阅读说明：本技术 具有密码子对去优化区域的重组病毒及其用于治疗癌症的用途 (Recombinant viruses with codon pair de-optimized regions and their use for treating cancer ) 是由 约翰·罗伯特·科尔曼 斯特芬·穆勒 杨晨 王颖 查尔斯·斯塔夫 于 2018-12-21 设计创作，主要内容包括：本发明为设计的重组病毒在治疗多种形式的恶性肿瘤中的用途。本发明的重组病毒为其中的野生型病毒的一个或多个区域被替换为合成的重新编码的序列的病毒,所述合成的重新编码的序列降低了相对于人密码子对偏性的密码子对分值,或增加了CpG二核苷酸的数量,或增加了UpA二核苷酸的数量。特别是,本发明的方法可用于治疗多种器官(例如：乳房、皮肤、结肠、支气管通道、胃肠道的衬里上皮、上呼吸道和生殖泌尿道、肝、前列腺和脑)的恶性肿瘤。实验动物的惊人缓解证实了对恶性多形性胶质母细胞瘤的治疗以及对乳腺癌和黑素瘤的治疗。(The invention relates to the use of the designed recombinant virus in the treatment of various forms of malignant tumors. The recombinant viruses of the invention are viruses in which one or more regions of the wild-type virus are replaced with a synthetic recoded sequence that decreases the codon pair score relative to human codon pair bias, or increases the number of CpG dinucleotides, or increases the number of UpA dinucleotides. In particular, the methods of the invention can be used to treat malignancies in a variety of organs such as the breast, skin, colon, bronchial passages, lining epithelium of the gastrointestinal tract, upper respiratory and genitourinary tracts, liver, prostate and brain. The surprising remission in experimental animals confirms the treatment of malignant glioblastoma multiforme and of breast cancer and melanoma.)

1. A method of treating a malignant tumor, the method comprising:

administering a modified virus to a subject in need thereof, wherein the modified virus is selected from the group consisting of:

a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a codon pair de-optimized region encoding a similar protein sequence, wherein the codon pair bias of the modified sequence is less than the codon pair bias of the parent virus and the codon pair bias of the modified sequence is reduced by at least about 0.05, or at least about 0.1, or at least about 0.2;

a modified virus derived from said wild type virus or a previously modified virus by replacing at least one genomic region of the wild type virus with a region encoding a similar protein sequence having an increased CpG dinucleotide, wherein the increase in CpG dinucleotide is at least 41 instances higher than the parental virus, or at least 21 instances higher than the parental virus genome;

a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of said virus with a region encoding a similar protein sequence having an increased UpA dinucleotide, wherein the increase in UpA dinucleotide is at least 26 instances higher than the parent, or at least 13 instances higher than the parent viral genome;

a modified virus derived from said wild type virus or a previously modified virus by replacing at least one genomic region of the wild type virus with a region having increased UpA and CpG dinucleotides encoding a similar protein sequence, wherein the increased combination of UpA and CpG dinucleotides is at least 42 instances higher than the parent; and

combinations thereof.

2. A method of treating a malignant tumor, the method comprising:

administering an initial dose of the modified virus to a subject in need thereof; and

administering to a subject in need thereof one or more booster doses of the modified virus,

wherein the initial dose and the booster dose of the modified virus are each independently selected from the group consisting of:

attenuated viruses produced by methods other than codon pair deoptimization;

a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a codon pair de-optimized region encoding a similar protein sequence, wherein the codon pair bias of the modified sequence is less than the codon pair bias of the parent virus and the codon pair bias of the modified sequence is reduced by at least about 0.05, or at least about 0.1, or at least about 0.2;

a modified virus derived from said wild type virus or a previously modified virus by replacing at least one genomic region of the wild type virus with a region encoding a similar protein sequence having an increased CpG dinucleotide, wherein the increase in CpG dinucleotide is at least 41 instances higher than the parental virus, or at least 21 instances higher than the parental virus genome;

a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of said virus with a region encoding a similar protein sequence having an increased UpA dinucleotide, wherein the increase in UpA dinucleotide is at least 26 instances higher than the parent, or at least 13 instances higher than the parent viral genome;

a modified virus derived from said wild type virus or a previously modified virus by replacing at least one genomic region of the wild type virus with a region encoding a similar protein sequence having increased UpA and CpG dinucleotides, wherein the increase in UpA and CpG dinucleotides in combination is at least 42 instances higher than the parent; and

combinations thereof.

3. The method of claim 2, wherein the initial dose is administered subcutaneously, intramuscularly, intradermally, intranasally, or intravenously.

4. The method of claim 2, wherein the one or more booster doses are administered intratumorally or intravenously.

5. The method of claim 2, wherein the first booster dose of said one or more booster doses is administered about 2 weeks after the initial dose; or if more than one initial dose, about 2 weeks after the last initial dose.

6. The method of claim 2, wherein the subject has cancer.

7. The method of claim 2, wherein the initial dose is administered when the subject does not have cancer.

8. The method of claim 7, wherein the subject is at higher risk of developing cancer.

9. The method of claim 7, wherein the one or more booster doses are administered about every 1 year, every 2 years, every 3 years, every 4 years, every 5 years, every 6 years, every 7 years, every 8 years, every 9 years, or every 10 years after the initial dose when the subject does not have cancer.

10. The method of claim 7, wherein the one or more booster doses are administered after the subject is diagnosed with cancer.

11. The method of any one of claims 1-10, wherein the method further comprises administering a PD-1 inhibitor or a PD-L1 inhibitor.

12. The method of claim 11, wherein the PD-1 inhibitor is an anti-PD 1 antibody.

13. The method of claim 12, wherein the anti-PD 1 antibody is selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cimiralizumab, AK105, BCD-100, BI754091, JS001, L ZM009, MGA012, Sym021, TSR-042, MGD013, AK104, XmAb20717, tiramerizumab, and combinations thereof.

14. The method of claim 11, wherein the PD-1 inhibitor is selected from the group consisting of: PF-06801591, pluripotent killer T lymphocytes expressing anti-PD 1 antibodies (PIK-PD-1), autologous anti-EGFRvIII 4SCAR-IgT cells, and combinations thereof.

15. The method of claim 11, wherein the PD-L1 inhibitor is an anti-PD-L1 antibody.

16. The method of claim 15, wherein the anti-PD-L1 antibody is selected from the group consisting of BGB-A333, CK-301, FAZ053, KN035, MDX-1105, MSB2311, SHR-1316, Attributab, Avermemab, Devolumab, BMS-936559, CK-301, and combinations thereof.

17. The method of claim 11, wherein the anti-PD-L1 inhibitor is M7824.

18. The method of any one of claims 1-17, wherein treating the malignancy reduces the likelihood of recurrence of the malignancy.

19. The method of any one of claims 1-17, wherein treating the malignancy reduces the likelihood of having a second cancer different from the malignancy.

20. The method of any one of claims 1-17, wherein, if the subject develops a second cancer different from the malignancy, treatment of the malignancy slows growth of the second cancer.

21. The method of any one of claims 1-17, wherein, after remission of the malignancy, if the subject develops a second cancer different from the malignancy, treatment of the malignancy slows growth of the second cancer.

22. The method of any one of claims 1-17, wherein treating the malignancy stimulates an inflammatory immune response in the tumor.

23. The method of any one of claims 1-17, wherein treating the malignant tumor recruits pro-inflammatory cells to the tumor.

24. The method of any one of claims 1-17, wherein treating the malignancy stimulates an anti-tumor immune response.

25. A method of treating a malignant tumor according to any one of claims 1 to 24, wherein the modified virus is a recombinant modified virus.

26. A method of treating a malignant tumor according to any one of claims 1 to 24, wherein the modified virus is modified from a picornavirus.

27. The method of claim 26, wherein the picornavirus is an enterovirus.

28. The method of claim 27, wherein the enterovirus is enterovirus C.

29. The method of claim 28, wherein enterovirus C is poliovirus.

30. A method of treating a malignant tumor according to any one of claims 1 to 24, wherein the modified virus is modified from an orthomyxovirus.

31. The method of claim 30, wherein the orthomyxovirus is influenza a virus.

32. The method of claim 31, wherein one or more segments of the influenza a virus are re-encoded.

33. The method of claim 31, wherein the HA segment, the NA segment, or both the HA segment and the NA segment are recoded.

34. The method of claim 31, wherein the modified virus is SEQ ID NO: 5 or SEQ ID NO: 6.

35. a method of treating a malignant tumor according to any one of claims 1 to 24, wherein the modified virus is modified from a flavivirus.

36. The method of claim 35, wherein the flavivirus is Zika virus.

37. The method of claim 35, wherein the envelope (E) coding region or the non-structural protein 3(NS3) coding region or both of said Zika virus is recoded.

38. The method of claim 35, wherein recoding comprises altering the frequency of CG and/or TA dinucleotides in the E and NS3 coding sequence.

39. The method of claim 37, wherein the re-encoded E protein coding sequence or the NS3 coding sequence, or both, has a codon pair bias of less than-0.1.

40. The method of claim 37, wherein the re-encoded E protein coding sequence or the NS3 coding sequence, or both, has a reduced codon usage of 0.1-0.4.

41. The method of claim 36, wherein the modified virus is SEQ ID NO: 2. SEQ ID NO: 3 or SEQ ID NO: 4.

42. a method of treating a malignant tumor according to any one of claims 1 to 24, wherein the malignant tumor is a solid tumor.

43. A method of treating a malignant tumor according to any one of claims 1 to 24, wherein the malignant tumor is glioblastoma, adenocarcinoma, melanoma, lung cancer, neuroblastoma, breast cancer, bladder cancer, colon cancer, prostate cancer, or liver cancer.

44. A method of treating a malignant tumor according to any one of claims 1 to 24, wherein the modified virus is administered intratumorally, intravenously, intracerebrally, intramuscularly, intraspinally, or intrathecally.

45. A method of treating a malignancy according to any one of claims 1 to 24 wherein the modified virus is PV1-MinY and is prepared from the wild-type virus or the previously modified virus in which the middle part of the P1 region of the wild-type virus or the previously modified virus is replaced with a synthetic sequence which is re-encoded according to human codon pair bias to have a reduced codon pair score.

46. A method of treating a malignancy according to any one of claims 1 to 24 wherein the recombinant modified virus is PV1-MinY and is prepared from the previously modified virus PV1(M) or the wild type virus PV1(M) and the middle portion of the P1 region of the previously modified virus PV1(M) or the wild type virus PV1(M) is replaced by a synthetic sequence recoded to have an increased UpA and/or CpG dinucleotide.

47. The method of claim 46, wherein the recombinant virus is PV1-MinY prepared from PV1(M) and the nucleotide fragment of PV1(M) comprising nucleotides 1513 to 2470 of the P1 region is substituted with a corresponding nucleotide fragment comprising nucleotides 1513 to 2470 of the P1 region recoded according to human codon pair bias to have a reduced codon pair score.

48. A method of treating a malignancy according to any one of claims 1 to 24, wherein the modified virus is PV1-MinY prepared from PV1(M) and the nucleotide fragment in the PV1(M) consisting of nucleotide 1513 to 2470 of the P1 region is substituted with the corresponding nucleotide fragment comprising nucleotide 1513 to 2470 of the P1 region recoded according to human codon pair bias to have a reduced codon pair score.

49. A method of treating a malignant tumor, the method comprising:

a recombinant modified picornavirus is prepared from a wild-type picornavirus by: at least the nucleotide fragment in the P1 domain of the wild-type picornavirus is replaced with a corresponding nucleotide fragment comprising a synthetic sequence re-encoded according to human codon pair bias to have a reduced codon pair score, and

optionally, replacing P1 of the wild-type picornavirus with synthetic P1 recoded according to human codon pair bias to have a reduced codon pair score, the P1 domain of the wild-type picornavirus being selected from the group consisting of PV1(S), PV2(S), and PV3 (S); and

administering the recombinant modified virus to a subject in need thereof.

50. The method of claim 49, wherein fragmenting at least one nucleotide comprises fragmenting at least one nucleotide comprising the nucleotide sequence of SEQ ID NO: 1 by a nucleotide fragment comprising a synthetic sequence recoded according to human codon pair bias to have a reduced codon pair score, the nucleotide fragment comprising the nucleotide sequence of nt #1513-nt #2470 of SEQ ID NO: 1 nt #1513-nt #2470 is the P1 domain of the modified virus.

51. The method of claim 49, wherein the recombinant virus is administered intratumorally, intravenously, intracerebrally, intramuscularly, intraspinally, or intrathecally, and causes cytolysis of tumor cells.

52. The method of claim 49, wherein the malignancy is selected from the group consisting of: glioblastoma multiforme, medulloblastoma, breast cancer, prostate cancer, colorectal cancer, hepatocellular carcinoma, bronchial cancer, and epidermoid cancer.

53. The method of claim 49, wherein the picornavirus is enterovirus C.

54. The method of claim 49, wherein the picornavirus is a poliovirus.

55. The method of claim 49, wherein the P1 domain is selected from the Sabin vaccine strains PV1(S), PV2(S) and PV3 (S).

Technical Field

The present invention relates to a modified virus comprising a modified viral genome, said modified genome comprising a plurality of nucleotide substitutions, for use in the treatment of cancer. Nucleotide substitutions result in codon substitutions with other synonymous codons and/or codon rearrangements and changes in codon pair bias (codon pair bias). These modified viruses are useful for treating malignancies.

Background

All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful for understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Virology of synthetic viruses

Rapid improvements in DNA synthesis technology are expected to revolutionize the traditional approach adopted in virology. One of the methods traditionally used to eliminate the function of different regions of the viral genome uses site-directed mutagenesis in large but laborious ways to explore the impact of small sequence variations in the viral genome. However, the genomes of viruses (especially RNA viruses) are relatively short, typically less than 10000 bases in length, which makes them suitable for whole genome synthesis using existing techniques. Recently developed microfluidic chip-based technologies can enable the de novo (de novo) synthesis of new genomes designed to specification, each of which is only a few hundred dollars. This allows the generation of entirely novel coding sequences or the adaptation of existing sequences to the extent that traditional cloning methods are hardly feasible. Such freedom of design provides tremendous incentive for large-scale redesign of DNA/RNA coding sequences to achieve the following goals: (1) studying the influence of parameter changes such as codon bias (codon bias), codon-pair bias (codon-pair bias) and RNA secondary structure on the translation and replication efficiency of the virus; (2) efficient whole genome scanning of unknown regulatory elements and other signals required for successful virus propagation; (3) the development of new biotechnology for genetic engineering of viral strains and for the design of antiviral vaccines; (4) synthetically modified viruses are used for oncolytic therapy.

Reverse genetics of Poliovirus (Poliovirus)

Reverse genetics generally involves experimental methods of discovering gene function, which proceed in the opposite direction to the so-called forward genetics method of classical genetics. That is, forward genetics approaches attempt to determine gene function by elucidating the genetic basis of phenotypic traits, while reverse genetics-based strategies begin with isolated genes and attempt to discover their function by studying the possible phenotypes resulting from expression of wt or mutated genes. As used herein in the context of viral systems, a "reverse genetics" system relates to the utility of techniques that allow genetic manipulation of a viral genome composed of RNA. Briefly, the viral genome is isolated from viral particles (virons) or from infected cells, converted to DNA ("cDNA") by an enzyme (reverse transcriptase), possibly modified as necessary, and reduced (typically via RNA intermediates) to infectious viral particles. This process is very simple in picornaviruses (picornaviruses); in fact, the first reverse genetics system developed for any animal RNA virus was with PV. The viral reverse genetics system is based on the following historical findings: naked viral genomic RNA is infectious when transfected into suitable mammalian cells. The discovery of reverse transcriptase in the 1970's and the development of molecular cloning technology enabled scientists to generate and manipulate cDNA copies of the RNA virus genome. Most commonly, an entire cDNA copy of the genome is cloned directly downstream of the bacteriophage T7RNA polymerase promoter (allowing in vitro synthesis of genomic RNA) and then transfected into cells to produce the virus. Alternatively, the same DNA plasmid can be transfected into cells that express T7RNA polymerase in the cytoplasm.

