have witnessed many new and more effective therapies that have revolutionized the treatment of malignant cancers. Data from cancer immunotherapy have demonstrated that supplementation and augmentation of existing anti-tumor immune responses offers excellent opportunities to enhance durable cancer remission. Among the various drugs recently approved by the FDA, the bispecific T cell engager (BiTE) blinatumomab (Blinatumomab) Due to its engineered structure and clinical efficacy against relapsed or refractory B-lineage leukemia or lymphoma, it represent...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/00A61K47/10A61K47/36A61K47/68A61P35/00
CPCA61P35/00A61K47/60A61K47/6849A61K47/6813A61K47/6851A61K47/6889
Inventor 刘树民乌德春文瑜雷杨吕卫东
Owner SHENZHEN ENDURING BIOTECH LTD
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