阅读说明：本技术 癌症靶向的、病毒编码的、可调节的t细胞（catvert）或nk细胞（catvern）接头 (Cancer-targeting, virus-encoded, regulatable T Cell (CATVET) or NK Cell (CATVERN) linkers ) 是由 蒂莫西·P·克里普 陈淳宇 布莱恩·胡特泽恩 马克·柯里尔 王品怡 道恩·钱德勒 于 2020-02-19 设计创作，主要内容包括：提供了在转基因中包含工程化(人工)外显子-内含子-外显子基因结构的重组多核苷酸和载体,其在靶细胞中表达时经历剪接。(Recombinant polynucleotides and vectors comprising engineered (artificial) exon-intron-exon gene constructs in transgenes that undergo splicing when expressed in target cells are provided.)

1. A polynucleotide or vector comprising:

(a) a first polynucleotide sequence comprising a first portion of an open reading frame encoding a first polypeptide;

(b) a second polynucleotide sequence comprising a second portion of the open reading frame encoding the first polypeptide;

(c) a third polynucleotide sequence encoding a second polypeptide; and

(d) a gene regulatory polynucleotide sequence located between the first polynucleotide and the second polynucleotide.

2. The polynucleotide or vector of claim 1, wherein the gene regulatory polynucleotide sequence comprises a splice donor site, an upstream intron, an exon including more than one stop codon sequence in their respective reading frames, a downstream intron, and a splice acceptor site.

3. A polynucleotide or vector according to claim 1 or 2 wherein the gene regulatory polynucleotide sequence comprises one or more of a binding sequence for an antisense oligonucleotide, a binding sequence for doxycycline, or a polynucleotide sequence encoding a riboswitch.

4. The polynucleotide or vector of any one of claims 1-3, wherein the antisense oligonucleotide is a morpholino oligonucleotide.

5. The polynucleotide or vector of claim 4, wherein the binding sequence for the morpholino oligonucleotide comprises a polynucleotide sequence that is at least 95% identical to SEQ ID No.24 or 25.

6. The polynucleotide or vector of claim 4, wherein said morpholino oligonucleotide comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO 27 or 28.

7. The polynucleotide or vector of claim 2, wherein the stop codon comprises:

an oligonucleotide of the group TAA, TAG or TGA;

a polynucleotide sequence of taaxtagxgatxgagxtaxtgax (SEQ ID number 1), wherein x is any nucleotide; or

TAATTAGTTGATTAGTTAATTGAT (SEQ ID number 2), or an equivalent thereof.

8. The polynucleotide or vector of any one of claims 1-7, wherein the gene regulatory polynucleotide comprises a polynucleotide sequence that is at least 95% identical to SEQ ID number 21.

9. The polynucleotide or vector of any one of claims 1-8, wherein the first polypeptide is a first antibody or antigen-binding fragment thereof and the second polypeptide is a second antibody or antigen-binding fragment thereof.

10. The polynucleotide or vector of any one of claims 1-9, wherein the first antibody or antigen-binding fragment thereof specifically binds to an activated antigen on immune effector cells and the second antibody or antigen-binding fragment thereof binds to a tumor antigen.

11. The polynucleotide or vector of any one of claims 1-9, wherein the first antibody or antigen-binding fragment thereof specifically binds to a tumor antigen and the second antibody or antigen-binding fragment thereof binds to an activating antigen on an immune effector cell.

12. The polynucleotide or vector of any one of claims 1-11, further comprising a fourth polynucleotide sequence encoding a third antibody or antigen-binding fragment thereof, wherein the third antibody or antigen-binding fragment thereof binds to an activating antigen or a tumor antigen on an immune effector cell.

13. The polynucleotide or vector of any one of claims 10-12, wherein the immune effector cell comprises a dendritic cell, a natural killer ("NK") cell, a macrophage, a T cell, a B cell, or a combination thereof.

14. The polynucleotide or vector of any one of claims 10-12, wherein the immune effector cell is a T cell or NK cell.

15. The polynucleotide or vector of any one of claims 10-12, wherein the activating antigen on the immune effector cell comprises CD3, CD2, CD4, CD8, CD19, LFA1, CD45, NKG2D, NKp44, NKp46, NKp30, DNAM, B7-H3, CD20, CD22, or a combination thereof.

16. The polynucleotide or vector of any one of claims 10-12, wherein the tumor antigen comprises one or more of: ephrin-a receptor 2 (EphA 2), Interleukin (IL) -13 ra 2, EGFR VIII, PSMA, EpCAM, GD3, fucosyl GM1, PSCA, PLAC1, sarcoma breakpoint, wilm's tumor 1, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, Epithelial Tumor Antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), blood differentiation antigen, surface glycoprotein, ganglioside (GM 2), growth factor receptor, stromal antigen, vascular antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR 1), mesothelin, CD38, CD123, human epidermal growth factor receptor 2 (HER 2), B Cell Maturation Antigen (BCMA), Fibroblast Activation Protein (FAP) alpha, or a combination thereof.

17. The polynucleotide or vector of any one of claims 1-16, wherein the vector is a recombinant vector, optionally a viral vector, and is capable of expressing a precursor mRNA that encodes a dimer when contacted with an antisense oligonucleotide (optionally a morpholino oligonucleotide).

18. The polynucleotide or vector of claim 17, wherein said dimer is a bispecific antibody.

19. The polynucleotide or vector of claim 17, wherein said dimer is a trispecific antibody.

20. The polynucleotide or vector of claim 18 or 19, wherein the bispecific or trispecific antibody comprises the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof.

21. The polynucleotide or vector of claim 18, wherein the bispecific antibody comprises a polypeptide sequence that is at least 95% identical to either of SEQ ID numbers 13 or 15.

22. The polynucleotide or vector of any one of claims 1-21, wherein the vector expresses a precursor mRNA that encodes a trispecific antibody when contacted with an antisense oligonucleotide (optionally a morpholino oligonucleotide).

23. The polynucleotide or vector of claim 22, wherein the trispecific antibody comprises the first antibody or antigen-binding fragment thereof, the second antibody or antigen-binding fragment thereof, and the third antibody or antigen-binding fragment thereof.

24. The polynucleotide or vector of claim 22, wherein the trispecific antibody comprises a polypeptide sequence at least 95% identical to SEQ ID number 11.

25. The polynucleotide or vector of any one of claims 1-24, wherein the first antibody and the second antibody are each independently a single chain variable fragment.

26. A polynucleotide or vector according to any one of claims 1-25, further comprising a polynucleotide sequence encoding a secretory peptide, optionally a secretory consensus sequence.

27. The polynucleotide or vector of any one of claims 1-26, further comprising a polynucleotide sequence encoding a dimerization domain.

28. The polynucleotide or vector of any one of claims 1-27, further comprising a 5 'Inverted Terminal Repeat (ITR) and a 3' ITR.

29. The polynucleotide or vector of any one of claims 1-28, wherein the vector comprises the sequence set forth in SEQ ID NOs 4, 6, 8, 12, 14, 16-23, 30-33, or 40-46.

30. The polynucleotide or vector of any one of claims 1-29, wherein the vector is a recombinant viral vector comprising a backbone vector selected from a retroviral vector, a lentiviral vector, a murine leukemia virus ("MLV") vector, an epstein-barr virus ("EBV") vector, an adenoviral vector, a herpes virus ("HSV") vector, or an adeno-associated virus ("AAV") vector.

31. The polynucleotide or vector of any one of claims 1-30, wherein the vector is an AAV vector, optionally a self-complementary AAV vector, further optionally an AAV rh74 vector.

32. A composition comprising the polynucleotide or vector of any one of claims 1-31 and a carrier, optionally a pharmaceutically acceptable carrier.

33. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the recombinant polynucleotide or vector of any one of claims 1-31 or the pharmaceutical composition of claim 32, wherein the polynucleotide or vector expresses a therapeutic anti-cancer antibody or antigen-binding fragment thereof.

34. The method of claim 33, further comprising administering to the subject an effective amount of an antisense oligonucleotide, optionally a morpholino oligonucleotide.

35. The method of claim 33 or 34, further comprising administering to the subject an anti-cancer agent.

36. The method of claim 35, wherein the anti-cancer agent comprises an agent selected from a peptide, a polypeptide, a nucleic acid molecule, a small molecule, a viral particle, or a combination thereof.

37. The method of claim 36, wherein the viral particle is an oncolytic HSV particle.

38. The method of any one of claims 33-37, wherein the subject is a mammal.

39. The method of any one of claims 33-38, wherein the subject is a human.

40. A method of producing a bispecific or trispecific antibody in a cell, comprising contacting a cell comprising the vector of any one of claims 1-31 with an effective amount of an antisense oligonucleotide, which is optionally a morpholino oligonucleotide.

41. The method of claim 40, wherein the morpholino oligonucleotide comprises a sequence that is at least 95% identical to a stereopure polynucleotide.

42. The method of claim 40, wherein the vector is introduced into the cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof.

43. The method of any one of claims 40-42, wherein the cells comprise fibroblasts, skeletal cells, epithelial cells, muscle cells, neural cells, endocrine cells, melanocytes, blood cells, or a combination thereof.

44. The method of any one of claims 40-43, wherein the bispecific antibody comprises a polypeptide sequence that is at least 95% identical to SEQ ID number 13 or 15.

45. The method of any one of claims 40-44, wherein the trispecific antibody comprises a polypeptide sequence at least 95% identical to SEQ ID No. 11.

46. The method of any one of claims 40-43, wherein the bispecific antibody is encoded by a polynucleotide sequence that is at least 95% identical to SEQ ID number 14, 16, 22, 23, 30-33, or 40-46.

47. The method of any one of claims 40-44, wherein the trispecific antibody is encoded by a polynucleotide sequence at least 95% identical to SEQ ID No. 12.

48. A kit comprising the polynucleotide or vector of any one of claims 1-31 or the pharmaceutical composition of claim 32.

49. The kit of claim 48, further comprising instructional materials.

Background

Gene transfer has now become a reality as a method of treating diseases (i.e., gene therapy). In 2017, the FDA approved Luxturna, an adeno-associated virus (AAV), designed to stably express normal copies of the RPE65 gene in retinal cells, for the treatment of congenital retinal dystrophies. In 2019, the FDA approved Zolgensma, an AAV designed for expression of SMN1 in motor neurons to treat spinal muscular atrophy. Currently ongoing clinical trials using AAV vectors to replace the function of other disease genes have also achieved very encouraging results, including the mini-version of the dystrophin gene for Duchenne (Duchenne) muscular dystrophy, and the factor VIII and IX genes for hemophilia a and B, respectively.

In all of the above examples, the goal was long-term gene expression to replace a missing or defective gene. However, there may be gene therapy applications where the transgene is expressed only for a short period of time (the gene is expressed as a therapeutic agent in gene therapy), for example in the treatment of time-limited diseases such as infections or cancer. Another illustrative example is the expression of the bacterial protein Cas9 to induce CRISPR/Cas9 gene editing, where only transient expression of Cas9 is desirable to minimize potential immune responses to Cas9 and minimize off-target gene mutations. Another application is where gene therapy may cause unwanted side effects and it is desirable to inactivate the gene. In these cases, a method of activating and/or inhibiting transgene expression is needed.

Therefore, a new approach to activating transgene expression is needed to utilize gene therapy platforms for short-term gene expression (weeks to months). Ideally, the system would place transgene expression under the control of the drug, so that administration of the drug would activate gene expression, and inactivation of the drug would revert to inactivation of gene expression. In this case, if side effects occur or transgene expression is no longer required, administration is no longer required. The present invention fulfills this need and provides related advantages.

Disclosure of Invention

The invention provides a method or vector for expressing a therapeutic gene for a disease or disorder, optionally under external control. In some cases, the disease or disorder is cancer. In certain instances, the vector expresses a polypeptide (referred to herein as "Dimert") that is used to bind the immune effector cell and the cancer cell together to trigger killing of the cancer cell.

The present invention provides a recombinant polynucleotide or vector comprising, consisting essentially of, or consisting of: (a) a first polynucleotide sequence comprising a first portion of an open reading frame encoding a first antibody or antigen-binding fragment thereof; (b) a second polynucleotide sequence comprising a second portion of the open reading frame encoding the first antibody or antigen-binding fragment thereof; (c) a third polynucleotide sequence encoding a second antibody or antigen-binding fragment thereof; and (d) a gene regulatory polynucleotide sequence located between the first polynucleotide and the second polynucleotide. In a further aspect, the gene regulatory polynucleotide sequence comprises, consists essentially of, or consists of: a splice donor site, an upstream intron, exons containing more than one stop codon sequence in their respective reading frames, a downstream intron, and a splice acceptor site. In a further aspect, the gene regulatory polynucleotide sequence comprises one or more of the binding sequences for the antisense oligonucleotide. In a further aspect, the antisense oligonucleotide is a morpholino (morpholino) oligonucleotide. In another aspect, the binding sequence of the morpholino oligonucleotide comprises a polynucleotide sequence that is at least 95% identical to SEQ ID number 24 (AATATGATCCAACAATAGAGGTAAATCTTG) or SEQ ID number 25 (GATCCAACAATAGAGGTAAATCTTGTTTTA). In another embodiment, the stop codon comprises an oligonucleotide of the group: TAA, TAG or TGA. In another aspect, the stop codon sequence comprises the polynucleotide sequence taaxtagxgatxgatxgtax (SEQ ID No. 1), wherein x is any nucleotide, or the stop codon sequence comprises the polynucleotide sequence TAATTAGTTGATTAGTTAATTGAT (SEQ ID No. 2) or an equivalent thereof. In another embodiment, the recombinant polynucleotide or vector encodes a bispecific or trispecific engager (engage). In another embodiment, the bispecific cell engager is a bispecific engager. In another embodiment, the bispecific cell engager is a trispecific engager.

In further embodiments, the first antibody or antigen-binding fragment thereof specifically binds to an activated antigen on an immune effector cell, and the second antibody or antigen-binding fragment thereof binds to a tumor antigen. In another aspect, the first antibody or antigen-binding fragment thereof specifically binds to a tumor antigen and the second antibody or antigen-binding fragment thereof binds to an activating antigen on an immune effector cell. In another aspect, the recombinant polynucleotide or vector further comprises a fourth polynucleotide sequence encoding a third antibody or antigen-binding fragment thereof, wherein the third antibody or antigen-binding fragment thereof binds to an activated antigen or tumor antigen on an immune effector cell. In one aspect, the immune effector cell comprises a dendritic cell, a natural killer ("NK") cell, a macrophage, a T cell, a B cell, a neutrophil, an eosinophil, a basophil, a mast cell, or a combination thereof. In one aspect, the immune effector cell is a T cell. In another aspect, the immune effector cell is an NK cell. In another embodiment, the third antibody or antigen-binding fragment thereof binds to an activating antigen on an NK cell and induces an immune response mediated by the NK cell.

In one embodiment, the activating antigen is a T cell surface molecule. In another embodiment, the activating antigen is an NK cell surface molecule. Non-limiting examples of activating antigens on immune effector cells include CD3, CD2, CD4, CD8, LFA1, CD45, NKG2D, NKp44, NKp46, NKp30, EphA2, DNAM, BT-H3, CD20, CD22, or a combination thereof.

In one aspect, the dimer (e.g., bispecific or trispecific cell engager) comprises a polypeptide sequence that is at least 95% identical to any one of SEQ ID nos. 7-10. In another embodiment, the polypeptide sequence encodes an antigen-binding fragment for CD3, CD2, CD4, CD8, LFA1, CD45, IL21R, NKG2D, NKp44, NKp46, NKp30, or DNAM. In another embodiment, the polypeptide sequence encodes an antigen-binding fragment for CD3, CD19, GD2, or NKG 2D. In another embodiment, the polypeptide encodes a first antigen-binding fragment and a second antigen-binding fragment. In another embodiment, the first antigen-binding fragment binds to CD3 and the second antigen-binding fragment binds to CD 19. In another embodiment, the first antigen-binding fragment binds to CD3 and the second antigen-binding fragment binds to GD 2. In another embodiment, the first antigen-binding fragment binds to NKG2D and the second antigen-binding fragment binds to GD 2.

In one embodiment, the dimer (e.g., a trispecific adapter or antibody) comprises a first antigen-binding fragment, a second antigen-binding fragment, and a third antigen-binding fragment. In another embodiment, the trispecific adaptor or antibody comprises three antigen-binding fragments that bind NKG2D, IL21R and GD2, respectively. In a further aspect, the trispecific adaptor or antibody comprises a polypeptide sequence at least 95% identical to SEQ ID No. 11.

In one embodiment, the antigen-binding fragment that binds to IL-21R is IL-21. The amino acid and cDNA sequences of IL-12 are shown in SEQ ID number 3 and SEQ ID number 4, respectively.

SEQ ID NO. 3: amino acid sequence of IL-21

MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

SEQ ID number 4: cDNA sequence of IL-21

ATGAGAAGCAGCCCCGGCAACATGGAGAGAATCGTGATCTGCCTGATGGTGATCTTCCTGGGCACCCTGGTGCACAAGAGCAGCAGCCAGGGCCAGGACAGACACATGATCAGAATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCCGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAGCTGAAGAGCGCCAACACCGGCAACAACGAGAGAATCATCAACGTGAGCATCAAGAAGCTGAAGAGAAAGCCCCCCAGCACCAACGCCGGCAGAAGACAGAAGCACAGACTGACCTGCCCCAGCTGCGACAGCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGATTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGAGCAGCAGAACCCACGGCAGCGAGGACAGC。

In one embodiment, an antigen-binding fragment that binds NKG2D comprises MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, Rae-1 α, Rae-1 β, Rae-1 γ, Rae-1 δ, Rae-1 ε, H60a, H60b, H60c, MULT1, or a fragment thereof. In one embodiment, the antigen-binding fragment that binds NKG2D is MICA or a fragment thereof. The amino acid and cDNA sequences of MICA are shown in SEQ ID number 5 and SEQ ID number 6, respectively.

SEQ ID NO. 5: amino acid sequence of MICA

EPHSLRYNLTVLSWDGSVQSGFLAEVHLDGQPFLRYDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETEEWTVPQSSRAQTLAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLESSVVLRRTVPPMVNVTRSEASEGNITVTCRASSFYPRNIILTWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICRGEEQRFTCYMEHSGNHSTHPVPS

SEQ ID number 6: cDNA sequence of MICA

gagccccaca gtcttcgtta taacctcacg gtgctgtcct gggatggatc tgtgcagtca gggtttcttg ctgaggtaca tctggatggt cagcccttcc tgcgctatga caggcagaaa tgcagggcaa agccccaggg acagtgggca gaagatgtcc tgggaaataa gacatgggac agagagacca gggacttgac agggaacgga aaggacctca ggatgaccct ggctcatatc aaggaccaga aagaaggctt gcattccctc caggagatta gggtctgtga gatccatgaa

gacaacagca ccaggagctc ccagcatttc tactacgatg gggagctctt cctctcccaa aacctggaga ctgaggaatg gacagtgccc cagtcctcca gagctcagac cttggccatg aacgtcagga atttcttgaa ggaagatgcc atgaagacca agacacacta tcacgctatg catgcagact gcctgcagga actacggcga tatctagaat ccagcgtagt cctgaggaga acagtgcccc ccatggtgaa tgtcacccgc agcgaggcct cagagggcaa catcaccgtg acatgcaggg cttccagctt ctatccccgg aatatcatac tgacctggcg tcaggatggg gtatctttga gccacgacac ccagcagtgg ggggatgtcc tgcctgatgg gaatggaacc taccagacct gggtggccac caggatttgc cgaggagagg agcagaggtt cacctgctac atggaacaca gcgggaatca cagcactcac cctgtgccct ct。

In one embodiment, the MICA sequence comprises a mutant of the wild type. In one embodiment, the MICA mutant is a sequence variant of MUC-30 comprising a methionine mutation other than alanine at position 129 of the wild-type MICA sequence. The amino acid and cDNA sequences of the MUC-30 variant are shown in SEQ ID number 7 and SEQ ID number 8, respectively.

SEQ ID NO. 7: amino acid sequence of MUC-30

EPHSLRYNLTVLSWDGSVQSGFLAEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETEEWTMPQSSRAQTLAMNIRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPS

SEQ ID number 8: cDNA sequence of MUC-30

gagccccaca gtcttcgtta taacctcacg gtgctgtcct gggatggatc tgtgcagtca

gggtttctcg ctgaggtaca tctggatggt cagcccttcc tgcgctgtga caggcagaaa

tgcagggcaa agccccaggg acagtgggca gaagatgtcc tgggaaataa gacatgggac

agagagacca gggacttgac agggaacgga aaggacctca ggatgaccct ggctcatatc

aaggaccaga aagaaggctt gcattccctc caggagatta gggtctgtga gatccatgaa

gacaacagca ccaggagctc ccagcatttc tactacgatg gggagctctt cctctcccaa

aacctggaga ctgaggaatg gacaatgccc cagtcctcca gagctcagac cttggccatg

aacatcagga atttcttgaa ggaagatgcc atgaagacca agacacacta tcacgctatg

catgcagact gcctgcagga actacggcga tatctaaaat ccggcgtagt cctgaggaga

acagtgcccc ccatggtgaa tgtcacccgc agcgaggcct cagagggcaa cattaccgtg

acatgcaggg cttctggctt ctatccctgg aatatcacac tgagctggcg tcaggatggg

gtatctttga gccacgacac ccagcagtgg ggggatgtcc tgcctgatgg gaatggaacc

taccagacct gggtggccac caggatttgc caaggagagg agcagaggtt cacctgctac

atggaacaca gcgggaatca cagcactcac cctgtgccct ct。

In a further aspect, a precursor mRNA (pre-mRNA) is expressed that encodes a trispecific antibody when contacted with a morpholino oligonucleotide.

In one aspect, the antigen binding domain is a single chain variable fragment or an antibody.

In a further aspect, the recombinant polynucleotide or vector comprises a polynucleotide sequence encoding a secretory peptide. In another aspect, the recombinant polynucleotide or vector further comprises a polynucleotide sequence encoding a dimerization domain. In another aspect, the recombinant polynucleotide or vector comprises a 5 'Inverted Terminal Repeat (ITR) and a 3' ITR. In another aspect, the vector comprises the sequences SEQ ID numbers 4, 6, 8, 12, 14, 16-23, 30-33, or 40-46. Non-limiting examples of such carriers include: a recombinant viral vector comprising a backbone vector selected from a retroviral vector, a lentiviral vector, a murine leukemia virus ("MLV") vector, an epstein-barr virus ("EBV") vector, an adenoviral vector, a herpes virus ("HSV") vector, an adeno-associated virus ("AAV") vector, an AAV vector, or optionally a self-complementary AAV vector.

The recombinant polynucleotide or vector may be comprised within a host cell, such as a prokaryotic or eukaryotic cell.

Recombinant polynucleotides, vectors, and cells can be included in compositions comprising vectors and/or host cells and vectors (e.g., pharmaceutically acceptable carriers). They can be formulated for various modes of administration and comprise an effective amount of the polynucleotide, vector and/or host cell, which is effective for the patient, disease or condition, vector and mode of administration. In one aspect, the mode of administration is systemic or intravenous. In another aspect, the topical administration is by direct injection. In one aspect, the morpholino oligonucleotide is contacted with the polynucleotide or vector simultaneously or subsequently, e.g., as a systemic or local injection. Alternatively, the contacting is prior to the polynucleotide or vector.

The recombinant polynucleotides or vectors may be used to treat a variety of diseases or disorders. In one aspect, a method for delivering a transgene is provided. The method comprises administering to a cell, tissue or patient to be treated an effective amount of a recombinant polynucleotide or vector comprising a transgene. In one aspect, an effective amount of a morpholino oligonucleotide is administered to a cell, tissue or patient to be treated. Non-limiting examples of providing and selecting transgenes according to the objectives of the method are provided. The cell or tissue may be a mammal, such as a human. In one aspect, the morpholino oligonucleotide is contacted with the vector simultaneously or subsequently thereto. Alternatively, the contacting is performed before the support. In another aspect, the vector is introduced into the cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof.

The present invention provides a method of treating cancer in a subject in need thereof. The method comprises, consists essentially of, or consists of: administering to the subject an effective amount of a recombinant polynucleotide or vector or cell as described herein. In further methods, the method further comprises administering to the subject an effective amount of a morpholino oligonucleotide. In one aspect, an effective amount of an anti-cancer agent is administered to a subject. Non-limiting examples of anti-cancer agents include anti-cancer peptides, polypeptides, nucleic acid molecules, small molecules, viral particles, or combinations thereof. In another aspect, the vector is introduced into the cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof.

In one aspect of the disclosed method, the viral particle is an oncolytic HSV particle.

Another method provided by the invention is a method of producing a bispecific or trispecific antibody in a cell, comprising, consisting essentially of, or consisting of: contacting a cell comprising a polynucleotide or vector as described herein with an effective amount of a morpholino oligonucleotide. In one aspect, the morpholino oligonucleotide is contacted with the vector simultaneously or after the vector. Alternatively, before the polynucleotide or vector. In one aspect, the morpholino oligonucleotide comprises a sequence that is at least 95% identical to SEQ ID No.27 or 28. A non-limiting example of a bispecific antibody comprises a polypeptide sequence that is at least 95% identical to SEQ ID No.13 or 15. In one embodiment, the bispecific antibody is encoded by a polynucleotide sequence that is at least 95% identical to SEQ ID No.14, 16, 22, 23, 30-33, or 40-46. In another aspect, the trispecific antibody comprises a polypeptide sequence at least 95% identical to SEQ ID No. 11. In one embodiment, the trispecific antibody is encoded by a polynucleotide sequence at least 95% identical to SEQ ID No. 12.

In another aspect, the polynucleotide or vector is introduced into the cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof. Non-limiting examples of cells include fibroblasts, skeletal cells, epithelial cells, muscle cells, nerve cells, endocrine cells, melanocytes, blood cells, or combinations thereof.

Further provided are kits comprising one or more of the polynucleotides, vectors, cells, or compositions as described herein, optionally with instructional materials.

Drawings

FIG. 1 shows a strategy overview of the general concept of splicing using the 3-exon gene structure. Exons are labeled 1, 2 or 3, and introns are shown as straight lines. The different splicing patterns are shown in lower case letters (a, b, c), and the structure of the resulting RNA is shown based on which of them are used. Oligonucleotides that can interfere with splice donor (1D, 2D) or acceptor (2A, 3A) sites are shown as blue dashes, and the expected expression of different possible RNA subtypes is shown as "+", depending on the site blocked by the oligonucleotide.

FIG. 2 shows a summary of the strategy of FIG. 1, using splicing type 1, where exon 2 is normally contained in the gene transcript. Transcripts containing all 3 exons spliced together are expected to predominate, but if oligonucleotides are present that block exon 2 splice acceptor or donor sites, transcripts with exon 1 fused to exon 3 will predominate.

Figure 3 shows an overview of the strategy #1 to create engineered intron-exon STOP (STOP) -intron in the transgene. Transcripts that retain introns will be non-functional, as introns will have premature stop codons and/or will be out of frame. The "a + b" transcript will be non-functional due to the stop codon in exon 2. Only transcripts with exon 2 skipped (using splicing c) are functional, will have a lower baseline, and will be activated by oligonucleotides that block exon 2 splice sites.

FIG. 4 shows a transgene regulatory map of the strategy shown in FIG. 1.

Figure 5 demonstrates a second strategy to create an engineered intron-exon STOP-intron within a transgene using "splice type 2", where exon 2 is not normally present in cancer cells, but is present in normal cells. In this case, the exon skipping oligonucleotide activates transgene expression in normal cells (exon 1+ 3), leaving it unchanged (or even higher) for some off-target skipping of cellular genes as a potential "extra" therapeutic effect.

Figure 6 shows a third strategy for creating an engineered intron-exon STOP-intron within a transgene using "splice type 3", where exon 2 is normally present in some cancer cells but not in normal cells. In this case, high levels of active transgene (exon 1+ 3) are present in normal cells, and the exon skipping oligonucleotide activates expression in tumor cells.

FIG. 7 shows an exemplary CD3xGD2-HDD AAV construct.

Figure 8 shows a cartoon of an experiment for determining the structure and function of Dimert as disclosed herein.

FIG. 9 shows SDS-PAGE of whole cell lysates from transfected 293T cells. The three constructs #1101, #1040 and #1124 (which included secreted peptides) of figure 7 retained less CD3xGD2-HDD dimer in transfected 293T cells relative to construct #1104 and control construct pcDNA3 GFP.

FIG. 10 shows the secretion of CD3xGD2-HDD Dimert by AAV vectors that bind to and activate human T cells.

FIG. 11 shows secreted CD3xGD2-HDD Dimert activating human T cells.

Figure 12A demonstrates that CD3xGD2-HDD binding to T cells is dose dependent.

Fig. 12B shows a bar graph normalized to the median staining level of the unstained control.

FIG. 13 shows a cartoon of the binding assay of anti-GD 2 arm of CD3xGD2-HDD dimer. This experiment confirmed that both GD2 and CD3 binding were present on a single molecule.

FIG. 14 shows an exemplary CD3xGD2-HDD dimer bound to CD3 and GD 2.

Figure 15 shows a flowchart of an assay to determine whether CD3xGD2-HDD induces GD2+ target cell killing of T cells.

Figure 16 shows that secreted CD3xGD2-HDD induces T cell killing of neuroblastoma cells.

Figure 17 demonstrates T cell mediated cytotoxicity of CD3xGD2-HDD Dimert, which correlates with GD2 expression.

Figure 18A shows a diagram of exemplary AAV constructs used to test CD19xCD3 Dimert expression.

Fig. 18B shows an exemplary CD19 transajoin genomic structure. Elements of the AAV-encoded transgene are shown in the figure.

Figure 18C shows a cartoon of CD19 Dimert interacting with cancer cells and T cells. CD19 Dimert was produced by cells containing CD19 transajoin. As shown, "Ca" represents cancer cells and "T" represents T cells.