De novo synthesis of viral genomes

Computer-based algorithms are used to design and synthesize viral genomes de novo. These synthetic genomes (in the synthetic examples of modified PVs described herein) encode proteins identical to wild-type (wt) virus, but by using alternative synonymous codons, various parameters (including codon bias, codon pair bias, RNA secondary structure and/or dinucleotide content) can be altered. The data presented indicate that these coding-independent changes produce highly modified viruses, usually due to protein dystranslations.

As used herein, "modified viruses" refer to viruses in which all or part of their genome have synonymous codons and/or codon rearrangements and codon pair bias variations, unless otherwise specified. The modified viruses of the invention are useful for treating cancer. Unless otherwise indicated, the recombinant viruses described herein are modified viruses.

By targeting the essential function of all viruses (i.e., protein translation), the inventive methods described herein have been developed for the predictable, safe, rapid, and inexpensive production of modified viruses that can be used for oncolytic therapy. It is shown herein that both codon deoptimization and codon pair deoptimization in the PV capsid coding region substantially reduces PV fitness (fittness). The present invention is not limited to any particular molecular mechanism by which viral attenuation is caused by a synonymous codon substitution.

Substitution coding

A given peptide may be encoded by a large number of nucleic acid sequences. For example, even a typical short 10-mer oligopeptide may consist of about 4¹⁰A (about 10)⁶One) and the PV protein may consist of about 10⁴⁴²A plurality of different nucleic acids. Natural selection eventually selects these possibilities 10⁴⁴²One of the nucleic acids serves as the PV genome. The primary amino acid sequence is the most important level of information encoded by a given mRNA, while other kinds of information are present in the different kinds of RNA sequences. These messages include RNA structural elements of different functions (e.g., for PV, cis-acting replication element or CRE), translational kinetic signals (pause site, frameshift site, etc.), polyadenylation signals, splicing signals, enzymatic functions (ribozymes), and, most likely, other yet unidentified messages and signals).

Even with the caveat that signals such as CRE must be retained, 10⁴⁴²The variety of possible coding sequences provides great flexibility to allow drastic changes to occur in the RNA sequence of polio (polio) while retaining the ability to encode the same protein. Password onSubportions or codon pair biases are altered, and nucleic acid signals and secondary structures in the RNA can be added or removed. Additional or novel proteins may even be encoded simultaneously in alternate frames.

Codon pair bias

The unique characteristic of the coding sequence is their codon pair bias, which can be illustrated by reference to the amino acid pair Ala-Glu, which can be encoded by 8 different codon pairs, and which pair can have a bias affecting the translation of the human and viral genes in human cells (Table 1). if no other factor than the frequency of each individual codon (as shown in Table 2) is responsible for the frequency of this codon pair, the expected frequencies of the 8 codes can be calculated by multiplying the frequencies of the two relevant codons.for example, by this calculation the expected codon pair GCA-GAA will occur at a frequency of 0.097 in all Ala-Glu coding pairs (0.23 ×.0.42; based on the frequencies in Table 2). in order to relate the expected (assumed) frequency of each codon pair to the observed frequency in the human genome, the Consensussus CDS (CCDS) database using human coding regions containing a consistent annotation of the coding sequence is calculated using the number of consistently annotated codon pairs of the coding sequence with the expected codon frequency of each other codon pair calculated by multiplying the expected codon pair frequency of the number of the coding sequence of the expected codon pairs with the expected codon pairs of the number of the expected codon pairs calculated by the number of the expected codon pairs in the coding sequence of the coding pairs (CCD). The. the expected codon pairs) is calculated by the number of the expected codon pairs as the expected codon pairs of the frequency of the coding pairs of the coding sequences of the coding pairs calculated by the frequency of the coding pairs of the coding sequences of the coding pairs of0.098 (equivalent to 0.97 in the first calculation using the Kazusa dataset). Finally, the actual codon pair frequencies observed in the 14,795 set of individual genes were determined by counting the total number of occurrences of each codon pair in the set and dividing by the number of all synonymous coding pairs in the set that encode the same amino acid pair (Table 2; observed frequencies). The frequency and observed/expected values of the amino acid pair Ala-Glu, which can be encoded by 8 different codon pairs, are shown in Table 1, based on 14795 set of individual genes, 3721 (61)²) A complete set of individual codon pairs (Coleman et al 2008).

TABLE 1 codon pair score for amino acids versus alanine-glutamine

Amino acid pair	Codon pair	Desired frequency	Frequency of observation	Observed/expected ratio
					AE	GCAGAA	0.098	0.163	1.65
AE	GCAGAG	0.132	0.198	1.51
					AE	GCCGAA	0.171	0.031	0.18
AE	GCCGAG	0.229	0.142	0.62
					AE	GCGGAA	0.046	0.027	0.57
AE	GCGGAG	0.062	0.089	1.44
					AE	GCTGAA	0.112	0.145	1.29
AE	GCTGAG	0.150	0.206	1.37
					Total		1.000	1.000

TABLE 2 codon bias in human genes

A codon pair is considered to be over-represented (overexpressed) if the ratio of observed/expected frequency of the codon pair is greater than 1. If the ratio is less than 1, a low degree of performance is indicated (underserved). In the examples, codons were over-represented by 1.65-fold for GCA-GAA, while codes were over-represented by 5-fold for GCC-GAA.

For example, codon pairs GCCGAA (AlaGlu) and GATCTG (Asp L eu) exhibit 3-6 times less low (preferred codon pairs are GCAGAG and GACCTG, respectively), while codon pairs GCCAAG (Ala L ys) and AATGA (AsnGlu) exhibit about 2 times more excess.

Codon pair bias was found in prokaryotic cells but was seen later in all other species examined including humans. This effect is statistically significant and certainly not just noise. However, its functional meaning (if any) is a puzzle. One proposal is that certain pairs of tRNAs interact well when they are on the ribosome, while others interact poorly. Since different codons are typically read by different trnas, codon pairs may be biased to avoid putting incompatible pairs of trnas together. Another concept is that many (but not all) codon pairs with low expression have a central CG dinucleotide (e.g., GCCGAA encoding AlaGlu), whereas CG dinucleotides are systemically low expressed in mammals. Thus, there may be two effects of codons on bias: one is an indirect effect of low CG expression in the mammalian genome and the other is related to the efficiency, speed and/or accuracy of translation. It is emphasized that the present invention is not limited to any particular molecular mechanism of codon pair bias.

Calculation of codon pair bias

Each individual codon pair (e.g., GTT-GCT) of the possible 3721 codon pairs containing a non "STOP" carries an assigned "codon pair score" or "CPS" that is specific to a given gene "training set". CPS for a given codon pair is defined as the log-ratio of the observed number of occurrences to the number expected for the gene set (in this example, the human genome). Determining the actual number of occurrences of a particular codon pair (or, in other words, the likelihood that a particular amino acid pair is encoded by a particular codon pair) simply counts the actual number of occurrences of codon pairs in a set of particular coding sequences. However, determining the expected number requires additional calculations. Similar to Gutman and Hatfield, the expected amount is calculated such that it is independent of neither amino acid frequency nor codon bias. That is, the expected frequency is calculated based on the relative proportion of the number of times an amino acid is encoded by a particular codon. A positive CPS value indicates that a given codon pair is statistically over-represented, whereas a negative CPS value indicates that the pair is statistically under-represented in the human genome.

To perform these calculations in the human context, the latest consensus annotated consensus cds (ccds) database of human coding regions, which contains a total of 14,795 genes, was used. The data set provides codons and codon pairs, and thus genome-scale amino acids and amino acid pair frequencies.

The Federov et al (2002) model was used to further enhance the Gutman and Hatfield (1989) methods. This allows calculation of the expected frequency for a given codon pair, while relying on codon frequency and non-random associations with adjacent codons encoding a particular amino acid pair. Detailed equations for calculating CPB are disclosed in WO 2008/121992 and WO 2011/044561, which are incorporated by reference.

In the calculations, Pij is the codon pair that appears in its synonymous set at the frequency of no (Pij). Ci and Cj are two codons containing Pij, appearing in their synonymous sets at frequencies F (Ci) and F (Cj), respectively. More specifically, f (Ci) is the frequency at which codon Ci codes for the corresponding amino acid Xi in all coding regions, and f (Ci) ═ no (cj)/no (Xi), where no (Ci) and no (Xi) are the observed number of occurrences of codon Ci and amino acid Xi, respectively. F (cj) is calculated accordingly. Further, no (Xij) is the number of occurrences of amino acid pair Xij in all coding regions. The codon pair bias score S (Pij) for Pij is calculated as the log ratio of the observed frequency No (Pij) to the expected number of occurrences of Ne (Pij).

Using the above formula, it was then determined whether each codon pair in each coding sequence was over-or under-represented when compared to the corresponding genomic ne (pij) value calculated by using the entire human CCDS dataset. This calculation yields a positive S (Pij) score value for codon pairs that are over-represented in the human coding region and a negative value for codon pairs that are under-represented.

The "combined" codon pair bias of an individual coding sequence is calculated by averaging all codon pair scores according to the following formula:

thus, codon pair bias for the entire coding region is calculated by adding all the individual codon pair scores that comprise the region and dividing the sum by the length of the coding sequence.

Calculating the codon pair bias and realizing the algorithm for changing the codon pair bias.

An algorithm was developed to quantify codon pair bias. There is one "codon pair score" or "CPS" for each possible individual codon pair. CPS is defined as the natural logarithm of the ratio of observed to expected number of occurrences of each codon in the coding region of all people representing the host species of the original vaccine virus to be re-encoded.

Although the calculation of the observed occurrence of a particular codon pair is simple (actual counts within the gene set), the expected number of occurrences of a codon pair requires additional calculations. Similar to Gutman and Hatfield, we calculated this expected number independent of amino acid frequency and codon bias. That is, the expected frequency is calculated based on the relative proportion of the number of times an amino acid is encoded by a particular codon. In the human genome, a positive CPS value indicates that a given codon pair is statistically over-represented, while a negative CPS indicates that the pair is statistically under-represented.

Using these calculated CPSs, any coding region can then be assessed by using either over-or under-represented codon pairs by taking the average of the codon pair scores, giving the Codon Pair Bias (CPB) for the entire gene.

As discussed further below, codon pair bias takes into account the score for each codon pair in the coding sequence, which is averaged over the entire length of the coding sequence. According to the invention, codon pair bias is determined by the following formula:

thus, for example, similar codon pair bias of a coding sequence can be obtained by minimizing codon pair scores over the entire subsequence or by appropriately reducing codon pair scores over the full length of the coding sequence.

Since all 61 sense codons and all sense codon pairs can certainly be used, it cannot be expected that the substitution of a single rare codon for a frequent codon or the substitution of a rare codon pair for a frequent codon pair will have a great effect. Regardless of the precise mechanism, the data indicate that large scale replacement of synonymous, de-optimized codons in the viral genome results in a severely attenuated virus. This procedure for generating modified viruses is called SAVE (synthetic attenuated virus engineering).

According to aspects of the invention, viral modification can be achieved by altering codon pair bias and codon bias in one or more portions of the viral genome. However, it is expected that regulatory codon pair bias is particularly advantageous. For example, attenuation of a virus by codon bias often requires elimination of common codons, and thus the complexity of the nucleotide sequence is reduced. Conversely, a reduction or minimization of codon pair bias can be achieved while maintaining greater sequence diversity, and thus better control of nucleic acid secondary structure, annealing temperature, and other physical and biochemical properties. The work disclosed herein includes modified sequences with reduced or minimized codon pair bias in which codons are shuffled but the codon usage profile is unchanged.

Malignant tumors are known to be caused by uncontrolled growth of cells in organs. Tumors grow to the point where they can severely impair normal organ function by tumor infiltration, replacement of functional tissues, competition for essential resources, and (often) metastatic spread to secondary sites. Malignant tumors are the second leading cause of death in the united states.

To date, methods for treating malignant tumors include surgical resection, radiation, and/or chemotherapy. However, many malignancies respond poorly to all of the traditionally available treatment options, and known practical approaches have serious side effects. Great progress has been made in reducing the severity of side effects while increasing the efficacy of the commonly used treatment regimens. However, there are still many problems and other therapeutic approaches need to be found. The research on the primary malignant tumor of the central nervous system is particularly urgent. Brain tumors (especially glioblastoma) remain one of the most difficult therapeutic challenges. Despite surgery, radiation therapy and chemotherapy, either alone or in combination, glioblastoma is almost always fatal, with median survival rates of less than one year and five-year survival rates of less than 5.5%. None of the available treatment modalities substantially changed the continued progression of glioblastoma.

Systematic studies of patients diagnosed with glioblastomas and subjected to surgery to remove the tumor in whole or in part, followed by chemotherapy and/or radiation, have shown that survival rates after one year are still low, particularly for patients over 60 years of age. Malignant gliomas have proven relatively resistant to radiation and chemotherapy regimens. Poor prognosis is exacerbated by the frequent occurrence of local recurrence following surgical resection of glioblastoma and adjuvant radiation/chemotherapy.

In recent years, the use of viruses to treat cancer has been proposed: (1) as gene delivery vectors; (2) as a direct oncolytic agent through the use of a virus genetically engineered to have its pathogenicity lost; or (3) as agents for selectively destroying malignant cells using viruses that have been genetically engineered for this purpose.

An example of the use of viruses against glioblastomas includes the herpes simplex virus dlsptk (HSVdlsptk) is a Thymidine Kinase (TK) negative mutant of HSV that has attenuated neurovirulence due to a deletion of 360 base pairs in the TK gene, the product of which is essential for normal viral replication.HSVdlsptk has been found to retain reproductive potential in rapidly dividing malignant cells, causing cell lysis and death^Ic(intracranial administration) is 10⁶pfu (fairly low dose). This limits the use of this mutant HSV. Other mutants of HSV have been proposed and tested. However, caused by viral encephalitisDeath remains a problem.

Another proposal has been to use retroviruses engineered to contain the HSV tk Gene to express thymidine kinase which causes phosphorylation of nucleoside analogues such as ganciclovir (gancylovir) or acyclovir (acyclovir) in vivo, blocks DNA replication and selectively kills dividing cells.Izquerdo, M. et al, Gene Therapy, 2: 66-69(1995) report the use of Moloney Murine leukemia Virus (Moloney Murine L eukamia Virus, MoM L V) engineered by insertion of the HSV tk Gene with its own promoter.

Similar systems have been developed to target upper respiratory tract malignancies that originate in and are readily accessible to tissues that are naturally susceptible to adenovirus infection. However, glioblastoma multiforme (a highly malignant tumor composed of a wide variety of cell types (hence the name polymorphic)) is characterized by a very variable genotype and is unlikely to respond to the oncolytic viral system against a homogeneous tumor with homogeneous genetic abnormalities.

The effect of our virus modification can be confirmed in a manner well known to those of ordinary skill in the art. Non-limiting examples induce plaque assays, growth measurements, reverse genetics of RNA viruses, and reduced lethality in test animals. The present application demonstrates that the modified viruses are capable of inducing a protective immune response in a host.

Disclosure of Invention

The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods, which are meant to be exemplary and illustrative, not limiting in scope.

It is an object of the present invention to develop modified viruses for the treatment of various types of cancer.

It is another object of the present invention to develop modified viruses for the treatment of various types of cancer that can be used in combination with anti-PD L-1 antibody therapeutics or other immune tumor therapies.

It is another object of the invention to treat cancer cells by infecting them with a modified virus to cause lysis and death of the cancer cells.

It is another object of the present invention to treat cancer cells by infecting them with a modified virus, thereby eliciting an anti-tumor immune response.

It is another object of the invention to treat cancer cells by infecting them with a modified virus to elicit an anti-tumor immune response by increasing or decreasing expression of anti-tumor immunity proteins (e.g., PD-1, CT L A-4, IDO1, TIM3, lag-3).

It is another object of the present invention to treat cancer cells by infecting them with a modified virus, thereby eliciting an innate immune response in the tumor cells by activating innate signal transduction receptors (RIG-1, STNG) and innate immune transcription factors (IRF3, IRF7, or NFkB) in the tumor.

It is another object of the invention to treat cancer cells by infecting them with a modified virus, thereby eliciting an innate immune response in the tumor.

It is another object of the present invention to treat cancer cells by infecting them with a modified virus, thereby eliciting a pro-inflammatory immune response in the tumor.

