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Mutant vaccinia viruses and use thereof

A technology of vaccinia virus and virions, applied in the direction of viruses, viral peptides, viruses/bacteriophages, etc., which can solve the problem of reduced ability of Ab binding to peptides

Pending Publication Date: 2021-09-03
ICELLKEALEX THERAPEUTICS LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Alanine substitutions of these residues resulted in a reduced ability of the Ab to bind the peptide

Method used

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  • Mutant vaccinia viruses and use thereof
  • Mutant vaccinia viruses and use thereof
  • Mutant vaccinia viruses and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] Materials and methods

[0135] Material

[0136] pUC57-Amp A27L, pUC57-Amp L1R, pUC57-Amp D8L, pUC57-Amp H3L, (GENEWIZ). CV-1 cells (ATCC, cat. #CCL-70). vSC20 vaccinia virus library. GeneJuice Transfection Reagent (Millipore, cat. #2703870). DMEM medium (GE Helathcare, cat. #SH30081.01), FBS (GE Healthcare, cat. #SH30070.03), DPBS (Sigma, cat. #8537). Dry ice / ethanol bath, 6-well tissue culture plates, 12 x 75–mm polystyrene tubes, disposable spatula or plunger for 1-ml syringes, sterile 2-ml sterile microcentrifuge tubes.

[0137] Cell preparation and infection with wild-type vaccinia virus

[0138] CV-1 cells (2×10 5 / well) in complete DMEM medium in the wells of a 6-well tissue culture plate and incubated to 50–80% confluence (37°C, 5% CO 2 overnight). Aliquots of parental virus were thawed and sonicated (30 sec) in ice water several times to remove clumps (cool on ice between each sonication). Dilute virus to 0.5 x 10 in complete DMEM 5 pfu / ml. The...

Embodiment 2

[0152] Neutralizing antibody (Nab) epitope determination for H3L–peptide array sequence analysis

[0153] To identify possible regions on H3L involved in NAb interactions, peptide arrays covering full-length H3L were synthesized and screened for peptides that bound anti-VV NAbs. The array starts at the N-terminus of H3L and spans the entire length of the protein sequence, with each contiguous spot containing 12 amino acids offset by 4 amino acids towards the C-terminus along the sequence, i.e., each spot in the array has 8 - The residue overlaps with a previous spot. Cellulose membranes containing synthetic H3L peptide arrays were then screened to identify peptides that bind to anti-VV polyclonal Nabs (Abeam, ab35219). Briefly, membranes were placed in Millipore H 2 Wash three times in O for 5 minutes each, and block with 5% (wt / vol) milk-PBS (MPBS) overnight at 4°C. 4 μg / mL NAb was incubated with the membrane in MPBS for 3 h at room temperature with gentle agitation. Af...

Embodiment 3

[0162] NAb epitope determination by H3L–alanine scanning of identified peptides

[0163] To further map the NAb epitopes and elucidate the key residues on the H3L peptides identified by our peptide array studies, a series of ELISAs were performed with the 9 identified peptides and their alanine-substituted variants (Figure 2). Nine peptide variants identified by peptide arrays were synthesized by alanine substitution (GenScript USA Inc. NJ, USA).

[0164] Table 4

[0165] A total of 80 variant peptides were synthesized

[0166]

[0167]

[0168]

[0169] Native peptides (unmutated, as shown in bold above, SEQ ID NO: 89-97) were biotin-tagged (N-terminal). 96-well Pierce™ NeutrAvidin-coated plates (Thermo Fisher, 15507) were rinsed with PBST and incubated overnight in MPBS (blocking buffer, 100 μL / well) at 4°C. The blocking buffer was discarded and 100 μL of biotinylated peptide at 200 ng / mL was added to the plate and incubated at 4°C for 90 min. Simultaneously...

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Abstract

The present invention discloses recombinant vaccinia virus (VV) virions that are resistant to antiviral defenses and have enhanced anti-tumor activities. In one embodiment, the recombinant VV comprise one or more variant VV proteins that have mutations at one or more neutralizing antibody epitopes, thereby conferring viral escape from the neutralizing antibodies. In another embodiment, the recombinant VV is resistant to complement-mediated neutralization due to the expression of a regulator of complement activation (e.g. CD55). In another embodiment, the recombinant VV has enhanced anti-tumor activities due to the expression of bi-specific antibodies co-targeting cancer cells and immune effector cells, or the expression of a polypeptide blocking the PD-1 pathway. The recombinant vaccinia virus virions can be used to treat cancer in a subject.

Description

[0001] Cross References to Related Applications [0002] This PCT application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 749,102, filed October 22, 2018, and U.S. Provisional Patent Application No. 62 / 912,344, filed October 8, 2019. See further description in the Summary of the Invention. Background technique [0003] Oncolytic viruses specifically infect, replicate and kill tumor cells while leaving normal cells undamaged. This preference for transformed cells makes oncolytic viruses ideal candidates for the development of new cancer therapies. Various oncolytic viruses have exploited their tumor-specific killing activity through direct (e.g., cytolysis due to viral replication and immune-mediated cytotoxicity) and indirect mechanisms (e.g., stimulation of bystander cell killing, induction of cytotoxicity, etc.). Oncolytic vaccinia virus (VV) is an attractive addition to current treatment options, demonstrating efficacy and safety in animal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/275C12N7/00C12N7/01C12N15/863C12N15/869A61K39/285A61K48/00C07K14/07
CPCC07K14/005A61K35/768C12N2710/24122C12N2710/24132C12N2710/24121C12N2710/24143C12N15/86A61K39/001129C07K16/2809A61K2039/53A61P35/00C07K16/081C07K16/40C07K2317/31C07K2317/622C07K16/2878A61K39/285C12N15/625
Inventor 宋晓彤M·维斯科夫斯卡M·L·戈麦斯梅达利亚
Owner ICELLKEALEX THERAPEUTICS LLC
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