Figure 19 shows that supernatants from cells transfected with AAV CD19xCD3 constructs comprising secretory sequences bind and activate T cells.

Figure 20 shows that supernatants from cells transfected with AAV vectors containing secretory peptides activate human T cells.

Figure 21 shows that AAV secreted CD19xCD3 specifically binds CD19, but not CD 45.

Figure 22A demonstrates that CD19xCD3 binding to T cells is dose-dependent.

Fig. 22B shows a bar graph normalized to the median staining level of the unstained control.

Figure 23A demonstrates that CD19xCD3 binding to B cells is dose-dependent.

Fig. 23B shows a bar graph normalized to the median staining level of the unstained control.

Figure 24 shows that CD3 Dimert activated T cells better than anti-CD 3 antibody by co-stimulation with anti-CD 28.

Figure 25 shows that 293T cells produced the highest transduction efficiency for AAV8 vector.

Figure 26 demonstrates that Dimert concentration in the supernatant of AAV 8-infected cells depends on AAV dose and transduction efficiency.

Figure 27 shows a single intravenous injection of CD19xCD3 transajoin, which selectively depletes B cells in humanized mice.

Figure 28 shows a single intravenous injection of CD19xCD3 transajoin, which resulted in prolonged depletion of B cells in humanized mice.

Figure 29 shows that a single intravenous injection of CD19xCD3 TransJoin abolished CD19+ lymphoma in humanized mice.

FIG. 30 shows an overview of the OncoSkip and TransSkip described herein.

Figure 31 shows that KRAS Oncoskip antisense morpholinyl induces exon skipping of endogenous KRAS in lung cancer cells.

FIG. 32 shows an exemplary AAV vector map of CD3xGD2-HDD TransSkip showing reverse engineered introns flanking exons inserted into the CD3xGD2-HDD Dimert coding sequence.

Fig. 33 shows an exemplary strategy for testing the activity of OncoSkip and TransSkip described herein.

FIG. 34 shows antisense morpholinyl to induce exon skipping of the CD3xGD2-HDD TransSkip transgene.

FIG. 35 shows KRAS OncoSkip induces secretory expression of CD3xGD2-HDD Dimert in cells transfected with CD3xGD2-HDD TransSkip AAV vector.

FIG. 36 shows that exon skipping of KRAS OncoSkip versus CD3xGD2-HDD TransSkip is an on-target effect.

Figure 37 demonstrates that induction of CD3xGD2-HDD Dimert expression as determined by T cell binding is an on-target effect.

Fig. 38 shows an AAV genomic map of an exemplary nsskip splice variant to reduce baseline nsskip "leakage" but maintain inducible exon skipping.

FIG. 39 shows CD3xGD2-HDD TransSkip splice variant K3, which eliminates baseline but retains inducible exon skipping.

FIG. 40 shows CD3xGD2-HDD TransSkip variant K3, which does not show baseline Dimert production, but is more easily induced by KRAS OncoSkip than other tested TransSkip variants.

FIG. 41 shows that OncoSkip-mediated induction of Dimert expression from CD3xGD2-HDD TransSkip variants K3 and K5 is an on-target effect.

Figure 42 demonstrates that oncoskip-induced secretion of Dimert from AAV CD3xGD2-HDD fransskip vector plays a role in mediating T cell killing of neuroblastoma cells.

FIG. 43 shows that transgene exon skipping in cells infected with AV CD3xGD2-HDD TransSkip variant K3 can be induced by KRAS OncoSkip targeting.

Figure 44A shows an AAV genomic map of an exemplary CD19xCD3 nsskip splice variant.

Fig. 44B shows an exemplary CD19 nsskip genomic structure. Elements of the AAV-encoded transgene are shown in the figure.

Figure 44C shows a cartoon representation of CD19 Dimert interaction with cancer cells and T cells. CD19 Dimer was produced by cells containing CD19 TransSkip. As shown, "Ca" represents cancer cells and "T" represents T cells.

FIG. 45 shows that OncoSkip morpholinyl KTS1 and KTS2 induce on-target exon skipping of CD19xCD3 TransSkip K1.

FIG. 46 shows KRAS OncoSkip induces secretory expression of CD19xCD3 Dimert in cells transfected with CD19xCD3 TransSkip K1 AAV vector.

Figure 47 shows that CD19xCD3 nsskip splice variant K3 eliminates baseline, but retains inducible exon skipping.

FIG. 48 demonstrates that induction of CD19xCD3 Dimert expression by CD19xCD3-K3 TransSkip as determined by T cell binding is an on-target effect.

FIG. 49 shows that induction of CD19xCD3 Dimert expression from CD19xCD3-K3 TransSkip is reproducible.

Detailed Description

Embodiments in accordance with the present invention are described more fully below. Aspects of the present invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Unless defined otherwise, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of this application and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Although not explicitly defined below, such terms should be construed according to their common meaning.

The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art.

The various features of the invention described herein may be used in any combination, unless the context indicates otherwise. Furthermore, the present invention also contemplates that in some embodiments, any feature or combination of features described herein may be excluded or omitted. For purposes of illustration, if the specification states that the compound includes components A, B and C, it is specifically intended that any one or combination of A, B or C can be omitted and disclaimed individually or in any combination.

Unless expressly stated otherwise, all specified embodiments, features and terms are intended to include the described embodiments, features or terms and their biological equivalents.

All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate and vary (+) or (-) by 1.0 or 0.1, or +/-15%, or 10%, or 5%, or 2%, as the case may be. It is to be understood that, although not always explicitly stated, all numerical designations are preceded by the term "about". It is also to be understood that, although not always explicitly stated, the reagents described herein are merely exemplary reagents and that equivalents of such reagents are known in the art.

Throughout the present invention, various publications, patents and published patent specifications are cited by identifying citations or arabic numerals. Full citations for publications identified by arabic numerals may be found before the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference in their entirety to more fully describe the state of the art to which this invention pertains.

Definition of

The practice of the present technology will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); current Protocols In Molecular Biology (F.M. Ausubel, et al. eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988); antibodies, a Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)).

As used in the description of the invention and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. As used herein, transitional phrases consisting essentially of … … (and grammatical variants thereof) are to be construed as including materials or steps that include those that do not materially affect the basic and novel characteristics of the described embodiments. Thus, the term "consisting essentially of … …" as used herein should not be construed as equivalent to "comprising". "consisting of … …" shall mean excluding trace elements in excess of other ingredients and the basic method steps for administering the compositions disclosed herein. Aspects defined by each of these transition terms are within the scope of the invention.

The term "about," as used herein, when referring to a measurable value (e.g., an amount or concentration, etc.), is meant to encompass a change of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the stated amount.

The term or "acceptable", "effective" or "sufficient" when used to describe the selection of any ingredient, range, dosage form, etc., disclosed herein means that the ingredient, range, dosage form, etc., is suitable for the purposes disclosed.

Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

As used herein, the term "adeno-associated virus" or "AAV" refers to a member of the viral class that is related to the name and belongs to the parvovirus-dependent, parvovirus family. Various serotypes of the virus are known to be suitable for gene delivery; all known serotypes can infect cells from a variety of tissue types. At least 11 sequentially numbered AAV serotypes are known in the art. Non-limiting exemplary serotypes that can be used in the methods disclosed herein include any of the 11 serotypes, such as AAV2, AAV8, AAV9, or variant serotypes, such as AAV-DJ and AAV php.b. AAV particles contain three major viral proteins: VP1, VP2 and VP 3. In one embodiment, AAV refers to serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV php.b, AAV rh74, or AAV-DJ (chimeras obtained by shuffling of eight different AAV wild types).

In certain instances, AAV serotypes preferentially target tissue types. In certain cases, the lower panel illustrates typical tissue types and AAV serotypes for each tissue type.

Tissue of	Serotype
		CNS	AAV1, AAV2, AAV4, AAV5, AAV8, AAV9
Heart and heart	AAV1, AAV8, AAV9
		Kidney (Kidney)	AAV2
Liver disease	AAV7, AAV8, AAV9
		Lung (lung)	AAV4, AAV5, AAV6, AAV9
Pancreas gland	AAV8
		Photoreceptor cell	AAV2, AAV5, AAV8
RPE (retinal pigment epithelium)	AAV1, AAV2, AAV4, AAV5, AAV8
		Skeletal muscle	AAV1, AAV6, AAV7, AAV8, AAV9

The term "cell" as used herein may refer to a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercially available source.

"eukaryotic cells" include all life kingdoms except anucleate. They can be easily distinguished by membrane-bound nuclei. Animals, plants, fungi and protists are eukaryotes or organisms whose cells are organized into complex structures by the inner membrane and cytoskeleton. The most typical membrane-bound structure is the nucleus. Unless otherwise specified, the term "host" includes eukaryotic hosts, including, for example, yeast, higher plant, insect, and mammalian cells. Non-limiting examples of eukaryotic cells or hosts include simians, bovines, porcines, murines, rats, avians, reptiles, and humans, such as HEK293 cells and 293T cells.

"prokaryotic cells", which generally lack the nucleus or any other membrane-bound organelle, are divided into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells may also contain genetic information in a loop called episome. The bacterial cells are very small, roughly corresponding to the size of the animal's mitochondria (about 1-2 μm in diameter, 10 μm long). Prokaryotic cells have three main shapes: rod-like, spherical and spiral. Bacterial cells do not undergo a complex replication process as eukaryotes do, but divide by binary division. Examples include, but are not limited to, bacillus, escherichia coli, and salmonella.

The term "encoding" as applied to a nucleic acid sequence refers to a polynucleotide that is described as "encoding" a polypeptide, which in its native state or when manipulated by methods well known to those skilled in the art, can be transcribed and/or translated to produce an mRNA for the polypeptide and/or fragments thereof. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

The terms "equivalent" or "bioequivalent" are used interchangeably in reference to a particular molecular, biological or cellular material and mean a material that has minimal homology while retaining a desired structure or function. Non-limiting examples of equivalent polypeptides include polypeptides having a polypeptide sequence that hybridizes under highly stringent conditions to a polynucleotide encoding the polypeptide sequence that has substantially the same or identical function as a reference polypeptide and that, in one aspect, encodes the reference polypeptide, or a polypeptide having at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96% identity, or at least 97% identity, or at least 98% identity, or at least 99% identity to the polypeptide sequence, or a polypeptide encoded by a polynucleotide or its complement. Highly stringent conditions are described herein and incorporated herein by reference. Alternatively, an equivalent thereof is a polypeptide encoded by a polynucleotide or its complement, having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% identity, or at least 96% identity, or at least 97% sequence identity, or optionally at least 98% identity, or optionally at least 99% identity to a reference polynucleotide (e.g., a wild-type polynucleotide or a reference polynucleotide).

Non-limiting examples of equivalent polynucleotides include polynucleotides that are at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96% identical, or at least 97% sequence identity, or at least 98% identity, or at least 99% identical to the reference polynucleotide. Equivalents also include polynucleotides that hybridize to a reference polynucleotide under highly stringent conditions, or the complement thereof.

A polynucleotide or polynucleotide region (or polypeptide region) has a percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" with another sequence, meaning that when aligned, the percentage of bases (or amino acids) are the same when comparing the two sequences. The alignment and percent homology or sequence identity can be determined using software programs known in the art, for example, the software programs described in Current Protocols in Molecular Biology (Ausubel et al, eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. In some embodiments, default parameters are used for alignment. Using default parameters, a non-limiting exemplary alignment program can be performed using BLAST. Specifically, exemplary programs include BLASTN and BLASTP using the following default parameters: genetic code = standard; filter = none; strand = two strands; cutoff = 60; desired value = 10; matrix = BLOSUM 62; =50 sequences are described; the sorting mode = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS transitions + SwissProtein + SPupdate + PIR. For detailed information about these programs, please access the following websites: ncbi.nlm.nih.gov/cgi-bin/BLAST. Sequence identity and percent identity can be determined by incorporating them into clustalW (available on the website: genome. jp/tools/clustalW/last visit date, 1/13/2017).

"homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which may be aligned for comparison. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence is one that has less than 40% identity, or less than 25% identity, to one of the sequences of the present invention.

"homology" or "identity" or "similarity" may also refer to two nucleic acid molecules that hybridize under stringent conditions.

"hybridization" refers to the reaction of one or more polynucleotides to form a complex that is stabilized by hydrogen bonding between nucleotide residue bases. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or any other sequence specific means. The complex may comprise two strands forming a double stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. The hybridization reaction may constitute a step in a broader process, such as the initiation of a PCR reaction, or enzymatic cleavage of a polynucleotide by a ribozyme.

Examples of stringent hybridization conditions include: an incubation temperature of about 25 ℃ to about 37 ℃; the hybridization buffer concentration is from about 6 × sodium citrate buffer (SSC) to about 10 × SSC; formamide concentrations of about 0% to 25%; and a wash solution of from about 4 XSSC to about 8 XSSC. Examples of moderately stringent hybridization conditions include: a culture temperature of about 40 ℃ to about 50 ℃; the buffer concentration is about 9 XSSC to 2 XSSC; formamide concentration of about 30% to 50%; and a wash solution of about 5 XSSC to about 2 XSSC. Highly stringent hybridization means that the oligonucleotide hybridizes to the target sequence without mismatches (or complete complementarity). Examples of high stringency conditions include: an incubation temperature of about 55 ℃ to about 68 ℃; the buffer concentration is about 1 XSSC to 0.1 XSSC; formamide concentrations of about 55% to 75%; and about 1 XSSC, 0.1 XSSC, or deionized water. Typically, the hybridization incubation time is 5 minutes to 24 hours with 1, 2 or more wash steps, and the wash incubation time is about 1, 2 or 15 minutes. SSC is 0.15M NaCl and 15 mM sodium citrate buffer. It should be understood that SSC equivalents using other buffering systems may be used.

As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and/or the process by which transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

"Gene" refers to a polynucleotide comprising at least one Open Reading Frame (ORF) and capable of encoding a particular polypeptide or protein after transcription and translation. "Gene product" or optionally "gene expression product" refers to the amino acids (e.g., peptides or polypeptides) produced when a gene is transcribed and translated.

"under transcriptional control" is a term well known in the art and indicates that transcription of a polynucleotide sequence (typically a DNA sequence) is dependent on its operative linkage with elements that help initiate or promote transcription. By "operably linked" is meant that the polynucleotides are arranged in a manner that allows them to function in a cell. In one aspect, the invention provides a promoter operably linked to a downstream sequence.

The term "exon" refers to a nucleic acid sequence comprising a protein coding sequence. The gene typically comprises more than one exon separated by introns.

The term "intron" as used herein refers to a nucleic acid sequence flanked by a 5 'splice donor site and a 3' splice acceptor site. In some embodiments, an intron is spliced or removed from an RNA or mRNA sequence expressed by a vector in which the intron is present.

The term "splice donor site" is a nucleic acid sequence or domain 5' to an intron. In one embodiment, the splice donor site marks the start of an intron and/or the boundaries of an intron with the immediately preceding coding sequence (or exon).

The term "splice acceptor site" as used herein refers to a nucleic acid sequence or domain at the 3' end of an intron. In one embodiment, the splice acceptor site marks the beginning of an intron and its boundary with a subsequent coding sequence (exon). In another embodiment, the splice acceptor site includes an intron branch point, which is the point to which the 5' end of an intron is attached during splicing. In some embodiments, the splice acceptor sequence and the intron branching site are placed adjacent to each other as a single unit. In some embodiments, the splice acceptor sequence and the intron branch site may be further separated by moving the branch site further 5' to the splice acceptor sequence.

The term "splice site" as used herein refers to a nucleic acid sequence or domain present at the 5 'or 3' end of an intron as defined above.

The term "exon skipping" as used herein refers to the modification of precursor mRNA splicing by targeting splice donor and/or acceptor sites within the precursor mRNA using one or more complementary antisense oligonucleotides. By blocking the proximity of the spliceosome to one or more splice donor or acceptor sites, one or more complementary antisense oligonucleotides may block the splicing reaction, resulting in deletion of one or more exons from the fully processed mRNA. In one embodiment, exon skipping is achieved in the nucleus during maturation of the precursor mRNA. It involves masking of key sequences involved in targeted exon splicing by the use of antisense oligonucleotides complementary to splice donor sequences within the pre-mRNA.

The term "gene regulatory sequence" as used herein refers to a nucleic acid sequence capable of controlling the transcription, splicing or modification of a gene, open reading frame or exon or intron. The gene regulatory sequences of the invention may include promoters, binding sites for antisense oligonucleotides, and/or enhancers. Thus, placing a gene under the regulatory control of a promoter or regulatory element means positioning the gene so that expression of the gene is controlled by the regulatory sequence. Thus, in the construction of a promoter-gene combination, the promoter is preferably located upstream of the gene and at a distance from the transcription start site that is close to the distance between the promoter and the gene it controls in its natural environment. This variation in distance can be tolerated without loss of promoter function. Similarly, the preferred positioning of a regulatory element (e.g., an enhancer) relative to a heterologous gene placed under its control reflects its natural position relative to the structural gene it naturally regulates. Enhancers are considered to be relatively position and orientation independent compared to promoter elements. In some embodiments, the gene regulatory sequence comprises one or more of a binding sequence for an antisense oligonucleotide, a binding sequence for doxycycline, or a polynucleotide sequence encoding a riboswitch. In some embodiments, the antisense oligonucleotide (ASO) comprises one or more modified nucleotides. In one embodiment, the antisense oligonucleotide is a morpholino oligonucleotide.

In some embodiments, an antisense oligonucleotide (ASO) described herein comprises about 8 to about 50 nucleotides in length. In certain instances, an ASO comprises nucleotides of about 8 to about 30, about 8 to about 25, about 8 to about 20, about 8 to about 18, about 8 to about 15, about 10 to about 50, about 10 to about 30, about 10 to about 25, about 10 to about 20, about 10 to about 18, about 10 to about 15, about 12 to about 50, about 12 to about 30, about 12 to about 25, about 12 to about 20, about 12 to about 18, or about 12 to about 15 in length. In some embodiments, the ASO comprises nucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 in length.

In certain instances, an ASO comprises one or more modified nucleotides. In certain instances, an ASO comprises about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% modified nucleotides. In other instances, an ASO comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, or more modified nucleotides. In some cases, the modification is located on the 2' hydroxyl group of the ribose moiety. In certain instances, the modifications include H, OR, R, halogen, SH, SR, NH2, NHR, NR2, OR CN, wherein R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogens, sulfur, thiols, thioethers, thioesters, amines (primary, secondary or tertiary), amides, ethers, esters, alcohols, and oxygen. In some cases, the alkyl moiety further comprises a modification. In some cases, the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic ring (e.g., an imidazole, a hydrazine group, or a hydroxylamine group), an isocyanate or cyanate group, or a sulfur-containing group (e.g., a sulfoxide, a sulfone, a sulfide, and a disulfide). In some instances, the alkyl moiety further comprises a hetero substitution. In some cases, the carbon of the heterocyclic group is substituted with nitrogen, oxygen, or sulfur. In certain instances, heterocyclic substitutions include, but are not limited to, morpholinyl, imidazole, and pyrrolidinyl. In some cases, the modification at the 2 'hydroxyl group is a 2' -O-methyl modification or a 2 '-O-methoxyethyl (2' -O-MOE) modification. In some cases, the modified nucleotide is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA), an Ethylene Nucleic Acid (ENA) (e.g., 2 '-4' -ethylene bridged nucleic acid), a peptide nucleic acid, or a morpholino. In some cases, the modified nucleotide further comprises one or more modified internucleotide linkages. Exemplary modified internucleotide linkages include, but are not limited to, phosphorothioate, phosphorodithioate, methylphosphonate, 5 '-alkylenephosphonate, 5' -methylphosphonate, 3 '-alkylenephosphonate, borazafluoride, borate phosphate and selenate of 3' -5 'or 2' -5 'linkages, phosphotriester, thioalkylphosphotriester, hydrophosphonate linkages, alkylphosphonate, alkylthiophosphonate, arylthiophosphonate, phosphoselenite, phosphonate, phosphoramidate, 3' -alkylphosphoramide, aminoalkylphosphoramide, thiophosphoryl amide, thiophosphorylaniline, phosphorylaniline, ketone, sulfone, sulfonamide, carbonate, carbamate, methylenehydrazine nitrogen, methylenedimethylhydrazine nitrogen, methylal, thiometal, oxime, methyleneimino, iodonium, or combinations thereof, and combinations thereof, Thioamides, bonds to a riboacetyl, aminoethylglycine, silicon-based or siloxane bond, alkyl or cycloalkyl bonds with or without heteroatoms, e.g., 1 to 10 carbons that are saturated or unsaturated and/or substituted and/or contain heteroatoms, bonds to a morpholino structure, amides, polyamides with an aza nitrogen wherein a base is directly or indirectly attached to the backbone, and combinations thereof.

The term "morpholino" as used herein refers to a polymeric molecule having a base backbone capable of forming hydrogen bonds with a polynucleotide. In some embodiments, the polymer on the morpholino group lacks pentose backbone moieties, more specifically lacks a ribose backbone linked by phosphodiester bonds, which are typical of nucleotides and nucleosides. In one embodiment, the morpholino oligonucleotide comprises a nitrogen ring. In another embodiment, the morpholino group is a stereopure oligonucleotide (e.g., see: wavelet, with last visit date of 2019, 1 month, 25 days) or a derivative thereof. In another embodiment, a morpholino comprises a sequence that is at least 95% identical to a stereopure polynucleotide.

In another embodiment, the morpholino comprises a structure of about 8 to about 50, about 8 to about 30, about 10 to about 50, about 10 to about 30, or about 12 to about 30 nucleotides, comprising a target base sequence that is complementary to a target region of a selected pre-treatment mRNA or precursor mRNA, e.g., an intron region of a precursor mRNA. In another embodiment, the morpholino antisense oligonucleotide facilitates splicing of the target exon, resulting in the lack of target exon in the transcript.

In certain instances, the antisense oligonucleotide (ASO) is referred to herein as OncoSkip. As used herein, the term "OncoSkip" refers to an ASO designed to induce skipping of an exon of interest during splicing of the transgene of interest, thereby inducing expression of the transgene of interest. In some cases, the transgene of interest encodes a polypeptide that binds to a surface polypeptide (e.g., a surface receptor) of the cell of interest. In some cases, the target cell is a tumor cell or an immune cell. In some cases, the transgene of interest is an oncogene. In this case, the use of OncoSkip induces skipping of the targeted exon during splicing, thereby inducing expression of the oncogene.

In some embodiments, the OncoSkip described herein comprises from about 8 to about 50 nucleotides in length. In some examples, the OncoSkip comprises nucleotides of about 8 to about 30, about 8 to about 25, about 8 to about 20, about 8 to about 18, about 8 to about 15, about 10 to about 50, about 10 to about 30, about 10 to about 25, about 10 to about 20, about 10 to about 18, about 10 to about 15, about 12 to about 50, about 12 to about 30, about 12 to about 25, about 12 to about 20, about 12 to about 18, or about 12 to about 15 in length. In some embodiments, the OncoSkip comprises nucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 in length.

In some embodiments, the OncoSkip comprises one or more modified nucleotides, e.g., comprises about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% modified nucleotides. In certain instances, the OncoSkip comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more modified nucleotides. In some cases, the OncoSkip contains one or more morpholino modified nucleotides. In some cases, the OncoSkip contains about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% morpholino modified nucleotides. In certain instances, the OncoSkip comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, or more morpholino modified nucleotides.

In some cases, the OncoSkip works in conjunction with the Transskip. As used herein, the term "TransSkip" refers to a recombinant vector (e.g., a recombinant viral vector, such as an AAV vector) comprising a transgene interrupted by an intron-exon-intron region, wherein the exon comprises a stop codon that prevents normal expression of the transgene-encoding polypeptide. In certain instances, the transgene is further encompassed by a construct comprising a dimer-encoding polynucleotide. In some cases, OncoSkip, in combination with OncoSkip, skips exons from intron-exon-intron regions during splicing to generate mRNA capable of expressing a transgene-encoded polypeptide. In the absence of OncoSkip, transgene expression from Transskip was silenced due to the presence of a stop codon in the intron-exon-intron region.

As used herein, the term "dimer" refers to an engineered protein molecule comprising two or more single chain variable fragments (scfvs), wherein each scFv recognizes a surface polypeptide (e.g., a surface receptor) expressed on a cell, and the two cells are different. In some cases, dimer targets two different cell types, e.g., cancer cells and immune cells, or two different immune cell types. Typical immune cell types include dendritic cells, Natural Killer (NK) cells, macrophages, T cells, B cells, monocytes or neutrophils. In some cases, the immune cell is an effector immune cell. In some cases, the effector immune cells include effector T (T) _eff) Cells (also referred to herein as tumor-infiltrating T-cells). Exemplary T_effCells include CD8+ T cells and non-regulatory CD4+ helper T cells. In some cases, dimert targets cancer cells and effector immune cells. In some cases, dimert targets cancer cells and T_effA cell. In some cases, dimer targets two different cancer cells, e.g., two different cancer cells from the same cancer.

In some cases, two or more scfvs of dimert are linked by a linker. In certain instances, the linker is a peptide linker that facilitates binding of each scFv to its respective target polypeptide. In some cases, the linker comprises a series of poly-Ala, poly-Gly, or combinations thereof. In certain instances, the poly-Ala linker, the poly-Gly linker, or the peptide linker comprising a combination of Ala and Gly, each independently is from about 2 residues to about 50 residues in length. In certain instances, the poly-Ala linker, the poly-Gly linker, or the peptide linker comprising a combination of Ala and Gly each independently has a length of from about 2 residues to about 45 residues, from about 4 residues to about 45 residues, from about 5 residues to about 45 residues, from about 8 residues to about 45 residues, from about 10 residues to about 45 residues, from about 15 residues to about 45 residues, from about 20 residues to about 45 residues, from about 30 residues to about 45 residues, from about 2 residues to about 40 residues, from about 4 residues to about 40 residues, from about 5 residues to about 40 residues, from about 8 residues to about 40 residues, from about 10 residues to about 40 residues, from about 15 residues to about 40 residues, from about 20 residues to about 40 residues, from about 30 residues to about 40 residues, from about 2 residues to about 30 residues, from about 4 residues to about 30 residues, or a combination of Ala and Gly, From about 5 residues to about 30 residues, from about 8 residues to about 30 residues, from about 10 residues to about 30 residues, from about 15 residues to about 30 residues, from about 20 residues to about 30 residues, from about 2 residues to about 20 residues, from about 4 residues to about 20 residues, from about 5 residues to about 20 residues, from about 8 residues to about 20 residues, from about 10 residues to about 20 residues, or from about 15 residues to about 20 residues. In certain instances, the poly-Ala linker, the poly-Gly linker, or the peptide linker comprising a combination of Ala and Gly, is each independently about 2, 4, 5, 6, 8, 10, 12, 14, 15, 16, 18, 20, 25, 30, 35, 40, 45, or 50 residues in length.

In some cases, the linker is (Gly)₄Ser) n linker, wherein n is an integer from 1 to 10. In some cases, n is an integer from 1 to 6, 1 to 4, or 1 to 3. In some cases, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain instances, the linker is about 10 to about 50 amino acid residues in length, optionally about 10 to about 30, about 10 to about 25, or about 10 amino acid residues in lengthTo about 20 amino acid residues. In some cases, the linker comprises one or more unnatural amino acids.

In some cases, dimer further comprises additional polypeptides. In some cases, the additional polypeptide enhances the affinity of dimer for the target cell. In some cases, the additional polypeptide is a dimeric domain of human hepatocyte nuclear factor 1 α (HNF 1 α). In certain instances, HNF1 a comprises a polypeptide sequence comprising at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to MVSKLSQLQTELLAALLESGLSKEALIQALGE (SEQ ID NO: 47). In some examples, HNF1 a is encoded by a polynucleotide that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to ATGGTGAGCAAGCTGAGCCAGCTGCAGACCGAGCTGCTGGCCGCCCTGCTGGAGAGCGGCCTGAGCAAGGAGGCCCTGATCCAGGCCCTGGGCGAG (SEQ ID NO: 48). In some cases, the additional polypeptide does not induce any additional immunogenicity or toxicity.

In some embodiments, the additional polypeptide is linked to the remainder of the dimer through a linker. In some cases, the linker comprises GSGGAP. As used herein, a GSGGAP peptide is also referred to herein as a spacer. In some cases, the joint includes TPLGDTTHTSG. In some cases, peptide TPLGDTTHTSG was from the hinge region of IgG 3.

As used herein, the term "transajoin" refers to a recombinant vector (e.g., a recombinant viral vector, such as an AAV vector) comprising a polypeptide encoding a dimert as described herein, but in the absence of an intron-exon-intron region, wherein the exon comprises a stop codon. Therefore, TransJoin differs from TransSkip in that TransJoin can express dimert without the need for OncoSkip.

In some embodiments, the transajoin described herein is optimized for delivery of dimer to a target cell or target tissue. In some cases, the TransJoin comprises a promoter and enhancer pair, or a promoter and regulatory element pair optimized for delivery and/or expression to a target cell or tissue. In some cases, TransJoin is optimized for constitutive expression of dimer over a period of time (e.g., using a promoter and enhancer pair, or a promoter and regulatory element pair). In some cases, TransJoin is optimized for constitutive and stable expression of dimert over a period of time (e.g., using a promoter and enhancer pair, or a promoter and regulatory element pair). In certain instances, the TransJoin comprises a promoter and regulatory elements for enhanced expression, constitutive expression, stable expression, or a combination thereof, such as the woodchuck hepatitis Virus (WHP) post-transcriptional regulatory element (WPRE).