It is another object of the invention to treat cancer cells by infecting them with a modified virus and thereby recruiting pro-inflammatory leukocytes into the tumor.

It is another object of the invention to treat cancer cells by infecting them with a modified virus and thereby reducing regulatory white blood cells from the tumor.

It is another object of the invention to pre-treat a recipient with a modified virus to elicit an immune response prior to administration of the virus to treat cancer.

It is another object of the invention to pre-treat a recipient with a modified virus to elicit an immune response prior to administering a natural isolate of the virus to treat cancer.

It is another object of the present invention to develop new wild-type virus modified virus chimeras which would be useful in the treatment and cure of gliomas, in particular glioblastoma.

It is another object of the present invention to develop new modified viruses that will be suitable for the treatment of adenocarcinomas, in particular cervical carcinomas.

It is another object of the present invention to develop new modified viruses that would be useful in the treatment of breast cancer.

It is another object of the invention to develop new modified viruses that would be useful in the treatment of cancer cells positive for keratin by immunoperoxidase staining.

It is another object of the present invention to develop additional novel modified viruses that would be useful in the treatment of cancer cells that are reported to have low or absent expression of the p53 gene.

It is another object of the present invention to develop additional novel modified viruses that would be useful in the treatment of tumors in which the cells are hypodiploid.

It is another object of the present invention to develop novel modified viruses which are useful in the treatment of lung cancer (lungcrcinomas), particularly lung cancer.

It is another object of the invention to develop novel modified viruses suitable for treating subthreloid cancers (e.g., chromosome counts of 64, 65, or 66 in about 4% of cells).

It is another object of the present invention to develop novel modified viruses suitable for treating cancers having a single copy of chromosomes N2 and N6 per cell.

It is another object of the present invention to develop a novel modified virus which is suitable for treating cancers expressing the isoenzyme G6PD-B of glucose-6-phosphate dehydrogenase (G6 PD).

It is another object of the present invention to develop new modified viruses which are suitable for the treatment of melanoma.

It is another object of the present invention to develop novel modified viruses that are useful in the treatment of malignant cells derived from melanocytes.

It is another object of the present invention to develop new modified viruses which are useful in the treatment of neuroblastoma.

It is another object of the invention to develop novel modified viruses suitable for treating cancers with at least 3-fold amplification of the MYCN oncogene (oncogene).

It is another object of the invention to develop modified viruses that are useful in the treatment of breast cancer.

It is another object of the invention to develop modified viruses which are suitable for use in the treatment of bladder cancer.

It is another object of the invention to develop modified viruses that are suitable for use in the treatment of colon cancer.

It is another object of the invention to develop modified viruses which are useful in the treatment of prostate cancer.

It is another object of the invention to develop modified viruses which are suitable for the treatment of peripheral nerve sheath tumors.

The invention provides modified viruses that comprise a modified viral genome comprising nucleotide substitutions engineered at one or more (e.g., 1, 2, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more) positions in the genome, wherein the substitutions introduce a plurality of synonymous codons into the genome. Such replacement of synonymous codons alters a variety of parameters including codon bias, codon pair bias, density of deoptimized codons and deoptimized codon pairs in the genome, RNA secondary structure, CpG dinucleotide content, C + G content, UpA dinucleotide content, translation frame shift sites (translation frame shift sites), translation pause sites, presence or absence of tissue specific microRNA recognition sequences, or any combination thereof. Due to the large number of deficiencies involved, the modified viruses of the present invention provide a means to generate stably modified oncolytic viruses against a variety of different tumor types.

In one embodiment, a modified virus is provided comprising a nucleic acid sequence encoding a viral protein or a portion thereof identical to a corresponding sequence of a parent virus, wherein the nucleotide sequence of the modified virus comprises codons from which the parent sequence of the modified virus is derived, and wherein the nucleotide sequence has less than 98% identity to the nucleotide sequence of the parent virus. In another embodiment, the nucleotide sequence has less than 90% identity to the sequence of the parent virus. The nucleotide sequence provided with substitutions for modifications is at least 100 nucleotides in length, alternatively at least 250 nucleotides in length, alternatively at least 500 nucleotides in length, alternatively at least 1000 nucleotides in length. The modified sequence has a codon pair bias less than that of the parent virus and is reduced by at least about 0.05, or at least about 0.1, or at least about 0.2.

In one embodiment, a modified virus is provided, the modified virus comprising a nucleic acid sequence encoding a viral protein or a portion thereof that is similar to a corresponding sequence of a parent virus, wherein the nucleotide sequence of the modified virus comprises a nucleotide sequence that is less than 98% identical to the nucleotide sequence of the parent virus. In another embodiment, the nucleotide sequence has less than 90% identity to the sequence of the parent virus. The nucleotide sequence provided with substitutions for modifications is at least 100 nucleotides in length, alternatively at least 250 nucleotides in length, alternatively at least 500 nucleotides in length, alternatively at least 1000 nucleotides in length. The CpG dinucleotide content of the modified sequence is increased by at least 19, alternatively by at least 41, compared to the parental virus.

In one embodiment, a modified virus is provided, the modified virus comprising a nucleic acid sequence encoding a viral protein or a portion thereof that is similar to a corresponding sequence of a parent virus, wherein the nucleotide sequence of the modified virus comprises a nucleotide sequence that is less than 98% identical to the nucleotide sequence of the parent virus. In another embodiment, the nucleotide sequence has less than 90% identity to the sequence of the parent virus. The nucleotide sequence provided with substitutions for modifications is at least 100 nucleotides in length, alternatively at least 250 nucleotides in length, alternatively at least 500 nucleotides in length, alternatively at least 1000 nucleotides in length. The UpA dinucleotide content of the modified sequence is increased by at least 13 compared to the parent virus, or at least about 26 compared to the parent virus.

Embodiments of the invention also provide a therapeutic composition for treating a subject, the therapeutic composition comprising a modified virus and a pharmaceutically acceptable carrier. The invention also provides a therapeutic composition for eliciting an immune response in a subject having cancer, the therapeutic composition comprising a modified virus and a pharmaceutically acceptable carrier. The invention further provides modified host cell lines that are specifically engineered to allow for modified viruses that are not viable in wild-type host cells.

According to the present invention, modified viruses are produced by transfecting modified viral genomes into host cells, thereby producing modified viral particles. The invention further provides pharmaceutical compositions comprising the modified viruses suitable for use in the treatment of cancer.

Various embodiments of the present invention provide methods of treating a malignant tumor, the methods comprising: administering to a subject in need thereof a modified virus, wherein the modified virus is selected from the group consisting of: a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a de-optimized region of codon pairs encoding a similar protein sequence, wherein the codon pair bias of the modified sequence is less than the codon pair bias of the parent virus and is reduced by at least about 0.05, or at least about 0.1, or at least about 0.2; a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region encoding a similar protein sequence having an increased CpG dinucleotide, wherein the increase in CpG dinucleotide is at least 41 instances higher than the parent, or at least 21 instances higher than the parent viral genome; a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region encoding a similar protein sequence having an added UpA dinucleotide, wherein the increase in the UpA dinucleotide is at least 26 instances higher than the parent, or at least 13 instances higher than the parent viral genome; a modified virus derived from a wild type virus or a previously modified virus by replacing at least one genomic region of the wild type virus with a region encoding a similar protein sequence having increased UpA and CpG dinucleotides, wherein the increase in UpA and CpG dinucleotides is at least 42 instances higher than the parent; and combinations thereof.

Various embodiments of the present invention provide methods of treating a malignant tumor, the methods comprising: administering an initial dose of the modified virus to a subject in need thereof; and administering one or more booster doses of the modified virus to a subject in need thereof, wherein the initial dose and the booster dose of the modified virus are each independently selected from the group consisting of: attenuated viruses produced by methods other than codon pair deoptimization; a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a de-optimized region of codon pairs encoding a similar protein sequence, wherein the codon pair bias of the modified sequence is less than the codon pair bias of the parent virus and is reduced by at least about 0.05, or at least about 0.1, or at least about 0.2; a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region having an increased CpG dinucleotide encoding a similar protein sequence, wherein the increase in CpG dinucleotide is at least 41 instances higher than the parent, or at least 21 instances higher than the parent viral genome; a modified virus derived from a wild type virus or a previously modified virus by replacing at least one genomic region of the wild type virus with a region encoding a similar protein sequence having an added UpA dinucleotide, wherein the increase in UpA dinucleotide is at least 26 instances higher than the parent, or at least 13 instances higher than the parent viral genome; a modified virus derived from a wild type virus or a previously modified virus by replacing at least one genomic region of the wild type virus with a region encoding a similar protein sequence having increased UpA and CpG dinucleotides, wherein the increase in UpA and CpG dinucleotides is at least 42 instances greater than the parent; and combinations thereof.

In various embodiments, the initial dose can be administered subcutaneously, intramuscularly, intradermally, intranasally, or intravenously. In various embodiments, one or more booster doses can be administered intratumorally or intravenously.

In various embodiments, the first dose of one or more booster doses may be administered about 2 weeks after the initial dose; or if more than one initial dose, about 2 weeks after the last initial dose.

In various embodiments, the subject may have cancer. In various embodiments, the subject may be at higher risk of developing cancer.

In various embodiments, an initial dose can be administered when the subject does not have cancer.

In various embodiments, when the subject is free of cancer, one or more booster doses can be administered approximately every 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years after the initial dose. In various embodiments, one or more booster doses may be administered after a diagnosis that the subject has cancer.

In various embodiments, the methods can further comprise administering a PD-1 inhibitor or a PD-L1 inhibitor.

In various embodiments, the anti-PD 1 antibody can be selected from the group consisting of pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cimipripilimumab, AK105, BCD-100, BI754091, JS001, L ZM009, MGA012, Sym021, TSR-042, MGD013, AK104, XmAb 17, tirelinizumab, and combinations thereof.

In various embodiments, the PD-1 inhibitor may be selected from the group consisting of: PF-06801591, a pluripotent killer T lymphocyte expressing an anti-PD 1 antibody (PIK-PD-1), autologous anti-EGFRvIII 4SCAR-IgT cells, and combinations thereof.

In various embodiments, the anti-PD-L antibody may be selected from the group consisting of BGB-A333, CK-301, FAZ053, KN035, MDX-1105, MSB2311, SHR-1316, aleuzumab, Avelumab, Dewar umab, BMS-936559, CK-301, and combinations thereof.

In various embodiments, the anti-PD-L1 inhibitor is M7824.

In various embodiments, treating a malignancy can reduce the likelihood of recurrence of the malignancy. In various embodiments, treating a malignancy can reduce the likelihood of having a second cancer different from the malignancy.

In various embodiments, if the subject develops a second cancer that is different from the malignancy, treatment of the malignancy can result in a slowing of the growth of the second cancer.

In various embodiments, wherein after remission of the malignancy, if the subject develops a second cancer different from the malignancy, treatment of the malignancy results in a reduction in growth of the second cancer.

In various embodiments, treating a malignant tumor can stimulate an inflammatory immune response in the tumor. In various embodiments, treating a malignant tumor recruits pro-inflammatory cells to the tumor. In various embodiments, treating a malignant tumor can stimulate an anti-tumor immune response.

Various embodiments of the present invention provide methods of the present invention for treating a malignant tumor, wherein the modified virus can be a recombinant modified virus.

Various embodiments of the present invention provide methods of the present invention for treating malignancies in which the modified virus can be modified from picornaviruses.

In various embodiments, the picornavirus is an enterovirus (enterovirus). In various embodiments, the enterovirus is enterovirus C. In various embodiments, enterovirus C is a poliovirus.

Various embodiments of the present invention provide methods of the present invention for treating malignancies in which the modified virus may be modified from orthomyxovirus (orthomyxovirus). In various embodiments, the orthomyxovirus may be Influenza a virus (Influenza a virus). In various embodiments, one or more segments of influenza a virus may be re-encoded. In various embodiments, the HA, NA, or both HA and NA segments are recoded (e.g., de-optimized).

In various embodiments, the modified virus can be SEQ ID NO: 5 or SEQ ID NO: 6.

various embodiments of the present invention provide methods of the present invention for treating a malignant tumor, wherein the modified virus is modified from a flavivirus.

In various embodiments, the flavivirus genus can be Zika virus (Zika virus).

In various embodiments, the pre-membrane/envelope (E) coding region, or the non-structural protein 3(NS3) coding region, or both, of zika virus may be re-encoded. In various embodiments, recoding can include altering the frequency of CG and/or TA dinucleotides in the E and NS3 coding sequences. In various embodiments, the re-encoded E protein coding sequence or the NS3 coding sequence, or both, may have a codon pair bias of less than-0.1. In various embodiments, the recoded E protein coding sequence or the NS3 coding sequence, or both, can have a reduced codon pair bias of 0.1-0.4.

In various embodiments, the modified virus can be SEQ ID NO: 2. SEQ ID NO: 3 or SEQ ID NO: 4.

various embodiments of the present invention provide a method of the present invention for treating a malignant tumor, wherein the malignant tumor may be a solid tumor.

Various embodiments of the present invention provide a method of treating a malignant tumor of the present invention, wherein the malignant tumor may be glioblastoma, adenocarcinoma, melanoma, lung cancer, neuroblastoma, breast cancer, bladder cancer, colon cancer, prostate cancer, or liver cancer.

Various embodiments of the present invention provide methods of the present invention for treating a malignant tumor, wherein the modified virus can be administered intratumorally, intravenously, intracerebrally, intramuscularly, intraspinally, or intrathecally.

Various embodiments of the present invention provide the method of treating malignant tumor of the present invention, wherein the modified virus may be PV1-MinY and prepared from a wild-type virus or a previously modified virus, wherein a middle portion of its P1 region is replaced with a synthetic sequence recoded according to human codon pair bias to have a reduced codon pair score.

Various embodiments of the present invention provide the method of the present invention for treating malignant tumor, wherein the recombinant modified virus may be PV1-MinY and is prepared from a previously modified virus PV1(M) or a wild-type virus PV1(M) and the middle portion of its P1 region is replaced with a synthetic sequence recoded to have an increased UpA and/or CpG dinucleotide.

In various embodiments, the recombinant virus may be PV1-MinY prepared from PV1(M), wherein the nucleotide fragment comprising nucleotide 1513 to 2470 of the P1 region is substituted with a corresponding nucleotide fragment comprising nucleotide 1513 to 2470 of the P1 region re-encoded according to human codon pair bias to have a reduced codon pair score.

Various embodiments of the present invention provide a method of treating a malignancy of the present invention, wherein the modified virus is PV1-MinY prepared from PV1(M), and wherein a nucleotide fragment comprising nucleotide 1513 to 2470 of the P1 region is substituted with a corresponding nucleotide fragment comprising nucleotide 1513 to 2470 of the P1 region recoded according to human codon pair bias to have a reduced codon pair score.

Various embodiments of the present invention provide methods of treating a malignant tumor, the methods comprising: preparing a recombinant modified picornavirus from a wild-type picornavirus; and, administering the recombinant modified virus to a subject in need thereof, the recombinant modified picornavirus prepared by: at least a fragment of a nucleotide in the P1 domain of a wild-type picornavirus selected from the group consisting of PV1(S), PV2(S) and PV3(S) is substituted with a corresponding fragment of a nucleotide comprising a synthetic sequence recoded according to human codon pair bias to have a reduced codon pair score, and optionally P1 of the wild-type picornavirus is substituted with a synthetic P1 recoded according to human codon pair bias to have a reduced codon pair score.

In various embodiments, replacing at least the nucleotide fragment comprises replacing at least the nucleotide fragment comprising SEQ ID NO: 1, which is the P1 structure of the modified virus, is replaced with a corresponding nucleotide fragment having a synthetic sequence recoded according to human codon pair bias to have a reduced codon pair score.

In various embodiments, the recombinant virus can be administered intratumorally, intravenously, intracerebrally, intramuscularly, intraspinally, or intrathecally, and causes cell lysis of tumor cells.

In various embodiments, the malignancy can be selected from the group consisting of: glioblastoma multiforme, medulloblastoma, breast cancer, prostate cancer, colorectal cancer, hepatocellular carcinoma, bronchial cancer, and epidermoid cancer.

In various embodiments, the picornavirus can be of enterovirus C species. In various embodiments, the picornavirus can be a poliovirus.

