Covalent protein drugs developed via proximity-enabled reactive therapeutics (PERX)

A protein and drug technology, applied in the field of covalent protein drugs developed through proximity-enabling response therapy, which can solve the problems of lack of covalent reactivity of natural amino acid residues, selective application of reactions, obstacles, etc.

Pending Publication Date: 2021-07-27
HANGZHOU BRANCH OF TECH INST OF PHYSICS & CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The challenges in developing covalent protein drugs are as follows: native proteins interact primarily through non-covalent interactions and lack covalent reactivity to native amino acid residues on the target
Additionally, the ubiquitous presence of amino acids in proteins, cells, and in vivo imposes a formidable barrier to achieving reaction selectivity against a target

Method used

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  • Covalent protein drugs developed via proximity-enabled reactive therapeutics (PERX)
  • Covalent protein drugs developed via proximity-enabled reactive therapeutics (PERX)
  • Covalent protein drugs developed via proximity-enabled reactive therapeutics (PERX)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0108] Cloning of human PD-1 and PD-L1 extracellular IgV domains

[0109] Protein stability aided by replacement of human PD-1 (hPD-1) IgV domain encoding gene (residues 32-160, Cys93 with Ser (C93S) 28 ) into the pET-26b vector with a carboxy-terminal 6x histidine tag to generate plasmid pET-26b-PD1(WT) (SEQ ID NO: 15 and 22). Briefly, plasmid pUC57-Kan-hPD-1 IgV domain (32 -160, C93S) to amplify the DNA of the hPD-1 IgV domain (32-160, C93S), which was codon-optimized and synthesized by GENEWIZ (China, Suzhou). The PCR product was digested with NdeI-HF and XhoI-HF and ligated into the pre-cut pET-26b vector using T4 DNA ligase (NEB, catalog number M0202L).

[0110] PD-1 WT-F:GTTAGACTcatatgTGGAATCCGCCGACCTTTAGC (SEQ ID NO:1)

[0111] PD-1 WT-R: ACTGctcgagCGGACTAGGACTCGGATGTGCG (SEQ ID NO: 2)

[0112] To introduce a TAG codon at site D77 or A129, overlap PCR was performed using Q5 high-fidelity DNA polymerase and using plasmid pET-26b-PD1(WT) as a template using the follow...

Embodiment 2

[0130] Expression and purification of human PD-1 IgV domain and PD-L1 IgV domain

[0131] Plasmids pET-26b-PD1(WT), pET-26b-PD-L1(WT) and pET-26b-PD-L1(H69A) were individually transformed into E. coli BL21(DE3) electrocompetent cells. Plasmid pET-26b-PD-1(Q75TAG), pET-26b-PD-1(D77TAG) or pET-26b-PD-1(A129TAG) each with plasmid pEvol-FSYRS 29 Co-transformation into Escherichia coli BL21 (DE3) electrocompetent cells.

[0132] For the expression of PD-1(WT), PD-L1(WT) and PD-L1(H69A), the transformed bacteria were cultured at 37°C in 2xYT medium containing 50 μg / mL kanamycin, and when OD 600 Induction with 1 mM IPTG when reaching 0.8. For FSY incorporation into proteins, the transformed bacteria were cultured at 37°C in 2xYT medium containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol, and when OD 600 At 0.8 it was induced with 1 mM IPTG, 0.2% arabinose and 1 mM FSY.

[0133]After induction of expression for 12 hours, bacteria were harvested by centrifugation at 7000 rpm...

Embodiment 3

[0140] Cross-linking of PD-1(FSY) and PD-L1

[0141] 3.1 In vitro cross-linking of PD-1(FSY) and PD-L1

[0142] Purified and refolded PD-1(WT), PD-1(Q75FSY), PD-1(D77FSY) or PD-1(A129FSY) and PD-L1(WT) or PD-L1(H69A) at a ratio of 1:1 The molar ratios were incubated in PBS buffer at 37 °C for 6 h. The amount of PD-L1 was 4 μg. After incubation, 5x reducing loading buffer (CWBio, cat# CW0027) was added to the incubation and heated at 100°C for 10 min. These samples were then separated by 15% SDS-PAGE gels followed by Coomassie blue staining. Then use a primary antibody specific for the His6x tag (mAb anti-6xHis tag, Abcam, Cat. No. 18184, 1:1000 dilution) and a secondary antibody goat pAb anti-mouse IgG (Abcam, Cat. No. 97023, 1:5000 dilution) Perform western blotting. Protein bands were visualized by chemiluminescence (Bio-rad, catalog #1705062).

[0143] 3.2 Mass spectrometry analysis

[0144] PD-1(FSY) and PD-1(FSY) / PD-L1 cross-linked protein samples were digested wit...

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Abstract

Provided a proximity-enabled reactive therapeutics (PERx) approach to generate covalent protein drugs. A latent bioreactive amino acid FSY was incorporated into human programmed cell death protein 1 (PD-1), which selectively reacted with a proximal histidine of human PD-L1 upon binding, enabling irreversible binding of PD-1 with PD-L1 in vitro, on cancer cells, and in tumor in mice. When administrated in humanized mouse models, the covalent PD-1 (FSY) exhibited robust antitumor effect over wildtype PD-1, achieving therapeutic efficacy equivalent to the FDA approved therapeutic monoclonal antibody atezolizumab. The PERx approach should provide a general method for converting interacting proteins into covalent protein drugs for high therapeutic efficacy.

Description

[0001] sequence listing [0002] This application is filed together with a Sequence Listing in electronic form. The entire content of the Sequence Listing is incorporated herein by reference. technical field [0003] The present application relates generally to methods of proximity-enabled response therapy for the generation of covalent protein drugs, and muteins comprising a potentially bioreactive unnatural amino acid (Uaa) that can be used to pass the proximity-enabled response therapy ( Proximity-enabled reactive therapeutics) developed covalent protein drugs. More specifically, the present application relates to a mutated PD-1 comprising o-fluorosulfate-L-tyrosine (fluorosulfate-L-tyrosine, FSY), its preparation method, and the application of the mutated PD-1. Background technique [0004] Small molecule drugs that covalently bind their targets have reemerged in recent years with great success, although historically shunned by pharmaceutical researchers and industry ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/197C07K1/113C07C381/02A61K31/198A61P35/00
CPCC07K1/113A61P35/00
Inventor 王谦
Owner HANGZHOU BRANCH OF TECH INST OF PHYSICS & CHEM CHINESE ACAD OF SCI
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