In some embodiments, a TransJoin described herein comprises a promoter and enhancer pair, or a promoter and regulatory element pair optimized for balanced expression of dimert in a target cell or tissue. In some cases, balanced expression refers to a range of expression above which expression induces toxicity, while expression below which expression does not produce a therapeutic effect. In some cases, promoters and enhancers, or promoters and regulatory elements (e.g., WPRE) act synergistically to balance expression of dimer, thereby achieving a target range. In some cases, balanced expression includes a broad range, e.g., providing a broad therapeutic window for dimert.

In some embodiments, the TransJoin described herein further comprises a consensus secretion (consensus) sequence, described below, that is optimized for balanced expression of dimer in a target cell or tissue. In some cases, balanced expression refers to a range of expression above which expression induces toxicity, while expression below which expression does not produce a therapeutic effect. In some cases, the secretory consensus sequence acts synergistically with promoters, enhancers, regulatory elements (e.g., WPRE), or combinations thereof to balance expression of dimer to achieve the target range. In some cases, balanced expression includes a broad range, e.g., providing a broad therapeutic window for dimert.

As described above, the time period includes one day, two days, three days, four days, five days, seven days, twenty-one day, twenty-eight days, one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, eight months, ten months, one year, two years, or longer.

The term "isolated" as used herein refers to a molecule, biological product, or cellular material that is substantially free of other materials.

As used herein, the term "functional" may be used to modify any molecule, biological, or cellular material to achieve a particular specific effect.

As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases.

As used herein, the term "promoter" refers to any sequence that regulates the expression of a coding sequence (e.g., a gene). For example, a promoter may be constitutive, inducible, repressible, or tissue specific. A "promoter" is a control sequence, which is a region of a polynucleotide sequence that controls the initiation and rate of transcription. It may contain genetic elements to which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors. Non-limiting exemplary promoters include the ROS Sarcoma Virus (RSV) LTR promoter (optionally with the RSV enhancer), the Cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β -actin promoter, the phosphoglycerate kinase (PGK) promoter, the U6 promoter, or the EF1 α short form (EFs) promoter.

In some embodiments, the promoter is the EF1 α short form (EFs) promoter. In certain instances, the EF1 α short form (EFS) promoter includes GTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGAATTCGCTAGCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGG (SEQ ID NO: 49), or an equivalent thereof.

Additional non-limiting exemplary promoters with specific target specificity are provided below, including but not limited to Cytomegalovirus (CMV), human polypeptide chain elongation factor (EF 1 a), SV40, phosphoglycerate kinase (PGK), e.g., PGK1 (human or mouse), P5, Ubc, human β -actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, Gal1, TEF1, GDS, ADH1, CaMV35S, ubiquitin (Ubi) such as ubiquitin c (ubic), H1, U6, α -1-antitrypsin, Splenomegalovirus (SFFV), and chicken β -actin (CBA). Synthetically derived promoters may be used for ubiquitous or tissue-specific expression. In addition, virus-derived promoters, some of which are described above, can be used in the methods disclosed herein, such as CMV, HIV, adenovirus, and AAV promoters.

In some embodiments, the promoter is a tissue-specific promoter. In some cases, the tissue-specific promoter is an endogenous promoter, or a promoter from a gene that is expressed only in the target cell type. Exemplary tissue-specific promoters include, but are not limited to, liver-specific promoters, such as ApoE/hAAT, LP1, SV 40/helb (invivogen); photoreceptor-specific promoters, such as human rhodopsin kinase (GRK 1) and somatostatin (CAR); b cell specific promoters, such as B29 (InvivoGen); hematopoietic cell-specific promoters such as the CD45 promoter and SV40/CD45 from InvivoGen; myocyte-specific promoters, such as the desmin promoter (InvivoGen); pancreatic acinar cell-specific promoters, such as the elastase-1 promoter (InvivoGen); endothelial cell specific promoters, such as the Flt-1 promoter (InvivoGen); and neuron-specific promoters, such as the SYN1 promoter (InvivoGen).

In some embodiments, the promoter is coupled to an enhancer to increase transcription efficiency. Non-limiting examples of enhancers include the RSV enhancer, CMV enhancer, and alpha-fetoprotein MERII enhancer.

An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter/enhancer" is a polynucleotide comprising sequences that provide both promoter and enhancer functions. For example, the long terminal repeat of a retrovirus contains both promoter and enhancer functions. Enhancers/promoters may be "endogenous" or "exogenous" or "heterologous". An "endogenous" enhancer/promoter is one that is naturally linked to a given gene in the genome. An "exogenous" or "heterologous" enhancer/promoter is an enhancer/promoter that is juxtaposed to a gene by genetic manipulation (i.e., molecular biology techniques) such that transcription of the gene is controlled by the linked enhancer/promoter.

In some embodiments, a vector (e.g., a viral vector, such as an AAV vector) for use herein further comprises one or more additional regulatory elements. Exemplary regulatory elements include, but are not limited to, transcription terminators, polyadenylation sites, and Inverted Terminal Repeats (ITRs), such as 5 'ITRs and 3' ITRs. In some cases, the regulatory element comprises a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE). In certain instances, an exemplary WPRE comprises the nucleic acid sequence of SEQ ID NO 50 or an equivalent thereof. The sequence of SEQ ID NO 50 is as follows:

TCGACAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTG (SEQ ID NO: 50)。

The terms "protein", "peptide" and "polypeptide" are used interchangeably and in their broadest sense refer to a compound consisting of two or more subunits of an amino acid, amino acid analog or peptidomimetic. The subunits may be linked by peptide bonds. In another aspect, the subunits may be linked by other linkages, such as esters, ethers, and the like. The protein or peptide must comprise at least two amino acids and is not limited to the maximum number of amino acids that can comprise the protein or peptide sequence. As used herein, the term "amino acid" refers to natural and/or unnatural or synthetic amino acids, including glycine and the D and L optical isomers, amino acid analogs, and peptidomimetics.

As used herein, the term "vector" refers to an nonchromosomal nucleic acid comprising an intact replicon, such that the vector can be replicated when placed in a cell, for example, by a transformation process. The vector may be a viral vector or a non-viral vector. Viral vectors include retroviruses, adenoviruses, herpesviruses, baculoviruses, modified baculoviruses, pasteuroviruses or other modified native viruses. Exemplary non-viral vectors for delivering nucleic acids include naked DNA; DNA complexed with cationic liposomes, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles, including DNA condensed with cationic polymers (e.g., isopolylysine, limited length oligopeptides, and polyethyleneimine), in some cases contained in liposomes; and the use of a ternary complex comprising a virus and polylysine DNA. In another embodiment, the vector is a recombinant viral vector comprising a backbone vector selected from a retroviral vector, a lentiviral vector, a murine leukemia virus ("MLV") vector, an epstein-barr virus ("EBV") vector, an adenoviral vector, a herpes virus ("HSV") vector, or an adeno-associated virus ("AAV") vector. In another embodiment, the vector is an AAV vector, or optionally a self-complementary AAV vector.

A "viral vector" is defined as a recombinantly produced virus or viral particle comprising a polynucleotide to be delivered to a host cell in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenoviral vectors, alphaviral vectors, and the like. Alphavirus vectors, such as those based on the Semliki Forest virus and those based on the Sindbis virus, have also been developed for gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) curr. Opin. Biotechnol. 5: 434-. In certain instances, the viral vector is an AAV vector, such as AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV php.b, AAV rh74, or AAV-DJ. In some cases, the viral vector is AAV rh 74. In some cases, AAV rh74 comprises vector sequence GenBank No. LP899424.1 (visited day 2/7 2020).

In some embodiments, the viral vector (e.g., an AAV vector) has limited carrying capacity. For example, the load bearing capacity of AAV vectors is limited to 4.7 kb. Thus, the combination of dimer with promoters, enhancers and other regulatory elements needs to be in the 4.7 kb capacity range. In this case, the promoter used herein is selected based on its nucleic acid length to enable packaging of the dimer and other regulatory elements into a viral vector (e.g., an AAV vector). In some cases, the promoter for such use is SFFV, EF1 α, PGK, Ubic, CMV, CBA or EFS. In some cases, the promoter is an EFS.

In another embodiment, the promoter is an inducible promoter. In a specific embodiment, the promoter is an inducible tetracycline promoter. The Tet-Off and Tet-On gene expression systems allow researchers to have ready access to regulated high level gene expression systems as described by Gossen & Bujard (1992; Tet-Off) and Gossen et al (1995; Tet-On). In the Tet-Off system, gene expression is turned on when tetracycline (Tc) or doxycycline (Dox; Tc derivative) is removed from the medium. In contrast, in the Tet-on system, expression is turned on by the addition of Dox. Both systems allow gene expression to be tightly regulated by varying concentrations of Tc or Dox. The maximum expression level in the Tet system is very high and performs well compared to that obtained with a strong constitutive mammalian promoter (e.g. CMV) (Yin et al, 1996). Unlike other inducible mammalian expression systems, gene regulation in the Tet system is highly specific, so interpretation of results is not complicated by pleiotropic effects or non-specific induction. In E.coli, the Tet repressor protein (TetR) negatively regulates the gene of the tetracycline resistance operator on the Tn10 transposable element. In the absence of Tc, TetR blocks transcription of these genes by binding to the tet operator (tetO). TetR and tetO provide the basis for regulation and induction in mammalian experimental systems. In the Tet-On system, the regulatory protein is based On the "reverse" Tet repressor (rTetR), which is generated by four amino acid changes in TetR (Hillen & Berens, 1994; Gossen et al, 1995). The resulting protein rtTA (reverse tTA is also known as tetracycline activator) is encoded by the pTet-On regulatory plasmid.

In a related embodiment, the vector further comprises, consists essentially of, or consists of: a nucleic acid encoding a tetracycline activator protein; and a promoter that regulates the expression of tetracycline activator protein.

Other inducible systems that can be used in the vectors, isolated cells, viral packaging systems, and methods described herein include modulation of ecdysone, estrogen, progesterone, dimerization chemistry inducers, and isopropyl-beta-D1-thiogalactopyranoside (EPTG).

As used herein, the term "recombinant expression system" or "recombinant vector" refers to a system for expressing one or more genetic constructs formed by recombination for the expression of certain genetic material.

A "gene delivery vector" is defined as any molecule capable of carrying a polynucleotide for insertion into a host cell. Examples of gene delivery vehicles include liposomes, micelles, biocompatible polymers, including natural and synthetic polymers; a lipoprotein; a polypeptide; a polysaccharide; a lipopolysaccharide; enveloping the artificial virus; metal particles; and bacterial or viral, such as baculoviruses, adenoviruses and retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombinant vectors commonly used in the art, which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and which are useful for gene therapy as well as simple protein expression.

The polynucleotides disclosed herein can be delivered to a cell or tissue using a gene delivery vector. As used herein, "gene delivery," "gene transfer," "transduction," and the like refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for introduction. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection/transfection or various other protein or lipid-based gene delivery complexes) and techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery and various other techniques for introducing polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance generally requires that the introduced polynucleotide comprise an origin of replication compatible with the host cell, or that the replicon integrate into the host cell, e.g., an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. Many vectors are known that are capable of mediating gene transfer into mammalian cells, as is known in the art and described herein.

A "plasmid" is an extrachromosomal DNA molecule that is separated from chromosomal DNA and is capable of replication independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within a microbial population, and generally offer a selective advantage under given environmental conditions. The plasmid may carry a gene that confers resistance to the natural antibiotic in a competitive environmental niche, or the protein produced may also act as a toxin under similar circumstances.

The "plasmid" used in genetic engineering is referred to as a "plasmid vector". Many commercial plasmids are available for such use. The gene to be replicated is inserted into a copy of the plasmid, which contains the gene conferring resistance to a particular antibiotic to the cell and a multiple cloning site (MCS or polylinker), a short region containing several commonly used restriction sites, allowing easy insertion of a DNA fragment at that location. Another major use of plasmids is the production of large quantities of protein. In this case, the researchers developed bacteria containing plasmids carrying the genes of interest. Just as bacteria produce proteins to confer antibiotic resistance, they can also be induced to produce large amounts of protein from an inserted gene.

In aspects in which gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV), vector constructs refer to polynucleotides and transgenes comprising a viral genome or portion thereof. Adenoviruses (Ad) are a relatively well characterized group of homologous viruses, including over 50 serotypes. Ad need not integrate into the host cell genome. Recombinant Ad-derived vectors, particularly those that reduce the potential for recombination and production of wild-type viruses, have also been constructed. These vectors are available from sources such as Takara Bio USA (Mountain View, CA), Vector Biolabs (Philadelphia, PA) and Creative Biogene (Shirley, NY). Wild-type AAV is highly infectious and specific and integrates into the host cell genome. See, world and Toth (2013) Current. Gene. Ther. 13(6): 421-.

Vectors comprising a promoter and a cloning site operably linked to a polynucleotide are common knowledge in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and may be obtained from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). To optimize expression and/or in vitro transcription, it may be desirable to remove, add, or alter the 5 'and/or 3' untranslated portions of the clones to eliminate additional, potential, inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, whether at the transcriptional or translational level. Alternatively, the consensus ribosome binding site can be inserted immediately 5' of the start codon to enhance expression.

As used herein, the term "adaptor", "adaptor molecule" or "activating antigen" refers to a molecule secreted from a cell capable of activating an immune cell. In particular embodiments, the adapter activates specific immune effector cells based on the domain present in the adapter. Illustrative examples of cells secreting a splicer include, but are not limited to, T cells, NK cells, NKT cells, CAR T cells, Mesenchymal Stem Cells (MSCs), neural stem cells, hematopoietic stem cells, or mixtures thereof. In another embodiment, the immune effector cell comprises a dendritic cell, a natural killer ("NK") cell, a macrophage, a T cell, a B cell, or a combination thereof.

The term "antigen recognition domain" or "antigen binding domain" refers to a portion of an adapter molecule that recognizes an antigen. In particular embodiments, the antigen may be of any nature, including but not limited to proteins, carbohydrates, and/or synthetic molecules.

As used herein, the term "activation antigen" or "activation domain" refers to a portion of an adapter molecule that interacts with an immune cell and induces a positive or negative immunomodulatory signal. Illustrative examples of positive immunomodulatory signals include signals that induce cell proliferation, cytokine secretion, or cytolytic activity. Illustrative examples of negative immune regulatory signals include signals that inhibit cell proliferation, inhibit secretion of immunosuppressive factors, or induce apoptosis.

As used herein, the term "innate immune cell" refers to an immune cell that occurs naturally in the immune system. Illustrative examples include, but are not limited to, T cells, NK cells, NKT cells, B cells, and dendritic cells.

As used herein, the term "engineered immune cell" refers to an immune cell that is genetically modified.

As used herein, the term "T cell" includes naive T cells, CD4+ T cells, CD8+ T cells, memory T cells, activated T cells, anergic T cells, T-tolerant cells, chimeric B cells, and antigen-specific T cells.

As used herein, the term "antibody" refers not only to intact antibody molecules, but also to fragments of antibody molecules that retain the ability to bind an immunogen. Such fragments are common knowledge in the art and are often used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules, but also to the well-known active fragment F (ab')₂And Fab. F (ab')₂And Fab fragments lacking the Fc fragment of the intact antibody are cleared more rapidly from the circulation and may bind less non-specific tissues of the intact antibody (Wahl et al, J. Nucl. Med. 24: 316-325 (1983)). The antibodies of the invention include all natural antibodies, monoclonal antibodies, human antibodies, humanized antibodies, camel antibodies, multispecific antibodies, bispecific antibodiesAntibodies, chimeric antibodies, Fab', single chain V region fragments (scFv), single domain antibodies (e.g., nanobodies and single domain camelid antibodies), VNAR fragments, bispecific T cell engager (BiTE) antibodies, minibodies, disulfide linked fvs (sdfv) and anti-idiotypic (anti-Id) antibodies, in vivo antibodies, fusion polypeptides, non-traditional antibodies, and antigen binding fragments of any of the foregoing. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA 2), or subclass.

In certain embodiments, the antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as V)_H) And constant heavy chain (C)_H) And (3) zone composition. The heavy chain constant region consists of three domains: CH1, CH2, and CH 3. Each light chain is composed of a light chain variable region (abbreviated herein as V)_L) And light chain constant C_LAnd (3) zone composition. The light chain constant region consists of a domain C_LAnd (4) forming. V_HAnd V_LThe regions can be further subdivided into regions of high variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each V_HAnd V_LConsists of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Cl q). As used interchangeably herein, the term "antigen-binding portion," "antigen-binding fragment," or "antigen-binding region" refers to a region or portion of an antibody that binds an antigen and confers antigen specificity to the antibody; fragments of antigen binding proteins, e.g., antibodies include one or more antibodies that retain the ability to specifically bind to an antigen Fragments (e.g., peptide/HLA complexes). It has been demonstrated that the antigen binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of antigen-binding moieties within the term "antibody fragment" comprising an antibody include: fab fragment from V_L、V_H、C_LAnd a CHI domain; (Fab)₂A fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; from V_HAnd a fragment of Fd consisting of the CHI domain; v with one arm consisting of antibody_LAnd V_H(iii) an Fv fragment consisting of a domain; dAb fragments (Ward et al, Nature 341: 544-546 (1989)) from V_HDomain composition; and an isolated Complementarity Determining Region (CDR).

Antibodies and antibody fragments can be derived in whole or in part from a mammal (e.g., human, non-human primate, goat, guinea pig, hamster, horse, mouse, rat, rabbit, and sheep) or non-mammalian antibody-producing animal (e.g., chicken, duck, goose, snake, tailed amphibian). Antibodies and antibody fragments can be produced in vivo in animals or in vitro, e.g., from yeast or phage (e.g., as individual antibodies or antibody fragments or as part of an antibody library).

Furthermore, despite the two domains V of the Fv fragment _LAnd V_HAre encoded by separate genes, but they can be joined into individual protein chains using recombinant methods by synthetic linkers, where V_LAnd V_HThe regions pair to form monovalent molecules. These are known as single chain fv (scFv); see, for example, Bird et al, Science 242: 423-. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for use in the same manner as intact antibodies.

An "isolated antibody" or "isolated antigen binding protein" refers to an antibody that has been identified, isolated, and/or recovered from a component of its natural environment. "synthetic antibodies" or "recombinant antibodies" are typically generated using recombinant techniques or synthetic peptide techniques known to those skilled in the art.

As used herein, the term "single-chain variable fragment" or "scFv" is covalently linked to form a V_H: V_LHeavy chain (V) of heterodimeric immunoglobulin (e.g., mouse or human)_H) And light chain (V)_L) Fusion proteins of variable regions. Heavy chain (V)_H) And light chain (V)_L) Either directly or through a peptide-encoding linker (e.g., about 10, 15, 20, 25 amino acids) that links V _HN terminal and V_LC terminal of (A), or V_HC terminal and V of_LThe N terminal of (1). The linker is typically rich in glycine to increase flexibility, serine or threonine to increase solubility. The linker may connect the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain.

Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies may be derived from antibodies comprising V, as described in Huston, et al (Proc. nat. Acad. Sci. USA, 85: 5879-_HAnd V_LExpression in nucleic acid of the coding sequence. See also, U.S. Pat. nos. 5,091,513, 5,132,405, and 4,956,778; and patent publication nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (see, e.g., Zhao et al, hybridoma (Larchmt) 27(6): 455-51 (2008), Peter et al, J Cachexia Sarcopenia Muscle (2012); Shieh et al, J. Imunol 183(4): 2277-85 (2009); Giomarelli et al, Thromb Haemost 97(6): 955-63 (2007); Fife et al, J Clin Invst 116(8): 2252-61 (2006); Brocks et al, Immunotechnology 3(3): 173-84 (1997)) and Moosmayer et al, Therr Imonol 2(10): 31-40 (1995); scFv for example J., Xemic et al, Piromo et al, 25278, and J. chemo 27 (11: 33, J. Iminorgan et al, Biomul et al, 14J. It, nat Biotech 15(8): 768-71 (1997); ledbetter et al, Crit Rev Immunol 17(5-6): 427-55 (1997); ho et al, Bio Chim biophysis Acta 1638(3): 257-66 (2003)).

As used herein, "F (ab)" refers to an antibody structural fragment that binds an antigen but is monovalent and does not have an Fc portion, e.g., a papain-digested antibody produces two F (ab) fragments and one Fc fragment (e.g., a heavy (H) chain constant region; an Fc region that does not bind antigen).

As used herein, "F (ab')₂"refers to an antibody fragment produced by pepsin digestion of an intact IgG antibody, wherein the fragment has two antigen binding (ab') (bivalent) regions, wherein each (ab1) region comprises two independent amino acid chains, a H chain and a portion of the light (L) chain linked by an S-S bond, for binding to an antigen, wherein the remaining H chain portions are linked together. A "F (ab')₂A "fragment" can be split into two separate Fab' fragments.

As used herein, a "CDR" is defined as the complementarity determining region amino acid sequence of an antibody that is a hypervariable region of an immunoglobulin heavy and light chain. See, for example, Kabat et al, Sequences of Proteins of Immunological Interest, 4th U.S. Department of Health and Human Services, National Institutes of Health (1987). Typically, an antibody comprises three heavy chain and three light chain CDRs or CDR regions in the variable region. The CDRs provide the majority of the contact residues for binding of the antibody to the antigen or epitope. In certain embodiments, CDR regions are delineated using the Kabat system (Kabat, E.A., et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication number 91-3242 (1991)).

As used herein, the term "affinity" refers to a measure of binding strength. Without being bound by theory, affinity depends on how closely the stereochemical fit between the antibody binding site and the antigenic determinant is, the size of the contact area between them and the distribution of charged and hydrophobic groups. Affinity also includes the term "avidity," which refers to the strength of an antigen-antibody bond after formation of a reversible complex (e.g., monovalent or multivalent). Methods for calculating the affinity of an antibody for an antigen are known in the art, including the use of binding assays to calculate affinity. Antibody activity in functional assays (e.g., flow cytometry assays) also reflects antibody affinity. Antibodies and affinities can be phenotypically characterized and compared by functional assays, such as flow cytometry. Nucleic acid molecules useful in the present subject matter include any nucleic acid molecule that encodes a polypeptide or fragment thereof. In certain embodiments, nucleic acid molecules useful in the inventive subject matter include nucleic acid molecules encoding antibodies or antigen-binding portions thereof. Such nucleic acid molecules need not be 100% identical to endogenous nucleic acid sequences, but will typically exhibit substantial identity. Polynucleotides having "substantial homology" or "substantial identity" to endogenous sequences are typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. By "hybridizing" is meant pairing under various stringent conditions to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein) or portions thereof (see, e.g., Wahl, G.M. and S.L. Berger, Methods enzyme. 152: 399 (1987); Kimmel, A.R. Methods enzyme. 152: 507 (1987)).

The term "substantially homologous" or "substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% or greater homology or identity to a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). For example, the sequence is at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 96%, or about 97%, or about 98%, or about 99% homologous or identical at the amino acid level or nucleic acid to the sequence being compared (e.g., wild-type or native sequence). In some embodiments, a substantially homologous or substantially identical polypeptide comprises one or more amino acid substitutions, insertions, or deletions relative to the sequence used for comparison. In some embodiments, a substantially homologous or substantially identical polypeptide comprises one or more unnatural amino acids or amino acid analogs, including D-amino acids and reverse transcribed amino acids, in place of the homologous sequence.

Sequence homology or Sequence identity is typically analyzed using Sequence Analysis Software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTOX YBprograms). Such software may be modified by substituting, deleting, and/or otherwise modifying the various types of software He modifies the assigned degree of homology to match the same or similar sequences. In an exemplary method of determining the degree of identity, the BLAST program can be used, where e^-3And e^-100The probability scores in between represent closely related sequences.

As used herein, the term "analog" refers to a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the presently disclosed engineered receptors (e.g., the extracellular antigen-binding domain of the engineered receptor) comprising the amino acid sequence. Conservative modifications may include amino acid substitutions, additions, and deletions. Modifications can be introduced into the presently disclosed human single chain antibodies that engineer the receptors by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified according to their physicochemical properties, such as charge and polarity. Conservative amino acid substitutions are those that replace an amino acid residue with an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, histidine, negatively charged amino acids include aspartic acid, glutamic acid, and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity: polar amino acids include arginine (basic polarity), asparagine, aspartic acid (acidic polarity), glutamic acid (acidic polarity), glutamine, histidine (basic polarity), lysine (basic polarity), serine, threonine, and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within a CDR region may be substituted with other amino acid residues from the same group, and the altered antibody may be tested for retained function (i.e., the function described in (c) to (1) above) using the functional analysis described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues within a given sequence or CDR region are altered.

As used herein, the term "ligand" refers to a molecule that binds to a receptor. In particular, the ligand binds to a receptor on another cell, allowing recognition and/or interaction between the cells.

As used herein, the term "co-stimulatory signaling domain" or "co-stimulatory domain" refers to an engineered receptor portion that comprises the intracellular domain of a co-stimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that, when bound to an antigen, provide a second signal required for efficient activation and function of T lymphocytes. Examples of such co-stimulatory molecules include CD27, CD28, 4-1BB (CD 137), OX40 (CD 134), CD30, CD40, PD-1, ICOS (CD 278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and ligands that specifically bind CD 83. Thus, while the present invention provides exemplary co-stimulatory domains derived from CD28 and 4-1BB, it is contemplated that other co-stimulatory domains may be used for the engineered receptors described herein. The inclusion of one or more costimulatory signal domains may enhance the efficacy and expansion of T cells expressing engineered receptors. The intracellular signaling and costimulatory signaling domains may be tandemly linked to the carboxy terminus of the transmembrane domain in any order.

As used herein, the term "chimeric co-stimulatory receptor" or "CCR" refers to a chimeric receptor that binds an antigen and provides a co-stimulatory signal but does not provide a T cell activation signal.

The term "bispecific antibody" or "trispecific antibody" refers to an antibody having two different antigen-binding regions (bispecific antibody) or three different antigen-binding regions (trispecific antibody). In some embodiments, bispecific antibodies comprise, e.g., Brinkmannet al., “The making of bispecific antibodies,” MABSFIG. two of FIG. 182 and FIG. 212 (2017); or a Labrijn,et al., “Bispecific antibodies: a mechanistic review of the pipeline,” Nature Reviews18: 585-Open antibody format (or antibody structure).

In some embodiments, at least one of the antigen binding regions of the bispecific or trispecific antibody binds to an activating antigen on an immune effector cell. This is understood to mean different targeted binding, but also binding to different epitopes in a target. Non-limiting examples of activating antigens include, but are not limited to, CD3, CD2, CD4, CD8, CD19, LFA1, CD45, NKG2D, NKp44, NKp46, NKp30, DNAM, B7-H3 (CD 276), CD20, CD22, or combinations thereof. Non-limiting examples of bispecific antibodies include, but are not limited to, CD3xCD19, CD3xGD2, CD3xeph 2, and NKG2DxGD2 antibodies.

In one embodiment, the aforementioned dimer comprises a bispecific antibody. In certain instances, dimer comprises a bispecific T cell engager ("BiTE") antibody, a bispecific killer cell engager ("BiKE") antibody, or other bispecific antibodies described herein, e.g., antibodies associated with different immune cells. In another embodiment, dimer comprises a trispecific antibody. In certain instances, dimert comprises a trispecific T cell engager ("TriTE") antibody or a trispecific killer cell engager ("TriKE") antibody, or other trispecific antibodies described herein, such as antibodies associated with different immune cells.

As used herein, the term "bispecific T cell engager" or "BiTE" refers to a bispecific monoclonal antibody comprising a first antigen-binding fragment that binds to a T cell receptor and a second antigen-binding fragment that binds to a tumor cell by a tumor-specific molecule. As used herein, the term "trispecific T-cell engager" ("TiTE" or "TriTE") refers to a trispecific monoclonal antibody comprising a first antigen binding fragment that binds to a T-cell engager, a second antigen binding fragment that binds to a tumor cell via a tumor specific molecule, and a third antigen binding fragment that binds to a T-cell engager or cytokine T-cell activation domain. In one embodiment, the tumor specific molecule comprises a protein selected from the group consisting of epinephrine a type receptor 2 (EphA 2), Interleukin (IL) -13 ra 2, EGFR VIII, PSMA, EpCAM, GD2 or GD3, fucosyl GM1, PSCA, PLAC1, sarcoma breakpoint, wilms 1, Alpha Fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, Epithelial Tumor Antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), blood differentiation antigen, surface glycoprotein, ganglioside (GM 2), growth factor receptor, stromal antigen, vascular antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR 1), mesothelin, CD38, CD123, human epidermal growth factor receptor 2 (HER 2), B Cell Maturation Antigen (BCMA), Fibroblast Activation Protein (FAP) alpha, or a combination thereof. Examples of BiTE include, but are not limited to Blinatumomab (MT 103) and Solitomab (MT 110).

As used herein, the term "bispecific killer cell engager" or "BiKE" refers to a bispecific monoclonal antibody comprising a first antigen-binding fragment that binds to a Natural Killer (NK) cell engager domain and a second antigen-binding fragment that binds to a tumor cell via a tumor-specific molecule. The term "trispecific NK cell engager" ("TiKE" or "TriKE") as used herein refers to a trispecific monoclonal antibody comprising a first antigen binding fragment that binds to NK cell engager, a second antigen binding fragment that binds to tumor cells via a tumor specific molecule, and a third antigen binding fragment that binds to NK cell engager or cytokine NK cell activation domain. The NKG2 dxl 21RxGD2 antibody is an exemplary TriKE antibody. Examples of NK cell engager domains include, but are not limited to, binding to CD16, CD16⁺CD2、CD16⁺DNAM and CD16⁺A ligand or molecule of NKp 46. Examples of cytokine NK activation domain including but not limited to IL-15, IL-12, IL-18, IL-21 or other NK cells enhance cytokines, chemokines and/or activation molecules. The NK-binding domain may comprise any portion that binds to and/or activates NK cells and/or any portion that prevents NK cell inhibition. In some embodiments, the NK-binding domain may comprise an antibody that selectively binds to a component on the surface of an NK cell. In other embodiments, the NK-binding domain may comprise a ligand or small molecule that selectively binds to NK cell surface components.