In various embodiments, the P1 domain may be selected from the Sabin vaccine strains PV1(S), PV2(S), and PV3 (S).

Other features and advantages of the present invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.

Drawings

Exemplary embodiments are illustrated in referenced figures of the drawings. The embodiments and figures disclosed herein are intended to be illustrative, not limiting.

FIG. 1 depicts the codon pair biases of all human ORFs and the synthetic P1 region. Codon Pair Bias (CPB) scores were calculated for all 14795 annotated human genes. Each gray point represents the calculated CPB score for the gene plotted against its amino acid length. CPB of the PV (M) -wt P1 region and CPB of the designed synthetic poliovirus capsids PV-Min, PV-MinXY, PV-MinZ and PV-Max are indicated with arrows and labels.

Figure 2 depicts the viability of the PV-Min subclones constructed (above) the poliovirus genome, translated as a single open reading frame (below) the cDNA of various chimeric synthetic poliovirus constructs constructed by molecular cloning, and their viability in He L a R19 cells in culture, a virus is obtained that contains at least a segment of PV-Min, so there is no apparent defect in any one of the synthetic regions (X, Y or Z), the nucleotide positions separating the regions are shown, the first 12 nucleotides (4 codons) in all reading frames are PV (m) -wt sequences to allow for the proper initiation of translation, the corresponding colors of the sequences: black PV (m) -wt; grey: PV-Min; Thatched: PV-Max.

After transfection of transcript RNA from all viable viral constructs (FIG. 4), the viruses were amplified by two passages in He L a R19 cells, then titers of growth of these viruses were measured by plaque assay on He L a R19 cell monolayers PV-Max and PV (M) -wt viruses grown to similar titers (-10)⁹) Whereas the PV-Min chimeric virus produced lower PFU/ml titers, with significantly stepwise reductions in titer. Addition of the PV-Min sequence to the P1 region reduced PFU titers. The figure shows the mean titres of three independent experiments, error bars indicate standard deviation values.

FIGS. 4A-4B depict the effect of altered codon bias on translation (FIG. 4A) the structure of a bicistronic reporter the first cistron uses the Hepatitis C Virus (HCV) Internal Ribosome Entry Site (IRES) to initiate translation of Renilla (Renilla) luciferase (R-L uc). the first cistron provides an internal control to normalize the amount of RNA input.the second cistron uses poliovirus IRES to initiate translation of firefly luciferase (F-L uc). the region labeled "P1" is replaced by the synthetic P1 region of the virus which alters CPB.the (+) RNA is transcribed from these constructs (FIG. 4B) in the presence of 2mMGuHCl, each bicistronic RNA is transfected into cells.6 hours after 6 hours, the activities of R-L uc and F-L uc are measured.F-L uc are set to have a slightly higher PV/Min-23 vs. Min-11/Min than Min-20. the translational efficiency of the Min-Met codon expressed by Min codon (-26) and the deviation from Min-P L Min-26. the translational efficiency of this shows a slight deviation from the normalized PV-9P vs. Min translational efficiency expressed by Min codon (Min-685).

Fig. 5A-5B depict control of tumor growth in NCR nude mice treated with modified poliovirus MV-MinY. When the tumor reaches 0.2cm³After sizing, 2 × 10 was used on days 0,3 and 5⁷Each mouse was treated with PV-MinY or mock (mock) injections of PFU (n-7; a).

FIG. 6 depicts the oncolytic effect of PV-MinY on astrocytomas in NCR nude mice (under) compared to untreated control mice (top). injection of 1 × 10 subcutaneously into NCR nude mice⁶HTB-14 cells, once tumor size reached 0.2cm³Experiment was started on day 0, day 3 and day 5, tumor was injected with either mock diluent (top) or 2 × 10⁷PV-MinY of PFU (bottom). The tumor size of the PV-MinY injected was maintained or reduced, while animals in the mock-treated group had to be euthanized after 7 days due to the tumor size.

FIG. 7 depicts the oncolytic effect of the modified virus PV-MinY on A549 cells. A549 cells are hypodiploid (chromosome count 64, 65 or 66 in 40% of cells) cells of human lung epithelial cancer cell lines (Giard et al, 1973). Chromosomes N2 and N6 as single copies per cell; there are typically 4 copies of N12 and N17. By immunoperoxidase staining, a549 cells were keratin positive. A549 expresses the isozyme G6PD-B of glucose-6-phosphate dehydrogenase (G6 PD). We used a549 cells as a lung cancer model. A549 cells were infected with PV-MinY at an MOI of 1.0. A549 cells were grown in DMEM + 10% FBS. After shaking at room temperature for 1 hour, the mixture was stirred with 10⁶PV-MinY of PFU infected 90% confluent cells; the inoculum was removed and replaced with DMEM + 2% FBS. Infected cells were incubated at 37 ℃ with 5% CO₂Is incubated in an incubator. As shown in FIG. 7, compared to non-infectedCompared with lung cancer cells, PV-MinY can lyse most cells within 36 hours after infection and all cells within 72 hours after infection.

Figures 8A-8B depict the rapid construction of six SAVE de-optimized, live attenuated zika virus vaccine candidates that grew in Vero cells without animal components in less than 1 month. (FIG. 8A) codon pair bias was optimized for the zika prM/E and NS3 genes of human ORFeome and their SAVE counterparts. Codon Pair Bias (CPB) is expressed as the average codon pair score of the Open Reading Frame (ORF) for a given gene. Positive and negative CPB values indicate the predominance of statistically over-or low-performing codon pairs in the ORF, respectively. The red circles represent the CPB of 14795 individual ORFs, representing most of the known annotated human genes (ORFeome) that we analyzed. The CPB of the wild-type prM/E and NS3 genes were within the normal range of the human host cell genes. After "deoptimization" of codon pairs by SAVE, the resulting deoptimized prM/E and NS3 gene segments are now predominantly encoded by low performing human codon pairs, as evidenced by their highly negative CPB, and are in contrast to any human gene. (FIG. 8B) cDNA genomes of wild-type and synthetic "de-optimized" chimeric Zika vaccine variants. SAVE-deoptimized synthetic prM/E and NS3 of FIG. 8A were synthesized de novo and subcloned into the WT PRVABC59(PR15) or MR766 genome, respectively, using overlapping PCR, to generate six independent cDNA genomes, each containing a synthetic "deoptimized" fragment. We constructed infectious cDNA genomes of wt PR15 and MR766 within 7 days, and then fully recovered infectious, replicating viruses for 6 de-optimized ZIKV vaccine candidates by transfection of RNA into BHK cells within 27 days.

Fig. 9 depicts a diagram of a subcloning strategy for reducing attenuation or increasing immunogenicity by reducing the length of a de-optimized sequence in the prM + E coding region. The second generation of Zika vaccine candidates use the flexibility of the SAVE platform to reduce the de-optimized region from 2014bp (E-Min) to 997bp (E-W/Min) or further to 664bp (E-W/W/Min) while maintaining 100% identity in the amino acid sequence.

FIG. 10 depicts the reduction of protein and RNA expression in ZIKV infected cells. After 24 hours incubation at 33 ℃, western blot was used to measure envelope glycoprotein expression in Vero cells infected with ZIKV strains PRVABC59(PR15) and E-min (min) and Wild Type (WT) variants of MR 766. The envelope glycoprotein expression of the E-Min variant was greatly reduced in both ZIKV strains.

Figure 11 depicts the viral phenotype in infected mice. Attenuation of SAVE ZIKV vaccine candidates in AG129 mice. AG129 mice were injected with any of the following: i) synthetic wild type virus MR766 and PR15 with dosage of 10²(positive control); ii) vaccine candidates PR15E-Min or MR766E-Min and NS3-Min at a dose of 10⁴Or 10²PFU; iii) vaccine candidate PR15NS3+ E-Min in a single dose of 10⁴All delivered subcutaneously at 100 μ L the injected mice were examined for mortality and morbidity (weight loss) and vaccinated with 10²wt virus of all mice and inoculated with 10⁴80% of the mice of MR766NS3-Min died from the infection. All other mice survived, including all vaccinated with 10⁴PFU as a primary candidate MR766E-Min and without any weight loss.

Fig. 12 depicts sav attenuated ZIKV vaccine candidates that induce high levels of neutralizing antibodies and protect against lethal challenge in AG129 mice. A) anti-PR 15 zika antibody after a single dose. Sera from immunized animals were harvested on day 28 and passed through PRNT on Vero cells₅₀The assay quantifies antibodies to ZIKV strain PR 15. By 10⁴Mice vaccinated with doses of PR15E-Min and MR766E-Min produced high levels of neutralizing antibodies (2.8 log)₁₀PRNT50)。

FIG. 13 depicts the immunogenicity of the E-W/W/Min vaccination of Cynomolgus macaques (Cynomolgus macaques). By 10⁵Or 10⁷E-W/W/Min of individual Plaque Forming Units (PFU) vaccinates Zika virus seronegative macaques (delivered subcutaneously in a volume of 0.5m L.) animals mock immunization were injected with 0.5m L vaccine diluent50% reduction of neutralization in Vero cells Using Foci (FRNT)₅₀) Assay the neutralizing activity against the wild-type ZIKV strain MR766 was tested. After the first immunization, 10 was found⁷Or 10⁵Immunization with PFU E-W/W/Min outperformed NIH inactivated ZIKV vaccine candidates, with two doses triggering comparable levels of neutralizing antibodies. On day 14 post inoculation, all animals vaccinated with E-W/Min serum were switched. No FRNT observed on day 28 after booster immunization₅₀An increase in titer, indicating that its bactericidal immunity prevents secondary infections from triggering recall responses (anamnestic responses). This further indicates that a single relatively low dose of E-W/WMin may be sufficient to trigger high levels of neutralizing antibodies (. gtoreq.1,028 FRNT₅₀)。

FIG. 14 depicts an experimental summary of testing the oncolytic performance of MR766E-W/W/Min Zika virus using an in vivo tumor model of CC L.1 murine epithelial melanoma cells implanted into immunocompetent DBA/2 mice day 0, day 14, day 26, day 71 and day 85 mice were immunized 5 times with MR766E-W/W/Min day 97 (11 days after final immunization) CC L53.1 cells implanted into immunized or mock-immunized mice day × 10 s.c. the right lower flank of all animals was injected subcutaneously⁶CC L53.1.1 cells to complete the implantation 2.5 × 10⁵CC L53.1.1 cells were injected subcutaneously into the lower left flank of some mice only on day 103 (10 days after tumor implantation) with 1 × 10⁷PFU MR766E-W/W/Min treated implanted mice or mock-treated by direct injection into right flank tumors with OptiPro SFM. Only right flank tumors were treated to observe immune-mediated oncolytic effects of the non-injected tumors. The same treatment was repeated twice on day 105 (12 days after tumor implantation) and 107 (14 days after tumor implantation). Mice were monitored daily for body weight and tumor size for 10 days post-treatment (days 107-117), and every other day until 20 days post-treatment (days 118-127).

Figure 15 depicts the reduction in tumor size in DBA/2 mice implanted with CC L53.1 murine epithelial melanoma cells in mice treated 3 times by intratumoral route injection of MR766E-W/Min by day 5, the tumors of the treated animals significantly decreased, while mock-treated DBA/2 mice did not improve and tumor size increased.

FIG. 16 depicts the effect of preimmunization with Zika virus MR766W-E-W/W/Min on tumor size of DBA/2 mice implanted with CC L53.1.1 murine epithelial melanoma cells on day 0, day 14, day 26, day 71 and day 85 mice were immunized with 5 times MR766E-W/W/Min or mock immunization with OptiPro medium by inoculating 1 × 10⁶CC L53.1.1 cells were injected subcutaneously into the lower right flank of all animals, and CC L53.1.1 cells were implanted into immunized or mock-immunized mice.1 tumors were injected directly into the right flank using 1 × 10⁷PFU MR766E-W/W/Min、1×10⁷PFU MR766WT treated implanted heat relieve, or mock treated implanted mice. Treatments were performed three times every two days and tumor size was measured daily for 10 days after treatment. Simulating immunity: mock-treated DBA/2 mice had steadily increased tumors within 10 days, however, treatment with MR766WT and MR766E-W/W/Min effectively reduced the average tumor volume of mice previously immunized with MR 766E-W/W/Min.

FIG. 17 depicts the effect of preimmunization with Zika viruses E-W/Min and E-W/W/Min on tumor size in DBA/2 mice implanted with CC L53.1.1 murine epithelial melanoma cells on days 0, 14, 26, 71 and 85, mice were immunized 5 times with MR766E-W/W/Min and MR766E-W/Min or mock immunization with OptiPro medium by inoculating 1 × 10⁶CC L53.1.1 cells were injected subcutaneously into the lower right flank of all animals, and CC L53.1.1 cells were implanted into immunized or mock-immunized mice by direct injection into right flank tumors using 1 × 10⁷PFUMR766E-W/W/Min、1×10⁷PFU MR766WT treated implanted mice or simulated implanted mice. Treatments were performed three times every two days and tumor size was measured daily for 10 days after treatment. Simulating immunity: mock-treated DBA/2 mice tumors steadily increased within 10 days; however, both MR766WT and MR766E-W/W/Min treated tumors decreased in size. Among the immunized mice, the MR766E-W/W/Min treatment outperformed the MR766WT treatment.

FIG. 18 depicts the effect of MR766WT treatment on tumor size in DBA/2 mice implanted with CC L53.1.1 murine epithelial melanoma cells on day 0, day 14, day 26, day 71 and day 85 mice were immunized 5 times with MR766-W-W-E-Min or mock-immunized with OptiPro medium by inoculating 1 × 10⁶CC L53.1.1 cells were injected subcutaneously into the lower right flank of all animals, and CC L53.1.1 cells were implanted into immunized or mock-immunized mice by direct injection into right flank tumors using 1 × 10⁷PFU MR766WT treated implanted mice or simulated implanted mice. Treatments were performed three times every two days and tumor size was measured daily for 10 days after treatment. Simulating immunity: mock-treated DBA/2 mice had steadily increased tumors within 10 days, as did MR766 WT-treated tumors in mock-immunized mice. In DBA/2 mice immunized with MR766E-W/W/Min, MR766WT treatment was effective to reduce tumor size significantly at day 10.

FIG. 19 depicts the effect of immunization on the efficacy of Zika virus MR766-E-W/W/Min treatment on tumor size in DBA/2 mice implanted with CC L53.1.1 murine epithelial melanoma cells on day 0, 14, 26, 71 and 85 mice were immunized with 5 times MR766E-W/W/Min, MR766E-W/W/Min and E-W/Min, or mice were mock immunized with OptiPro medium by inoculating 1 × 10.13510⁶CC L53.1.1 cells were injected subcutaneously into the lower right flank of all animals, and CC L53.1.1 cells were implanted into immunized or mock-immunized mice by direct injection into right flank tumors using 1 × 10⁷PFU MR766E-W/W/Min treated the implanted mice or simulated the implanted mice. Treatments were performed three times every two days and tumor size was measured daily for 10 days after treatment. Simulating immunity: mock-treated DBA/2 mice had steadily increased tumors within 10 days, as did MR 766E-W/W/Min-treated tumors in mock-immunized mice. In DBA/2 mice immunized with MR766E-W/W/Min and DBA/2 mice immunized with the combination of MR766E-W/W/Min and E-W/Min, the tumor size disappeared after treatment with MR 766E-W/W/Min.

FIG. 20 depicts the effect of MR766E-W/W/Min treatment on off-side tumor size in DBA/2 mice mock immunized with OptiPro medium or with MR766E-W/W/Min and implanted with CC L53.1.1 mouse epithelial melanoma cells in this study, 1 × 10⁶An individual CC L53.1.1 murine epithelial melanoma cell was injected subcutaneously into the right lower flank of the mouse, either treated by intratumoral injection of MR766E-W/W/Min or mock treated with OptiPro medium, untreated 2.5 × 10 was also injected⁵The size of these tumors was also measured to assess immune-mediated offsite clearance of viral oncolytic, in mock-immunized, mock-treated left flank tumors, the size of which steadily increased within 10 days post-treatment, in mock-immunized MR 766E-W/Min-treated mice, the left flank tumor size was maintained, whereas in MR 766E-W/Min-immunized E-W/Min-treated mice, the left flank tumor size increased several-fold and then decreased again<0.001)。

FIGS. 21A-21B show that animals treated with Zika virus were protected from tumor re-challenge the DBA/2 mice were initially immunized with diluent (mock), MR766WT virus (WT), MR766E-W/Min (EW) or MR766E-W/W/Min (EWW) () 21A) in the contralateral flank of mice cured with CC L53.1 tumor (melanoma) treated with WT or EWW, 1 × 10⁶The individual CC L53.1.1 cells were again challenged and matched with the blankControl DBA/2 mice (n-5; RC01-13-1, RC01-13-2, RC01-13-3, RC01-13-4, RC 01-13-5). For each group, tumor growth in the flank was assessed within 22 days after tumor cell administration. Y-axis mean tumor size in mm³Measured, X-axis Days Post Challenge (DPC); 21B) tumors were taken from each mouse on day 22 post-injection.

