Tumor immunotherapy composition based on antigen-presenting cells activated by attenuated
A tumor immunotherapy composition based on modified cells, in particular antigen-presenting cells activated by means of attenuated Listeria monocytogenes , a preparation method therefor and an application thereof. Attenuated Listeria monocytogenes carrying a specific antigen plasmid is used to activate antigen-presenting cells, thereby activating MHC antigen presenting properties and a series of cellular immune responses in vivo so as to achieve the purpose of anti-tumor therapy. The described technical solution may specifically activate macrophages and/or dendritic cells, thereby eliciting a series of specific anti-tumor immune responses. The operation process does not require genetic modification of autologous cells, is not limited by tumor type, and operations of the overall process are simple, easy-to-implement and reproducible. The tumor immunotherapy composition of the present disclosure may activate a series of anti-tumor immune responses in vivo, thereby greatly shortening the treatment process and significantly improving targeting ability and safety.
1 . A population of antigen-presenting cells obtained by contacting said antigen-presenting cells with a recombinant Listeria that is attenuated, wherein said recombinant Listeria comprises: (i) a recombinant nucleic acid molecule, (ii) a recombinant plasmid, or (iii) a recombinant expression vector; wherein said recombinant Listeria expresses (iv) a recombinant protein; wherein
said (i) recombinant nucleic acid molecule comprises an open reading frame encoding a recombinant polypeptide, said recombinant polypeptide comprises a heterologous antigen fused to a derived Listeriolysin O (LLO) polypeptide, said recombinant nucleic acid molecule further comprises a first promoter; wherein the amino acid sequence of said derived Listeriolysin O (LLO) polypeptide comprises a first polypeptide sequence of amino acids 1-28 of SEQ ID NO: 2, a second polypeptide sequence of amino acids 22-523 of SEQ ID NO: 2, and a third polypeptide sequence of amino acids 524-529 of SEQ ID NO: 2; wherein said heterologous antigen or a fragment thereof is presented on the surface of at least one of the antigen-presenting cells; said (ii) recombinant plasmid or said (iii) recombinant expression vector comprises the sequence of said (i) recombinant nucleic acid molecule; and
said (iv) recombinant protein is encoded by said (i) recombinant nucleic acid molecule, or is expressed by said (ii) recombinant plasmid or said (iii) recombinant expression vector.
2 . The antigen-presenting cells of claim 1 , wherein the antigen-presenting cells comprise macrophages or dendritic cells.
3 . The antigen-presenting cells of claim 2 , wherein the antigen-presenting cells comprise macrophages.
4 . The antigen-presenting cell of claim 2 , wherein the antigen-presenting cells comprise dendritic cells.
5 . The antigen-presenting cells of claim 1 , wherein said derived Listeriolysin O (LLO) polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 2.
6 . The antigen-presenting cells of claim 1 , wherein said heterologous antigen is ovalbumin (OVA) or a fragment thereof that comprises an amino acid sequence as set forth in SEQ ID NO: 7.
7 . The antigen-presenting cells of claim 1 , wherein said (i) recombinant nucleic acid molecule further comprises a linking sequence, wherein said linking sequence links a nucleotide sequence encoding said derived Listeriolysin O (LLO) polypeptide and a nucleotide sequence encoding said heterologous antigen.
8 . The antigen-presenting cells of claim 7 , wherein said (i) recombinant nucleic acid molecule comprises a nucleotide sequence that is connected to said nucleotide sequence encoding said derived Listeriolysin O (LLO) polypeptide, wherein said nucleotide sequence comprises said linking sequence and said nucleotide sequence encoding said heterologous antigen, wherein said nucleotide sequence encodes an amino acid sequence that is as set forth in SEQ ID NO: 11.
9 . The antigen-presenting cells of claim 7 , wherein in said (i) recombinant nucleic acid molecule, said linking sequence comprises a nucleotide sequence encoding an amino acid sequence as set forth in SEQ ID NO: 10.
10 . The antigen-presenting cells of claim 1 , wherein in said (i) recombinant nucleic acid molecule, said first promoter is a Phly promoter.
11 . The antigen-presenting cells of claim 10 , wherein said recombinant nucleic acid molecule further comprises a tag sequence for detection or a gene encoding a metabolite.