In some embodiments, the NK-binding domain can selectively bind to a receptor located at least partially on the surface of NK cells. In certain embodiments, the NK-binding domain may function to bind NK cells, thereby bringing NK into spatial proximity to selective binding of the targeting domain. However, in certain embodiments, the NK binding domain may selectively bind to a receptor that activates NK cells, and thus also have an activating function. As described above, activation of the CD16 receptor can cause antibody-dependent cell-mediated cytotoxicity. Thus, in certain embodiments, the NK binding domain may comprise at least a portion of an anti-CD 16 receptor antibody effective to selectively bind to the CD16 receptor. In other embodiments, the NK engager cell domain may interrupt the mechanism of NK cell inhibition. In such embodiments, the NK-binding domain may include, for example, anti-PDl/PDLl, anti-NKG 2A, anti-TIGIT, anti-Killer Immunoglobulin Receptor (KIR), and/or any other blockade-inhibition domain.

In particular embodiments, cells (including immune cells) are genetically modified with an adaptor molecule comprising at least an antigen recognition domain and an activation domain and optionally a cytokine, a co-stimulatory domain and/or a domain of a negative regulatory molecule that inhibits T cell activation. The antigen recognition domain of the adaptor molecule binds to one or more molecules present within and/or on or secreted by the target cell. In a particular aspect, the target cell is a cancer cell, including at least a solid tumor cell. Once the adaptor molecules bind to the target molecules, they can activate cells expressing molecules recognized by the activation domains. The adaptor molecule may activate cells that are genetically modified with the adaptor molecule, or may activate unmodified cells.

Activation may produce a positive or negative signal depending on the desired effect. Examples of positive signals include signals that induce cell proliferation, cytokine secretion, or cytolytic activity. Examples of negative signals include signals that inhibit T cell proliferation, inhibit secretion of immunosuppressive factors, or induce cell death.

In particular aspects, immune cells secreting the adaptor molecule are capable of redirecting resident (naturally endogenous to a particular individual) immune cells to cancer cells.

Embodiments of the invention provide for the delivery of modified immune cells that secrete adaptor molecules to individuals in need of adaptor molecules (known to have cancer or suspected of having cancer, including particular cancers) rather than merely delivering the adaptor molecules to the individual itself (in the absence of production of the modified immune cells). In the present invention, the individual receives modified immune cells that allow for the production of adaptor molecules. In particular embodiments, the cell produces an immunostimulatory cytokine; proliferating in an antigen-specific manner; killing the appropriate target cell; redirecting bystander immune cells (including at least T cells or NK cells) to cancer cells; secreting the adaptor molecule upon activation; and/or effective against cancer in a localized area or systemic manner. Figure 3 shows an example of a modified T cell or NK cell secreting an adaptor molecule. Although a particular T cell or NK cell may produce an adaptor that can be directed against the same cancer cell-specific antigen, the activation domains of the T cell or NK cell must be different because NK cells do not express CD 3. Examples of NK cell activation domains include at least, for example, CD16, NKG2D, or NKp 30.

Gene delivery vectors also include DNA/liposome complexes, micelles, and targeted viral protein DNA complexes. Liposomes comprising the targeting antibody or fragment thereof can be used in the methods disclosed herein. In addition to delivering the polynucleotide to a cell or population of cells, the proteins described herein can be introduced directly into the cell or population of cells by non-limiting protein transfection techniques, or other non-limiting techniques can enhance the expression of and/or promote the culture conditions for the activity of the proteins disclosed herein.

As used herein, the term "signal peptide" or "signal polypeptide" means an amino acid sequence that is typically present at the N-terminus of a newly synthesized secreted or membrane polypeptide or protein. Its function is to direct the polypeptide to a specific cellular location, e.g., across the cell membrane, into the cell membrane, or into the nucleus. In some embodiments, the signal peptide is removed after localization. Examples of signal peptides are well known in the art. Non-limiting examples are those described in U.S. patent nos. 8,853,381, 5,958,736, and 8,795,965.

As used herein, the term "viral capsid" or "capsid" refers to the proteinaceous coat or shell of a viral particle. The function of the capsid is to encapsulate, protect, transport and release the viral genome into the host cell. The capsid is typically composed of oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term "encapsidation" refers to encapsulation within a viral capsid.

As used herein, the term "helper" with respect to a virus or plasmid refers to a virus or plasmid, such as a modified AAV disclosed herein, that is used to provide additional components required for replication and packaging of the viral particle or recombinant viral particle. The components encoded by the helper virus may include any genes required for virion assembly, encapsidation, genome replication, and/or packaging. For example, a helper virus may encode an enzyme required for replication of the viral genome. Non-limiting examples of helper viruses and plasmids suitable for use in AAV constructs include pHELP (plasmid), adenovirus (virus), or herpes virus (virus).

As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells, where some of its functions are provided by co-infected helper viruses. General information and review of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp., 228, and Berns, 1990, Virology, pp., 1763, Raven Press, (New York). It is fully anticipated that the same principles described in these reviews will apply to other AAV serotypes characterized after the date the review was published, as it is well known that the various serotypes are very closely related in structure and function, even at the genetic level. (see, e.g., Blacklowe, 1988, pp. 165, 174 of Parvoviruses and Human Disease, J.R. Pattison, ed.; and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, all AAV serotypes apparently display very similar replication properties mediated by homologous rep genes; they all carry three related capsid proteins, such as those expressed in AAV 2. Heteroduplex analysis further indicates the degree of correlation, which reveals extensive cross-hybridization between serotypes along the length of the genome; and similar self-annealing fragments were present at the ends corresponding to the "inverted terminal repeats" (ITRs). Similar infection patterns also indicate that the replication function of each serotype is under similar regulation.

As used herein, "AAV vector" refers to a vector comprising one or more polynucleotides of interest (or transgenes) flanked by AAV terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a vector host cell that has been transfected with vectors encoding and expressing rep and cap gene products.

An "AAV virion" or "AAV vector particle" refers to a virion composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is often referred to as an "AAV vector particle," or simply an "AAV vector. Thus, production of AAV vector particles necessarily includes production of AAV vectors, as such vectors are contained within AAV vector particles.

In some embodiments, the AAV is a replication-defective parvovirus having a single-stranded DNA genome of about 4.7 kb in length comprising two 145-nucleotide Inverted Terminal Repeats (ITRs). There are many serotypes of AAV. The nucleotide sequence of the AAV serotype genome is known. For example, the complete genome of AAV-1 is provided in GenBank Accession number NC-002077; the complete genome of AAV-2 is provided in GenBank Accession number NC-001401 and Srivastava et al, J.Virol., 45: 555-564 (1983); the complete genome of AAV-3 is provided in GenBank Accession number NC-1829; the complete genome of AAV-4 is provided in GenBank Accession number NC-001829; AAV-5 genome is provided in GenBank Accession number AF 085716; the complete genome of AAV-6 is provided in GenBank Accession number NC-001862; at least part of the AAV-7 and AAV-8 genomes are provided in GenBank Accession number AX753246 and AX753249, respectively; AAV-9 genome is provided in Gao et al, J.Virol, 78: 6381-; AAV-10 genomes are provided in mol. ther., 13(1): 67-76 (2006); AAV-11 genome is provided in Virology, 330(2), 375-383 (2004). The sequences of the AAV rh.74 genome are provided in U.S. patent 9,434,928, which is incorporated herein by reference. U.S. patent No. 9,434,928 also provides capsid protein sequences and self-complementary genomes. In one aspect, the genome is a self-complementary genome. Cis-acting sequences that direct viral DNA replication (rep), encapsidation/packaging, and chromosomal integration of host cells are included in AAV ITRs. Three AAV promoters (designated by relative map positions as p5, pl9, and p 40) drive expression of two AAV internal open reading frames encoding rep and cap genes. Differential splicing of the two rep promoters (p 5 and pi 9) to a single AAV intron (at nucleotides 2107 and 2227) resulted in the production of four rep proteins (rep 78, rep 68, rep 52 and rep 40). The Rep proteins have a variety of enzymatic properties and are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP 3. Alternative splicing and non-uniform translation initiation sites are responsible for the production of three related capsid proteins. A single consensus polyadenylation site is located at position 95 of the AAV genome map. AAV has a life cycle and genetics reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

AAV has unique properties that make it an attractive vector for delivering foreign DNA to cells, for example in gene therapy. AAV infection of cultured cells is non-cytopathic, and natural infection of humans and other animals is asymptomatic. In addition, AAV infects many mammalian cells, thereby making it possible to target many different tissues in vivo. In addition, AAV transduces slowly dividing and non-dividing cells and persists throughout the life cycle of these cells as a transcriptionally active nuclear episome (extrachromosomal component). The AAV proviral genome is inserted into a plasmid as cloned DNA, which makes it possible to construct a recombinant genome. In addition, since the signals directing AAV replication and encapsidation of the genome are contained in ITRs of the AAV genome, a portion or all of about 4.3 kb inside the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced by foreign DNA. To generate AAV vectors, rep and cap proteins can be provided in trans. Another notable feature of AAV is that it is a very stable and powerful virus. It is easily tolerant of the conditions used to inactivate adenovirus (56 ℃ to 65 ℃ for several hours), making cryopreservation of AAV less important. AAV may even be lyophilized. Finally, AAV infected cells are not resistant to repeated infection.

Several studies have demonstrated long-term (> 1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al, Hum Gene Ther, 8: 659-; and Xiao et al, J Virol, 70: 8098-. See also Chao et al, Mol Ther, 2: 619-. Furthermore, due to the high vascularization of muscle, recombinant AAV transduction has resulted in the appearance of transgene products in the systemic circulation after intramuscular injection, as described in Herzog et al, Proc Natl Acad Sci USA, 94: 5804-. Furthermore, Lewis et al, J Virol, 76: 8769-8775 (2002) demonstrated that skeletal muscle fibers have the correct antibody glycosylation, folding and secretion of the required cytokines, indicating that muscle is capable of stable expression of secreted protein therapies. The AAV DNA in the rAAV genome may be from any AAV serotype from which a recombinant virus may be derived, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV PHP.B, AAV rh74, and AAV-DJ. For example, the production of pseudotyped rAAV is disclosed in WO 01/83692. Other types of rAAV variants are also contemplated, such as rAAV with capsid mutations. See, e.g., Marsic et al, Molecular Therapy, 22(11): 1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

As used herein, the term "external" with reference to a viral capsid protein refers to the surface, domain, region or end of the capsid protein that faces outward in the assembled viral capsid. The term "internal" with reference to a viral capsid protein refers to the surface, domain, region or terminus (amino-or carboxy-terminus) of the capsid protein that faces internally in the assembled viral capsid. The term "interior" when used with respect to an assembled viral capsid refers to the encapsidation space within the viral capsid and the inward facing surface of the capsid exposed to the enclosed space. The interior space is surrounded by viral capsid proteins and may include nucleic acids, such as viral genomes, viral proteins, proteins of the host or packaging cell, and any other components or factors packaged or embedded during replication, virion assembly, encapsidation, and/or packaging.

As used herein, the term "label" refers to a directly or indirectly detectable compound or composition, e.g., a polynucleotide or protein, e.g., an antibody, coupled directly or indirectly to a composition to be detected to produce a "labeled" composition. The term also includes sequences coupled to the polynucleotide that provide a signal upon expression of the inserted sequence, such as Green Fluorescent Protein (GFP), and the like. The label may be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The label may be suitable for small scale detection or more suitable for high throughput screening. Thus, suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label can simply be detected or quantified. A response that is simply detected typically includes a response that merely confirms its presence, while a quantified response typically includes a response having a quantifiable (e.g., digitally reportable) value, such as an intensity, polarization, and/or other characteristic. In luminescence or fluorescence assays, a luminophore or fluorophore associated with an assay component actually involved in binding that directly produces a detectable response may be used, or a luminophore or fluorophore associated with another (e.g., reporter or indicator) component may be used indirectly.

Examples of luminescent labels that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. The detectable luminescent response typically includes a change or occurrence of a luminescent signal. Suitable methods and luminophores for luminescent labelling of analytical components are known in the art, for example as described in Haughland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, haematochrome, coumarin, methylcoumarin, pyrene, apple green, stilbene, Lucy Falset, Cascade blue TM., and Texas Red. Other suitable optical dyes are described, for example, in Haughland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

In another aspect, the fluorescent label is functionalized to facilitate covalent attachment to cellular components present in or on the surface of the cell or tissue, such as cell surface markers. Suitable functional groups include, but are not limited to, isothiocyanate, amino, haloacetyl, maleimide, succinimidyl ester, and sulfonyl halide, all of which can be used to attach a fluorescent label to a second molecule. The choice of the fluorescent-labeled functional group depends on the attachment site of the linker, reagent, label, or second label.

Attachment of the fluorescent label may be directly to the cellular component or compound, or may be through a linker. Suitable binding pairs for indirectly linking a fluorescent label to an intermediate include, but are not limited to, antigens/antibodies such as rhodamine/anti-rhodamine, biotin/avidin, and biotin/streptavidin.

The phrase "solid support/support" refers to a non-aqueous surface, such as a "culture plate", "gene chip" or "microarray". Such gene chips or microarrays can be used for diagnostic and therapeutic purposes by a number of techniques known to those skilled in the art. In one technique, oligonucleotides are ligated and arrayed on a gene chip for determination of DNA sequences by hybridization methods, such as the methods outlined in U.S. Pat. Nos. 6,025,136 and 6,018,041. The polynucleotides of the invention may be modified into probes and may be used to detect genetic sequences. Such techniques are described, for example, in U.S. Pat. nos. 5,968,740 and 5,858,659. Probes can also be attached or immobilized to the surface of an electrode for electrochemical detection of Nucleic acid sequences, as described by Kayem et al U.S. Patent No. 952,172 and Kelley et al (1999) Nucleic Acids Res.27: 4830-4837.

"composition" refers to the active polypeptide, polynucleotide or antibody and another inert (e.g., detectable label) or active (e.g., gene delivery vector) compounds or compositions of combination.

"pharmaceutical composition" is meant to include the combination of an active polypeptide, polynucleotide or antibody with a carrier (inert or active carrier, e.g., a solid carrier) that renders the composition suitable for diagnostic or therapeutic use ex vivo, in vivo or in vitro.

As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate buffered saline solutions, water, and emulsions, such as oil/water or water/oil emulsions, as well as various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's pharm. sci., 15th ed. (Mack pub. co., Easton).

As used herein, the term "cancer" includes solid tumors and hematological malignancies. Exemplary solid tumors include, but are not limited to, bladder cancer, bone cancer, brain cancer (e.g., glioblastoma), breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, or stomach cancer. Exemplary hematological malignancies include, but are not limited to, Acute Myeloid Leukemia (AML), Chronic Lymphocytic Leukemia (CLL), Small Lymphocytic Lymphoma (SLL), Follicular Lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), Mantle Cell Lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, lymph node marginal zone B-cell lymphoma, Burkitt's lymphoma, non-Burkitt's high grade B-cell lymphoma, primary mediastinal B cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B lymphocyte lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphoma-like granuloma. In some cases, the cancer is a metastatic cancer (e.g., a metastatic solid tumor or a metastatic hematological malignancy). In certain instances, the cancer is a relapsed or refractory cancer (e.g., a relapsed or refractory solid tumor or a relapsed or refractory hematologic malignancy).

In some embodiments, the tumor is characterized by up-regulated expression of Fibroblast Activation Protein (FAP) (wherein the amino acid sequence of human FAP is disclosed in GenPept access Number I38593, obtained 2, 10 days 2020). FAP, also known as FAP α and prolyl endopeptidase FAP, is a membrane-bound glycoprotein that is part of the dipeptidyl peptidase (DPP) family. FAP has both proline exopeptidase and gelatinase activities. In certain instances, cancers characterized by upregulation of FAP expression (FAP-positive cancers) include, but are not limited to, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, gastric cancer, liver cancer, lung cancer, oral cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, and renal cancer. In certain instances, FAP-positive cancers comprise high levels of fibrosis. In certain instances, the level is compared to an equivalent cancer that is not upregulated by FAP. In other cases, the level is compared to the level of fibrosis in a normal subject.

In certain instances, dimer described herein binds to the extracellular portion of FAP α. In certain instances, dimer binds to human FAP α (e.g., binds to the extracellular portion of FAP α having GenPept access Number I38593 or an equivalent thereof). In certain instances, dimert binds FAP α and a cell surface polypeptide expressed on an immune cell target, e.g., a T cell or NK cell. In certain instances, dimer binds to FAP α and optionally binds to an immune cell target (e.g., a cell surface polypeptide expressed on a T cell or NK cell) for use in treating FAP-positive cancer, optionally pancreatic cancer, further optionally FAP-positive cancer characterized by a high level of fibrosis.

In some embodiments, the cancer is characterized by the expression and/or upregulation of B Cell Maturation Antigen (BCMA), a cell surface receptor of the TNF receptor superfamily (also referred to as tumor necrosis factor receptor superfamily member 17, TNFRSF17, and BCM). In some cases, the amino acid sequence of human BCMA is disclosed in GenPept access Number BAB60895.1 (obtained 2 months and 10 days 2020). In certain instances, the cancer characterized by BCMA expression and/or upregulation is myeloma (or multiple myeloma).

In certain instances, a dimer described herein binds to the extracellular portion of BCMA. In certain instances, dimer binding binds to human BCMA (e.g., to the extracellular portion of BCMA with GenPept access Number BAB60895.1, or an equivalent thereof). In certain instances, dimert binds to BCMA and a cell surface polypeptide expressed on an immune cell target, such as a T cell or NK cell. In certain instances, dimert binds BCMA and optionally binds an immune cell target (e.g., a cell surface polypeptide expressed on T cells or NK cells) to treat myeloma.

In some embodiments, the cancer is characterized by expression or upregulation of Epidermal Growth Factor Receptor (EGFR) mutants (e.g., EGFR variant iii (egfrviii)). EGFRvIII refers to EGFR mutations that include a deletion of exons 2-7 of the EGFR gene. In certain instances, cancers characterized by expression or upregulation of EGFRvIII (EGFRvIII-positive cancers) include, but are not limited to, glioblastoma, bladder cancer, breast cancer, colorectal cancer, esophageal cancer, Head and Neck Squamous Cell Carcinoma (HNSCC), lung cancer, melanoma, ovarian cancer, Peripheral Nerve Sheath Tumor (PNST), prostate cancer, sarcoma, and thyroid cancer.

In certain instances, dimert described herein binds to the extracellular portion of EGFRvIII. In some cases, dimert binds to the extracellular portion of human EGFRvIII. In certain instances, dimert binds to EGFRvIII and an immune cell target, such as a cell surface polypeptide expressed on a T cell or NK cell. In certain instances, dimert binds to EGFRvIII, and optionally to an immune cell target (e.g., a cell surface polypeptide expressed on a T cell or NK cell), for use in treating an EGFRvIII positive cancer, optionally a glioblastoma.

In some embodiments, the cancer is characterized by an upregulation of human epidermal growth factor receptor 2 (HER 2) (also known as HER2/neu, receptor tyrosine-protein kinases erbB-2, CD340, and erbB 2). In certain instances, the amino acid sequence of human HER2 is disclosed in GenPept Access Number NP-004439.2 (obtained at 10/2/2020). In certain instances, HER 2-positive cancers include, but are not limited to, breast, ovarian, gastric, colorectal, pancreatic, and endometrial cancers.

In certain instances, dimer described herein binds to the extracellular portion of HER 2. In certain instances, dimer binds to an extracellular portion of human HER2 (e.g., comprising the amino acid sequence set forth in GenPept access Number NP-004439.2 or an equivalent thereof). In certain instances, dimert binds to HER2 and an immune cell target, such as a cell surface polypeptide expressed on a T cell or NK cell. In certain instances, dimert binds to HER2 and optionally binds to an immune cell target (e.g., a cell surface polypeptide expressed on a T cell or NK cell) to treat HER2 positive cancer, optionally breast cancer.

In some embodiments, the cancer is characterized by the upregulation of CD123 (also known as interleukin-3R or IL-3 RA). In some cases, the amino acid sequence of human CD123 is disclosed in GenPept access Number NP _002174.1 (obtained 2 months and 10 days 2020). In certain instances, CD 123-positive cancers include, but are not limited to, Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL), blastic plasmacytoid dendritic cell tumors, and hairy cell leukemia.

In certain instances, a dimer as described herein binds to the extracellular portion of CD 123. In certain instances, dimer binds to an extracellular portion of human CD123 (e.g., comprising the amino acid sequence set forth in GenPept access Number NP _002174.1, or an equivalent thereof). In certain instances, dimert binds to CD123 and a cell surface polypeptide expressed on an immune cell target, such as a T cell or NK cell. In certain instances, dimert binds to CD123 and optionally to an immune cell target (e.g., a cell surface polypeptide expressed on T cells or NK cells) to treat CD 123-positive cancer, optionally AML.

In some embodiments, the cancer is characterized by an upregulation of CD38 (also referred to as ADP ribosyl cyclase 1 or ADPRC 1). In certain instances, the amino acid sequence of human CD38 is disclosed in GenPept access Number BAA18966.1 (obtained at 10/2/2020). In certain instances, CD 38-positive cancers include, but are not limited to, multiple myeloma, acute myeloid leukemia, prostate cancer, and lung cancer.

In certain instances, dimer described herein binds to the extracellular portion of CD 38. In certain instances, dimer binds to an extracellular portion of human CD38 (e.g., comprising an amino acid sequence listed in GenPept access Number BAA18966.1 or an equivalent thereof). In certain instances, dimert binds to CD38 and a cell surface polypeptide expressed on an immune cell target, such as a T cell or NK cell. In certain instances, dimert binds to CD38 and optionally to an immune cell target (e.g., a cell surface polypeptide expressed on T cells or NK cells) to treat CD38 positive cancer, optionally AML.

In some embodiments, the cancer is characterized by an upregulation of mesothelin (also referred to as MSLN). In some cases, the amino acid sequence of human mesothelin is disclosed in GenPept access Number AAV87530.1 (obtained 2 months and 10 days 2020). In certain instances, mesothelin-positive cancers include, but are not limited to, mesothelioma, pancreatic cancer, ovarian cancer, endometrial cancer, cholangiocarcinoma, gastric cancer, lung adenocarcinoma, and childhood acute myeloid leukemia.

In certain instances, a dimer as described herein binds to the extracellular portion of mesothelin. In certain instances, dimer binds to an extracellular portion of human mesothelin (e.g., comprising the amino acid sequence set forth in GenPept access Number AAV87530.1 or an equivalent thereof). In some cases, dimert binds to mesothelin and an immune cell target, such as a cell surface polypeptide expressed on a T cell or NK cell. In certain instances, dimert binds to mesothelin and optionally to an immune cell target (e.g., a cell surface polypeptide expressed on T cells or NK cells) to treat mesothelin-positive cancer, optionally mesothelioma.

In some embodiments, the cancer is characterized by an upregulation of interleukin-13 receptor alpha (IL 13R alpha). In certain instances, IL13R α comprises IL13R α 1 (IL 13R α 1) and IL13R α 2 (IL 13R α 2). In certain instances, the amino acid sequence of human IL13R α 1 is disclosed in GenPept access Number P78552.1 (obtained at 10/2/2020). In certain instances, the amino acid sequence of human IL13R α 2 is disclosed in GenPept access Number Q14627.1 (obtained at 10/2/2020). In certain instances, IL13R α -positive cancers include, but are not limited to, brain cancer (e.g., glioblastoma) and Renal Cell Carcinoma (RCC).

In certain instances, dimer described herein binds to the extracellular portion of IL13R α. In certain instances, dimer binds to an extracellular portion of human IL13R α (e.g., comprising the amino acid sequence set forth in GenPept access Number P78552.1, Q14627.1, or equivalents thereof). In certain instances, dimer binds to IL13R α and a cell surface polypeptide expressed on an immune cell target, such as a T cell or NK cell. In certain instances, dimer binds to IL13R α and optionally to an immune cell target (e.g., a cell surface polypeptide expressed on a T cell or NK cell) to treat IL13R α -positive cancer, optionally brain cancer.

In some embodiments, the cancer is characterized by an upregulation of B7-H3 (also referred to as CD276, a member of the immune checkpoint). In certain instances, the amino acid sequence of human B7-H3 is disclosed in GenPept Access Number CAE47548.1 (obtained 2, 10 days 2020). In certain instances, B7-H3-positive cancers include, but are not limited to, lung cancer (e.g., non-small cell lung cancer), breast cancer, prostate cancer, renal cell carcinoma, brain cancer, pancreatic cancer, renal cancer, gastric cancer, ovarian cancer, melanoma, and thyroid cancer.

In certain instances, dimer described herein binds to the extracellular portion of B7-H3. In certain instances, dimer binds to an extracellular portion of human B7-H3 (e.g., comprising the amino acid sequence set forth in GenPept access Number CAE47548.1 or an equivalent thereof). In certain instances, dimert binds to B7-H3 and a T cell or NK cell target, e.g., a cell surface polypeptide expressed on a T cell or NK cell. In certain instances, dimert binds to B7-H3 and optionally to a T cell or NK cell target (e.g., a cell surface polypeptide expressed on a T cell or NK cell) to treat a B7-H3 positive cancer, optionally lung cancer (e.g., non-small cell lung cancer), breast cancer, prostate cancer, renal cell carcinoma, brain cancer, pancreatic cancer, renal cancer, gastric cancer, ovarian cancer, melanoma, or thyroid cancer.

In some embodiments, the cancer is characterized by upregulation of receptor tyrosine kinase-like orphan receptor 1 (ROR 1) (also known as neurotrophic tyrosine kinase, receptor-associated 1, or NTRKR 1). In certain instances, the amino acid sequence of human ROR1 is disclosed in GenPept access Number NP _005003 (obtained 10/2/2020). In certain instances, ROR 1-positive cancers include, but are not limited to, breast cancer, lung cancer, gastric cancer, ovarian cancer, Chronic Lymphocytic Leukemia (CLL), and Acute Lymphocytic Leukemia (ALL).

In certain instances, a dimer as described herein binds to the extracellular portion of ROR 1. In certain instances, dimer binds to an extracellular portion of human ROR1 (e.g., comprising the amino acid sequence set forth in GenPept access Number NP _005003 or an equivalent thereof). In certain instances, dimer binds to ROR1 and a cell surface polypeptide expressed on an immune cell target, such as a T cell or NK cell. In certain instances, dimer binds to ROR1 and optionally binds to an immune cell target (e.g., a cell surface polypeptide expressed on a T cell or NK cell) to treat ROR 1-positive cancer, optionally breast, lung, gastric, ovarian, CLL, or ALL.

In some embodiments, the cancer is characterized by upregulation of the adrenergic a-type receptor 2 (EphA 2) (also known as EphA2 receptor, tyrosine protein kinase receptor ECK, epithelial cell receptor protein tyrosine kinase). In some cases, the amino acid sequence of human EphA2 is disclosed in GenPept access Number NP _004422.2 (obtained 10/2/2020). In certain instances, EphA 2-positive cancers include, but are not limited to, breast cancer, bladder cancer, prostate cancer, skin cancer, lung cancer, ovarian cancer, brain cancer, mesothelioma, thyroid cancer, colorectal cancer, gastric cancer, esophageal cancer, endometrial cancer, cervical cancer, pancreatic cancer, melanoma, renal cell carcinoma, and liver cancer.

In certain instances, dimer described herein binds to the extracellular portion of EphA 2. In certain instances, dimer binds to an extracellular portion of human EphA2 (e.g., comprising the amino acid sequence set forth in GenPept access Number NP _004422.2 or an equivalent thereof). In certain instances, dimer binds to EphA2 and an immune cell target, such as a cell surface polypeptide expressed on a T cell or NK cell. In certain instances, dimert binds to EphA2 and optionally to an immune cell target (e.g., a cell surface polypeptide expressed on a T cell or NK cell) to treat EphA2 positive cancer, optionally breast, bladder, prostate, skin, lung, ovarian, brain, mesothelioma, thyroid, colorectal, gastric, esophageal, endometrial, cervical, pancreatic, melanoma, renal cell, or liver cancer.

In some embodiments, the cancer is a B cell leukemia or a B cell lymphoma. Exemplary B cell leukemias include B cell chronic lymphocytic leukemia (or B cell small lymphocytic lymphoma); acute lymphocytic leukemia, mature B cell type; b cell prolymphocytic leukemia; precursor B lymphocyte leukemia; hairy cell leukemia. In certain instances, the B cell leukemia, B cell lymphoma, or a combination thereof is characterized by high expression of CD20 and/or CD22 on B cells. In certain instances, a dimer described herein binds to CD20 or CD 22. In some cases, dimert binds to CD20 expressed on B cells. In some cases, dimert binds to CD22 expressed on B cells. In some cases, dimert further binds to another cellular target, such as a cell surface polypeptide expressed on cancer cells or a cell surface polypeptide expressed on T cells or NK cells. In certain instances, dimert binds to CD20 or CD22 and optionally to another cellular target (e.g., a cell surface polypeptide expressed on cancer cells or a cell surface polypeptide expressed on T cells or NK cells) to treat B cell leukemia or B cell lymphoma.

As used herein, "first line therapy" includes primary treatment of a subject, optionally a subject with cancer. In some cases, the cancer is a primary cancer. In other cases, the cancer is metastatic or recurrent cancer. In some cases, the first line therapy includes chemotherapy. In other cases, the first line therapy includes radiation therapy. The skilled person will readily appreciate that different first line treatments may be applicable to different types of cancer.

As used herein, second line therapy includes therapy used after cessation of primary therapy or first line therapy. Three-line therapy, four-line therapy or five-line therapy includes follow-up therapy. As the naming convention dictates, third line therapy involves a course of therapy in which the primary therapy and second line therapy have ceased.