22A-22C show that Zika virus treated animals were protected from re-challenge with a xenogenic tumor. Initially using MR766E-W/MinDBA/2 mice were immunized and boosted 3 times with MR766E-W/Min both flanks (tumor size pooled from both flanks) were treated with 1 × 10 at 22A) in mice cured of CC L53.1.1 tumor by treatment with WT or EWW⁶Each CR L2947 cell (renal carcinoma cell) (N ═ 4) was rechallenged, or 22B) was rechallenged in the right flank with K L N-205 cells (lung carcinoma cells) (N ═ 5) and compared to placebo DBA/2 mice (N ═ 3)³Measurements were taken with X-axis Day Post Challenge (DPC). immunized mice had significantly smaller CR L2947 tumor sizes at day 11 (p 0.0064) and day 21 (p 0.0012), and therefore "tumor resistance". K L N-205 tumor sizes also decreased at day 11 (p 0.0179) and day 14 (p 0.0478) post challenge, and other time points were also close to significance (p 0.0532). 22C) tumors were taken from each mouse at day 21 (CR L2947) and day 17 (K L N-205) post-injection.

Fig. 23 shows an exemplary treatment protocol.

FIG. 24 depicts that no mortality and morbidity was observed in mice vaccinated with CodaVax-H1N1M 101/V6.

FIGS. 25A-25C depict the efficacy of oncolytic CodaVax-H1N1M101/V6 in treatment of implanted syngeneic 4T1TNCB cells in Balb/C mice delivered 8 times 10 times on days 8, 10, 12, 14, 16, 18, 26 and 28 post-implantation (DPI)⁷Observations were made during the course of the PFU treatment. Fig. 25A depicts 4T1 cells implanted and mock treated (n-5, red) or with 10 on days 8, 10, 12, 14, 16, 18, 26 and 28 (DPI) post-implantation⁷Mean tumor volume (in mm) over time in PFU CodaVax-H1N1M101/V6 (N-8, blue) treated Balb/C mice³Meter). FIG. 25B depicts survival rates and uses tumor volumes ≧ 400mm³The humanoid early end point (human early end point) of (1) was calculated. Figure 25C depicts 4T1 cells implanted and mock treated (n-5, red) or with 10 at day 8, day 10, day 12, day 14, day 16, day 18, day 26 and day 28 (DPI) post-implantation⁷PFU CoMean tumor volume (as a percentage of starting volume) over time in Balb/C mice treated with daVax-H1N1M101/V6(N ═ 8, blue).

FIG. 26 depicts a treatment plan for DBA/2 mice subcutaneously implanted with CC L.1 cells, mice implanted on day 0 and treated with CodaVax-H1N1M101/V6 on days 8, 10, 12, 14 and 16.

FIG. 27 shows that no mortality or morbidity was observed in mice vaccinated with CodaVax-H1N 1.

Figure 28 depicts tumor size (mm) at the start of 8DPI treatment³) At the beginning of the treatment with CodaVax-H1N1M101/V6, mice implanted with CC L53.1.1 cells treated with 0.1mg of anti-PD-1 antibody on day 3 and day 7 had smaller tumors (p 0.0466, student t test).

FIG. 29 shows the efficacy of oncolytic CodaVax-H1N1M101/V6 in treating implanted syngeneic CC L53.1.1 melanoma cells in DBA/2 mice delivered 5 times 8 × 10 at 8, 10, 12, 14 and 16DPI⁵Observations were made during the course of the PFU treatment. A) The tumor volume is more than or equal to 400mm³The survival rate was calculated at the early end of the human track. Tumor size was measured in volume (mm)³) Reports were made and plotted as B) mean ± standard deviation.

FIG. 30 shows induction of differentiation of cultured human monocytes (THP-1) into macrophages by adding 100nM PMA to the medium, infection with the indicated virus, followed by 5% CO at 37 ℃₂The effect at 1 and 3 days of incubation and secretion of I L-1B in the supernatant was measured by E L ISA.

FIGS. 31A-31F show immune cell activation in DBA/2 mice with 10 on days 0, 14, 28, and 42⁷FFU MR766E-W/Min immunization or mock immunization with viral diluent on day 63, delivered by subcutaneous route, 1 × 10⁶Individual CC L53.1.1 melanoma cells were implanted into immunized and mock-immunized mice, and on the second dayTreatment started on day 73 with intratumoral delivery of 10 on days 73, 75 and 77⁷PFUMR766EW/W/Min or viral diluent tumors were harvested and fixed in 4% paraformaldehyde for immunohistochemistry and staining on days 1 and 7 after treatment (days 74 and 80) to count the immune markers and measure CD4+/CD8+ cell infiltration fig. 31A) percentage of CD45+ cells present in implanted CC L.1 tumors after treatment with MR766 84-W/Min alone or 1 day after E-W/Min immunization again after E-W/Min treatment fig. 31B) percentage of CD 6325 + cells present in implanted CC L.1 tumors after treatment with MR766E-W/Min alone or 1 day after E-W/Min immunization and 1 day and 7 after E-W/Min treatment after E-W/Min immunization, percentage of CD 34 + cells present in implanted CC L.1 tumors was visualized by immunohistochemistry on days 3/W/Min 9, CD 6353 + cells present in implanted CD 6353.31B 53) tumor implantation map 31A graph that the percentage of CD 34 + cells present in implanted CC 9653.1 tumor was greatly increased by immunohistochemistry on days 2, CD 6353, CD 3, CD 53.

Detailed Description

Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs Singleton et al, Dictionary of Microbiology and Molecular Biology, 3 rd edition, Revised, J.Wiley & Sons (New York, NY 2006), and Sambrook and Russel, Molecular cloning: A L laboratory, 4 th edition, Cold Spring Harbor L laboratory Press (Cold Spring Harbor, NY 2012), provide one of ordinary skill in the art with a general guide to many of the terms used in this application.

Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. Indeed, the invention is in no way limited to the methods and materials described.

Viral modification by de-optimization of codon pair bias

According to aspects of the invention, codon pair bias can be altered independently of codon usage. For example, in a protein coding sequence of interest, codon pair bias can be altered simply by directional rearrangement of its codons. In particular, the same codons that occur in the parental sequences (which may have different frequencies in the host organism) may be used in the altered sequences, but at different positions. In the simplest form, the codon usage in the coding region of the protein in question remains unchanged (as does the encoded amino acid sequence) because the same codons as the parent sequence are used. However, certain codons occur in a new context, i.e., preceded and/or followed by codons that encode the same amino acid as in the parent sequence, but employ a different nucleotide triplet. Ideally, rearrangement of codons results in a lower frequency of codon pairs than in the parental sequence. In practice, rearranged codons will generally result in a lower frequency of codon pairs at one position and a higher frequency at a second position. By judicious rearrangement of codons, the bias in codon pair usage over a given length of the coding sequence can be reduced relative to the parental sequence. Alternatively, the codons can be rearranged to produce a sequence that utilizes codon pairs more frequently in the host than the parent sequence.

Codon pair bias is evaluated by considering each codon pair in turn, scoring each pair according to the frequency with which codon pairs are observed in the protein coding sequence of the host, and then determining the codon bias of that sequence, as disclosed herein. It will be appreciated that many different sequences can be created with the same codon pair bias. At the same time, the bias of the codon pairs may vary more or less depending on the way the codons are rearranged. Codon pair bias of a coding sequence can be altered by recoding the entire coding sequence, or by recoding one or more subsequences. As used herein, "codon pair bias" can also be evaluated over the length of the coding sequence, even though mutations may be made to only a portion of the sequence. Because codon pairs are scored against the context of codon usage in the host organism, codon pair bias values can be assigned to wild-type viral sequences and mutant viral sequences. According to aspects of the invention, the virus may be modified by recoding all or part of its protein coding sequence, thereby reducing its codon usage.

According to an aspect of the invention, codon pair bias is a quantitative characteristic determined from codon pair usage of the host. Thus, for any given viral protein coding sequence, an absolute codon pair bias value can be determined. Alternatively, the relative change in codon pair bias values can be determined to correlate the de-optimized viral protein coding sequence with a "parental" sequence from which the optimized viral protein coding sequence is derived. Since viruses are of various types (i.e., types I through VII in the Baltimore classification), and natural (i.e., virulent) isolates of different viruses produce different absolute codon pair bias values, the relative changes in codon pair bias are often more relevant to determining the desired level of attenuation. Accordingly, the present invention provides modified viruses comprising a viral genome in which codon pair bias of one or more protein-encoding nucleotide sequences is reduced by mutation, and methods of making the same, and uses these viruses and therapies for malignancies. In viruses that encode only a single protein (i.e., a multimeric protein), codon pair bias can be reduced by mutating all or part of the multimeric protein to a desired degree, and all or part of the mutated sequence can be provided in the recombinant viral construct. For viruses that encode multiple proteins individually, the codon pair bias of all protein-encoding sequences may be reduced simultaneously, or only one or more protein-encoding sequences may be selected for modification. The reduction in codon pair bias is determined over the length of the protein coding sequence and is at least about 0.05, or at least about 0.1, or at least about 0.15, or at least about 0.2, or at least about 0.3, or at least about 0.4. Depending on the virus, the absolute codon pair bias based on the codon pair usage of the host may be about-0.05 or less, or about-0.1 or less, or about-0.15 or less, or about-0.2 or less, or about-0.3 or less, or about-0.4 or less.

Obviously, codon pair biases can also be superimposed on other sequence variations. For example, a coding sequence may be altered to encode a protein or polypeptide containing one or more amino acid changes, and may also have altered codon usage. Also, in some cases, codons can be shuffled to maintain exactly the same codon usage profile as the parent protein coding sequence in protein coding sequences with reduced codon bias. The program emphasizes the power of codon vs. bias changes, but does not require adherence. Alternatively, codon usage may result in an overall change in codon usage in the coding sequence. In this regard, it should be noted that in certain examples provided herein, (e.g., the design of PV-Min) even if the codon usage profile does not change during the generation of the codon pair bias minimizing sequence, when a portion of that sequence is subcloned into the unmutated sequence (e.g., PV-MinXY or PV-MinZ), the codon usage profile on the subcloned portion and in the resulting hybridization will not match the profile of the original unmutated protein coding sequence. However, changes in these codon usage profiles have minimal impact on codon bias.

Similarly, it should be noted that altering the nucleotide sequence alone to encode a protein or polypeptide having one or more amino acid substitutions is also highly unlikely to result in a sequence with significant changes in codon pair bias. Thus, even in nucleotide sequences that have been further modified to encode mutated amino acid sequences, codon pair bias changes may be recognized. It is also noteworthy that mutations which are intended to increase codon bias themselves may have little effect on codon bias. For example, as disclosed herein, codon pair bias for a modified viral mutant that is re-encoded to maximize the use of non-preferred codons (PV-AB) is reduced by about 0.05 from wild-type (PV-1(M)) (Mueller et al, 2006). It is also noteworthy that such protein coding sequences have greatly reduced sequence diversity. In contrast, considerable sequence diversity is maintained in the codon pair bias modified sequences of the invention. Moreover, the significant reduction in codon pair bias that can be achieved without increasing rare codon usage indicates that, rather than maximizing the use of non-preferred codons, it would be beneficial to rearrange the non-preferred codons with a sufficient number of preferred codons to more effectively reduce codon pair bias.

The degree and intensity of mutation may vary depending on the length of the nucleic acid encoding the protein, whether all or part of the mutation is possible, and the desired codon pair bias is reduced. In embodiments of the invention, the protein coding sequence is modified over a length of at least about 100 nucleotides, or at least about 200 nucleotides, or at least about 300 nucleotides, or at least about 500 nucleotides, or at least about 1000 nucleotides.

The term "parent" viral or "parent" protein coding sequence is used herein to refer to viral genomes and protein coding sequences from which novel sequences may be derived that are more or less modified. Thus, a parental virus may be a "wild-type" or "naturally occurring" prototype or isolate or variant, or a mutant specifically created or selected based on the properties that are authentic or deemed desirable.

Using de novo DNA synthesis, the capsid coding region of PV (M) (region P1 from nucleotide 755 to nucleotide 3385) was redesigned to introduce the largest possible number of rarely used codon pairs (viral PV-Min). Cells transfected with PV-Min mutant RNA were not killed and no viable virus could be recovered. Fragmenting the PV-Min capsid region (PV-Min)^755-2470、PV-Min^2470-3386) Subcloning into wt background resulted in very weak but not dead virus. This result demonstrates the effectiveness of altering the extent to which codon pairs substituted into the genome of the wild-type parental virus de-optimize the sequence to alter the codon pair bias of the overall sequence and attenuation of the viral product.

Viruses with de-optimized codon pair bias are attenuated. As exemplified in the reference by Coleman et al 2008, CD155tg mice survived the challenge by intracerebral injection of attenuated virus, in an amount 1000-fold more lethal than the wild-type virus. These findings demonstrate the ability of codon pair bias de-optimization to minimize viral lethality. Furthermore, by selecting the extent to which codons are de-optimized for bias, a balance can be struck between reduced viability and infectivity of the virus. In addition, once the degree or range of degrees of codon pair bias de-optimization that provides the desired attenuation characteristics is determined, additional sequences can be designed to obtain the degree of codon pair bias. For example, SEQ ID NO: 1 provides a modified viral sequence having a codon pair bias of about-0.2, and the mutations are distributed over a region comprising the mutated portion of PV-MinY.

Sequence design algorithm

The inventors have developed several algorithms for gene design that optimize a DNA sequence for a particular desired property while encoding a given amino acid sequence. In particular, algorithms have been developed to maximize or minimize short-haired secondary structure of the desired RNA in the sequence (Cohen and Skiena, 2003) and to maximize the addition and/or removal of a specified set of patterns (Skiena, 2001). The former problem arises when designing live viruses, while the latter can be used for technical reasons to optimally insert restriction sites. The extent to which overlapping genes can be designed that encode two or more genes simultaneously in alternate reading frames has also been investigated. This property of encoding different functional polypeptides in different reading frames of a single nucleic acid is common in viruses and can be used for technical purposes, for example to encode antibiotic resistance genes.

First generation design tools for synthetic biology have been established, as described by Jayaraj et al (2005) and Richardson et al (2006.) these focus primarily on the design for optimization of manufacturability (i.e., oligonucleotides without local secondary structure and terminal repeats) rather than optimizing sequences for biological activity.

As exemplified herein, computer-based algorithms can be used to manipulate codon pair bias of any coding region. The algorithm has the ability to shuffle existing codons and evaluate the resulting CPB, and then to shuffle the sequence, optionally locking in a particular "valuable" codon pair. The algorithm also takes the form of "simulated annealing" to avoid trapping local minima. Other parameters (e.g., free energy of RNA folding) may also optionally be under control of the algorithm to avoid generating undesirable secondary structures. The algorithm can be used to find sequences with the least bias in codon pairs, and in the case where such sequences do not provide live virus, the algorithm can be adjusted to find sequences with reduced but not minimized bias. Of course, it is also possible to generate viable viral sequences using only subsequences of the computer-minimized sequence.

Whether performed with the aid of a computer or not, for example, using gradient descent, or simulated annealing or other minimization routines. An example of a procedure to rearrange the codons present in the starting sequence can be shown by the following steps:

1) obtaining the genome sequence of the wild type virus.

2) Protein coding sequences were selected for targeted modification design.

3) Non-coding functions are used to lock known or predicted DNA segments.

4) The desired codon distribution was chosen for the remaining amino acids in the redesigned protein.

