Virus vectors expressing multiple epitopes of tumor associated antigens for inducing antitumor immunity

A technology of tumor-related antigens and epitopes, applied in the field of cases, can solve the problem of inability to effectively target viruses

Inactive Publication Date: 2019-03-01
NEW YORK UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Furthermore, to date, some tumor cells may not be effectively targeted by viruses used in cancer therapy, thus emphasizing the need to develop new therapies and other approaches to enhance anticancer treatments

Method used

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  • Virus vectors expressing multiple epitopes of tumor associated antigens for inducing antitumor immunity
  • Virus vectors expressing multiple epitopes of tumor associated antigens for inducing antitumor immunity
  • Virus vectors expressing multiple epitopes of tumor associated antigens for inducing antitumor immunity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0282] Example 1 - Method

[0283] Vector Preparation: Construction of recombinant viral vectors is carried out using standard techniques well known to those of ordinary skill in the field of molecular biology, including but not limited to plasmid purification, restriction enzyme digestion, ligation, transformation, polymerase chain reaction, and DNA Sequencing (for example, Current Protocols in Molecular Biology, EM. Ausubel et al. (Eds), John Wiley and Sons, Inc., NY, USA. (1998) and Molecular Cloning: A Laboratory Manual (2nd Ed.), J.Sambrook, E.F. Fritsch and T. Maniatis (Eds), Cold Spring Harbor Laboratory Press, NY, USA. (1989)).

[0284] For experiments using Sindbis virus vectors encoding LacZ (SV / LacZ) as immunogenic SV / TAA agents, and SV / Fluc and SV / GFP as control vectors, vectors were prepared as previously described. (Tseng J.C. et al., 2004, Nat. Biotechnol., 22:70-77). Briefly, plasmids carrying replicons (SinRep5-LacZ, SinRep5-GFP, or SinRep5-Fluc) or DHBB hel...

Embodiment 2

[0289] Example 2 - Construction of Sindbis virus mediator expressing multiple epitopes to induce anti-tumor immunity

[0290] Polynucleotides (DNA sequences; minigenes) encoding multiple T cell recognition epitopes separated by furin cleavage sites were synthesized by GeneArt (Life Technologies Corp., Waltham MA) using standard molecular biology methods. Synthetic polynucleotides contain a ribosome binding site, a translation initiation codon, an endoplasmic reticulum signal sequence, followed by a furin cleavage site interspersed with epitope coding sequences, a stop codon, and a polynucleotide sequence allowing insertion The XbaI / ApaI restriction endonuclease site (WO 2015 / 035213A2) of the Sindworth replicon pT7StuI-RLacZ#202 was used to replace the restriction enzyme site of the LacZ gene. The Hindworth replicon contains the viral subgenomic promoter sequence upstream of the XbaI site and the mRNA polyadenylation sequence downstream of the ApaI site. The synthetic DNA sequ...

Embodiment 3

[0317] Example 3 - Sindbis virus mediators encoding multiple epitopes of tumor-associated antigens produce polyepitopic mRNA

[0318] Experiments were performed to determine whether the Sindbis virus vector (SV MG-CT26) encoding multiple epitopes of tumor-associated antigens (i.e., NY-ESO-1, gp70 and survivin as described above in Example 2), produced correct polyepitopic mRNA. For the experiment, a 10-fold serial dilution of a Sindbis virus vector encoding multiple epitopes (referred to herein as "SV / MG-CT.26" (100 to 10 -11 )) to infect 2×10 4 Kidney cells of a baby hamster. After overnight culture, cells were harvested by centrifugation and RNA was isolated using a Qiagen kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA was quantified using a nanodrop spectrophotometer.

[0319] One microgram (1 μg) of each sample was reverse transcribed using ThermoScript (Life Technologies, CA) according to the manufacturer's instructions. The cDNA5_R r...

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Abstract

Provided are polynucleotides and viral vectors, particularly, alphavirus vectors such as Sindbis viral vectors, which encode multiple, e.g., two or more, epitopes of at least one tumor associated antigen in which each epitope is separated by a processing or enzyme cleavage site. The multiple epitopes of the two or more tumor associated antigens encoded by the described polynucleotides and viral vectors may be the same or different. Methods of treating mammalian subjects having a cancer or tumor expressing the tumor associated antigen epitopes are provided, in which the viral vectors encoding the multiple epitopes, as well as other immunostimulatory or immunomodulatory components, generate an anti-cancer or anti-tumor immune response in which high levels of effector T cells increase the survivability of tumored mammalian subjects and result in epitope spreading, thus providing a further enhancement of the immune response.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 303,923, filed March 4, 2016, which is hereby incorporated by reference in its entirety. Background technique [0003] Despite the implementation of available cancer treatments (which can include aggressive surgical approaches and combination chemotherapy regimens) over the past two decades, many cancers routinely evade detection and destruction by cells of the immune system, and the patients with a grim prognosis. [0004] Anticancer immunity, including protective immunity, is thought to be based on the magnitude of the immune response and the phenotype of the memory immune response, including T central memory cells (Tcm) and T effector memory cells (Tem). Tcm is characterized by a CD62L+CD127+ phenotype, whereas Tem is defined by a CD62L-CD127+ phenotype. Tems are transported through non-lymphoid tissues and exert immediate effector fun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18C07K14/47A61K39/12A61K39/21C12N15/10
CPCA61K39/00C07K14/005C07K2319/50C12N2770/36143C12N2840/20A61K39/0011C12N9/12C12Y207/12002A61P35/00A61P35/02A61P37/04A61P43/00A61K39/001193A61K39/001161A61K39/001194A61K39/00117A61K39/001191A61K39/00115A61K39/001163A61K39/001124A61K39/001157A61K39/001166A61K39/001164A61K39/001182A61K39/001197A61K39/001156A61K39/001189A61K39/001152A61K39/001184A61K39/001188A61K39/001106A61K39/001139A61K39/001151A61K39/001154A61K39/001181A61K39/001149A61K39/001102A61K39/00119A61K39/001144A61K39/001153A61K39/001162A61K39/001168A61K39/001186A61K39/001195C07K14/4748C12N7/00A61K2039/5256A61K2039/645C07K14/4747C07K2319/40C12N2770/36122
Inventor D·梅鲁埃洛C·潘佩诺A·赫尔达多马丁尼兹
Owner NEW YORK UNIV
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