A "subject" for diagnosis or treatment is a cell or an animal, such as a mammal or a human. Subjects are not limited to a particular species, and include non-human animals that are diagnosed or treated, as well as animals that are affected by infection or animal models, e.g., simians, murines (e.g., rats, mice, chinchillas), canines (e.g., dogs), lagomorphs (e.g., rabbits), livestock, sport animals, and pets. Human patients are also included in the term.

As used herein, the term "tissue" refers to the tissue of a living or dead organism, or any tissue derived from or designed to mimic a living or dead organism. The tissue may be healthy, diseased, and/or have a genetic mutation. Biological tissue may include any single tissue (e.g., a collection of cells that may be connected to one another) or tissues that make up an organ or body part or region of an organism. The tissue may comprise homogeneous cellular material or may be a composite structure, such as found in a body region including the breast, which may include, for example, lung tissue, skeletal tissue, and/or muscle tissue. Exemplary tissues include, but are not limited to, tissues derived from the liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidney, brain, biliary tract, duodenum, abdominal aorta, iliac vein, heart, and intestinal tract, including any combination thereof.

As used herein, the term "specifically binds" or "specifically binds to" or "specific target" refers to a polypeptide or fragment thereof that recognizes and binds a biological molecule of interest (e.g., a polypeptide) but does not substantially recognize and bind other molecules in a sample (e.g., a biological sample that contains or expresses a tumor antigen).

As used herein, "treating" or "treatment" of a disease in a subject refers to (1) preventing the appearance of symptoms or disease in a subject susceptible to or not yet exhibiting symptoms of the disease; (2) inhibiting or arresting the development of the disease; or (3) ameliorating or causing regression of the disease or disease symptoms. As understood in the art, "treatment" is a method for obtaining beneficial or desired results, including clinical results. For purposes of the present technology, beneficial or desired results may include one or more of, but are not limited to: alleviating or ameliorating one or more symptoms; reduction in the extent of the disorder (including disease); a stable (i.e., not worsening) condition (including disease); delay or alleviation of disorders (including diseases); progression, amelioration, or remission of a disorder (including disease); status and remission (whether partial or total), whether detectable or not. In one aspect, the term "treating" does not include preventing.

As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired effect. In the case of therapeutic or prophylactic use, the effective amount will depend on the type and severity of the disease in question and on the characteristics of the individual subject, such as general health, age, sex, body weight and tolerance to pharmaceutical compositions. In the context of gene therapy, in some embodiments, an effective amount is an amount sufficient to result in the recovery of some or all of the function of a gene that is lacking in a subject. In other embodiments, an effective amount of a recombinant polynucleotide, vector, or AAV viral particle is an amount sufficient to result in expression of a gene in a subject. In some embodiments, the effective amount is an amount required to increase galactose metabolism in a subject in need thereof. One skilled in the art will be able to determine appropriate amounts based on these and other factors.

In some embodiments, the effective amount will depend on the size and nature of the application. But also on the nature and sensitivity of the target object and the method used. One skilled in the art will be able to determine an effective amount based on these and other considerations. According to an embodiment, an effective amount may comprise one or more administrations of the composition.

As used herein, the term "administering" or "administration" refers to the delivery of a substance to a subject (such as an animal or human). Administration may be once, continuously or intermittently throughout the course of treatment. Methods of determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for treatment, the purpose of the treatment, and the age, health, or sex of the subject being treated. Single or multiple administrations may be carried out with the dose level and pattern being selected by the treating physician, and in the case of pets and animals, by the treating veterinarian. Appropriate dosage formulations and methods of administration are known in the art. The route of administration can be determined, and the method of determining the most effective route of administration is known to those skilled in the art and will vary with the composition used for treatment, the purpose of the treatment, the health or disease stage of the subject being treated, and the target cell or tissue. Non-limiting examples of routes of administration include intravenous injection, intra-arterial injection, intramuscular injection, intracardiac injection, intrathecal injection, sub-ventricular injection, epidural injection, intracerebral injection, intracerebroventricular injection, subretinal injection, intravitreal injection, intra-articular injection, intraocular injection, intraperitoneal injection, intrauterine injection, intradermal injection, subcutaneous injection, transdermal injection, submucosal injection, and inhalation.

Modes for carrying out the invention

Cancer is the second leading cause of death worldwide, with 960 million estimated to die in 2018. There are many different types of cancer treatments in clinical development and on the market, such as immunotherapy, hormonal therapy, targeted drug therapy, adoptive cell therapy and chemotherapy. Despite advances in many therapeutic areas, challenges remain related to factors such as half-life and toxicity that affect therapeutic efficacy. For example, oncolytic virus-based therapies can target the delivery of a payload to a tumor microenvironment of interest. However, these oncolytic viruses express their payload in tumor cells and induce a cell killing effect, thereby limiting the expression of the payload to a maximum of several days, typically to several hours. Thus, to achieve a sustained and long lasting therapeutic effect, multiple administrations are required, which increases toxicity, resulting in an adverse immune response. Adoptive cell therapy, such as Chimeric Antigen Receptor (CAR) -T cell therapy, provides individualized treatment options for cancer patients. However, the most common side effects of CAR T cell therapy include cytokine release syndrome; neurological events such as encephalopathy, aphasia, epilepsy, and loss of balance; neutropenia and anemia. Furthermore, the production of CAR-T cells requires several weeks of culture and expansion prior to administration, a time that is particularly important for patients with advanced cancer.

In certain embodiments, disclosed herein are methods of delivering a therapeutic transgene (e.g., a dimert disclosed herein) using a gene therapy vector. In some embodiments, the gene therapy vector provides for stable sustained expression of a therapeutic transgene (e.g., dimer). In other embodiments, the gene therapy vector provides constitutive expression. In further embodiments, the gene therapy vector provides for modulated expression. In some cases, the gene therapy vector is transduced in normal cells (e.g., in organ cells such as liver cells or muscle cells). In some cases, a single administration of the gene therapy vector is sufficient to induce stable sustained expression of the therapeutic transgene (e.g., dimert). In other cases, the gene therapy vector provides for sustained, long-term expression of a therapeutic transgene (e.g., dimer), thereby providing long-term stress to the cancer cells.

In certain embodiments, disclosed herein is a method of delivering a therapeutic transgene (e.g., a dimert disclosed herein) using a transaxin. In some cases, TransJoin provides constitutive expression of a therapeutic transgene (e.g., dimer). In some cases, TransJoin provides for sustained, stable, long-term expression of a therapeutic transgene (e.g., dimer). In some cases, long-term expression includes about 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 1 year, or longer. In some cases, the TransJoin is transduced in normal cells (e.g., in organ cells such as liver cells or muscle cells). In some cases, a single administration of TransJoin is sufficient to induce stable sustained expression of a therapeutic transgene (e.g., dimert). In other cases, TransJoin provides sustained, long-term expression of a therapeutic transgene (e.g., dimer), thereby providing long-term stress to cancer cells.

In certain embodiments, disclosed herein is a novel method of activating transgene expression (e.g., dimer disclosed herein) that can be used as a gene therapy platform to modulate expression, such as short-term gene expression (e.g., weeks to months, further optionally 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months or longer). In some cases, the method uses TransSkip. In one aspect, transgene expression is placed under the control of a drug or external agent (e.g., OncoSkip) such that administration of the drug or agent modulates (activates or inactivates) gene expression. On the other hand, the original state of gene expression before administration is restored when the administration or withdrawal of the drug or agent is absent. For example, where a drug or agent can activate gene expression, drug withdrawal can inactivate gene expression if there is a side effect or if transgene expression is no longer desired, thereby minimizing side effects, if any. In some cases, TransSkip is transduced in normal cells (e.g., in organ cells such as liver cells or muscle cells). In some cases, a single administration of TransSkip is sufficient to induce stable sustained expression of a therapeutic transgene (e.g., dimert). In other cases, TransSkip provides for regulated but sustained expression of a therapeutic transgene (e.g., dimer), thereby providing long-term stress to cancer cells.

Exon skipping is a technique used to treat genetic disorders that are deficient in short regions of certain genes. DNA mutations result in protein damage either because the wrong amino acids are present in the protein, or because they generate stop mutations that result in truncation of the protein, or because they alter the reading frames in which both are generated. Since mammalian genes are usually encoded in exons, which means that they are divided into multiple gene fragments (exons) that are spliced together during mRNA processing, DNA mutations (base pair changes or deletions) that cause many diseases are contained in a single exon. If the exon can be skipped and not included in the final spliced mRNA, the mutated region will not be included in the final protein. Although the protein will shorten, possibly lacking some parts, it will still be "in frame" and may retain some of its functions.

For example, Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD) are the most common childhood muscular dystrophies, caused by a genetic defect in the DMD gene encoding the dystrophin protein, the muscle protein required for the interaction of the cytoskeleton and the extracellular matrix to maintain muscle fiber stability during contraction. DMD mutations of the dystrophin gene are characterized by frameshift insertions or deletions or nonsense point mutations, resulting in a loss of functional dystrophin. BMD mutations generally maintain the reading frame intact, allowing the synthesis of partially functional dystrophin proteins. Exon skipping-based treatment resulted in the conversion of an out-of-frame mutation in DMD patients to an in-frame mutation encoding a partially functional dystrophin protein. In 2016, the FDA approved the first exon skipping drug Eteplirsen ™ polypeptide (Sarepta therapy) for Duchenne muscular dystrophy with exon 51 mutations in the muscular dystrophy gene. When a drug (this is a short segment of modified DNA, also known as an oligonucleotide) is administered, exon 51 is "skipped" to recover a nearly full-length and more functional protein. Similar techniques are being developed to skip over other dystrophin exons as well as exons in other pathogenic genes.

In contrast to exon skipping, the present invention utilizes transgene activation in gene therapy applications. Most, if not all, gene therapy approaches currently use complementary (cDNA) gene sequences; that is, the genetic sequence of the transgene is only the coding sequence (only exons), and does not include any intervening (intronic) sequences. Thus, the gene does not undergo any RNA splicing.

The present invention and technique forces normal exon splicing of transgenes using intervening sequences containing splice donor and acceptor sites and RNA spliceosome binding sites. Thus, a "reverse engineered" (artificial) exon-intron-exon gene construct in a transgene is provided which undergoes splicing when expressed in a target cell. However, splicing is regulated according to the principle of exon skipping. In one aspect, a deliberately inserted exon containing a stop codon in the middle of the gene regulates gene expression, i.e., an artificial construct will express a normal functional transgene only when an exon is skipped. In one embodiment, the functional transgene encodes an antibody. In another embodiment, the antibody is a bispecific or trispecific antibody (e.g., dimer). In another embodiment, the antibody (e.g., dimer) is a bispecific T cell engager (BiTE), a bispecific NK cell engager (BiTE), a trispecific T cell engager (TriTE), or a trispecific NK cell engager (TriKE).

On the other hand, the degree and type of splicing varies depending on the cell type, as many genes typically undergo alternative splicing, sometimes differing from cell type to cell type. In addition, splicing is sometimes altered in certain cancer cells (certain exons of certain genes may be included or excluded in normal and cancer cells). This technique can take advantage of these features to achieve different transgene control than in normal and cancer cells. In addition, antibodies encoded by functional transgenes, e.g., bispecific or trispecific antibodies, can be used to treat cancer. Thus, in one aspect, the modulation of transgene splicing in the present invention provides a method of treating cancer.

Construction examples

In certain embodiments, provided herein is a polynucleotide or vector comprising, consisting essentially of, or consisting of: (a) a first polynucleotide sequence comprising a first portion of an open reading frame encoding a first polypeptide; (b) a second polynucleotide sequence comprising a second portion of an open reading frame encoding the first polypeptide; (c) a third polynucleotide sequence encoding a second polypeptide; and (d) a gene regulatory polynucleotide sequence located between the first polynucleotide and the second polynucleotide. In certain instances, the first polypeptide binds to a surface polypeptide (e.g., a surface receptor) of a first target cell and the second polypeptide binds to a surface polypeptide (e.g., a surface receptor) of a second target cell. In some cases, the first target cell and the second target cell are different. For example, the first target cell can be a tumor cell and the second target cell can be an immune cell. In a second example, the first target cell can be an immune cell and the second target cell can be a tumor cell. In a third example, the first target cell can be a first immune cell, the second target cell can be a second immune cell, and the first immune cell is a different cell type than the second immune cell. In a fourth example, the first target cell is a first cancer cell, the second target cell is a second cancer cell, and the first cancer cell and the second cancer cell are from the same type of cancer, e.g., associated with the same genetic defect or optionally having the same tissue type.

In some embodiments, the first polypeptide is a first antibody or binding fragment thereof and the second polypeptide is a second antibody or binding fragment thereof. In certain instances, provided herein is a polynucleotide or vector comprising, consisting essentially of, or consisting of: (a) a first polynucleotide sequence comprising a first portion of an open reading frame encoding a first antibody or antigen-binding fragment thereof; (b) a second polynucleotide sequence comprising a second portion of the open reading frame encoding the first antibody or antigen-binding fragment thereof; (c) a third polynucleotide sequence comprising a sequence encoding a second antibody or antigen-binding fragment thereof; and (d) a gene regulatory polynucleotide sequence located between the first polynucleotide and the second polynucleotide. Complementary sequences of the polynucleotides are also provided. In one aspect, the polynucleotide, its complement and/or the vector are detectably labeled. In some cases, the first antibody binds to a first target and the second antibody binds to a second target. In some cases, the first target is a surface polypeptide (e.g., a surface receptor) on a first cell and the second target is a surface polypeptide (e.g., a surface receptor) on a second cell. In some cases, the first target cell and the second target cell are different. For example, the first target cell can be a tumor cell and the second target cell can be an immune cell. In a second example, the first target cell can be an immune cell and the second target cell can be a tumor cell. In a third example, the first target cell can be a first immune cell, the second target cell can be a second immune cell, and the first immune cell is a different cell type than the second immune cell. In a fourth example, the first target cell is a first cancer cell, the second target cell is a second cancer cell, and the first cancer cell and the second cancer cell are from the same type of cancer, e.g., associated with the same genetic defect or optionally having the same tissue type. In some cases, the first target is a first epitope and the second target is a second epitope, both epitopes being present on the same antigen. In some cases, the first antibody and the second antibody have different amino acid sequences. In one aspect, the polynucleotide is contained within a gene expression vector, non-limiting examples of which include plasmids, DNA viral vectors, or gene delivery vectors.

In some embodiments, also disclosed herein is a vector for gene therapy comprising: a first polynucleotide sequence encoding a first antibody or antigen-binding fragment thereof; and a second polynucleotide sequence encoding a second antibody or antigen-binding fragment thereof. Complementary sequences of the polynucleotides are also provided. In one aspect, the polynucleotide, its complement and/or the vector are detectably labeled. In some cases, the first antibody binds to a first target and the second antibody binds to a second target. In some cases, the first target is a surface polypeptide (e.g., a surface receptor) on a first cell and the second target is a surface polypeptide (e.g., a surface receptor) of a second cell. In some cases, the first target cell and the second target cell are different. For example, the first target cell can be a tumor cell and the second target cell can be an immune cell. In a second example, the first target cell can be an immune cell and the second target cell can be a tumor cell. In a third example, the first target cell can be a first immune cell, the second target cell can be a second immune cell, and the first immune cell is a different cell type than the second immune cell. In a fourth example, the first target cell is a first cancer cell, the second target cell is a second cancer cell, and the first cancer cell and the second cancer cell are from the same type of cancer, e.g., associated with the same genetic defect or optionally having the same tissue type. In some cases, the first target is a first epitope and the second target is a second epitope, both epitopes being present on the same antigen. In some cases, the first antibody and the second antibody have different amino acid sequences.

In another aspect, the gene regulatory polynucleotide sequence includes a splice donor site, an upstream intron, an exon containing a stop codon sequence in all three reading frames, a downstream intron, and a splice acceptor site. In another aspect, the gene regulatory polynucleotide sequence comprises one or more binding sequences for an antisense oligonucleotide. In another aspect, the antisense oligonucleotide is morpholino. In another aspect, the binding sequence of the morpholino oligonucleotide comprises a polynucleotide sequence that is at least 95% identical to, or has at least 96%, or at least 97%, or 98%, or at least 99% identity to, SEQ ID NO. 24 (AATATGATCCAACAATAGAGGTAAATCTTG) or SEQ ID number 25 (GATCCAACAATAGAGGTAAATCTTGTTTTA). In one embodiment, the morpholino oligonucleotide comprises a polynucleotide sequence that is at least 95% identical to SEQ ID number 27 (CAAGATTTACCTCTATTGTTGGATCATATT) or SEQ ID number 28 (TAAAACAAGATTTACCTCTATTGTTGGATC), or has at least 96%, or at least 97%, or 98%, or at least 99% identity to each thereof. Splice donor sites and splice acceptor sites are well known in the art. Sequences, such as consensus sequences, for the splice donor site and the splice acceptor site are known to those of ordinary skill in the art. Exemplary splice site consensus sequences for the U2 intron may include the 5' splice site MAG- GTRAGT, wherein M is A or C; r is A or G, the underlined nucleotides indicate that "GT" is invariant; the dash "-" indicates a splice site. The 3' splice site of the U2 intron may be CAG-G, wherein the underlined nucleotides indicate that "AG" is invariant; the dash "-" indicates a splice site. Other examples of consensus splice site sequences include, but are not limited to, the following:

p53 exon 105' splice site (donor): CAG-gtgagt, wherein the dash "-" indicates a splice site;

brd2 exon 35' ss: AAG-gtgagt, wherein the dash "-" indicates a splice site;

BRCA1 exon 225' splice site: CAG-gtaagt, wherein the dash "-" indicates a splice site;

SMN1 exon 15' ss: CAG-gtgagg, wherein the dash "-" indicates a splice site;

BRD 23' splice site acceptor intron 1 (lower case)/exon 2 (upper case): cccatctttacag-GCTCCC, wherein the dash "-" indicates a splice site;

BCL-X3' splice acceptor intron 2 (lower case)/exon 3 (upper case): tctctccctgcag-GATACT, wherein the dash "-" indicates a splice site;

fibronectin 3' splice acceptor intron 28 (lower case)/exon 29 (upper case): ctttttcatacag-GAGGAA, wherein the dash "-" indicates a splice site; and

Survivin 3' splice acceptor intron 2 (lower case)/exon 3 (upper case): tctttatttccagGCAAAG, wherein the dash "-" indicates a splice site.

In some embodiments, the splice site consensus sequence is obtained from// science. umd. edu/labs/mount/RNAinfo/matrices. html.

In some embodiments, the stop codon comprises an oligonucleotide of TAA, TAG, or TGA. In another aspect, the stop codon sequence comprises the polynucleotide sequence of taaxtagxgatxgatxgaxtgax (SEQ ID number 1), wherein x is any nucleotide, or the stop codon sequence comprises the polynucleotide sequence of TAATTAGTTGATTAGTTAATTGAT (SEQ ID number 2). In a further aspect, the gene regulatory polynucleotide comprises a polynucleotide sequence at least 95% identical to SEQ ID No.2, or a polynucleotide sequence at least 96%, or at least 97%, or 98%, or at least 99% identical to SEQ ID No. 2.

In further embodiments, the first antibody or antigen-binding fragment thereof specifically binds to an activated antigen on an immune effector cell and the second antibody or antigen-binding fragment thereof binds to a tumor antigen. In another aspect, the first antibody or antigen-binding fragment thereof specifically binds to a tumor antigen, and the second antibody or antigen-binding fragment thereof binds to an activated antigen on an immune effector cell. In another aspect, the vector further comprises a fourth polynucleotide sequence encoding a third antibody or antigen-binding fragment thereof, wherein the third antibody or antigen-binding fragment thereof binds to an activating antigen or a tumor antigen on an immune effector cell. In one aspect, the immune effector cell comprises a dendritic cell, a natural killer ("NK") cell, a macrophage, a T cell, a B cell, or a combination thereof. Non-limiting examples of immune effector cells include T cells or NK cells.

Non-limiting examples of activating antigens on immune effector cells include CD3, CD2, CD4, CD8, CD19, LFA1, CD45, NKG2D, NKp44, NKp46, NKp30, DNAM, or a combination thereof.

Non-limiting examples of target antigens on antigen presenting cells include, but are not limited to, B7-H3 (CD 276).

Non-limiting examples of target antigens on B cells include, but are not limited to, CD20 and CD 22.

Non-limiting examples of tumor antigens include one or more of the following: epinephrine a type receptor 2 (EphA 2), Interleukin (IL) -13 ra 2, EGFR VIII, PSMA, EpCAM, GD3, fucosyl GM1, PSCA, PLAC1, sarcoma breakpoint, wilms tumor 1, Alpha Fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, Epithelial Tumor Antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), blood differentiation antigen, surface glycoprotein, ganglioside (GM 2), growth factor receptor, stromal antigen, vascular antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR 1), mesothelin, CD38, CD123, human epidermal growth factor receptor 2 (HER 2), B Cell Maturation Antigen (BCMA), Fibroblast Activation Protein (FAP) alpha, or a combination thereof. Other examples may be found in the art, see, e.g., incorporated herein by reference. In another aspect, the recombinant vector expresses a precursor mRNA encoding a dimer described herein. In certain instances, when the precursor mRNA is contacted with a morpholino oligonucleotide, the dimer is a bispecific antibody or a trispecific antibody. Non-limiting examples of dimert are: a bispecific T cell engager (BiTE) or a bispecific NK cell engager (BiKE); trispecific antibodies include trispecific T cell engagers (trites) or trispecific NK cell engagers (trikes). In one aspect, a trispecific antibody comprises a first antibody or antigen-binding fragment thereof and a second antibody or antigen-binding fragment thereof.

In one aspect, a dimer (e.g., a bispecific or trispecific cell engager) comprises a polypeptide sequence having at least 95% sequence identity to SEQ ID No. 11, optionally a polypeptide sequence having at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID No. 11. In another embodiment, the polypeptide sequence encodes an antigen binding fragment for: CD3, CD2, CD4, CD8, CD 19; lymphocyte function-associated antigen 1 (LFA 1); CD 45; interleukin 21 receptor (IL 21R); natural killer 2 group, member D (NKG 2D); natural Cytotoxic Receptors (NCRs), such as NKp44, NKp46, or NKp 30; or DNAX helper molecule-1 (DNAM or DNAM-1; also known as CD226, or platelet and T cell activating antigen 1 (PTA 1)). In another embodiment, the polypeptide sequence encodes an antigen-binding fragment for CD3, CD19, GD2, or NKG 2D. In another embodiment, the polypeptide encodes a first antigen-binding fragment and a second antigen-binding fragment. In another embodiment, the first antigen-binding fragment binds to CD3 and the second antigen-binding fragment binds to CD 19. In another embodiment, the first antigen-binding fragment binds to CD3 and the second antigen-binding fragment binds to GD 2. In another embodiment, the first antigen-binding fragment binds to NKG2D and the second antigen-binding fragment binds to GD 2.

In one embodiment, a trispecific adaptor or antibody comprises a first antigen-binding fragment, a second antigen-binding fragment and a third antigen-binding fragment. In another embodiment, the trispecific adaptor or antibody comprises three antigen binding fragments that bind to NKG2D, IL21R and GD2, respectively. In another aspect, the trispecific adaptor or antibody has a polypeptide sequence with at least 95% sequence identity to SEQ ID No.11 or has at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID No. 11.

In one embodiment, the antigen-binding fragment that binds to IL-21R is IL-21. The amino acid and cDNA sequences of IL-12 are shown in SEQ ID number 3 and SEQ ID number 4, respectively. In one embodiment, an antigen-binding fragment that binds NKG2D comprises MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, Rae-1 α, Rae-1 β, Rae-1 γ, Rae-1 δ, Rae-1 ε, H60a, H60b, H60c, MULT1, or a fragment thereof. In one embodiment, the antigen binding fragment that binds NKG2D is MICA (SEQ ID number 5) or a fragment thereof or an equivalent thereof.

In one embodiment, the MICA sequence comprises a wild-type mutant. In one embodiment, the MICA mutant is a sequence variant of wild-type MICA (e.g., the wild-type MICA sequence set forth in SEQ ID NO: 5). In another embodiment, the MICA mutant is a sequence variant of MUC-30 (SEQ ID NO. 7) comprising a methionine mutation instead of an alanine at position 129 of the wild-type MICA sequence (MICA-129 Met). An equivalent of the MICA mutant (MICA-129 Met) retained the methionine mutation at position 129 of the wild-type MICA. In another embodiment, the antigen-binding fragments of the bispecific or trispecific engagers or antibodies are separated by a linker sequence. One embodiment of a linker sequence comprises, consists essentially of, or consists of the sequence: GGGGSGGGGSGGGGS (SEQ ID number 9) or an equivalent thereof. The linker is encoded by polynucleotide sequence GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGC (SEQ ID number 10) or an equivalent thereof. In one embodiment, the two antigen-binding fragments separated by the linker sequence are IL21 and MICA (e.g., wild-type MICA or a MICA mutant, such as MUC-30 or MICA-129 Met) or fragments or equivalents of each thereof. In one embodiment, the two antigen-binding fragments separated by the linker sequence are GD2 and MICA (e.g., wild-type MICA or MICA mutant, such as MUC-30 or MICA-129 Met) or respective fragments or equivalents thereof. In one embodiment, the two antigen binding fragments separated by the linker sequence are IL21 and GD2, or respective fragments or equivalents thereof. In another embodiment, the linker is inserted between antigen-binding fragments of any of the following trispecific adapters:

IL21-MICA129-GD2

MICA129-IL21-GD2

GD2-IL21-MICA129

GD2-MICA129-IL21

IL21-MICA/V129M-GD2-HDD

MICA/V12M-IL21-GD2-HDD

GD2-IL21-MICA129-HDD

GD2-MICA129-IL21-HDD。

In another embodiment, a dimer (e.g., a bispecific or trispecific adaptor) comprises a secretory consensus sequence (also referred to herein as sec 0A). In certain instances, the secretory consensus sequence (sec 0A) comprises at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 51), or consists of SEQ ID NO: 51. In certain instances, sec0A is encoded by a polynucleotide comprising ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCC (SEQ ID NO: 52) or an equivalent thereof.

In some cases, the secretory consensus sequence further comprises one, two, three, four, or more residues at the C-terminus of the sequence. In some cases, one, two, three, four, or more residues are residues with aliphatic side chains (e.g., Ala, Met, Ile, Val, or Leu). In certain instances, one, two, three, four, or more residues are Ala residues, Gly residues, Val residues, Ile residues, or a combination thereof. In certain instances, one, two, three, four, or more residues are Ala residues, Gly residues, Val residues, or a combination thereof. In some cases, one, two, three, four, or more residues are Ala residues, Gly residues, or a combination thereof. In some cases, the secretory consensus sequence further comprises one, two, three, four, or more Ala residues at the C-terminus of the sequence. In some cases, the secretory consensus sequence further comprises one, two, three, four, or more Gly residues at the C-terminus of the sequence. In some cases, the secretory consensus sequence further comprises one, two, three, four, or more Val residues at the C-terminus of the sequence. In some cases, the secretory consensus sequence further comprises one, two, three, four, or more Ile residues at the C-terminus of the sequence.

In some cases, the secretory consensus sequence further comprises one, two, or three residues with aliphatic side chains (e.g., Ala, Met, Ile, Val, or Leu). In some cases, one, two, or three residues are Ala residues, Gly residues, Val residues, Ile residues, or a combination thereof. In some cases, one, two, or three residues are Ala residues, Gly residues, Val residues, or a combination thereof. In some cases, one, two, or three residues are Ala residues, Gly residues, or a combination thereof. In some cases, the secretory consensus sequence further comprises one, two, or three Ala residues at the C-terminus of the sequence. In some cases, the secretory consensus sequence further comprises one, two, or three Gly residues at the C-terminus of the sequence. In some cases, the secretory consensus sequence further comprises one, two, or three Val residues at the C-terminus of the sequence. In some cases, the secretory consensus sequence further comprises one, two, or three Ile residues at the C-terminus of the sequence.

In some cases, the secretory consensus sequence further comprises one or two residues with aliphatic side chains (e.g., Ala, Met, Ile, Val, or Leu). In some cases, one or both residues are Ala residues, Gly residues, Val residues, Ile residues, or a combination thereof. In some cases, one or both residues are Ala residues, Gly residues, Val residues, or a combination thereof. In some cases, one or both residues are Ala residues, Gly residues, or a combination thereof. In some cases, the secretory consensus sequence further comprises one or two Ala residues at the C-terminus of the sequence. In some cases, the secretory consensus sequence further comprises one or two Gly residues at the C-terminus of the sequence. In some cases, the secretory consensus sequence further comprises one or two Val residues at the C-terminus of the sequence. In some cases, the secretory consensus sequence further comprises one or two Ile residues at the C-terminus of the sequence.

In some cases, the secretory consensus sequence further comprises a residue having an aliphatic side chain (e.g., Ala, Met, Ile, Val, or Leu). In some cases, the residue is an Ala residue, a Gly residue, a Val residue, an Ile residue, or a combination thereof. In some cases, the secretory consensus sequence further comprises an Ala residue at the C-terminus of the sequence. In some cases, the secretory consensus sequence further comprises a Gly residue at the C-terminus of the sequence. In some cases, the secretory consensus sequence further comprises a Val residue at the C-terminus of the sequence. In some cases, the secretory consensus sequence further comprises an Ile residue at the C-terminus of the sequence.

In some cases, the secretory consensus sequence further comprises one or two Ala residues at the C-terminus of the sequence, and such sequence is referred to as secrecon1A (or sec 1A), where one Ala is located at the C-terminus and secrecon2A (or sec 2A) has two Ala residues located at the C-terminus. In certain instances, sec1A has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to MWWRLWWLLLLLLLLWPMVWAA (SEQ ID NO: 53). In certain instances, sec1A is encoded by a polynucleotide comprising ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCCGCC (SEQ ID NO: 54) or an equivalent thereof. In certain instances, sec2A has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to MWWRLWWLLLLLLLLWPMVWAAA (SEQ ID NO: 55). In certain instances, sec2A is encoded by a polynucleotide comprising ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCCGCCGCC (SEQ ID NO: 56) or an equivalent thereof.