12 . A pharmaceutical composition comprising a therapeutically effective amount of the antigen-presenting cells of claim 1 , wherein said pharmaceutical composition further comprises a second therapeutic agent or a pharmaceutically acceptable carrier.
13 . The antigen-presenting cells of claim 1 , wherein the heterologous antigen is a tumor antigen.
14 . The antigen-presenting cells of claim 1 , wherein the heterologous antigen is a non-tumor antigen.
15 . The antigen-presenting cells of claim 1 , wherein the open reading frame encoding the recombinant polypeptide comprises a sequence encoding the heterologous antigen inserted between the sequence encoding the first polypeptide sequence and the sequence encoding the second polypeptide sequence.
16 . The antigen-presenting cells of claim 1 , wherein the open reading frame encoding the recombinant polypeptide comprises a sequence encoding the heterologous antigen inserted between the sequence encoding the second polypeptide sequence and the sequence encoding the third polypeptide sequence.
17 . The antigen-presenting cells of claim 1 , wherein said recombinant Listeria is a recombinant Listeria L monocytogenes.
18 . A method for slowly and continuously killing cells, comprising contacting said cells with the antigen-presenting cells of claim 1 .
19 . A method for inducing an immune response in a subject, wherein said method comprises
(a) contacting a population of antigen-presenting cells with a recombinant Listeria that is attenuated, wherein said recombinant Listeria comprises: (i) a recombinant nucleic acid molecule, (ii) a recombinant plasmid, or (iii) a recombinant expression vector; wherein said recombinant Listeria expresses (iv) a recombinant protein; wherein
said (i) recombinant nucleic acid molecule comprises an open reading frame encoding a recombinant polypeptide, said recombinant polypeptide comprises a heterologous antigen fused to a derived Listeriolysin O (LLO) polypeptide, said recombinant nucleic acid molecule further comprises a first promoter; wherein the amino acid sequence of said derived Listeriolysin O (LLO) polypeptide comprises a first polypeptide sequence of amino acids 1-28 of SEQ ID NO: 2, a second polypeptide sequence of amino acids 22-523 of SEQ ID NO: 2, and a third polypeptide sequence of amino acids 524-529 of SEQ ID NO: 2; wherein said heterologous antigen or a fragment thereof is presented on the surface of at least one of the antigen-presenting cells when the contacting step is finished;
said (ii) recombinant plasmid or said (iii) recombinant expression vector comprises the sequence of said (i) recombinant nucleic acid molecule;
said (iv) recombinant protein is encoded by said (i) recombinant nucleic acid molecule, or is expressed by said (ii) recombinant plasmid or said (iii) recombinant expression vector; and
(b) administering said antigen-presenting cells obtained in step (a) to the subject.
20 . A method for activating a population of antigen-presenting cells, wherein said method comprises
contacting the antigen-presenting cells with a recombinant Listeria that is attenuated, wherein said recombinant Listeria comprises: (i) a recombinant nucleic acid molecule, (ii) a recombinant plasmid, or (iii) a recombinant expression vector; wherein said recombinant Listeria expresses (iv) a recombinant protein; wherein
said (i) recombinant nucleic acid molecule comprises an open reading frame encoding a recombinant polypeptide, said recombinant polypeptide comprises a heterologous antigen fused to a derived Listeriolysin O (LLO) polypeptide, said recombinant nucleic acid molecule further comprises a first promoter; wherein the amino acid sequence of said derived Listeriolysin O (LLO) polypeptide comprises a first polypeptide sequence of amino acids 1-28 of SEQ ID NO: 2, a second polypeptide sequence of amino acids 22-523 of SEQ ID NO: 2, and a third polypeptide sequence of amino acids 524-529 of SEQ ID NO: 2; wherein said heterologous antigen or a fragment thereof is presented on the surface of at least one of the antigen-presenting cells when the contacting step is finished;
said (ii) recombinant plasmid or said (iii) recombinant expression vector comprises the sequence of said (i) recombinant nucleic acid molecule;
said (iv) recombinant protein is encoded by said (i) recombinant nucleic acid molecule, or is expressed by said (ii) recombinant plasmid or said (iii) recombinant expression vector;
wherein said contacting occurs in vitro; wherein the antigen-presenting cells comprise macrophages or dendritic cells.