5) The unlocked codon positions were randomly shuffled and codon pair scores were calculated.

6) Optionally, simulated annealing procedures are employed to further reduce (or increase) the codon pair score.

7) The resulting design was checked for excess secondary structure and unnecessary restriction sites:

a. if so, go to step (5) or modify the design by replacing the problematic region with the wild-type sequence and go to step (8).

8) DNA sequences corresponding to the virus design were synthesized.

9) Virus constructs were created and expression was assessed:

a. if the attenuation is excessive, a subclone construct is made and turned to 9;

b. if attenuation is insufficient, go to 2.

Alternatively, a program can be designed that allows each pair of amino acids to be de-optimized by selecting codon pairs without requiring the codons to be swapped out from elsewhere in the protein coding sequence.

Viruses and modified viruses

Viruses may be dsDNA viruses (e.g., Adenoviruses (Adenoviruses), Herpes viruses (Herpesviruses), Poxviruses (Poxviruses)), single-stranded "positive" sense DNA viruses (e.g., Parvoviruses (subvierusses)), double-stranded RNA viruses (e.g., Reoviruses (Reoviruses)) or single-stranded "negative" sense RNA viruses (e.g., Orthomyxoviruses (Orthomyxoviruses), Rhabdoviruses (Rhabdoviruses)), in certain non-limiting embodiments of the invention, the viruses are Poliovirus (PV), rhinoviruses (rhinoviruses), influenza viruses (inflenza viruses), dengue viruses (Virus), West Nile Virus (West disease), varicella (varicella-zoster Virus), Herpes Virus (Herpes Simplex Virus), mumps Virus (Herpes Simplex Virus) (Herpes Simplex Virus L)), or Herpes Simplex Virus (Herpes Simplex Virus) (Herpes Simplex Virus (HSV) (Herpes Simplex Virus), Herpes Simplex Virus (HSV) (Herpes Simplex Virus) (HSV) (manvirus L)).

In various embodiments, the modified virus belongs to the Picornaviridae (Picornaviridae) family of viruses and all related genera, strains, types, and isolates.

Polio is a disease of the central nervous system caused by poliovirus infection. Poliovirus is a human enterovirus (enterovirus) belonging to the family picornaviridae and classified into three stable serotypes. It is spherical, 20nm in size, and comprises an RNA core surrounded by an envelope (consisting of proteins). It is transmitted through the mucosa of the mouth, throat or digestive tract. All three modified virus serotypes are reported to be causative agents of paralytic polio, although occurring at different frequencies (type 1 > type 2 > type 3).

However, modified viral infection does not necessarily lead to the development of polio. In contrast, most infections (98% -99%) result in local replication of the virus in the gastrointestinal tract, causing only mild symptoms, or no symptoms at all. The modified virus is less invasive to the CNS, where it selectively targets anterior spinal cord horns and medullary motor neurons for destruction. Bodian, D.in, Diseases of the New vous System, Minckler, J.ed., McGraw-Hill, New York, pp.2323-2339 (1972).

The abnormally limited cellular tropism of modified viruses leads to unique pathological features. It is characterized by the loss of motor neurons in the spinal cord and medulla, leading to the hallmark clinical signs of polio, flaccid paralysis. Other neuronal components of the central nervous system, as well as glial cells, often escape infection. In the brain tissue infected under an electron microscope, severe changes were observed in motor neurons, while no significant changes were observed in glial components. Normal astrocytes and oligodendrocytes can be seen next to degenerated neurons or axons, with no signs of infection or response. Bodian d. The restrictive tropism of the modified virus is not known. In addition to the restrictive cell and tissue tropism, the modified virus only infects primate and primate cell cultures. Other mammals remain unaffected.

The isolation of the modified virus in 1908 led to intensive research efforts to understand the mechanisms of infection. Early work required the use of monkeys and chimpanzees as animal models. Such animals have a long life cycle and are very expensive and difficult to use in research. The discovery of the human poliovirus receptor (PVR), also known as CD155, a cell docking molecule for poliovirus, has led to the development of transgenic mice expressing human modified viral receptors as a new animal model for poliomyelitis. Transgenic mice can be used to study the pathogenicity of the modified virus.

Early research efforts have also led to the development of attenuated PV strains lacking the potential for neuropathogenesis and soon were tested as potential vaccine candidates for the prevention of polio. Among the most effective are the modified viruses developed by a. Sabin, the type 1, type 2 and type 3 Sabin strains. In rare cases, vaccine-related paralytic poliomyelitis was observed after oral administration of live attenuated strains of modified virus (Sabin strain). The occurrence of vaccine-associated paralytic poliomyelitis was associated with the emergence of neurovirulent variants of the attenuated Sabin strain after immunization.

For understanding the present invention, it is also important to understand the structure of poliovirus. All picornaviruses (including enteroviruses, cardioviruses, rhinoviruses, aphtoviruses, hepatoviruses, and parechoviruses) contain 60 copies, each of which comprises four polypeptide chains: VP1, VP2, VP3 and VP 4. These chains are elements of protein subunits called mature "promoters". Promoters are defined as the smallest identical subunit of the virus. Traces of the fifth protein VP0, cleaved into VP2 and VP4, were also observed. These proteins together form the coat or coat of poliovirus.

The picornavirus genome has single-stranded messenger active RNA. The "+" strand of the genomic messenger active RNA is polyadenylated at the 3 'end and carries a small protein VPg covalently attached to the 5' end. The first picornavirus RNA that was completely sequenced and cloned into DNA was poliovirus type 1. However, poliovirus lacks 5' m⁷The gppppg cap structure, and efficient translation of RNA requires ribosome binding, which is achieved by a ribosome entry site (IRES) within the 5 'untranslated region (5' NTR).

The common tissue pattern of the modified virus is schematically represented in fig. 2, comprising 5'NTR, P1, P2, P3 and 3' NTR with polya tail. The 5' NTR comprises 6 domains, designated I, II, III, IV, V and VI, respectively. The IRES comprises domain II-domain VI. P1 is the coding region for a structural protein (also known as capsid protein). P2 and P3 encode non-structural proteins.

In nature, there are three immunologically distinct modified virus types: serotype 1, serotype 2 and serotype 3. These types differ in the specific sequences in their capsid proteins that interact with a specific set of neutralizing antibodies. All three types are present in different strains, and all naturally occurring types and strains can cause polio. Therefore, they are neurotoxic. The genetic organization and replication mechanisms of the serotypes are identical; the nucleotide sequences of their genomes are > 90% identical. Moreover, all polioviruses (even attenuated vaccine strains) use the same cellular receptor (CD155) to enter and infect host cells; they exhibit the same tropism for human and susceptible transgenic animal tissues.

The neuropathogenicity of the modified virus can be attenuated by mutations in the regions designated P1 and P3 proteins and the ribosome entry site (IRES) within the 5' NTR. The Sabin vaccine strains type 1, 2 and 3 each carry a mutation in domain V of their IRES element, which correlates with the attenuated phenotype. Despite its effectiveness as a vaccine, Sabin strains remain neuropathogenic in animal models of polio. These strains in vaccines may cause disease despite low morbidity.

Indeed, single point mutations in IRES elements of each Sabin vaccine strain can be recovered in the vaccine within a period of 36 hours to days. Overall, vaccine-related acute polio occurs with a probability of 1 out of 530000 vaccines in the united states. Poliovirus isolated from a patient immunized with polio may also recover mutations at different positions in its genome.

In other embodiments, the modified virus is derived from an influenza a virus, an influenza b virus, or an influenza c virus. In other embodiments, the influenza a virus belongs to, but is not limited to, the following subtypes: H10N, H11N, H12N, H13N, H14N, H15N, H16N, H1N, H2N, H3N, H6N, H4N, H6N, H4H 6N, H7N, H6H 4H 6N, H6H 4H 6N, H7H 6N, H6. In various embodiments, the modified virus is H1N1M101/V6 as disclosed herein. In various embodiments, one or more segments of influenza a virus are re-encoded (e.g., de-optimized). In various embodiments, the HA segments are re-encoded. In various embodiments, the NA segments are recoded. In various embodiments, both the HA and NA segments are recoded. In various embodiments, the recoded influenza a virus has reduced codon bias or codon pair bias as discussed herein with respect to other viruses.

In various embodiments, the modified virus belongs to the Flaviviridae (Flaviviridae) family of viruses and all related genera, strains, types, and isolates. In various embodiments, the modified virus is a zika virus species, as discussed further herein.

In various embodiments, the modified virus belongs to the family of Adenoviridae (Adenoviridae) and all related genera, strains, types, and isolates, such as, but not limited to, human adenovirus type a, B, C.

In various embodiments, the modified virus belongs to the family of Herpesviridae (Herpesviridae) viruses and all related genera, strains, types, and isolates, such as, but not limited to, herpes simplex virus.

In various embodiments, the modified virus belongs to the Reoviridae (Reoviridae) family of viruses and all related genera, strains, types, and isolates.

In various embodiments, the modified virus belongs to the papillomavirus family (papillomanaviridae) virus and all related genera, strains, types, and isolates.

In various embodiments, the modified virus belongs to the family of Poxviridae (Poxviridae) viruses and all related genera, strains, types, and isolates.

In various embodiments, the modified virus belongs to the family of Paramyxoviridae (Paramyxoviridae) viruses and all related genera, strains, types, and isolates.

In various embodiments, the modified virus belongs to the Orthomyxoviridae (Orthomyxoviridae) family of viruses and all related genera, strains, types, and isolates.

In various embodiments, the modified virus belongs to the Bunyaviridae (Bunyaviridae) family of viruses and all related genera, strains, types, and isolates.

In various embodiments, the modified virus belongs to the family of viruses of the order Nidovirales (Nidovirales) and all related genera, strains, types, and isolates.

In various embodiments, the modified virus belongs to the Caliciviridae (Caliciviridae) family of viruses and all related genera, strains, types, and isolates.

In various embodiments, the modified virus belongs to the Rhabdoviridae (Rhabdoviridae) family of viruses and all related genera, strains, types, and isolates.

In various embodiments, the modified virus belongs to the Togaviridae (Togaviridae) family as well as all related genera, strains, types and isolates.

In various embodiments, the modified virus is SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5 or SEQ ID NO: 6.

Modified Zika virus

Embodiments of the present invention use attenuated Zika viruses having reduced viral protein expression therein, which have excellent growth performance useful for vaccine production, yet possess extraordinary safety and enhanced protective properties₅₀Values reveal) that are generally very effective in providing protective immunity against challenge with the same strain of zika virus, and also against challenge with other strains of zika virus.

In certain embodiments of the invention, the attenuated Zika virus of the invention comprises a recoded pre-membrane/envelope (E) coding region, a recoded non-structural protein 3(NS3) coding region, or E and NS3 coding regions. C. The NS1, NS2, NS4, or NS5 protein coding regions are not recoded and do not exclude mutations and other variations in those sequences, but merely mean that any mutation or variation made in those sequences has little or no effect on attenuation. Little or no effect on attenuation, including one or both of: 1) when a variant of C, NS1, NS2, NS4, or NS5 coding region is the only variant in the test zika virus, a mutation or variation in C, NS1, NS2, NS4, or NS5 coding regions does not reduce viral replication or viral infectivity by more than 20%; 2) c, NS1, NS2, NS4, or NS5 coding region, wherein a mutation or variation in any one of the coding regions represents less than 10% of the nucleotides in the coding sequence.

Zika virus used in the present invention is highly attenuated. In an embodiment of the invention (compared to the wild type), the zika virus is attenuated at least 5000-fold, or at least 10000-fold, or at least 20000-fold, or at least 33000-fold, or at least 50000-fold, or at least 100000-fold in the AG129 mouse model compared to a wild type virus having the same sequence but a protein encoded by a different nucleotide sequence.

The attenuated Zika virus used in the present invention also showed a large safety margin (i.e., L D)₅₀And PD₅₀The difference between) and therefore has a high safety factor, defined herein as L D₅₀/PD₅₀The ratio of (a) to (b). In certain embodiments of the invention, the safety factor is at least 10²Or at least 10³Or at least 10⁴Or at least 10⁵Or at least 2 × 10⁵Or at least 5 × 10⁵Or at least 10⁶Or at least 2 × 10⁶Or at least 5 × 10⁶. In certain embodiments, the factor of safety is 10²-10³Or 10³-10⁴Or 10⁴-10⁵Or 10⁵-10⁶。

Recoding of the E and NS3 protein coding sequences of the attenuated viruses of the invention can be performed using any algorithm or program known in the art or newly designed for recoding protein coding sequences. According to the present invention, nucleotide substitutions were engineered at multiple positions in the E and NS3 coding sequences, wherein the substitutions introduced multiple synonymous codons into the genome. In certain embodiments, the synonymous codon substitution alters codon bias, codon pair bias, density of infrequent codons or infrequently occurring codon pairs, RNA secondary structure, CG and/or TA (or UA) dinucleotide content, C + G content, presence or absence of a translational frameshift site, a translational pause site, a microRNA recognition sequence, or any combination thereof, in the genome. Codon substitution engineering can be performed at multiple positions distributed throughout the E and NS3 coding sequence or at multiple positions limited to portions of the E and NS3 coding sequence. Due to the large number of defects (i.e., nucleotide substitutions) involved, the present invention provides methods for generating stable attenuated viruses and live vaccines.

As discussed herein, in some embodiments, a viral coding sequence is re-encoded by replacing one or more codons with synonymous codons that are used less frequently in a zika host (e.g., human, mosquitoes (mosquitoes)). In some embodiments, the viral coding sequence is re-encoded by replacing one or more codons with synonymous codons that are less frequently used in zika virus. In certain embodiments, the number of codons replaced with synonymous codons is at least 5. In some embodiments, at least 10 or at least 20 codons are substituted with synonymous codons.

In some embodiments, the viral codon pair is re-encoded to reduce (i.e., decrease the codon pair bias value) the codon pair bias. In certain embodiments, codon pair bias is reduced by: identifying a codon pair in the E or NS3 coding sequence having a reduced codon pair score and reducing codon pair bias by replacing the codon pair with a codon pair having a lower codon pair score. In some embodiments, such replacement of codon pairs takes the form of rearranging existing codons of the sequence. In some such embodiments, the subset of codon pairs is replaced by rearranging the subset of synonymous codons. In other embodiments, the codon pairs are replaced by maximizing the number of synonymous codons rearranged. It should be noted that while codon rearrangement results in an overall decrease in codon pair bias (making negative values greater) for viral coding sequences, and rearrangement results in a decrease in CPS at many positions, other positions may be accompanied by an increase in CPS, but on average, the codon pair score, and thus CPB, of the modified sequence decreases. In some embodiments, recoding of a codon or codon pair may allow for alteration of the G + C content of the E and NS3 coding sequences. In some embodiments, recoding of a codon or codon pair may allow for alteration of the frequency of CG and/or TA dinucleotides in the E and NS3 coding sequence.

In certain embodiments, the recoded E protein coding sequence has a codon pair bias of less than-0.1, or less than-0.2, or less than-0.3, or less than-0.4. In certain embodiments, the recoded (i.e., reduced expression) NS3 protein coding sequence has a codon usage of less than-0.1, or less than-0.2, or less than-0.3, or less than-0.4. In certain embodiments, the codon pair bias of the recoded HA protein coding sequence is reduced by at least 0.1, or at least 0.2, or at least 0.3, or at least 0.4 as compared to the parental E protein coding sequence from which the recoded HA protein coding sequence is derived. In certain embodiments, the codon pair bias of the recoded NS3 protein-encoding sequence is reduced by at least 0.1, or at least 0.2, or at least 0.3, or at least 0.4 as compared to the parent NS3 protein-encoding sequence from which the recoded NS3 protein-encoding sequence is derived. In certain embodiments, the rearrangement of synonymous codons of the E protein coding sequence provides a reduction in codon usage of at least 0.1, or at least 0.2, or at least 0.3, or at least 0.4 of the parent E protein coding sequence from which the E protein coding sequence is derived. In certain embodiments, the rearrangement of synonymous codons of the NS3 protein coding sequence provides a reduced codon pair bias of at least 0.1, or at least 0.2, or at least 0.3, or at least 0.4 of the parent NS3 protein coding sequence from which the NS3 protein coding sequence is derived.