In some embodiments, the secretory consensus sequence modulates the expression and/or secretion of dimert. In some cases, a secretory consensus sequence (e.g., sec1A or sec 2A) enhances expression and/or secretion of dimert. In some cases, the secretory consensus sequence (e.g., sec1A or sec 2A) modulates (e.g., enhances) expression of dimer by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold or more. In certain instances, the secretory consensus sequence (e.g., sec1A or sec 2A) modulates (e.g., enhances) secretion of dimer by about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 50-fold or more.

In some embodiments, a dimer (e.g., a trispecific adaptor) described herein comprises a secretion consensus sequence (e.g., sec0A, sec1A, or sec 2A), an IL21 sequence (e.g., SEQ ID NO: 3), a MICA sequence (e.g., a wild-type sequence, MUC-30, or MICA-129 Met), an anti-GD 2 peptide sequence, or equivalents of one or more thereof. In certain instances, the dimer (e.g., a trispecific adaptor) comprises a secrecon1A sequence, an IL21 sequence, a MICA129 sequence, an anti-D2 peptide sequence, or one or more equivalents thereof. In one embodiment, the dimer (e.g., trispecific adaptor) comprises secrecon 1A-linker-IL 21-linker-MICA 129-linker-anti-GD 2 peptide or an equivalent of one or more thereof. In one embodiment, the dimer (e.g., trispecific adaptor) comprises the polypeptide sequence of SEQ ID NO: 11.

SEQ ID NO. 11

MWWRLWWLLLLLLLLWPMVWAARSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSGGGGSEPHSLRYNLTVLSWDGSVQSGFLAEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETEEWTMPQSSRAQTLAMNIRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGGGGSGGGGSGGGGSQVQLQQSGPELVKPGASVKISCKTSGYKFTEYTMHWVKQSHGKCLEWIGGINPNNGGTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARDTTVPYAYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASSVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPLTFGCGTKLEIKRASTKGP or an equivalent thereof.

In one embodiment, the dimer (e.g., trispecific adaptor) is encoded by the sequence of SEQ ID number 12.

SEQ ID NO. 12

ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCCGCCAGAAGCAGCCCCGGCAACATGGAGAGAATCGTGATCTGCCTGATGGTGATCTTCCTGGGCACCCTGGTGCACAAGAGCAGCAGCCAGGGCCAGGACAGACACATGATCAGAATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCCGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAGGCCCAGCTGAAGAGCGCCAACACCGGCAACAACGAGAGAATCATCAACGTGAGCATCAAGAAGCTGAAGAGAAAGCCCCCCAGCACCAACGCCGGCAGAAGACAGAAGCACAGACTGACCTGCCCCAGCTGCGACAGCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGATTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGAGCAGCAGAACCCACGGCAGCGAGGACAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGAGCCCCACAGTCTTCGTTATAACCTCACGGTGCTGTCCTGGGATGGATCTGTGCAGTCAGGGTTTCTCGCTGAGGTACATCTGGATGGTCAGCCCTTCCTGCGCTGTGACAGGCAGAAATGCAGGGCAAAGCCCCAGGGACAGTGGGCAGAAGATGTCCTGGGAAATAAGACATGGGACAGAGAGACCAGGGACTTGACAGGGAACGGAAAGGACCTCAGGATGACCCTGGCTCATATCAAGGACCAGAAAGAAGGCTTGCATTCCCTCCAGGAGATTAGGGTCTGTGAGATCCATGAAGACAACAGCACCAGGAGCTCCCAGCATTTCTACTACGATGGGGAGCTCTTCCTCTCCCAAAACCTGGAGACTGAGGAATGGACAATGCCCCAGTCCTCCAGAGCTCAGACCTTGGCCATGAACATCAGGAATTTCTTGAAGGAAGATGCCATGAAGACCAAGACACACTATCACGCTATGCATGCAGACTGCCTGCAGGAACTACGGCGATATCTAAAATCCGGCGTAGTCCTGAGGAGAACAGTGCCCCCCATGGTGAATGTCACCCGCAGCGAGGCCTCAGAGGGCAACATTACCGTGACATGCAGGGCTTCTGGCTTCTATCCCTGGAATATCACACTGAGCTGGCGTCAGGATGGGGTATCTTTGAGCCACGACACCCAGCAGTGGGGGGATGTCCTGCCTGATGGGAATGGAACCTACCAGACCTGGGTGGCCACCAGGATTTGCCAAGGAGAGGAGCAGAGGTTCACCTGCTACATGGAACACAGCGGGAATCACAGCACTCACCCTGTGCCCTCTGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATCAGCTGCAAGACCAGCGGCTACAAGTTCACCGAGTACACCATGCACTGGGTGAAGCAGAGCCACGGCAAGTGCCTGGAGTGGATCGGCGGCATCAACCCCAACAACGGCGGCACCAACTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGAAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGACACCACCGTGCCCTACGCCTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGCATCAGCTACATGCACTGGTACCAGCAGAAGCCCGGCACCAGCCCCAAGAGATGGATCTACGACACCAGCAAGCTGGCCAGCAGCGTGCCCGCCAGATTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCACCAGAGAAGCAGCTACCCCCTGACCTTCGGCTGCGGCACCAAGCTGGAGATCAAGAGAGCCAGCACCAAGGGCCCCTAG or an equivalent thereof.

In one embodiment, the dimer (e.g., bispecific adapter) comprises a secrecon1A sequence (sec 1A), a CD3 sequence, and an anti-GD 2 peptide sequence, or equivalents of one or more thereof. In one embodiment, the bispecific adapter comprises a polypeptide sequence of sequence ID No.13 arranged as sec 1A-anti-CD 3-linker-anti-GD 2-HDD. The sec1A portion is underlined. The anti-CD 3 portion is bold. The anti-GD 2 portion is underlined and italicized. The gray shaded region represents the HDD portion, which contains the HDD peptide shown in bold, the lower case HDD peptide upstream hinge region, and the lower case HDD peptide downstream spacer region.

SEQ ID NO. 13

MWWRLWWLLLLLLLLWPMVWAAQVQLQQSGPELVKPGASVKISCKTSGYKFTEYTMHWVKQSHGKCLEWIGGINPNNGGTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARDTTVPYAYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASSVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPLTFGCGTKLEIKRASTKGPGGGGSGGGGSGGGGS QVQLVQSGGGVVQP GRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRFTISRDNSKNTAFLQMDSLRPED TGVYFCARYYDDHYCLDYWGQGTPVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYM NWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITR Or an equivalent thereof.

In another embodiment, a sec1A sequence, an anti-CD 3 sequence, and an anti-GD 2 peptide sequence, or equivalents of one or more thereof, are included. dimer (e.g., bispecific adapter) is encoded in one aspect by the sequence of SEQ ID No. 14.

SEQ ID NO. 14

ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCCGCCCAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATCAGCTGCAAGACCAGCGGCTACAAGTTCACCGAGTACACCATGCACTGGGTGAAGCAGAGCCACGGCAAGTGCCTGGAGTGGATCGGCGGCATCAACCCCAACAACGGCGGCACCAACTACAACCAGAAGTTCAAGGGCAAGGCCACCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAGCTGAGAAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGAGACACCACCGTGCCCTACGCCTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCGAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGCATCAGCTACATGCACTGGTACCAGCAGAAGCCCGGCACCAGCCCCAAGAGATGGATCTACGACACCAGCAAGCTGGCCAGCAGCGTGCCCGCCAGATTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCACCAGAGAAGCAGCTACCCCCTGACCTTCGGCTGCGGCACCAAGCTGGAGATCAAGAGAGCCAGCACCAAGGGCCCCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGCGGCGGCGTGGTGCAGCCCGGCAGAAGCCTGAGACTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGATACACCATGCACTGGGTGAGACAGGCCCCCGGCAAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGTTCAAGGACAGATTCACCATCAGCAGAGACAACAGCAAGAACACCGCCTTCCTGCAGATGGACAGCCTGAGACCCGAGGACACCGGCGTGTACTTCTGCGCCAGATACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCCCCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGCAGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGACCCCCGGCAAGGCCCCCAAGAGATGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTGCCCAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTACACCTTCACCATCAGCAGCCTGCAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCTTCACCTTCGGCCAGGGCACCAAGCTGCAGATCACCAGAACCCCCCTGGGCGACACCACCCACACCAGCGGCATGGTGAGCAAGCTGAGCCAGCTGCAGACCGAGCTGCTGGCCGCCCTGCTGGAGAGCGGCCTGAGCAAGGAGGCCCTGATCCAGGCCCTGGGCGAGGGCAGCGGCGGCGCCCCCTAG or an equivalent thereof.

In another embodiment, a dimer (e.g., a bispecific adaptor) comprises a secrecon1A sequence, an anti-CD 19 sequence, and an anti-CD 3 sequence arranged as sec 1A-anti-CD 19-linker-anti-CD 3. The bispecific adapter comprises the polypeptide sequence of SEQ ID No. 15. The sec1A portion is underlined.

SEQ ID NO. 15

MWWRLWWLLLLLLLLWPMVWAADIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKHHHHHHTPLGDTTHTSGMVSKLSQLQTELLAALLESGLSKEALIQALGEGSGGAP or an equivalent thereof.

In another embodiment, a dimer (e.g., a bispecific adaptor) comprises a secrecon1A sequence, an anti-CD 19 sequence, and an anti-CD 3 sequence arranged as sec 1A-anti-CD 19-linker-anti-CD 3, or an equivalent of one or more thereof. In one aspect, it is encoded by the polynucleotide sequence of SEQ ID number 16.

SEQ ID NO. 16

ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCCGCCGACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGAGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGAGACCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGAGACCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCAGAAGAGAGACCACCACCGTGGGCAGATACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCAGACCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCAGATACACCATGCACTGGGTGAAGCAGAGACCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGATACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGAGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGAGATGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACAGATTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGCACCACCACCACCACCACACCCCCCTGGGCGACACCACCCACACCAGCGGCATGGTGAGCAAGCTGAGCCAGCTGCAGACCGAGCTGCTGGCCGCCCTGCTGGAGAGCGGCCTGAGCAAGGAGGCCCTGATCCAGGCCCTGGGCGAGGGCAGCGGCGGCGCCCCCTA or an equivalent thereof.

In some embodiments, dimer comprises a secretion signal referred to as secreconAA (sec 2A). In some cases, dimert comprises secreconAA in tandem with an amino acid sequence from Blinatumomab (targeting CD3 and CD 19). In certain instances, secreconAA-BlinatumAb (or sec2A-CD19xCD 3) comprises the polypeptide sequence of SEQ ID NO:29, with the secreconAA portion underlined.

SEQ ID NO: 29

MWWRLWWLLLLLLLLWPMVWAAADIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKHHHHHH or an equivalent thereof.

In some embodiments, the dimert comprising sec2A-CD19xCD3 is encoded by the polynucleotide sequence of SEQ ID NO: 30.

SEQ ID NO: 30

ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCCGCCGCCGACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGAGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGAGACCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGAGACCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCAGAAGAGAGACCACCACCGTGGGCAGATACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCAGACCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCAGATACACCATGCACTGGGTGAAGCAGAGACCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGATACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGAGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGAGATGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACAGATTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGCACCACCACCACCACCACTAG or an equivalent thereof.

In some embodiments, dimer comprises the secrenona (sec 1A) sequence, CD19 and CD3, or equivalents of one or more thereof, arranged in sec1A-CD19xCD 3. In one aspect, it is encoded by the polynucleotide sequence of SEQ ID NO. 31.

SEQ ID NO: 31

ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCCGCCGACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGAGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGAGACCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGAGACCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCAGAAGAGAGACCACCACCGTGGGCAGATACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCAGACCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCAGATACACCATGCACTGGGTGAAGCAGAGACCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGATACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGAGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGAGATGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACAGATTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGCACCACCACCACCACCACTAG or an equivalent thereof.

In some embodiments, dimer comprises a secrecon (sec 0A) sequence, CD19, and CD3, or equivalents of one or more thereof, arranged in sec0A-CD19xCD 3. In one aspect, it is encoded by the polynucleotide sequence of SEQ ID NO. 32.

SEQ ID NO: 32

ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCCGACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGAGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGAGACCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGAGACCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCAGAAGAGAGACCACCACCGTGGGCAGATACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCAGACCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCAGATACACCATGCACTGGGTGAAGCAGAGACCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGATACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGAGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGAGATGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACAGATTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGCACCACCACCACCACCACTAG or an equivalent thereof.

In some embodiments, dimer comprises CD19 and CD3 sequences, or equivalents of either or both, arranged as CD19xCD3, that do not contain secrecon sequences. In one aspect, it is encoded by the polynucleotide sequence of SEQ ID NO. 33.

SEQ ID NO: 33

ATGGACATCCAGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGAGAGCCACCATCAGCTGCAAGGCCAGCCAGAGCGTGGACTACGACGGCGACAGCTACCTGAACTGGTACCAGCAGATCCCCGGCCAGCCCCCCAAGCTGCTGATCTACGACGCCAGCAACCTGGTGAGCGGCATCCCCCCCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGAAGGTGGACGCCGCCACCTACCACTGCCAGCAGAGCACCGAGGACCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGTGAGACCCGGCAGCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAAGCAGAGACCCGGCCAGGGCCTGGAGTGGATCGGCCAGATCTGGCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACGAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGGCCAGCGAGGACAGCGCCGTGTACTTCTGCGCCAGAAGAGAGACCACCACCGTGGGCAGATACTACTACGCCATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGACATCAAGCTGCAGCAGAGCGGCGCCGAGCTGGCCAGACCCGGCGCCAGCGTGAAGATGAGCTGCAAGACCAGCGGCTACACCTTCACCAGATACACCATGCACTGGGTGAAGCAGAGACCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGATACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCGTGGAGGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCAGCGGCGGCGTGGACGACATCCAGCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGGCGAGAAGGTGACCATGACCTGCAGAGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGAGATGGATCTACGACACCAGCAAGGTGGCCAGCGGCGTGCCCTACAGATTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAGCACCACCACCACCACCACTAG or an equivalent thereof.

In one aspect, the bispecific T cell engager comprises a polypeptide sequence having at least 95% identity to any one of SEQ ID nos. 12 and 13, or at least 96%, or at least 97%, or 98%, or at least 99% identity. In one embodiment, a trispecific antibody comprises a first antibody or antigen-binding fragment thereof, a second antibody or antigen-binding fragment thereof, and a third antibody or antigen-binding fragment thereof. In another aspect, the trispecific antibody has a polypeptide sequence with at least 95% sequence identity, or at least 96%, or at least 97%, or 98%, or at least 99% sequence identity to SEQ ID No. 11.

In a further aspect, the recombinant polynucleotide expresses a precursor mRNA that encodes a trispecific antibody when contacted with a morpholino oligonucleotide.

In one aspect, the antigen binding domain is a single chain variable fragment of an antibody.

In a further aspect, the recombinant polynucleotide or vector further comprises a polynucleotide sequence encoding a secretory peptide. In another aspect, the vector further comprises a polynucleotide sequence encoding a dimerization domain. In another aspect, the vector comprises a 5 'Inverted Terminal Repeat (ITR) and a 3' ITR. In another aspect, the vector has at least 95%, or at least 96%, or at least 97%, or any 98%, or at least 99% identity to a sequence of SEQ ID number 4, 6, 8, 12, 15, 16, 30-33, or an equivalent thereof, or a polynucleotide. Non-limiting examples of such carriers include: a recombinant viral vector comprising a backbone vector selected from a retroviral vector, a lentiviral vector, a mouse leukemia virus ("MLV") vector, an epstein-barr virus ("EBV") vector, an adenoviral vector, a herpes virus ("HSV") vector, an adeno-associated virus ("AAV") vector, an AAV vector, or optionally a self-complementary AAV vector. These are optionally detectably labeled. Also provided are complementary sequences of the optionally detectably labeled polynucleotides.

The recombinant polynucleotide vector may be comprised within a host cell, such as a prokaryotic cell or a eukaryotic cell. Cells containing a polynucleotide can be used to recombinantly express or replicate the polynucleotide by culturing the cell under conditions that allow the polynucleotide to replicate and, optionally, express the polynucleotide. The polynucleotide and/or expression product is optionally isolated from the cell culture.

The present invention also provides a virus packaging system comprising: the vector as described above, wherein the backbone is derived from a plasmid, a virus; packaging the plasmid; and an envelope plasmid. The packaging plasmid contains a nucleoside, a capsid protein, and a matrix protein. Examples of packaging plasmids are also described in the patent literature, for example, U.S. Pat. nos. 7,262,049, 6,995,258, 7,252,991 and 5,710,037. The system further comprises a plasmid encoding an envelope protein provided by the envelope plasmid.

The invention also provides suitable packaging cell lines. In one aspect, the packaging cell line is the HEK-293 cell line. Other suitable cell lines are known in the art, for example, as described in U.S. patent nos. 7,070,994, 6,995,919, 6,475,786, 6,372,502, 6,365,150, and 5,591,624.

The invention also provides a method of producing an AAV particle, the method comprising, consisting essentially of, or consisting of: the packaging cell line is transduced with the viral system as described above under conditions suitable for packaging of the viral vector. Such conditions are known in the art and are briefly described herein. Viral particles can be isolated from the cell supernatant using methods known to those skilled in the art, such as centrifugation. The invention further provides such isolated particles.

The invention further provides isolated AAV viral particles produced by this method. The viral particle comprises, consists essentially of, or consists of a polynucleotide as described herein.

Host cell

Further provided is an isolated cell or population of cells comprising, consisting essentially of, or consisting of an isolated polynucleotide, viral particle, vector and packaging system, as described above and incorporated herein by reference. In one aspect, the isolated cell is a packaging cell line.

Also provided is an isolated cell or population of cells comprising, consisting essentially of, or consisting of a polynucleotide sequence as described herein.

The isolated cell described herein may be any cell of the following species: mouse, rat, rabbit, simian, bovine, ovine, porcine, canine, feline, farm animal, sport animal, pet, equine, and primate, particularly human cells.

The vectors and cells can be included in a composition comprising the vector and/or the host cell and the vector (e.g., a pharmaceutically acceptable carrier). They can be formulated for various modes of administration and include an effective amount of a vector and/or host cell effective for the patient, disease or condition, vector, and mode of administration. In one aspect, the mode of administration is systemic or intravenous injection. In another aspect, topical administration is by direct injection. In one aspect, the morpholino oligonucleotide is contacted with the vector simultaneously or after the vector. Alternatively, the morpholino oligonucleotide is contacted prior to the vector.

Application method

The polynucleotides and vectors are useful for treating a variety of diseases or disorders. In one aspect, a method of delivering a transgene is provided. The method comprises administering to a cell, tissue or patient to be treated an effective amount of a polynucleotide or vector comprising a transgene. In one aspect, an effective amount of an antisense oligonucleotide (e.g., a morpholino oligonucleotide) is administered to a cell, tissue, or patient to be treated. Non-limiting examples of transgenes are provided and selected for the purpose of the method. The cell or tissue may be a mammal, such as a human. In one aspect, the antisense oligonucleotide (e.g., a morpholino oligonucleotide) is contacted with the vector simultaneously or after the vector. Alternatively, the contacting is performed before the support. In another aspect, the vector is introduced into the cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof.

Also provided herein are methods of treating cancer in a subject in need thereof. The method comprises, consists essentially of, or consists of: administering to the subject an effective amount of a recombinant viral vector or cell as described herein. In another aspect, the method further comprises administering to the subject an effective amount of an antisense oligonucleotide (e.g., a morpholino oligonucleotide). In one aspect, an effective amount of an anti-cancer agent is administered to a subject. Non-limiting examples of anti-cancer agents include anti-cancer peptides, polypeptides, nucleic acid molecules, small molecules, viral particles, or combinations thereof. In another aspect, the vector is introduced into the cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof. The therapy may be administered as a first line, second line, third line, fourth line or fifth line therapy. The therapy may be adjuvant therapy or may be combined with other cancer therapies.

In one aspect of the disclosed method, the viral particle is an oncolytic HSV particle.

Administration of

Administration of the recombinant polynucleotides and/or vectors (e.g., AAV), viral particles, or compositions of the invention can be carried out in a single dose, continuously, or intermittently throughout the course of treatment. Administration may be by any suitable mode of administration, including but not limited to: intravenous, intraarterial, intramuscular, intracardiac, intrathecal, subcentricular, epidural, intracerebral, intracerebroventricular, subretinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation. In some cases, the mode of administration includes parenteral administration. In one embodiment, the recombinant polynucleotide or vector or composition is administered by intramuscular injection or intravenous injection. In another embodiment, the recombinant polynucleotide or vector or composition is administered systemically. In another embodiment, the recombinant polynucleotide or vector or composition is administered parenterally by injection, infusion or implantation.

Methods of determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for treatment, the purpose of the treatment, and the subject being treated. Dosage levels and patterns can be selected by the treating physician for single or multiple administrations. Notably, the dosage may be affected by the route of administration. Appropriate dosage formulations and methods of administration are known in the art. Non-limiting examples of such suitable doses can be as low as 1E +9 vector genomes per administration to as high as 1E +17 vector genomes.

In some embodiments of the methods described herein, the number of viral particles (e.g., AAV) administered to the subject ranges from about 10⁹To about 10¹⁷. In particular embodiments, about 10 is administered to a subject¹⁰To about 10¹⁶About 10¹⁰To about 10¹⁵About 10¹⁰To about 10¹²About 10¹¹To about 10¹³About 10¹¹To about 10¹²About 10¹¹To about 10¹⁴About 10¹¹To about 10¹⁵About 10¹¹To about 10¹⁶About 5x10¹¹To about 5x10¹²Or about 10¹²To about 10¹³And (c) viral particles. In certain instances, about 10 is administered to a subject¹¹To about 10¹²And (c) viral particles. In certain instances, about 10 is administered to a subject¹³To about 10¹⁵And (c) viral particles. In certain instances, about 10 is administered to a subject⁹To about 10¹²And (c) viral particles. In certain instances, about 10 is administered to a subject⁹To about 10¹¹And (c) viral particles. In certain instances, the amount of viral particles administered is based on the weight of the subject. One skilled in the art will understand how to adjust the amount of viral particles delivered such that the total amount delivered to the subject ranges from about 10⁹To about 10¹⁷Optionally about 10¹⁰To about 10¹⁶About 10¹⁰To about 10¹⁵About 10¹⁰To about 10¹²About 10¹¹To about 10¹³About 10¹¹To about 10¹²About 10¹¹To about 10¹⁴About 10¹¹To about 10¹⁵About 10¹¹To about 10¹⁶About 5x10 ¹¹To about 5x10¹²Or about 10¹²To about 10¹³And (c) viral particles. In certain instances, the subject is a pediatric subject (e.g., a subject under 18 years of age). In some cases, about 10 is administered to a pediatric subject⁹To about 10¹²About 10¹⁰To about 10¹²About 10¹¹To about 10¹²Or about 10⁹To about 10¹⁰And (c) viral particles.

In another aspect, the viral particles and compositions of the present invention can be administered in conjunction with other treatments, e.g., approved treatments for cancer and its associated diseases or disorders.

Determining successful treatment and/or repair when one or more of the following is detected: alleviating or ameliorating one or more diseases, disorders or conditions in a subject; alleviating the extent of the disease, disorder or condition in the subject, stabilization (i.e., not worsening) of the disease, disorder or condition; delaying or slowing the progression of the disease, disorder or condition in the subject; and amelioration or palliation of the disease, disorder or condition in the subject. In some embodiments, the success of the treatment is determined by detecting the presence of the repaired target polynucleotide in one or more cells, tissues or organs isolated from the subject. In some embodiments, the success of the treatment is determined by detecting the presence of a polypeptide encoded by the repaired target polynucleotide in one or more cells, tissues or organs isolated from the subject.

Reagent kit

In some embodiments, the reagents, vectors, or compositions described herein can be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic, or research applications. In some embodiments, the kits of the invention comprise one or more of the following: a modified viral capsid protein, an isolated polynucleotide, a vector, a host cell, a recombinant viral particle, a recombinant expression system, a modified AAV, a modified cell, an isolated tissue, a composition or a pharmaceutical composition as described herein.

In some embodiments, the kit further comprises instructions for use. In particular, such kits can include one or more of the reagents described herein, as well as instructions describing the intended use and proper use of the reagents. For example, in one embodiment, a kit can include instructions for mixing one or more components of the kit and/or separating and mixing samples and applying to a subject. In certain embodiments, the agents in the kit are pharmaceutical formulations and dosages suitable for the particular application and method of administration. Kits for research purposes may contain the appropriate concentrations or amounts of the components for conducting the various experiments.

The design of the kit may facilitate the use of the methods described herein and may take a variety of forms. Where applicable, each composition of the kit may be provided in liquid form (e.g., a solution) or solid form (e.g., a dry powder). In some cases, some compositions may be combinable or otherwise processable (e.g., into an active form), such as by addition of a suitable solvent or other species (e.g., water or cell culture medium), which may or may not be provided with a kit. In some embodiments, the composition can be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde, and CryoStor @ (Stem Cell Technologies, Canada, Vancouver). In some embodiments, the preservation solution comprises an amount of a metalloprotease inhibitor.

As used herein, "instructions" may define parts of instructions and/or promotions and generally relate to written instructions for or relating to the packaging of the method, recombinant vector or composition. The instructions may also include any verbal or electronic instructions provided in any manner such that the user will clearly recognize that the instructions will be associated with the kit, e.g., audiovisual (e.g., videotape, DVD, etc.), internet and/or network-based communications, etc. In some embodiments, the written instructions are in a format prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflect manufacture, use or sale approved by an animal regulatory agency.

In some embodiments, a kit comprises any one or more of the components described herein in one or more containers. Thus, in some embodiments, a kit can include a container holding a reagent described herein. These agents may be in the form of a liquid, gel or solid (powder). The formulations may be prepared aseptically, packaged in syringes and shipped refrigerated. Alternatively, it may be stored in a vial or other container. The second container may contain other reagents prepared by sterilization. Alternatively, the kit may include the active agents premixed and shipped in syringes, vials, tubes, or other containers. The kit may have one or more or all of the components required to administer the medicament to the subject, such as a syringe, a topical administration device, or an IV needle and bag.

The treatments described herein can be combined with appropriate diagnostic techniques to identify and select patients for the treatment.

Production method

In an additional method provided by the invention is a method of producing a bispecific or trispecific antibody in a cell comprising contacting a cell containing a vector described herein with an effective amount of a morpholino oligonucleotide. In one embodiment, the morpholino oligonucleotide has a polynucleotide sequence that is at least 95% identical to SEQ ID number 27 (CAAGATTTACCTCTATTGTTGGATCATATT) or SEQ ID number 28 (TAAAACAAGATTTACCTCTATTGTTGGATC) or equivalent thereof. In one aspect, the morpholino oligonucleotide is contacted with the vector simultaneously or after the vector. Alternatively, the contacting is performed before the support. Non-limiting examples of such bispecific antibodies have a polypeptide sequence that is at least 95% identical to any one of SEQ ID NOs 13 and 15, or have a sequence that is at least 96%, or at least 97%, or at least 98%, or at least 99% identical to any one of SEQ ID NOs 13 and 15. In another aspect, the trispecific antibody has a polypeptide sequence at least 95% identical to SEQ ID No. 11, or has a sequence at least 96%, or at least 97%, or at least 98%, or at least 99% identical to SEQ ID No. 11.

In another aspect, the vector is introduced into the cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof. Non-limiting examples of cells include fibroblasts, skeletal cells, epithelial cells, muscle cells, nerve cells, endocrine cells, melanocytes, blood cells, or combinations thereof.

Further provided are kits comprising one or more vectors, cells, or compositions as described herein, optionally with a specification document or material.

Detailed description of the preferred embodiments

There are several approaches to regulate gene expression by the exon skipping principle.

In one aspect, at least three exons are required to skip an exon in this strategy. For the three exon structure, there are two splice donors and two splice acceptor sites, as shown in FIG. 1. The first donor site is spliced to a first acceptor site ("a" in fig. 1), or, alternatively, to a second acceptor site ("c" in fig. 1). The second donor site can only be spliced to the second acceptor site ("b" in FIG. 1). Thus, this three exon/two intron structure can give rise to 5 possible RNA structures depending on whether splicing has occurred, whether only one splicing event (a, b or c) has occurred, or whether two splicing events (a + b) have occurred.

Sequence-specific oligonucleotides ("oligonucleotides") are constructed that specifically interfere with each splicing event, usually at the splice donor or acceptor site, or at one of the binding sites of the spliceosome components (sometimes in the middle of an intron). For the purposes of this example and as shown in FIG. 1, each oligonucleotide is classified as a 1-donor (1D), 2-acceptor (1A), 2-donor (2D) or 3-acceptor (3D) based on whether it interferes with the donor or acceptor.

For example, exon 2 of the gene is skipped in cancer cells due to alternative splicing or interference of oligonucleotides, but remains in normal cells. In this example, the normal gene comprising the skipped exon is not expressed in cancer cells, but the protein is expressed in normal cells. Intron sequences flanking exon 2 in this construct can be used to replicate the same splicing pattern in the transgene. Thus, constructs containing the transgene in the typical intron-exon-intron construct that are spliced in normal and tumor cells remain relatively efficiently spliced and produce high levels of fully spliced transcripts (exon 1+2+3, with splicing a + b). In addition, when the oligonucleotide blocks splicing of either intron a or intron b (or both), exon 2 is skipped to generate a transcript that fuses exon 1 to exon 3. Thus, the relative expected abundances of the "theoretically possible" transcripts shown in FIG. 1 are modified in FIG. 2 to show transcripts that may be abundant.

