TEMPERATURE-CONTROLLABLE, RNA IMMUNOTHERAPEUTIC FOR CANCER
The present disclosure relates to mRNA, self-replicating RNA, and temperature-sensitive, self-replicating RNA encoding a cancer antigen. The RNA constructs are suitable for cancer immunotherapy in a mammalian subject, such as a human subject.
1 . A composition for stimulating an immune response against a cancer antigen in a mammalian subject, comprising an excipient, and a temperature-sensitive self-replicating RNA comprising an open reading frame (ORF) encoding a fusion protein, and an Alphavirus replicon lacking a viral structural protein coding region, wherein the ORF comprises from 5′ to 3′:
(i) a nucleotide sequence encoding a mammalian signal peptide; and
(ii) a nucleotide sequence encoding a cancer antigen,
wherein the temperature-sensitive self-replicating RNA is capable of expressing the fusion protein at a permissive temperature but not at a non-permissive temperature.
2 . The composition of claim 1 , wherein the cancer antigen comprises a tumor-associated antigen (TAA).
3 . The comprises of claim 2 , wherein the TAA comprises a WT1 antigen, a NY-ESO-1 antigen, a MAGEA3 antigen, a BIRC5 (SURVIVIN) antigen, a PRAME antigen, or a combination thereof.
4 . The composition of claim 2 , wherein the TAA comprises a WT1 antigen.
5 . The composition of claim 4 , wherein the amino acid sequence of the WT1 antigen comprises SEQ ID NO:2, or the amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2.
6 . The composition of claim 2 , wherein the TAA is a TAA fusion protein comprising a WT1 antigen, a NY-ESO-1 antigen, a MAGEA3 antigen, a BIRC5 antigen, and a PRAME antigen.
7 . The composition of claim 6 , wherein the amino acid sequence of the TAA fusion protein comprises SEQ ID NO:7, or the amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:7.
8 . The composition of claim 2 , wherein the TAA comprises a BIRC5 antigen.
9 . The composition of claim 8 , wherein the amino acid sequence of the BIRC5 antigen comprises SEQ ID NO:3, or the amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:3.
10 . The composition of claim 2 , wherein the TAA comprises a NY-ESO-1 antigen.
11 . The composition of claim 10 , wherein the amino acid sequence of the NY-ESO-1 antigen comprises SEQ ID NO:4, or the amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4.
12 . The composition of claim 2 , wherein the TAA comprises a MAGEA3 antigen.
13 . The composition of claim 12 , wherein the amino acid sequence of the MAGEA3 antigen comprises SEQ ID NO:5, or the amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5.
14 . The composition of claim 2 , wherein the TAA comprises a PRAME antigen.
15 . The composition of claim 14 , wherein the amino acid sequence of the PRAME antigen comprises SEQ ID NO:6, or the amino acid sequence at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:6.
16 . The composition of claim 1 , wherein the cancer antigen comprises a neoantigen.
17 . The composition of any one of claims 1-16 , wherein the mammalian signal peptide is a signal peptide of a surface protein expressed in mammalian antigen presenting cells.
18 . The composition of claim 17 , wherein the mammalian signal peptide is a CDS signal peptide and the amino acid sequence of the CDS signal peptide comprises SEQ ID NO:1, or the amino acid sequence at least 90% or 95% identical to SEQ ID NO:1.
19 . The composition of any one of claims 1-18 , wherein the Alphavirus is selected from the group consisting of a Venezuelan equine encephalitis virus, a Sindbis virus, and a Semliki Forrest virus.
20 . The composition of claim 19 , wherein the Alphavirus is a Venezuelan equine encephalitis virus.
21 . The composition of any one of claims 1-20 , wherein the Alphavirus replicon comprises a nonstructural protein coding region with an insertion of 12-18 nucleotides resulting in expression of a nonstructural Protein 2 (nsP2) comprising from 4 to 6 additional amino acids between beta sheet 5 and beta sheet 6 of the nsP2.
22 . The composition of claim 21 , wherein the additional amino acids comprise the sequence of SEQ ID NO:14 (TGAAA).
23 . The composition of claim 22 , wherein the amino acid sequence of the nsP2 comprises SEQ ID NO:12.
24 . The composition of claim 23 , wherein the amino acid sequence of the nsP2 comprises one sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO: 11.
25 . The composition of claim 24 , wherein the amino acid sequence of the nsP2 comprises SEQ ID NO:11.
26 . The composition of any one of claim 1-25 , wherein the permissive temperature is from 30° C. to 36° C., or 31° C. to 35° C., or 32° C. to 34° C., or 33° C.±0.5° C., and the non-permissive temperature is 37° C. #0.5° C., optionally wherein the permissive temperature is from 31° C. to 35° C. and the non-permissive temperature is at least 37° C. #0.5° C.
27 . The composition of any one of claim 1-26 , wherein the composition does not comprise lipid nanoparticles.
28 . The composition of any one of embodiments 1 - 27 , wherein the composition further comprises chitosan.
29 . A method for stimulating an immune response against a cancer antigen in a mammalian subject, comprising administering the composition of any one of claims 1-28 to a mammalian subject so as to stimulate an immune response against the cancer antigen in the mammalian subject.
30 . The method of claim 29 , wherein the composition is administered intradermally.
31 . The method of claim 29 or claim 30 , wherein the immune response comprises a cellular immune response reactive with mammalian cells expressing the cancer antigen.
32 . The method of claim 31 , wherein the cellular immune response comprises one or both of a cancer antigen-specific cytotoxic T lymphocyte response and a cancer antigen-specific helper T lymphocyte response.
33 . The method of claim 32 , wherein the immune response further comprises a humoral immune response reactive with the cancer antigen.
34 . The method of any one of claims 29-33 , wherein the mammalian subject is a human subject.
35 . A kit comprising:
(i) the composition of any one of claims 1-28 ; and
(ii) a device for intradermal delivery of the composition to a mammalian subject.
36 . The kit of claim 35 , wherein the device comprises a syringe and a needle.