Typically, these substitutions and alterations are made and the expression of the encoded viral protein is reduced without altering the amino acid sequence of the encoded protein. In certain embodiments, the invention also includes alterations in the E and/or NS3 coding sequences that result in the substitution of non-synonymous codons and amino acid substitutions in the encoded protein, which may or may not be conservative.

For example, alanine is encoded by GCU, GCC, GCA and GCG.three amino acids (L eu, Ser and Arg) are encoded by six different codons, while only Trp and Met have unique codons.

TABLE 6 genetic code

a the first nucleotide in each codon encoding a particular amino acid is shown in the leftmost column; the second nucleotide is shown in the first row; the third nucleotide is shown in the rightmost column.

In accordance with the present invention, viral attenuation is achieved by reducing the expression of viral proteins through codon pair de-optimization of the E and NS3 coding sequences. One way to reduce the expression of a coding sequence is by reducing codon pair bias, but other methods may be used alone or in combination. Although codon bias can be varied, adjusting codons is particularly advantageous for bias. For example, attenuation of a virus by codon bias often requires elimination of common codons, and thus the complexity of the nucleotide sequence is reduced. Conversely, a reduction or minimization of codon pair bias and thus better control of nucleic acid secondary structure, annealing temperature, and other physical and biochemical properties can be achieved while maintaining more sequence diversity.

Codon pair biases for protein coding sequences (i.e., open reading frames) were calculated as described above and described in Coleman et al, 2008.

Viral attenuation and induction or protective immune responses can be confirmed in a manner well known to those of ordinary skill in the art, including but not limited to the methods and assays disclosed herein. Non-limiting examples include plaque assay, growth measurement, reduction in lethality of test animals, and protection against subsequent wild-type viral infection.

In various embodiments, the invention uses highly attenuated viruses. Such Zika virus variants include viruses in the so-called African and Asian lineages. Examples of attenuated zika protein coding sequences include SEQ ID nos: 2. SEQ ID No: 3 and SEQ ID No: 4.

codon substitution

In certain embodiments, the synonymous codon substitution for the modified virus alters codon bias, codon pair bias, density of deoptimized codons and deoptimized codon pairs, RNA secondary structure, CpG dinucleotide content, C + G content, translational frameshift sites, translational pause sites, presence or absence of microRNA recognition sequences, or any combination thereof, in the genome. Codon replacement engineering can be performed at multiple positions distributed throughout the genome or at multiple positions restricted to portions of the genome.

In a further embodiment, the portion of the genome is a capsid coding region.

In a further embodiment, the portion of the genome is a structural protein coding region of the genome.

In a further embodiment, the portion of the genome is a non-structural protein coding region of the genome.

Modified oncolytic viral compositions

The invention further provides a method of synthesizing any of the modified viruses described herein, the method comprising (a) identifying codons at a plurality of positions within at least one non-regulatory portion of the viral genome, which codons can be replaced by synonymous codons; (b) selecting a synonymous codon for replacing each identified codon; and (c) replacing each identified codon with a synonymous codon.

In certain embodiments of the method, steps (a) and (b) are directed by a computer-based algorithm for Synthetic Attenuated Virus Engineering (SAVE) that allows the design of a viral genome by altering the specified de-optimized codon distribution and/or the pattern set of the de-optimized codon pair distribution within preferred limits. The present invention also provides a method wherein the set of modes alternatively or additionally comprises: the density of deoptimized codons and deoptimized codon pairs, RNA secondary structure, CpG dinucleotide content, UpA dinucleotide content, C + G content, overlapping coding boxes, restriction site distribution, frameshift sites, or any combination thereof.

In other embodiments, step (c) is accomplished by de novo synthesis of DNA comprising synonymous codons and/or codon pairs, and replacing the corresponding region of the genome with the synthesized DNA. In a further embodiment, the entire genome is replaced with synthetic DNA. In other embodiments, portions of the genome are replaced with synthetic DNA. In other embodiments, the portion of the genome is a capsid coding region.

By "subject" is meant any animal or artificially modified animal. Animals include, but are not limited to, humans, non-human primates, cows, horses, sheep, pigs, dogs, cats, rabbits, ferrets, rodents (e.g., mice, rats, and guinea pigs), and birds. Artificially modified animals include, but are not limited to, SCID mice with a human immune system and CD155tg transgenic mice expressing the human poliovirus receptor CD 155. In a preferred embodiment, the subject is a human. Preferred embodiments of birds are domesticated poultry species, including but not limited to chickens, turkeys, ducks, and geese.

In various embodiments, a modified virus is derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a codon pair de-optimized region encoding a similar protein sequence, wherein the codon pair bias of the modified sequence is less than the codon pair bias of the parent virus and the codon pair bias of the modified sequence is reduced by at least about 0.05, or at least about 0.1, or at least about 0.2.

In various embodiments, the modified virus is derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region encoding a similar protein sequence having an increased CpG dinucleotide, wherein the increase in CpG dinucleotide is at least 41 instances higher than the parental or at least 21 instances higher than the parental viral genome.

In various embodiments, the modified virus is derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region encoding a similar protein sequence having an increased UpA dinucleotide, wherein the increase in UpA dinucleotide is at least 26 instances higher than the parent, or at least 13 instances higher than the parent viral genome.

In various embodiments, the modified virus is derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region having increased UpA and CpG dinucleotides encoding a similar protein sequence, wherein the increase in UpA and CpG dinucleotides in combination is at least 42 instances greater than the parent.

Synthesis of recombinant poliovirus

Recombinant modified virus chimeras can be synthesized by well-known recombinant DNA techniques. Any standard manual on DNA technology provides detailed protocols for the generation of the modified virus chimeras of the present invention.

Assays for reduced pathogenicity of modified viruses

PV-Min chimeras XY and Z and PV-Max were tested for neurovirulence by intracerebral injection of increasing doses of virus in CD155tg mice. Specifically, 4-6-8 week old CDs 155 are groupedtg mice were vaccinated with different doses and then observed for the onset of polio.in parallel experiments a control group of mice was injected with pv (m) -wt. injected dose is based on particles instead of PFU to normalize the amount of virions inserted into the brain.mice were monitored daily for the onset of flaccid paralysis (characteristic symptoms of polio). the standard value used to quantify viral virulence was lethal dose 50 (L D)₅₀) The synthetic viruses PV-MinXY and PV-MinZ have L D higher than PV (M) -wt₅₀Thus the pathogenicity was 1500 times smaller (particle based) or 20 times smaller (PFU based) (table 4).

Evaluation of poliovirus oncolytic Performance

The oncolytic properties of the modified virus chimeras of the invention can also be evaluated in vivo as follows. Experimental tumors were generated in athymic mice by subcutaneous or stereotactic intracerebral implantation of malignant cells. Clinical observations and pathological examinations were performed following tumor progression in athymic and untreated athymic mice given oncolytic modified viral recombinants following multiple treatment regimens. The technique for implanting tumors into athymic mice is a standard procedure described in detail in Fogh, j.

Pharmaceutical composition

The modified virus chimeras of the invention are useful in prophylactic and therapeutic compositions for the treatment of malignancies in various organs, such as the breast, colon, bronchial passages, lining of the gastrointestinal tract, upper and genitourinary tract, liver, prostate, brain or any other human tissue. In various embodiments, the modified virus chimeras of the present invention are useful for treating solid tumors. In a particular embodiment, the tumor to be treated is glioblastoma, adenocarcinoma, melanoma or neuroblastoma. In various embodiments, the tumor treated is a triple negative breast cancer.

The pharmaceutical composition of the present invention may further comprise other therapies for preventing malignant tumors. For example, the modified virus chimeras of the present invention can be used in combination with surgery, radiation therapy, and/or chemotherapy. In addition, one or more modified virus chimeras can be used in combination with two or more of the foregoing therapeutic procedures. Such combination therapies may advantageously utilize lower doses of the administered therapeutic agents, thereby avoiding possible toxicity or side effects associated with multiple monotherapies.

The pharmaceutical compositions of the invention comprise a therapeutically effective amount of one or more modified virus chimeras according to the invention, and a pharmaceutically acceptable carrier. By "therapeutically effective amount" is meant an amount capable of causing lysis of cancer cells to cause necrosis of the tumor. By "pharmaceutically acceptable carrier" is meant a carrier that does not cause an allergic reaction or other adverse effect in the patient to whom it is administered.

Suitable pharmaceutically acceptable carriers include, for example: one or more of water, saline, phosphate buffered saline, dextran, glycerol, ethanol, and the like, and combinations thereof. The pharmaceutically acceptable carrier may further comprise minor amounts of auxiliary substances (e.g., wetting or emulsifying agents, preservatives or buffers) which may extend the shelf-life or effectiveness of the modified virus chimera.

The compositions of the present invention may take a variety of forms. These include, for example, liquid dosage forms such as liquid solutions, dispersions or suspensions, injectable solutions and infusible solutions. The preferred form depends on the intended mode of administration and prophylactic or therapeutic application. Preferred compositions are in the form of injectable solutions or infusible solutions.

Prophylactic and therapeutic cancer treatment

The present invention relates to the production of modified viruses useful as oncolytic therapies to treat different tumor types, and methods of treating tumors and cancers by administering the modified viruses described herein.

Accordingly, various embodiments of the present invention provide a modified virus comprising a modified viral genome comprising nucleotide substitutions engineered at one or more positions in the genome, wherein the substitutions introduce multiple synonymous codons into the genome and/or changes in existing codon sequences for the same amino acid (changing codon pair usage). In both cases, within 98% of the original wild-type amino acid sequence of the viral gene product is retained.

For example, three amino acids (L eu, Ser, and Arg) are encoded by six different codons, while only Trp and Met have unique codons.

Treatment of existing cancers

Various embodiments of the present invention provide methods of inducing oncolytic effects on a tumor or cancer cell. In various embodiments, this type of treatment can be performed when the subject has been diagnosed with cancer. The method comprises administering a modified virus to a subject in need thereof, wherein the modified virus is derived from the wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a codon pair de-optimized region encoding a similar protein sequence, wherein the codon pair bias of the modified sequence is less than the codon pair bias of the parent virus and the codon pair bias of the modified sequence is reduced by at least about 0.05, or at least about 0.1, or at least about 0.2.

Various embodiments of the present invention provide methods of inducing oncolytic effects on a malignant tumor, the method comprising: administering a modified virus to a subject in need thereof, wherein the modified virus is derived from the wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region encoding a similar protein sequence having an increased CpG dinucleotide, wherein the increase in CpG dinucleotide is at least 41 instances higher than the parental virus or at least 21 instances higher than the parental virus genome.

Various embodiments of the present invention provide methods of inducing oncolytic effects on a malignant tumor, the method comprising: administering a modified virus to a subject in need thereof, wherein the modified virus is derived from the wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region encoding a similar protein sequence having an increased UpA dinucleotide, wherein the increase in UpA dinucleotide is at least 26 instances higher than the parent, or at least 13 instances higher than the parent viral genome.

Various embodiments of the present invention provide methods of inducing oncolytic effects on a malignant tumor, the method comprising: administering a modified virus to a subject in need thereof, wherein the modified virus is derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region having increased UpA and CpG dinucleotides encoding a similar protein sequence, wherein the increase in UpA and CpG dinucleotides in combination is at least 42 instances greater than the parent.

Examples of other attenuated viruses that may also be used as an initial dose and/or booster dose include the family of viruses and all related genera, strains, types, and isolates described herein (e.g., in the "viruses and modified viruses" section above); and attenuated viruses belonging to the Picornaviridae (Picornaviridae) family of viruses and all related genera, strains, types and isolates; attenuated viruses belonging to the family of viruses of the family herpesviridae and all related genera, strains; attenuated viruses belonging to the family Rhabdoviridae and all related genera, strains, types and isolates; attenuated viruses belonging to the reoviridae family of viruses and all related genera, strains; attenuated viruses belonging to the family of viruses of the poxviridae and all related genera, strains; attenuated viruses belonging to the virus family of togaviridae and all related genera, strains.

In various embodiments, inducing an oncolytic effect on the malignant tumor results in treating the malignant tumor.

In various embodiments, the treatment further comprises administering a PD-1 inhibitor, in other embodiments, the treatment further comprises administering a PD-L1 inhibitor, in other embodiments, the treatment further comprises administering both a PD-1 inhibitor and a PD-L1 inhibitor.

In various embodiments, the PD-1 inhibitor is an anti-PD 1 antibody in various embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody examples of PD-1 inhibitors and PD-L1 inhibitors are provided herein.

In various embodiments, treatment of a malignant tumor reduces the likelihood of recurrence of the malignant tumor. It may also reduce the likelihood of having a second cancer different from the malignancy. If the subject develops a second cancer that is different from the malignancy, treatment of the malignancy slows the growth of the second cancer. In some embodiments, following remission of the malignancy, the subject develops a second cancer different from the malignancy, and treatment of the malignancy slows growth of the second cancer.

Prime-boost therapy

Various embodiments of the present invention provide methods for eliciting an immune response and inducing oncolytic effects on tumor or cancer cells using a prime-boost treatment regimen. In various embodiments, eliciting an immune response and inducing an oncolytic effect on a tumor or cancer cell results in the treatment of a malignant tumor.

An initial dose of the attenuated virus or modified virus of the invention is administered to elicit an initial immune response. Thereafter, a booster dose of the attenuated virus or modified virus of the invention is administered to induce oncolytic effects and/or elicit an immune response that includes oncolytic effects on the tumor.

In various embodiments, the initial dose and booster dose comprise the same attenuated virus or modified virus of the invention. In other embodiments, the initial dose and the booster dose are different attenuated viruses or modified viruses of the invention.

In various embodiments, the method comprises administering an initial dose of the modified virus to a subject in need thereof; and administering one or more booster doses of the modified virus to a subject in need thereof, wherein the initial dose and the booster dose of the modified virus are each independently selected from the group consisting of: (1) attenuated viruses produced by methods other than codon pair deoptimization; (2) a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a codon-pair deoptimized region encoding a similar protein sequence, wherein the codon-pair bias of the modified sequence is less than the codon-pair bias of the parent virus and the codon-pair bias of the modified sequence is reduced by at least about 0.05, or at least about 0.1, or at least about 0.2; (3) a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region encoding a similar protein sequence having an increased CpG dinucleotide, wherein the increase in CpG dinucleotide is at least 41 instances higher than the parent, or at least 21 instances higher than the parent viral genome; (4) a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region encoding a similar protein sequence having an increased UpA dinucleotide, wherein the increase in UpA dinucleotide is at least 26 instances higher than the parent, or at least 13 instances higher than the parent viral genome; (5) a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region having increased UpA and CpG dinucleotides encoding a similar protein sequence, wherein the increase in UpA and CpG dinucleotides in combination is at least 42 instances higher than the parent; and (6) combinations of the above.

In various embodiments, the initial dose is administered subcutaneously, intramuscularly, intradermally, intranasally, or intravenously.

In various embodiments, one or more booster doses are administered intratumorally, intravenously, intrathecally, or intratumorally (directly into the tumor). The preferred mode of administration is directly to the tumor site.

The timing between the initial dose and the booster dose may vary, for example, depending on the type of cancer, the stage of the cancer, and the health of the patient. In various embodiments, the first of the one or more booster doses is administered about 2 weeks after the initial dose. That is, an initial dose is administered, and about two weeks thereafter, a booster dose is administered.

In various embodiments, one or more booster doses are administered about 2 weeks after the initial dose. In various embodiments, 2, 3, 4, or 5 booster doses are administered. In various embodiments, the interval between booster doses can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 or 10 weeks. In further embodiments, the interval between booster doses may be 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. As non-limiting examples, an initial dose may be administered; about two weeks thereafter, a first booster dose may be administered; about one month after the first booster dose, a second booster dose can be administered; about 6 months after the second booster dose, a third booster dose can be administered. As another non-limiting example, an initial dose may be administered; about two weeks thereafter, a first booster dose may be administered; about six months after the first booster dose, a second booster dose can be administered; about 12 months after the second booster dose, a third booster dose can be administered. In further embodiments, additional booster doses may be administered periodically; for example, every year, every other year, every 5 years, every 10 years, etc.

In various embodiments, the dosage may vary between the initial dose and the booster dose. As a non-limiting example, the initial dose may contain fewer copies of the virus than the booster dose.