In one embodiment, based on the splicing concept described above, the transgene is converted from a transcript containing exon 1, exon 2 and exon 6 to a transcript containing exon 1 and exon 3 using oligonucleotides and "exon skipping" techniques. Thus, the construct or vector produces a functional polypeptide encoded by a transcript having exon 1 fused to exon 3. In one embodiment, the stop codon is designed to be operably linked to exon 2 such that transcription stops at the stop codon before exon 3 translation without oligonucleotide interference to skip exon 2. Premature stop codons result in non-functional transcripts containing exon 1, exon 2 and exon 3. In one embodiment, stop codons are placed in all three reading frames to ensure complete termination. In this case, the baseline of the non-functional transcript (exon 1+2+ 3) was switched to the functional transcript (exon 1+ 2), as shown in fig. 3, because the ribosome stops the translation of the transcript after the exon 3 stop codon is included.

Construction of such a transgene is quite simple, but an intron-exon STOP-intron structure is chosen that is normally properly "spliced". Intron-exon STOP-introns can be engineered with sequences flanking the normally spliced exons and cloned into specific sites in the transgene to form splice donor and acceptor consensus sequences in the flanking bases. Since exons of sequences extracted from normal genes will also be skipped, exons should be carefully selected. For example, an intron-exon-intron may be from a spliced DNA viral gene, in which case normal cellular genes would not be expected to be affected. Alternatively, if the gene therapy application is directed to cancer, it may be considered to utilize an oncogene intron-exon-intron boundary, which may lead to a defect in the oncogene. This strategy would utilize non-targeted hopping of cellular genes as a potential "extra" therapeutic effect.

Some exons are differentially regulated in cancer cells compared to normal cells. For example, the splicing type 2 exon is "spliced in" in normal cells, but excluded from the same gene in some cancer cells. In this case, if the intron-exon-intron boundary of the splicing type 2 exon is used in the construction (see FIG. 4), the baseline and skipped expression of the transgene will differ in normal and cancer cells, as shown in FIG. 5. Because engineered exon STOPs are not normally present in cancer cells, the transgene will be activated in these cells, but not in normal cells (FIG. 5). Such exons can be used to selectively express transgenes in cancer cells, but cannot be expressed in normal cells even without exon skipping. This may be desirable, for example, for toxin genes or drug precursor enzymes. Skipping exon 2 using the oligonucleotide will activate the transgene in normal cells, and if the baseline skipping in these cells is less than 100%, it is possible to increase expression in cancer cells.

In contrast, the splicing type 3 exon is excluded in normal cells, but is "spliced in" in some cancer cells. Therefore, regulated splicing using such exons would produce a result opposite to splicing type 2, as shown in FIG. 6.

In view of the above, the following strategies have been disclosed for the purpose of down-regulating transgene expression in the context of gene therapy:

strategy # 1: activation of transgene expression is dependent on the administration of exon skipping oligonucleotides. This effect can be seen in both normal and cancer cells.

Strategy # 2: in the absence of any drug or external additive, the activation of transgene expression is generally higher in certain cancer cells (only in certain cancers) and lower in normal cells. This application can be used for cancer-selective expression in cancers with appropriately modulated splicing. In addition to specific cancer cells, administration of exon skipping oligonucleotides will activate expression in normal cells.

Strategy # 3: activation of transgene expression is higher in normal cells but lower in some cancer cells. The expression of these cancer cells can be activated by administration of exon skipping oligonucleotides.

Gene therapy for cancer therapy is a particular application, although the principle of controlled gene expression can be applied to any gene therapy application. In this embodiment, therapeutic agents expressed and secreted in normal cells can be activated as needed by administering appropriate exon skipping oligonucleotides.

For these purposes, the applicant has devised a medicament fromKRASThe intron-exon STOP-intron cassette of exon 1 of the gene, as an example of a strategy #1 construct, is inserted into a transgene of interest to achieve regulatable gene expression. Oligonucleotides that induce exon skipping are selected for therapeutic applications.

On the one hand, the applicant utilizesKRASThe structure of a gene, one of the most common mutant genes in cancer. KRAS is not an enzyme, and has no apparent drug binding pocket on its surface, and therefore cannot be a drug target. Thus, to the best of the applicant's knowledge, this skipping technique is the first approach to target KRAS in cancer. For these purposes, applicants 'constructs utilize a 5' untranslated regionKRASThe ATG initiation codon for protein translation is located at the first exon of (1)Within two exons. (thus, the second exon is commonly referred to as exon 1 and the first exon is referred to as exon 0). An oligonucleotide that induces exon skipping of an ATG-containing exon will result in a transcript that is not translated normally because of the lack of the ATG start codon.

KRAS-based intron-exon STOP-intron

According to the Genbank sequence (NCBI reference sequence: NG-007524.1, last visit date 2.20.2019), the exons of KRAS are located:

4990..5170,10526..10647,28509..28687,30148..30307, 46010..51132, and cDNA added to: 10537..10647,28509..28687,30148..30307,46010..46126.

Is normalKRASThe first ATG of the gene is located at position 10537.

The exons to be skipped are located between 10526 and 10647 (122 bp).

This construct contains a dummy version with a STOP codon in each reading frame.

In one embodiment, forKRAS1 exon, shaded and underlined nucleotides (includingKRASATG) by a stop codon:

exemplary stop codons are: TAA, TAG and TGA. In one aspect, all three reading frames are: taaxtagxgtga, where x is any nucleotide.

In order to design all 3 stop codons twice in succession, a stop codon may be inserted. Furthermore, 11 base pair repeats can be avoided by using the sequence TAAxTAGxTGAxTAAxTGAX (24 bp) (SEQ ID No: 1), where x is any nucleotide. The specific embodiment is as follows: TAATTAGCTGAGTAGATAAGTGAT (SEQ ID No: 2). Thus, one example of a STOP comprising exo kras1 (the STOP codon is underlined) is:

GCCTGCTGAAATAATTAGCTGAGTAGATAAGTGATGTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAG (SEQ ID NO. 18)。

the normal upstream intron is 5171-10525 (5355 bp) of NCBI reference sequence NG _ 007524.1: "gtacg … ataag", wherein the remainder of the upstream intron is shown as "…".

The normal downstream intron is 10648-28508 (17861 bp) of NCBI reference sequence NG _ 007524.1: "gtaaa … ctcag", wherein the remainder of the downstream intron is shown as "…".

In one aspect, approximately 75 base pairs ("bp") on the upstream and downstream ends of the two introns are included. In one embodiment, the upstream intron sequence is as follows:

GTACGGAGCGGACCACCCCTCCTGGGCCCCTGCCCGGGTCCCGACCCTCTTTGCCGGCGCCGGGCGGGGCCGGCGGAGTATTTGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAG (SEQ ID NO. 19)。

in one embodiment, the downstream intron sequence is as follows:

GTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGACCATTTTGAGTTGTATATAACACCTTTTTTGAAGTAAAAGGTGCACTGTAATAATCCAGACTGTGTTTCTCCCTTCTCAG (SEQ ID NO. 20)。

thus, in one embodiment, the following sequences are intron-exon Kras1 STOP-intron sequences (the sequence not highlighted is the upstream intron, the grey sequence is the intron comprising the STOP sequence, and the underlined sequence is the downstream intron):

in one aspect, a series of oligonucleotides spanning the boundaries of exon-downstream intron boundaries can be added because of their ability to cause exon skipping. Exon skipping oligonucleotides are typically 20-30 base pairs, antisense to DNA.

In one embodiment, the morpholino binding site is a Kras1 derived intron-exon STOP-intron morpholino binding site (SEQ ID number 26). The sequence of the K1 Exonsotpintron 3' linker sequence is shown below (exons marked in grey and introns marked underlined):

Applicants created and tested two morpholinyl binding sites, named KTS1 (SEQ ID number 24) and KTS2 (SEQ ID number 25). The KTS1 morpholino sequence comprises the sequence of SEQ ID number 27. And the KTS2 morpholinyl sequence comprises the sequence of SEQ ID number 28. Applicants used KTS2 morpholinyl reverse sequence (SEQ ID number 29) as a negative control.

SEQ ID number 24-KTS 1 (KRAS TransSkip 1) (exon markers in grey, intron underlined)

Morpholinyl of SEQ ID number 27-KTS 1

CAAGATTTACCTCTATTGTTGGATCATATT

SEQ ID NO. 25—KTS2 (KRAS TransSkip 2)

Morpholinyl of SEQ ID number 28-KTS 2

TAAAACAAGATTTACCTCTATTGTTGGATC

Morpholinyl reverse sequence of SEQ ID number 29-KTS 2

CTAGGTTGTTATCTCCATTTAGAACAAAAT。

In some cases, the splice donor sites include a/C AG × GT a/G AGT (SEQ ID NO: 34), wherein "×" indicates the exon-intron boundaries and the sites of insertion of the intron-exon STOP-intron sequences.

In some cases, the splice acceptor comprises (Py) XCAG G/T (SEQ ID NO: 35), wherein "×" indicates an intron-exon boundary.

In certain instances, exemplary splice donor sites for insertion of an intron-exon STOP-intron sequence include AAG-GG (SEQ ID NO: 36), CAG-GG (SEQ ID NO: 37), AAG-GT (SEQ ID NO: 38), or CAG-GT (SEQ ID NO: 39), wherein "-" indicates the insertion site.

CATAAVERTConstruct

To create cancer-targeted modulated AAV expression T: (Cancer Targeted AAVexpressed, regulated TCATAAVERT) a linker (also referred to as "TransSkip") that inserts the intron-exon Kras1 Stop-intron sequence into the coding region at a site that creates a consensus splice donor and acceptor site. In one embodiment, the vector comprising regulated CATAAVERT expresses CD3 and GD 2.

In another embodiment, the vector comprises a sequence encoding secrecon-AA-CD3xGD2-HDD dimer. For this construct, the secrecon-AA-CD3xGD2-HDD dimer sequence contains all potential sites (highlighted in grey) for insertion of the intron-exon Kras1 stop-intron to generate a consensus splice donor/acceptor (the insertion is located after the third of one or more of the 5 bp's) provided below:

in one aspect, the most upstream site is inserted to minimize the length of CD3xGD2 dimer translated before the STOP codon (underlined sequences are splice junctions remaining in mRNA at splicing of the insert, double underlined sequences are splice donor sites at the beginning of the upstream insert sequence, grey sequences are exons containing the STOP sequence, and bold sequences are downstream introns). In some instances, this sequence is referred to as CD3xGD 2K 1 dimer:

In some cases, the upstream splicing factor binding site is modified (modified regions shown in italics and bold). Underlined sequences are splice junctions that remain in the mRNA upon splicing of the insert, double underlined sequences are splice donor sites at the beginning of the upstream intron sequence, gray sequences are exons containing the STOP sequence, and bold sequences are the downstream introns. In some instances, this sequence is referred to as CD3xGD 2K 2 dimer:

in certain instances, the CD3xGD2 dimert construct comprises one or more additional modifications in the polynucleotide sequence. In some cases, the sequence of the CD3xGD2 dimert construct sec2A-CD3xGD2-HDD-K3, sec2A-CD3xGD2-HDD-K3 is shown in SEQ ID NO: 41. Underlined sequences are splice junctions that remain in the mRNA upon splicing of introns, double underlined sequences are splice donor sites at the beginning of upstream insert sequences, grey sequences are exons containing the STOP sequences, and bold sequences are downstream introns.

SEQ ID NO: 41

In some cases, the sequence of the CD3xGD2 dimert construct sec2A-CD3xGD2-HDD-K4, sec2A-CD3xGD2-HDD-K4 is shown in SEQ ID NO: 42. Underlined sequences are splice junctions that remain in the mRNA upon splicing of the intron, double underlined sequences are splice donor sites at the beginning of the upstream intron sequence, gray sequences are exons containing the STOP sequence, and bold sequences are the downstream introns.

SEQ ID NO: 42

In some cases, the sequence of the CD3xGD2 dimert construct sec2A-CD3xGD2-HDD-K5, sec2A-CD3xGD2-HDD-K5 is shown in SEQ ID NO 43. Underlined sequences are splice junctions that remain in the mRNA upon splicing of the intron, double underlined sequences are splice donor sites at the beginning of upstream intron sequences, grey sequences are exons containing termination sequences, and bold sequences are downstream introns.

SEQ ID NO: 43

In some embodiments, the dimer described herein comprises a CD3 sequence, a CD19 sequence, and optionally a secrecon sequence. In some cases, dimert comprises the CD3xCD19 construct as set forth in SEQ ID NO:44, highlighting all potential sites for insertion of the intron-exon-Kras 1 stop-intron sequence to create a consensus splice donor/acceptor site. Each potential insertion site is highlighted in gray.

SEQ ID NO: 44

In some cases, the insertion is made to the most upstream site. In some cases, dimer further comprises a secrecon sequence. In some cases, the CD3xCD19 construct is sec1A-CD3xCD19-K1, the sequence of which is set forth in SEQ ID NO: 45. As shown below, underlined sequences are splice junctions that remain in the mRNA when introns are spliced, italicized and bold sequences are upstream intron sequences, gray sequences are exons containing the STOP sequence, and bold sequences are downstream introns.

SEQ ID NO: 45

In some cases, the CD3xCD19 construct is sec1A-CD3xCD19-K3, the sequence of which is set forth in SEQ ID NO: 46. As shown below, underlined sequences are splice junctions remaining in the mRNA when introns are spliced, italicized and bold regions are modified regions of the upstream intron binding site, gray sequences are exons containing the STOP sequence, and bold sequences are downstream introns.

SEQ ID NO: 46

Examples

These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

Example 1 Generation of CD3xGD2-HDD Dimert Using an exemplary CD3xGD2-HDD TransJoin

Maps of exemplary AAV constructs used to test CD3xGD2-HDD expression.

As proof of principle, the previously described bispecific molecule was expressed that targets human CD3 on T cells and the bis-sialylganglioside GD2 on neuroblastoma and other types of cancer cells. The bispecific protein has the amino acid sequences for the heavy and light chain variable regions of human CD3 derived from clone OKT3 (using single chain variable fragment format scFv) fused via a short linker (L) to the scFv construct for GD2 derived from clone 5F11, linked as a dimer via HNF1a dimerization domain (HDD), such as Ahmed et al., OncoImmunology4:4, e989776, DOI 10.4161/2162402 X.2014.989776. The optimized human DNA coding sequence for CD3xGD2-HDD was reverse engineered using vectorbiuder.com codon optimization tool (//en. vectorbiuder.com/tool/code-optimization. html). The resulting DNA sequence starting with the ATG start codon was synthesized and cloned into an adeno-associated virus expression cassette downstream of the chicken actin b globin promoter (CAGp) with an inverted terminal repeat from AAV 2. Based on the discovery that alanine enhances secretion according to protein (Gu ̈ ler-Gane)et al., PLoS ONE11(5) e0155340. doi 10.1371/journal. bone.0155340), three other versions were made which contained a common secretion signal domain ("secrecon", based on Barash @)et al., Biochem Biophys Res Commun2002; 294: 835-842) downstream of the ATG start site and ending with 0, 1 or 2 alanine. Proteins derived from these constructs are called heterodimeric scfvs or Dimert. FIG. 7 illustrates 4 exemplary CD3xGD2-HDD dimer constructs.

And (5) determining the structure and the function of the Dimert.

293T cells were transduced with AAV expression vectors (or controls) and supernatants were collected and stored. Supernatants were tested for the presence of correctly sized proteins on electrophoresis (SDS-PAGE), bound to CD3 as determined by binding competition using flow cytometry, T cells were activated by flow cytometry, and tumor cells were killed upon co-incubation with T cells. See fig. 8, which shows a cartoon of an assay for determining the structure and function of a dimert as disclosed herein.

The three constructs containing the secretory peptide showed less retention of CD3xGD2-HDD Dimert in the cells.

Whole cell lysates of 293T cells transfected with different AAV CD3xGD2-HDD expression plasmids were collected 48 hours after transfection. Polyacrylamide electrophoresis (PAGE) was performed using 50ug of total protein per lane and the gel was stained with ponceau S. The results show that there is more protein using construct #1104 lacking the secretory sequence, while all 3 constructs with the secretory sequence show less protein. Controls included cells alone or cells transfected with a control plasmid (pcDNA 3-GFP). KDa, kilodalton; sec, secretory domain; GFP, green fluorescent protein; MW, molecular weight. See fig. 9.

Only supernatants from cells transfected with AAV CD3xGD2-HDD constructs containing secretory sequences bound and activated T cells.

The supernatants were tested for binding and activation of human T cells. Binding was determined by competition with fluorescently labeled anti-CD 3 antibody that was previously bound to t (jurkat) cells. Stained cells were 77.6%, 79.24%, and 78.7% (Q2 + Q3) CD3 positive in the control group (DMEM, control, GFP), respectively, and 77.7% in AAV vector #1104 lacking the secretory peptide. In contrast, binding of fluorescently labeled anti-CD 3 antibody was reduced to 2.15%, 3.67%, and 3.99% (Q2 + Q3) by the supernatant of cells transfected with each vector containing secretory peptide. In addition, cells were co-stained with anti-CD 69 as a marker of T cell activation. Both control and AAV vector #1104 showed less than 13.4% positive for CD69 (Q1 + Q2), while three different AAV vectors containing secretory peptides showed 58.87-68.5% positive for CD 69. See fig. 10.

Details of the method: 400ul of Jurkat 2e5 cells/well in RPMI +10% FBS were seeded in 24-well plates and then 100ul of supernatant from 293T transfected cells (20% of total culture) was added to each well. After 4 hours of incubation, Jurkat cells were centrifuged, washed 1 time with PBS, and then stained on ice for 30 minutes with PE-anti-hCD 69 (1: 100) and PerCP-anti-hCD 3e (1: 300 # OKT 3). After washing with FACS buffer, each sample was fixed in 1% PFA and analyzed by flow cytometry. DMEM is medium added to Jurkat alone, controls are supernatant added from untransfected 293T cells, and GFP is supernatant added from 293T cells transfected with AAV plasmids expressing GFP.

Only supernatants from cells transfected with AAV vectors containing secretory peptides activate human T cells.

Human T (jurkat) cells were incubated with supernatants of 293T cells transfected with various AAV vectors, stained with PE-labeled antibody for CD69, and examined under a fluorescent microscope. See fig. 11, where the left panel is phase contrast and the right panel is fluorescence. Neither the EGFP vector control nor the secreted peptide-lacking vector #1104 showed positive staining, while the other three vectors showed high levels of staining (red). Details of the method: 400ul of Jurkat 2e5 cells/well in RPMI +10% FBS were seeded into 24-well plates and 100ul of supernatant from 293T transfected cells (20% of total culture) was added to each well. After 48 hours of incubation, Jurkat cells were centrifuged, washed 1 time with PBS, and then stained with Phycoerythrin (PE) -anti-hCD 69 (1: 100) on ice for 30 minutes. After washing with FACS buffer, each sample was fixed in 1% PFA for 5 minutes. Scale bar: 100 um.

CD3xGD2-HDDBinding to T cells is dose dependent.

Different amounts of supernatants from 293T cells transfected with different AAV plasmids containing secretory peptides were incubated and tested for competition with Peridinin chlorophyll protein complex (PerCP) -conjugated anti-hCD 3 on Jurkat T cells. See fig. 12A, where the left panel shows FACS plots and the right panel shows median staining level numbers. The grey shades in the left panel were unstained and the dark grey lines in each panel represent the most stained cells without supernatant added. Figure 12B shows a bar graph of median staining levels normalized to unstained controls.

Details of the method: jurkat 2e5 cells/well in 400ul RPMI +10% FBS were seeded into 24-well plates, and then 10ul, 25ul, 50ul or 100ul of supernatant from 293T transfected cells (2-20% of total culture) was added to each well. After 24 hours of incubation, Jurkat cells were centrifuged, washed 1 time with PBS, and then stained with PerCP-anti-hCD 3e (1: 300 # OKT 3) on ice for 30 minutes. After washing with FACS buffer, each sample was fixed in 1% PFA for 5 minutes and then analyzed by flow cytometry. Sec = secretory peptide, a = alanine.

CD3xGD2-HDD DimerBinding assays against GD2 arm and confirmed that GD2 and CD3 binding were both present on a single molecule.

The binding of CD3xGD2-HDD to GD2 was measured indirectly by first incubating supernatants from 293T transfected cells with GD2 positive or GD2 negative cells, and then repeating the CD 3T cell binding competition assay. Proteins that bind GD2 should be taken up by GD2 positive cells but not by GD2 negative cells, resulting in loss of competition for T cell binding. The assay was designed to confirm that GD2 binding was co-associated with CD3 binding, in other words, the single molecule was bispecific. See FIG. 13, cartoon of binding assays.

CD3xGD2-HDDBinds to CD3 and GD 2.

As shown in FIG. 13, supernatants from 293T cells transfected with #1101 CD3xGD2-HDD AAV vector were collected and pre-incubated with GD2 positive SK-N-Be (2) or GD2 negative Raji cells. After spinning and washing, CD 3t (jurkat) cells were subjected to a binding competition assay.

FIG. 14 illustrates an exemplary CD3xGD2-HDD Dimert in conjunction with both CD3 and GD 2. The grey shading in the top panel with black outline is unstained Jurkat T cells and the dark grey line is Jurkat T cells that were fully stained for CD3 without the addition of supernatant. Sample Jurkat _ CD3e showed fully competitive CD3 binding using supernatant without pre-incubation, almost overlapping with supernatant pre-incubated with GD2 negative Raji cells. In contrast, sample Jurkat _ CD3e sknbe2 exhibited a loss of competition when the supernatant was preincubated with GD2 positive SK-N-Be (2) neuroblastoma cells, confirming that the same molecule binds to CD3 and GD 2.

Test to determine whether CD3xGD2-HDD induces T cells to kill GD2+ target cells.

FIG. 15 illustrates a flowchart of an assay to determine whether CD3xGD2-HDD induces killing of G2+ target cells by T cells. As shown in fig. 15, GD2+ neuroblastoma (SK-N-Be (2)) cells were seeded into wells, followed by primary human T cells (purchased from StemExpress) and supernatant from AAV vector-transfected 293T cells. Cell viability was assessed using CellTiter-Glo from Promega (Madison, Wis.).

Secreted CD3xGD2-HDD induced T cell killing of neuroblastoma cells.

The cytotoxicity of human T cells plus supernatant (from 293T cells transfected with various AAV vector plasmids) was tested on GD2+ SK-N-Be (2) neuroblastoma cells using the assay shown in fig. 15. A T cell to target cell ratio of 10:1 was used. Cell viability was determined after 48 hours of co-culture. The supernatant from cells transfected with constructs expressing unrelated Dimert (CD 19xCD 3) or a vector lacking the secretory peptide (# 1104) was not significantly cytotoxic compared to the supernatant from untransfected cells ("Dimert-free") (fig. 16). In contrast, supernatants from cells transfected with each vector containing the secretory peptide induced statistically significant cytotoxicity (p < 0.001), killing 25-30% of the cells (fig. 16).

TCell-mediated cytotoxicity of CD3xGD2-HDD dimer was associated with GD2 expression.

A panel of neuroblastoma cell lines were tested for sensitivity to human T cell killing, and supernatants from AAV vector #1101 (upper panel of fig. 17) were bound and their GD2 expression was measured by flow cytometry (lower panel of fig. 17, shaded curve is isotype control). CHP-134 cells showed the greatest cytotoxicity and the highest GD2 expression.

Example 2 Generation of CD19xCD3 Dimert Using an exemplary CD19xCD3 TransJoin

Maps of exemplary AAV constructs used to test CD19xCD3 Dimert expression.

FDA approved protein therapeutics, known as bornatemumab (blinatumomab), are a so-called bispecific T-cell engager (BiTE) used to target human CD19 on B-cell/B-cell malignancies and human CD3 on T-cells. The optimal human DNA coding sequence for CD19xCD3 was reverse engineered using the publicly available bornauzumab amino acid sequence (//www.drugbank.ca/drugs/DB 09052) and further using vectorbiurer com codon optimization tool (//en. vectorbiurer. com/tool/codon-optimization. html). The resulting DNA sequence starting with the ATG start codon was synthesized and cloned into an adeno-associated virus expression cassette downstream of the chicken-actin-b-globin promoter (CAGp), with an inverted terminal repeat from AAV 2. Based on the discovery that alanine enhances secretion according to protein (Gu ̈ ler-Gane) et al., PLoS ONE11(5) e0155340. doi 10.1371/journal. bone.0155340), three other versions were prepared which contained a common secretion signal domain ("secrecon", based on Barash @)et al., Biochem Biophys Res Commun2002; 294: 835-842) downstream of the ATG start site and ending with 0, 1 or 2 alanine. Proteins derived from these constructs are called heterodimeric scfvs or Dimert. Fig. 18A and 18B illustrate 5 exemplary CD19xCD3 constructs.

FIG. 18C illustrates a cartoon representation of the interaction of CD19 dimer with cancer cells and T cells. CD19 dimer was produced by cells containing CD19 transaxin. When administered to a subject, e.g., intravenously, AAV transaxin (e.g., AAV CD19 transaxin as shown in this figure) enters normal cells (e.g., liver or muscle) and expresses the polypeptide encoded thereby. The secretory signal peptide is cleaved during secretion, leaving an active dimer, which binds to cancer cells (Ca) at one end and T immune cells at the other end.

Only the supernatant of cells transfected with the AAV CD19xCD2 construct containing the secretory sequence bound and activated T cells.

The supernatants were tested for binding and activation of human T cells. Binding was determined by competition with a fluorescently labeled anti-CD 3 antibody that previously bound to human t (jurkat) cells. Stained cells showed 77.6%, 79.24% and 78.7% (Q2 + Q3) CD3 positivity in the control group (DMEM, control, GFP), respectively, and 79.4% in vector #1323 lacking secretory peptide. In contrast, binding of fluorescently labeled anti-CD 3 antibody was reduced to 23.88%, 18.59%, and 30.76% (Q2 + Q3) by the supernatant of cells transfected with each secretory peptide-containing vector. In addition, cells were co-stained with anti-CD 69 as a marker of T cell activation. Both control and #1323 showed less than 13.38% positive for CD69 (Q1 + Q2), while three different secretory peptide-containing vectors showed 63.9%, 67.3% and 66.6% positive for CD69 (fig. 19).

Details of the method: 400ul of Jurkat 2e5 cells/well in RPMI +10% FBS were seeded in 24-well plates and then 100ul of supernatant from 293T transfected cells (20% of total culture) was added to each well. After 48 hours of incubation, Jurkat cells were centrifuged, washed 1 time with PBS, and then stained on ice for 30 minutes with PE-anti-hCD 69 (1: 100) and PerCP-anti-hCD 3e (1: 300 # OKT 3). After washing with FACS buffer, each sample was fixed in 1% PFA and analyzed by flow cytometry. DMEM is medium added to Jurkat alone, controls are supernatant added from untransfected 293T cells, and GFP is supernatant added from 293T cells transfected with AAV plasmids expressing GFP.

Only supernatants from cells transfected with AAV vectors containing secretory peptides activate human T cells.

Human T (jurkat) cells were incubated with supernatants of 293T cells transfected with various AAV vectors, stained with PE-labeled antibody for CD69, and examined under a fluorescent microscope. See fig. 20, left panel is phase contrast and right panel is fluorescence. Neither EGFP vector control nor AAV vector #1323 lacking secretory peptide showed positive staining, while the other three vectors showed high levels of staining (red).

Details of the method: 400ul of Jurkat 2e5 cells/well in RPMI +10% FBS were seeded in 24-well plates and then 100ul of supernatant from 293T transfected cells (20% of total culture) was added to each well. After 48 hours of incubation, Jurkat cells were centrifuged, washed 1 time with PBS, and then stained with Phycoerythrin (PE) -anti-hCD 69 (1: 100) on ice for 30 minutes. After washing with FACS buffer, each sample was fixed in 1% PFA for 5 minutes. Scale bar: 100 um.

AAVSecreted CD19xCD3 specifically binds to CD19 but not to CD 45.

Binding competition assays were used to determine whether supernatant from AAV vector-transfected 293T cell supernatant would interfere with staining of epstein-barr virus (EBV) -transformed human B cells with two different antibodies, one staining for the B cell marker CD19 and the other staining for the leukocyte marker CD 45.

See fig. 21, where cells in 3 control groups (DMEM, control, GFP) showed 93%, 93.1%, and 93.2% positive staining of CD19 (Q1 + Q2), respectively, while cell supernatants transfected with AAV vector #1323 lacking secretory peptide did not compete for staining, showing 93.2% positive for CD 19. In contrast, each AAV vector containing a secretory peptide competed for CD19 signaling to decrease to 33.33%, 31.07%, and 35.88%. Note that the cut-off was set so that unstained cells showed 14.58% positive CD19, indicating that the supernatant competition from cells transfected with these three AAV vectors was reduced to 2-fold over background. In contrast, none of the vectors competed for CD45 staining, was 78.89%, 79.14% and 78.32% positive in 3 controls (Q2 + Q3), respectively, and 82.9% in the vector lacking the secretory peptide, while the supernatants used from cells transfected with the other three vectors were 80.5%, 78.5% and 79.4% positive.

Details of the method: b cells were transformed with EBV called NB122R (Gene Ther, 2013 Jul;20(7):761-9. doi: 10.1038/gt.2012.93) at 2e5 cells/well in 400ul RPMI +10% FBS, seeded in 24-well plates and 100ul 293T transfected cell supernatant (2-20% of total culture) was added per well. After 24 hours of incubation, the cells were centrifuged, washed 1 time with PBS, and then stained with PEcy 7-anti-hCD 45 (1: 100) and APC-anti-hCD 19 (1: 300) on ice for 30 minutes. After washing with FACS buffer, each sample was fixed in 1% PFA for 5 minutes before flow cytometry analysis. DMEM is medium added to Jurkat alone, controls are supernatant added from untransfected 293T cells, and GFP is supernatant added from 293T cells transfected with AAV plasmids expressing GFP.