In other embodiments, the type of attenuated virus or modified virus of the invention produced by methods other than codon pair deoptimization may vary between the priming dose and the boosting dose. In one non-limiting example, the modified virus of the invention can be used in an initial dose; and the same or different family, genus, species, group or purpose of the attenuated virus (produced by methods other than codon pair de-optimization) can be used in booster doses.

In other embodiments, the type of attenuated virus or modified virus of the invention produced by methods other than codon pair deoptimization may also be used as the priming dose and the boosting dose. In one non-limiting example, attenuated viruses may be used in the initial dose, while attenuated viruses of the same or different family, genus, species, group, or purpose (produced by methods other than codon pair de-optimization) may be used in the booster dose.

Examples of other attenuated viruses that may also be used as initial and/or booster doses include the families of viruses described herein (e.g., in the "viruses and modified viruses" sections above) as well as all related genera, strains, types, and isolates; and, attenuated viruses belonging to the family of picornaviridae viruses and all related genera, strains, types, and isolates; attenuated viruses belonging to the family of viruses of the family herpesviridae and all related genera, strains; attenuated viruses belonging to the family Rhabdoviridae and all related genera, strains, types and isolates; attenuated viruses belonging to the reoviridae family of viruses and all related genera, strains; attenuated viruses belonging to the family of viruses of the poxviridae and all related genera, strains; attenuated viruses belonging to the virus family of togaviridae and all related genera, strains.

In other embodiments, the route of administration may vary between the initial dose and the booster dose. In a non-limiting example, the initial dose can be administered subcutaneously and a booster dose can be administered into the tumor by injection; for inaccessible or inaccessible tumors, booster doses may be administered intravenously.

In various embodiments, the treatment further comprises administration of a PD-1 inhibitor, in other embodiments, the treatment further comprises administration of a PD-L1 inhibitor, in other embodiments, the treatment further comprises administration of both a PD-1 inhibitor and a PD-L1 inhibitor, in particular embodiments, the PD-1 inhibitor, the PD-L1 inhibitor, or both are administered during the treatment (potentiation) phase, but not during the priming phase.

In various embodiments, the PD-1 inhibitor is an anti-PD 1 antibody in various embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody examples of PD-1 inhibitors and PD-L1 inhibitors are provided herein.

Prime-boost therapy prior to having cancer

Various embodiments of the present invention provide a method of eliciting an immune response in a subject that does not have cancer and inducing oncolytic effects on the tumor or cancer cells if and when the tumor or cancer cells develop in the subject. The method uses a prime-boost treatment regimen. In various embodiments, eliciting an immune response and inducing an oncolytic effect on a tumor or cancer cell if and when the subject develops cancer results in treating the malignancy.

An initial dose of the attenuated virus or modified virus of the invention is administered to elicit an initial immune response when the subject does not have cancer or when the subject is deemed not to have cancer. The latter may be due to undetectable or undetected cancer.

The initial dose of attenuated virus may be an attenuated virus produced by methods other than codon pair de-optimization when the subject does not have cancer or when the subject is deemed to have no cancer, or the modified virus of the invention may also be used as an initial dose and a booster dose. Also, the latter may be due to undetectable or undetected cancers.

Thereafter, in some embodiments, a booster dose of an attenuated virus or modified virus of the invention is administered periodically to continue to elicit an immune response. For example, booster doses may be administered about every 1 year, every 2 years, every 3 years, every 4 years, every 5 years, every 6 years, every 7 years, every 8 years, every 9 years, or every 10 years. In particular embodiments, booster doses may be administered about every 5 years.

Alternatively, in other embodiments, a booster dose of an attenuated virus or modified virus of the invention is administered after the subject has been diagnosed with cancer. For example, once a subject is diagnosed with cancer, a treatment regimen comprising administration of a booster dose may be initiated shortly thereafter to induce an oncolytic effect and/or elicit an immune response comprising an oncolytic effect on the tumor. In further embodiments, additional booster doses may be administered to continue treatment of the cancer.

While not wishing to be bound by any particular theory or set of protocols, it is believed that the initial and booster doses "teach" the subject's immune system to recognize virus-infected cells. Thus, when a subject develops cancer and is given a booster dose, the subject's immune system recognizes the virus-infected cells; this time, the virus-infected cells were cancer cells. During the immune response against virus infected cancer cells, the immune system is also primed by cancer antigens, thus enhancing anti-cancer immunity, as the immune system will also target cells expressing cancer antigens.

Thus, in various embodiments, treatment of a malignant tumor reduces the likelihood of recurrence of the malignant tumor. It may also reduce the likelihood of having a second cancer different from the malignancy. If the subject develops a second cancer that is different from the malignancy, treatment of the malignancy slows the growth of the second cancer. In some embodiments, after remission of a malignancy, the subject develops a second cancer different from the malignancy, and the treatment of the malignancy is a decrease in the growth of the second cancer.

The initial and booster doses can be considered as anti-cancer vaccines, preparing the immune system to target the treated tumor cells as the cancer progresses.

In various embodiments, the initial dose and booster dose comprise the same attenuated virus or modified virus of the invention. In other embodiments, the initial dose and the booster dose are different attenuated viruses or modified viruses of the invention.

In various embodiments, the method comprises administering an initial dose of the modified virus to a subject in need thereof; and administering one or more booster doses of the modified virus to a subject in need thereof, wherein the initial dose and the booster dose of the modified virus are each independently selected from the group consisting of: (1) attenuated viruses produced by methods other than codon pair deoptimization; (2) a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a codon-pair deoptimized region encoding a similar protein sequence, wherein the codon-pair bias of the modified sequence is less than the codon-pair bias of the parent virus and the codon-pair bias of the modified sequence is reduced by at least about 0.05, or at least about 0.1, or at least about 0.2; (3) a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region encoding a similar protein sequence having an increased CpG dinucleotide, wherein the increase in CpG dinucleotide is at least 41 instances higher than the parent, or at least 21 instances higher than the parent viral genome; (4) a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region encoding a similar protein sequence having an increased UpA dinucleotide, wherein the increase in UpA dinucleotide is at least 26 instances higher than the parent, or at least 13 instances higher than the parent viral genome; (5) a modified virus derived from a wild-type virus or a previously modified virus by replacing at least one genomic region of the wild-type virus with a region having increased UpA and CpG dinucleotides encoding a similar protein sequence, wherein the increase in UpA and CpG dinucleotides in combination is at least 42 instances higher than the parent; and (6) combinations of the above.

In various embodiments, the initial dose is administered subcutaneously, intramuscularly, intradermally, intranasally, or intravenously.

In various embodiments, one or more booster doses are administered subcutaneously, intramuscularly, intradermally, intranasally, or intravenously when administered to a subject that does not have cancer or is not suspected of having cancer.

In various embodiments, one or more booster doses are administered intratumorally, intravenously, intrathecally, or intratumorally (directly into the tumor) when administered to a subject diagnosed with cancer. The preferred mode of administration is directly to the tumor site.

The time interval between the initial dose and the booster dose may vary, for example, depending on the type of cancer, the stage of the cancer, and the health of the patient. In various embodiments, if the subject does not have cancer or is not suspected of having cancer, the first of the one or more booster doses is administered about every 1 year, every 2 years, every 3 years, every 4 years, every 5 years, every 6 years, every 7 years, every 8 years, every 9 years, or every 10 years after the initial dose. In particular embodiments, booster doses may be administered about every 5 years.

In various embodiments, for example, when a subject is diagnosed with cancer, one or more booster doses are administered after the diagnosis of cancer. In various embodiments, 2, 3, 4, or 5 booster doses are administered. In various embodiments, the interval between booster doses can be 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 or 10 weeks. In further embodiments, the interval between booster doses may be 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. As non-limiting examples, an initial dose may be administered; about five years thereafter, a first booster dose may be administered; about one year after the first booster dose, the subject is diagnosed with cancer, and a second booster dose can be administered; about 2 weeks after the second booster dose, a third booster dose may be administered; about 2 weeks after the third booster dose, a fourth booster dose may be administered; about 1 month after the fourth booster dose, a fifth booster dose can be administered. Once cancer remission is determined, additional periodic booster doses may be administered; for example, every 6 months, year, every 2 years, every 3 years, every 4 years, or every 5 years.

In various embodiments, the dosage may vary between the initial dose and the booster dose. As a non-limiting example, the initial dose may contain fewer copies of the virus than the booster dose.

In other embodiments, the type of attenuated virus or modified virus of the invention produced by methods other than codon pair deoptimization may vary between the priming dose and the boosting dose. In one non-limiting example, the modified virus of the invention can be used in an initial dose; and the same or different family, genus, species, group or purpose of the attenuated virus (produced by methods other than codon pair de-optimization) can be used in booster doses.

Examples of other attenuated viruses that may also be used as initial and/or booster doses include the families of viruses described herein (e.g., in the "viruses and modified viruses" sections above) as well as all related genera, strains, types, and isolates; and, attenuated viruses belonging to the family of picornaviridae viruses and all related genera, strains, types, and isolates; attenuated viruses belonging to the family of viruses of the family herpesviridae and all related genera, strains; attenuated viruses belonging to the family Rhabdoviridae and all related genera, strains, types and isolates; attenuated viruses belonging to the reoviridae family of viruses and all related genera, strains; attenuated viruses belonging to the family of viruses of the poxviridae and all related genera, strains; attenuated viruses belonging to the virus family of togaviridae and all related genera, strains.

In other embodiments, the route of administration may vary between the initial dose and the booster dose. In a non-limiting example, the initial dose can be administered subcutaneously and a booster dose can be administered into the tumor by injection; for inaccessible or inaccessible tumors, booster doses may be administered intravenously.

In various embodiments, subjects receiving such treatment (e.g., initial dose prior to having cancer, or initial and booster doses prior to having cancer, and then booster doses after having cancer) can be subjects at higher risk of developing cancer examples of such subjects include, but are not limited to, subjects with genetic predisposition (e.g., BRCA1 or BRCA2 mutation, TP53 mutation, PTEN mutation, KRAS mutation, c-Myc mutation, any mutation deemed a cancer susceptibility mutation by national cancer research, etc.), family history of cancer, advanced age (e.g., age 40, age 45, age 55, age 65, or older), higher than normal radiation exposure, prolonged sun exposure, tobacco use history (e.g., smoking, chewing), history of alcohol abuse, history of drug abuse, body mass index >25, history of chronic inflammatory disease (e.g., inflammatory bowel disease, ulcerative colitis, Crohn's disease, asthma, rheumatoid arthritis, etc.), history of immunosuppression, history of infections known to be associated with increased risk of cancer (e.g., CMV, chronic hepatitis, hepatitis C-H L, PyVal H, MCrV, and other).

In various embodiments, subjects receiving these treatments (e.g., an initial dose and booster dose prior to having cancer, and then a booster dose after having cancer) can be subjects who are not listed in the higher risk category, but the clinician prescribes the initial dose and booster dose as a measure to prevent future cancer risk.

In various embodiments, the treatment further comprises administration of a PD-1 inhibitor, in other embodiments, the treatment further comprises administration of a PD-L1 inhibitor, in other embodiments, the treatment further comprises administration of both a PD-1 inhibitor and a PD-L1 inhibitor, in particular embodiments, the PD-1 inhibitor, the PD-L1 inhibitor, or both are administered during the treatment (booster) phase, but not during the priming phase.

In various embodiments, the PD-1 inhibitor is an anti-PD 1 antibody in various embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody examples of PD-1 inhibitors and PD-L1 inhibitors are provided herein.

Inflammatory reaction

Administration of the modified viruses of the present invention stimulates I L-1 b although not wishing to be bound by any particular theory, the modified viruses of the present invention provide stimulation of RIG-1, STNG, IRF3, IRF7 and NFkB that promotes a sustained inflammatory response and, in part, provides therapeutic efficacy.

Although not wishing to be bound by any particular theory, the modified viruses of the present invention provide stimulation of the innate immune receptors RIG-1 and STNG, which stimulate and promote a sustained inflammatory response and, in part, provide therapeutic efficacy.

In various embodiments, the modified viruses of the present invention are administered to stimulate endogenous I L-1B production in a subject although not wishing to be bound by any particular theory, the modified viruses of the present invention provide stimulation of the innate immune transcription factors IRF3, IRF7 and NF κ B, which stimulate and promote a sustained inflammatory response and provide, in part, therapeutic efficacy.

In various embodiments, the modified viruses of the present invention are administered to maintain a therapeutically effective amount of I L-1B production in a subject to promote a sustained inflammatory response and provide, in part, therapeutic efficacy.

In various embodiments, the modified viruses of the invention are administered to stimulate the production of endogenous type 1 interferon in a subject, which in part provides therapeutic efficacy.

In various embodiments, the modified viruses of the invention are administered to maintain a therapeutically effective amount of type 1 interferon production in a subject, which in part provides therapeutic efficacy.

In other embodiments, the modified viruses of the invention are administered to activate type I interferon in a subject to maintain ionizing radiation and chemotherapy sensitization of the subject.

In various embodiments, the modified viruses of the invention are administered to recruit pro-inflammatory immune cells (including CD45+ leukocytes, neutrophils, B cells, CD4+ T cells, and CD8+ immune cells) to the site of cancer, which in part provides therapeutic efficacy.

In various embodiments, the modified viruses of the invention are administered to reduce anti-inflammatory immune cells (e.g., FoxP3+ T regulatory cells or M2-macrophages) from the site of cancer, which in part provide therapeutic efficacy.

In various embodiments, treatment of a malignant tumor reduces the likelihood of recurrence of the malignant tumor. It may also reduce the likelihood of having a second cancer different from the malignancy. If the subject develops a second cancer that is different from the malignancy, treatment of the malignancy slows the growth of the second cancer. In some embodiments, after remission of a malignancy, the subject develops a second cancer different from the malignancy, and treatment of the malignancy slows growth of the second cancer.

PD-1 inhibitors and PD-L1 inhibitors

Examples of anti-PD 1 antibodies that can be used as discussed herein include, but are not limited to, pembrolizumab, nivolumab, pidilizumab, AMP-224, AMP-514, spartalizumab, cimeprinimab, AK105, BCD-100, BI754091, JS001, L ZM009, MGA012, Sym021, TSR-042, MGD013, AK104, XmAb20717, and tiramerizumab.

Other examples of PD-1 inhibitors include, but are not limited to, PF-06801591, pluripotent killer T lymphocytes expressing anti-PD 1 antibodies (PIK-PD-1), and autologous anti-EGFRvIII 4SCAR-IgT cells.

Examples of anti-PD-L1 antibodies include, but are not limited to, BGB-A333, CK-301, FAZ053, KN035, MDX-1105, MSB2311, SHR-1316, Attributab, Avermemab, Devolumab, BMS-936559, and CK-301 Another example of an anti-PD-L1 inhibitor is M7824.

Route of administration

In addition to those discussed above, therapeutic oncolytic modified viruses can be delivered intratumorally, intravenously, intrathecally, or intratumorally (directly into the tumor). The preferred mode of administration is directly to the tumor site.

It will be apparent to those skilled in the art that a therapeutically effective amount of a modified virus chimera of the invention may depend on: schedule of administration, unit dose of modified viral chimera administered, whether the modified viral chimera is administered in combination with other therapeutic agents, condition and health of the patient.

The therapeutically effective amount of oncolytic recombinant viruses can be determined empirically and depends on the maximum amount of recombinant virus that can be safely administered, as well as the minimum amount of recombinant virus that produces an effective oncolytic effect.

Therapeutic vaccination with oncolytic modified viruses can be administered repeatedly, depending on the effect of the initial treatment regimen. If the host's immune response to the particular oncolytic modified virus initially administered limits its effectiveness, additional injections of oncolytic modified viruses having a different modified viral serotype can be made. The immune response of a host to a particular modified virus can be readily determined by serology. However, it will be appreciated that lower or higher doses than those prescribed above may be used depending on the chosen administration regimen.

To this end, serological data on the immune status for any given modified virus can be used to make informed decisions about which modified virus variant to use. For example, if high titers to modified virus serotype 1 are evident by serological analysis of candidate patients for treatment with oncolytic viruses, an alternative modified virus preparation should be used for tumor therapy.

TABLE 7 sequences