CD19xCD3Binding to T cells is dose dependent and vectors containing a single alanine downstream of the secretory peptide consensus sequence are superior to other test vectors.

Different amounts of supernatants from 293T cells transfected with different AAV plasmids containing secretory peptides were incubated and tested for competition with Peridinin chlorophyll protein complex (PerCP) -conjugated anti-hCD 3 on Jurkat T cells. See fig. 22A, where the left panel shows FACS plots and the right panel shows median staining level numbers. The grey shading in each left panel is unstained cells and the dark grey line in each left panel is the largest stained cell to which supernatant was not added. Figure 22B shows a bar graph normalized to the median staining level of the unstained control. Vector #1325, which had a single alanine downstream of the secretory peptide consensus sequence, exhibited the greatest competition compared to the other test vectors and was selected for further experiments.

Details of the method: jurkat 2e5 cells/well in 400ul RPMI +10% FBS were seeded into 24-well plates, and then 10ul, 25ul, 50ul or 100ul of supernatant from 293T transfected cells (2-20% of total culture) was added to each well. After 24 hours of incubation, Jurkat cells were centrifuged, washed 1 time with PBS, and then stained with PerCP-anti-hCD 3e (1: 300 # OKT 3) on ice for 30 minutes. After washing with FACS buffer, each sample was fixed in 1% PFA for 5 minutes and then analyzed by flow cytometry. MFI = mean fluorescence intensity, Sec = secretory peptide, a = alanine.

CD19xCD3Binding to B cells is dose dependent and vectors containing a single alanine downstream of the secretory peptide consensus sequence are superior to other test vectors.

Different amounts of supernatants from 293T cells transfected with different AAV plasmids containing secretory peptides were incubated and tested for competition with Allophycocyanin (APC) -conjugated anti-hCD 19 on human B cells. See fig. 23A, where the left panel shows FACS plots and the right panel shows median staining level numbers. The grey shades in each left panel were unstained and the dark grey lines in each left panel were the most stained cells without supernatant added. Figure 23B shows a bar graph of median staining levels normalized to unstained controls. Vector #1325, which had a single alanine at the end of the secretory peptide, showed the strongest competition, consistent with the T cell binding results shown in fig. 22A and 22B, and was selected for further experiments.

Details of the method: NB122R human B cells in 400ul RPMI +10% FBS were seeded into 24-well plates at 2e5 cells/well, and then 10ul, 25ul, 50ul or 100ul of supernatant from 293T transfected cells (2-20% of total culture) were added to each well. After 24 hours of incubation, cells were centrifuged, washed 1 time with PBS, and then stained with APC-anti-hCD 19 (1: 300) on ice for 30 minutes. After washing with FACS buffer, each sample was fixed in 1% PFA for 5 minutes and then analyzed by flow cytometry. MFI = mean fluorescence intensity, Sec = secretory peptide, a = alanine.

CD3 DimertCo-stimulation of T cells by anti-CD 28 was better than the anti-CD 3 antibody.

Human T (jurkat) cells were incubated with either anti-CD 28 antibody and the supernatant derived from AAV vector-transfected 293T cells (left panel of fig. 24) or increased concentrations of anti-CD 3 antibody (right panel of fig. 24). Cellular mRNA was harvested and IL-2 (upper panel of FIG. 24) and IL-8 (lower panel of FIG. 24) mRNA were subjected to quantitative reverse transcription polymerase chain reaction (RT-PCR) and expression relative to housekeeping mRNA GAPDH was calculated. Only supernatants from cells transfected with AAV vector constructs containing the secretory domain showed stimulation of either gene above control (gfp, 293 t) and above the vector construct lacking the secretory domain (# 1104 for CD3xGD2-HDD and #1323 for CD19xCD 3).

293TThe cells produced the highest transduction efficiency for AAV 8.

Four different cell lines were infected with 2 different concentrations of AAV8 expressing Green Fluorescent Protein (GFP) at multiplicity of infection (MOI) =1e4 or 1e5 genomic copies (gc) in a medium containing 2% fetal bovine serum. GFP signal was assessed by fluorescence microscopy 48 hours after virus infection. See fig. 25. AML-12: normal mouse hepatocytes; 293T: human embryonic kidney cells transformed with SV40-T antigen; H441-CRM: human lung cancer cells having a KRAS mutation; SK-N-Be (2): NMYC expanded human neuroblastoma cells.

AAV8The Dimert concentration in the supernatant of infected cells depends on AAV dose and transduction efficiency.

Supernatants from 293T and H441 cells transfected with either TransJoin vector or AAV-GFP controls were subjected to a T (Jurkat) binding assay. See fig. 26, where the grey shading in the panels is unstained control cells and the dark grey lines in each panel are T cells fully stained with anti-CD 3 antibody. Although CD19xCD3 dimer appears to be much more effective in competing for binding than CD3xGD2-HDD dimer, this effect is dose-dependent (curve moves to the left as MOI increases) and is not observed in cell lines with poor AAV8 transduction efficiency (H441 cells). MOI, multiplicity of infection.

A single intravenous injection of CD19xCD3 TransJoin selectively eliminated B cells from humanized mice.

Immunodeficient mice (NSG-SGM 3, Jackson Labs) were purchased and irradiated and injected intravenously with human CD34+ hematopoietic stem cells. By 12 weeks, mice were shown to be engrafted with human blood cells, including T and B lymphocytes, by flow cytometry of peripheral blood. A single injection of CD19xCD3 TransJoin (AAV 8-Sec1A-CAG-193, vector #1325 packaged into AAV8 capsid) was administered at different doses in mice and blood of human lymphocytes collected at different time points was analyzed by flow cytometry, as shown in figure 27. While low doses (5 e9 and 5e10 vector genomes (vg) per kilogram (kg) body weight) had no effect, higher doses (5 e11 and 5e12 vg/kg) were sufficient to eliminate circulating B cells without affecting CD4+ or CD8+ T cells.

A single intravenous injection of CD19xCD3 TransJoin resulted in an extended time of B cell depletion in the humanized mice.

Humanized mice were treated with a single dose of CD19xCD3 AAV8 transajoin and lymphocyte subpopulations in blood were subsequently measured. As shown in figure 28, for a single mouse, prolonged and selective B cell depletion was observed in all mice analyzed, as long as these mice were still alive.

A single intravenous injection of CD19xCD3 TransJoin abolished CD19+ lymphoma in humanized mice.

Human CD19+ Raji cells (from patients with burkitt's lymphoma) were implanted into the flanks of two humanized mice and allowed to reach more than 250 mm³The size of (2). The tail vein of one animal was then injected with control AAV virus AAVGFP and the tail vein of a second animal was injected with CD19xCD3 AAV8 transoxin. Tumors in control mice eventually grow rapidly, requiring euthanasia of the animals. Tumors in the TransJoin treated mice grew slowly and eventually shrank almost completely. See fig. 29. Animals need to be sacrificed due to symptoms of graft versus host disease, a known result of humanized mice, but this experiment demonstrates proof of principle that a single TransJoin injection can treat cancer.

Example 3-use of OncoSkip and TransSkip to simultaneously target oncogene expression and activate therapeutic transgene expression

Overview of OncoSkip and Transskip described herein.

OncoSkip is an antisense morpholino that induces exon skipping in oncogenes. In some embodiments, the OncoSkip is designed as an antisense morpholinyl that induces exon skipping of a key exon in an oncogene (left side of figure 30). For proof of principle, KRAS (exons 1, 2, 3, indicated by black-gold-grey on the left of figure 30) was used and morpholinyl designed to skip KRAS exon 2 (shown as the light grey bar on top of the oncogene in figure 30) was tested, which contains the ATG start site, so normal expression of the oncogene would be reduced. Derivatives of the same exon to be skipped are then created, but mutated to include multiple stop codons in each reading frame, as well as flanking intron sequences. A new intron-exon (STOP) -intron is then inserted into the transgene coding sequence at a site that reconstitutes the donor and acceptor splice sites, splicing it into the transgene mRNA and disrupting the normal coding sequence. In the presence of antisense morpholinyl (light grey bars on top of oncogenes), the newly inserted exon is skipped, rejoining the coding sequence to generate a functional product. A class of AAV vectors containing genes interrupted by intron-exon-intron sequences is known as TransSkip viruses, while morpholinyl designed to down-regulate oncogenes is known as OncoSkip.

KRAS OncoSkipAntisense morpholinyl induces exon skipping of endogenous KRAS in lung cancer cells.

Lung cancer cell lines a549 and H441 (not shown) were incubated with KTS1 and KTS2 OncoSkip antisense morpholinyl and the reverse control morpholinyl of KTS 2. Both KTS1 and KTS2 bind at the 3-prime (prime) end of the KRAS exon 2 exon-intron junction and are designed to induce exon 2 skipping since it contains the ATG initiation site. Endogenous KRAS mRNA was then analyzed by reverse transcriptase RT-PCR for the presence of exon 2 by PCR using primers present in exon 1 (forward) and exon 2 (reverse). Primers in exon 4 were used as controls for total KRAS mRNA. Dose-dependent reductions in transcripts containing exon 2 ("exon 1+ 2") were observed for both OncoSkip morpholinyl groups. See fig. 31.

CD3xGD2-HDD TransSkipThe AAV vector map of (a) shows that the reverse engineered intron is flanked by exons inserted into the CD3xGD2-HDD Dimert coding sequence.

The 3-priming sequence of the human intron (Ki 1) upstream of KRAS exon 2 (a derivative of KRAS exon 2 containing mutations to multiple STOP codons (STOP) in all three reading frames) and the 5-priming sequence of the human intron (Ki) downstream of KRAS exon 2 were synthesized and this cassette was cloned into the gene sequence encoding GD2 Dimert, a specific sequence at the repeated splice donor and acceptor sites. See fig. 32. When a new STOP exon is spliced into the transcript, no functional Dimert is produced. When cells were exposed to antisense morpholinyl binding to the 3-prime exon-intron junction, the STOP exon was skipped and full length Dimert mRNA was expressed. The KRAS sequence was selected such that antisense morpholinyl designed to cause exon 2 skipping simultaneously altered mRNA splicing of the nsskip transgene, resulting in expression of full-length Dimert in the transduced cell, and reduced or eliminated native KRAS expression in the cancer cell, since exon 2 contains the KRAS ATG start codon.

Exemplary strategies for testing the activity of OncoSkip and Transskip.

As shown in fig. 33, cell pellets and supernatants were collected from 293T cells transduced with AAV Dimert vectors. mRNA was isolated from the cell pellet and subjected to reverse transcriptase RT-PCR to determine the extent to which the artificial exon in the transgene spliced into mRNA. Supernatants from Dimert expression were analyzed by T cell binding and killing assays.

The antisense morpholinyl induces exon skipping of the CD3xGD2-HDD TransSkip transgene.

293T cells were transfected with CD3xGD2-HDD Dimert plasmid # 1042. Cells were harvested 48 hours post transfection for total RNA isolation. Approximately 1ug of RNA was used for RT-PCR. See FIG. 34, where lane #9 shows full-length transgenic RNA with no splicing between primers, using DNA plasmid as template. Lanes 7 and 8 and the control lane are free of antisense morpholinyl and show that most transcripts include internal exons (striped rectangles). There were some transcripts that did not include exons (minimal bands), indicating that there was some "leakage" of activating transcripts for this construct. Addition of either morpholino group (KTS 1, KTS 2) reduced the proportion of inactivated exon-containing transcripts (219 bp) and increased activation transcripts (97 bp) excluding exons in a dose-dependent manner.

KRAS OncoSkipSecretory expression of CD3xGD2-HDD Dimert was induced in cells transfected with CD3xGD2-HDD TransSkip AAV vector.

Supernatants were collected from transfected 293T cells and tested for T cell binding (interference of fluorescently labeled anti-CD 3 antibody). As shown in fig. 35, the gray line in the top panel is unstained T (jurkat) cells and the dark gray line in the top panel is fully stained T cells with anti-CD 3 antibody. As a positive control, the supernatant of cells transfected with constitutively expressed CD3xGD2-HDD Transjoin #1011 almost completely eliminated anti-CD 3 staining. Supernatants from cells transfected with CD3xGD2-HDD TransSkip #1042 showed moderate competition, consistent with some "leakage" of exon-skipped Dimert mRNA, and were further induced by OncoSkip morpholinyl (KTS 1, KTS 2).

KRAS OncoSkipExon skipping on CD3xGD2-HDD TransSkip is an on-target effect.

Exon skipping induced by the targeted antisense morpholinyl KTS2 was compared to morpholinyl containing the same bases but of the opposite sequence (fig. 36). KTS2 induced exon skipping to almost 100% of transcripts (97 bp), while the reverse control morpholinyl had no effect compared to the internal transporter only (vector for morpholinyl) control.

Induction of CD3xGD2-HDD Dimert expression as determined by T cell binding is an on-target effect.

Supernatants from 293T cells transfected with CD3xGD2-HDD TransJoin (positive control) and CD3xGD2-HDD TransSkip without or incubated with different antisense morpholinyl were collected and human T (Jurkat) cell binding assays were performed by flow cytometry (competition for binding of fluorescently labeled anti-CD 3 antibody). See fig. 37, where the gray lines in the top panel are unstained T cells and the dark gray lines in the top panel are fully stained T cells. The line indicating #1101 CD3xGD2-HDD TransJoin is almost a complete competitive signal from constitutively expressed GD3 TransJoin. As before, supernatants from CD3xGD2-HDD TransSkip transfected cells showed slight competition at baseline (# 1042 CD3xGD2-HDD TransSkip without morpholinyl) which was not altered by the control morpholinyl (CD 3xGD2-HDD TransSkip + "KTS 2-reverse control") but was induced to compete more by the medium target "OncoSkip" morpholinyl KTS2 (CD 3xGD2-HDD TransSkip + KTS 2).

AAV genomic maps of exemplary nsskip splice variants were created and tested to reduce baseline nsskip "leakage" but maintain inducible exon skipping.

Splice variants of TransSkip were designed to reduce the baseline exon skipping observed with CD3xGD2-HDD TransSkip. Woodchuck hepatitis virus post-transcriptional regulatory elements (WPRE) downstream of the coding sequence are used to increase transgene expression. Due to AAV packaging size limitations, it is desirable to use shorter promoters to contain the WPRE sequences, thus creating a series of new vectors whose promoters are derived from the short form of the human eukaryotic translation elongation factor 1 α 1 promoter (EFSp). These variants were created by altering specific base pairs in the binding site for the U2 cofactor (U2 AF) located in the poly-pyrimidine orbital at the 3-priming end of the first intron (Ki 1-5). See fig. 38.

CD3xGD2-HDD TransSkipThe splice variant K3 eliminated the baseline, but still maintained inducible exon skipping.

293T cells were transfected with the new 5 different CD3xGD2-HDD TransSkip constructs in the absence and presence of the KTS2 antisense "OncoSkip" morpholinyl and compared to the "internal transporter only" control with a morpholinyl vector but no morpholinyl groups. mRNA transcripts were analyzed by reverse transcriptase RT-PCR and found to be of different phenotypes. As shown in fig. 39, variant K4 showed similar (or even slightly more) baseline exon skipping levels as K1 and could induce skipping even beyond K1. The variants K2, K3 and K5 showed virtually no baseline jump. Of these 3, the variant K3 was most induced to skip exons.

CD3xGD2-HDD TransSkipVariant K3 showed no baseline Dimert production, but was most easily induced by KRAS OncoSkip.

The CD3xGD2-HDD transajoin splice variant panel was tested for production of secreted CD3xGD2-HDD Dimert expression without or with KRAS KTS2 OncoSkip antisense morpholinyl using a t (jurkat) cell binding assay (far right). As shown in FIG. 40, the gray line in the right panel is unstained T cells, dark gray is fully stained T cells, blue is constitutively expressed CD3xGD2-HDD TransJoin, green is the corresponding CD3xGD2-HDD TransSkip without OncoSkip ("internal transporter only"). This sequence shows a polypyrimidine U2AF binding site, with the base pair variants designed in gray (underlined and italicized) relative to the wild-type KRAS intron sequence (K1). The top band in the RT-PCR gel is the transcript containing the exon and the bottom band is the transcript with the exon skipped. Variants K1 and K4 showed some detectable baseline exon skipping and labeled baseline Dimert expression by RT-PCR (green, rightmost), while variants K2, K3 and K5 showed no baseline exon skipping by RT-PCR and did not show Dimert expression by binding competition (green, rightmost). All three variants showed a small amount of inducible exon skipping by RT-PCR expression in the presence of KRAS antisense morpholinyl OncoSkip KTS2 and a different degree of dose-dependent Dimert expression in the presence of KRAS OncoSkip (light purple and dark purple represent lower and higher concentrations, respectively). The K3 variant appeared to be most inducible, so K3 was chosen as the lead construct for further studies.

OncoSkipThe mediated induction of Dimert expression of CD3xGD2-HDD transcskip variants K3 and K5 is an on-target effect.

The RT-PCR and T (Jurkat) binding assays were repeated with CD3xGD2-HDD TransSkip variants K2, K3 and K5 and included "reverse KTS 2" antisense morpholinyl as controls. As shown in FIG. 41, inverted KTS2 contains the same nucleotide bases as KTS2, but in the opposite order. The reverse KTS2 failed to induce exon skipping in K3 and K5 (lower band on the gel), while the correct KTS2 sequence induced exon skipping in K3 and K5 (not induced in the K2 variant). In the Dimert-expressing T cell binding assay (right-most panel), gray is unstained T cells, dark gray is fully stained T cells, blue is constitutively expressed CD3xGD2-HDD transajoin, purple is KRAS OncoSkip KTS2 (light color is lower dose, dark color is higher dose), yellow is reverse KTS2 control. The detection of the Dimert protein is consistent with RT-PCR; none of the morpholinyl groups induced Dimert expression from the K2 variant, only KTS2 OncoSkip and not the reverse control induced Dimert expression of the K3 and K5 variants.

The OncoSkip-induced secretion of Dimert from the AAV CD3xGD2-HDD fransskip vector plays a role in mediating T cell killing of neuroblastoma cells.

Human T cells plus supernatants from 293T cells transfected with various AAV CD3xGD2-HDD TransSkip variants were tested for cytotoxicity against GDS2+ SK-N-Be (2) neuroblastoma cells in the absence or presence of KRAS OncoSkip KTS2 (FIG. 42). A T cell to target cell ratio of 10:1 was used. Cell viability was determined after 48 hours of co-culture. Results were normalized to the supernatant from untransfected cells. CD3xGD2-HDD transcskip variants K1 and K4 showed cytotoxicity in the absence of KRAS OncoSkip KTS2, consistent with baseline exon skipping, and could further induce cytotoxicity using K4. The CD3xGD2-HDD TransSkip variant K2 exhibited no baseline or inducible cytotoxicity consistent with its known efficient splicing and exon inclusion. In contrast, the CD3xGD2-HDD TransSkip variants K3 and K5 did not show baseline, but did show inducible cytotoxicity, with the K3 variant being dose-dependent.

Transgene exon skipping in cells infected with AAV CD3xGD2-HDD Transskip variant K3 can be induced by targeting in KRAS Oncoskip.

Exon skipping was examined in the context of AAV viral infection to determine whether it reflects what was seen when AAV plasmids were transfected in other studies. CD3xGD2-HDD fransskip variants K1 and K3 were packaged into AAV8 and analyzed for exon inclusion ("inactivation") and exclusion ("activation") by RT-PCR 48 hours after AAV infection of 293T cells. Cells were incubated without ("internal transporter only") or with antisense morpholinyl KRAS OncoSkip KTS2 or a reverse control ("invttkts 2 control"). As shown in figure 43 and by the vector plasmid, AAV GD 2K 1 variants showed exon skipping at baseline, which was further induced by KRAS OncoSkip KTS2, but not by the control. In contrast, no baseline exon skipping was observed with the K3 variant, but induction by OncoSkip KTS2 (alone or in complex with the vector "Vivo-KTS 2") was observed. In contrast, it was not induced by the control morpholinyl.

CD19xCD3 TransSkipAAV genomic profile of splice variants.

Based on the results of the CD3xGD2-HDD transcskip study, in which variant K3 showed a lower baseline compared to K1, but still induced Dimert expression, K1 and K3 intron variants were created that inserted the CD19xCD3 Dimert transgene. As with the GD2 series, WPRE downstream of the coding sequence is used to increase transgene expression, requiring the use of a shorter promoter EFSp. Ki, sequence derived from a partial KRAS intron. STOP, exon 2 from KRAS, was mutated to contain a STOP codon in all three reading frames. See fig. 44A and fig. 44B, which are illustrative examples of CD19xCD3 TransSkip constructs.

Fig. 44C illustrates a cartoon representation of the interaction of CD19 dimer with cancer cells and T cells. CD19 dimer was produced by cells containing CD19 nsskip. When administered (e.g., intravenously) to a subject, AAV transaskip (e.g., AAV CD19 transaskip shown in this figure) enters normal cells, e.g., liver or muscle, but does not express the polypeptide due to the insertion of introns and exons containing stop codons into the normal coding sequence of the transgene. In the presence of antisense polynucleotides that induce exon skipping (e.g., morpholino antisense oligonucleotides), mRNA transcripts processed by the antisense oligonucleotides comprise the complete transgene uninterrupted by the STOP exon. In this case, the polypeptide is synthesized and secreted, and the secretory signal peptide is cleaved during secretion, leaving the active dimer bound to cancer cells (Ca) at one end and T immune cells at the other end.

OncoSkipBoth morpholinyl KTS1 and KTS2 induced targeted exon skipping in CD19xCD3 nsskip K1.

As shown in figure 45, 293T cells were transfected with AAV vectors including CD19xCD3 transajoin (positive control, # 1325) and CD19xCD3 transaskip K1 (# 1098), without co-incubation (lane # 4) or with a control morpholinyl (lane # 3) or two different KRAS OncoSkip morpholinyl (lanes 1 and 2). CD19xCD3 TransSkip K1 showed some baseline exon skipping (lower "activating" bands in lanes 3 and 4), consistent with our finding in the parallel construct of CD3xGD2-HDD TransSkip. These results indicate that the engineered TransSkip design is similar to a variety of different transgenes.

KRAS OncoSkipInduction of differentiation of CD19xCD3 Dimert in cells transfected with CD19xCD3 TransSkip K1 AAV vectorAnd (4) secreting and expressing.

Supernatants were collected from transfected 293T cells and tested for T cell binding (interference of fluorescently labeled anti-CD 3 antibody). As shown in fig. 46, the gray line in the top panel is unstained T (jurkat) cells and the dark gray line in the top panel is fully stained T cells with anti-CD 3 antibody. As a positive control, cell supernatants transfected with constitutively expressed CD19xCD3 Transjoin #1325 compete for anti-CD 3 staining (# 1325). Supernatants from cells transfected with CD19xCD3 TransSkip #1098 in the presence of morpholino control showed some baseline competition (KTS 2 reverse control), consistent with "leakage" of exon-skipped Dimert mRNA, and further induced by OncoSkip morpholino (KTS 1; KTS 2) to levels achieved using constitutive CD19xCD3 TransJoin.

CD19xCD3 TransSkipThe splice variant K3 eliminated the baseline, but still maintained inducible exon skipping.

As shown in figure 47, 293T cells were transfected with K1 and K3 CD19xCD3 nsskip constructs in the absence and presence of KTS2 antisense "OncoSkip" morpholinyl or control morpholinyl ("invttkts 2 control") and compared to "internal transporter only" controls with morpholinyl vector but no morpholinyl. mRNA transcripts were analyzed by reverse transcriptase RT-PCR. Consistent with the parallel CD3xGD2-HDD TransJoin construct, variant K1 showed baseline exon skipping. In contrast, variant K3 showed no baseline jump ("internal transporter only") or jump by morpholinyl control. The K3 (# 1168) variant observed skipping using KTS2 OncoSkip, which was confirmed by analysis of protein expression by T cell binding assays.

Induction of CD19xCD3 Dimert expression by CD19xCD3 TransSkip K3 as determined by T cell binding is an on-target effect.

Supernatants were collected from 293T cells transfected with CD3xGD2-HDD TransJoin (positive control) and CD19xCD3 TransSkip, incubated with no or different antisense morpholinyl groups, and subjected to human T (Jurkat) cell binding assays by flow cytometry (competition for fluorescent-labeled anti-CD 3 antibody binding). As shown in figure 48, grey in each top panel is unstained T cells, dark grey is fully stained T cells, pink is fully stained T cells in the presence of supernatant from untransfected 293T cells, and blue is a signal from supernatant competition from cells transfected with constitutively expressed CD19xCD3 TransJoin # 1073. The supernatant of CD19xCD3 TransSkip K1 #11166 showed high expression at baseline and using the control morpholinyl, comparable to the positive control CD19xCD3 transajoin, indicating that this construct is not suitable for controlling gene expression. In contrast to the experience of CD3xGD2-HDD transcskip, CD19xCD3 TransSkip variant K3 #1168 showed no baseline expression (green) or expression induced by control morpholinyl (light and dark orange, "IntCtl"), but dose-dependent expression with KRAS OncoSkip morpholinyl KTS2 (light and dark purple).

The induction of CD19xCD3 Dimert expression from CD19xCD3 TransSkip K3 was reproducible.

The exon skipping assay and T cell binding assay were repeated to confirm the absence of leakage and the presence of inducibility of K3 CD19xCD3 nsskip relative to K1 CD19xCD3 nsskip (figure 49). Please refer to fig. 48.

Detailed description of the preferred embodiments

Embodiment 1: a vector for gene therapy comprising: a first polynucleotide sequence encoding a first antibody or antigen-binding fragment thereof; and a second polynucleotide sequence encoding a second antibody or antigen-binding fragment thereof.

Embodiment 2: the vector of embodiment 1, wherein the vector is a recombinant vector.

Embodiment 3: the vector of embodiment 1 or 2, wherein the vector is a viral vector.

Embodiment 4: the vector according to any one of embodiments 1-3, wherein the viral vector is a retroviral vector.

Embodiment 5: the vector of any one of embodiments 1-4, wherein the viral vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a lentiviral vector, a murine leukemia virus ("MLV") vector, an Epstein-Barr virus ("EBV") vector, or a herpes virus ("HSV") vector.

Embodiment 6: the vector of any one of embodiments 1-5, wherein the AAV vector is an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV PHP.B, AAV rh74, or AAV-DJ vector.

Embodiment 7: the vector of embodiment 6, wherein the AAV vector is AAV rh74 (GenBank accession LP 899424.1).

Embodiment 8: the vector according to any one of embodiments 1-7, wherein the first antibody or antigen-binding fragment thereof specifically binds to an activated antigen on immune effector cells and the second antibody or antigen-binding fragment thereof binds to a tumor antigen.

Embodiment 9: the vector according to any one of embodiments 1-7, wherein the first antibody or antigen-binding fragment thereof specifically binds to a tumor antigen and the second antibody or antigen-binding fragment thereof binds to an activated antigen on immune effector cells.

Embodiment 10: the vector of any one of embodiments 1-9, further comprising a third polynucleotide sequence encoding a third antibody or antigen-binding fragment thereof, wherein the third antibody or antigen-binding fragment thereof binds to an activated antigen or a tumor antigen on an immune effector cell.

Embodiment 11: the vector according to any one of embodiments 8-10, wherein the immune effector cell comprises a dendritic cell, a natural killer ("NK") cell, a macrophage, a T cell, or a B cell.

Embodiment 12: the vector according to any one of embodiments 8-11, wherein the immune effector cell is a T cell or an NK cell.

Embodiment 13: the vector of any one of embodiments 8-12, wherein the activating antigen on the immune effector cell comprises CD3, CD2, CD4, CD8, CD19, LFA1, CD45, NKG2D, NKp44, NKp46, NKp30, DNAM, B7-H3, CD20, CD22, or a combination thereof.

Embodiment 14: the vector according to any one of embodiments 8-13, wherein the tumor antigen comprises one or more of: ephrin-a receptor 2 (EphA 2), Interleukin (IL) -13 ra 2, EGFR VIII, PSMA, EpCAM, GD3, fucosyl GM1, PSCA, PLAC1, sarcoma breakpoint, wilm's tumor 1, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, Epithelial Tumor Antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), blood differentiation antigen, surface glycoprotein, ganglioside (GM 2), growth factor receptor, stromal antigen, vascular antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR 1), mesothelin, CD38, CD123, human epidermal growth factor receptor 2 (HER 2), B Cell Maturation Antigen (BCMA), Fibroblast Activation Protein (FAP) alpha, or a combination thereof.

Embodiment 15: the vector according to any one of embodiments 1-14, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof form a dimer.

Embodiment 16: the vector of embodiment 15, wherein said dimer is a bispecific antibody.

Embodiment 17: the vector of embodiment 15, wherein said dimer is a trispecific antibody.

Embodiment 18: the vector of embodiment 15 or 16, wherein the bispecific antibody comprises a polypeptide sequence at least 95% identical to either of SEQ ID NOs 13 or 15.

Embodiment 19: the vector of embodiment 15 or 17, wherein the trispecific antibody comprises a polypeptide sequence at least 95% identical to SEQ ID No. 11.

Embodiment 20: the vector according to any one of embodiments 1-19, wherein said vector further comprises a polynucleotide sequence encoding a secretory peptide.

Embodiment 21: the vector of embodiment 20, wherein the secretory peptide comprises a secretory consensus sequence.

Embodiment 22: the vector of embodiment 20 or 21, wherein the secretory consensus sequence comprises at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 51.

Embodiment 23: the vector of embodiment 20 or 21, wherein the secretory consensus sequence consists of SEQ ID NO 51.

Embodiment 24: the vector according to any one of embodiments 20-23, wherein said secretory consensus sequence is encoded by a polynucleotide comprising SEQ ID NO: 52 or an equivalent thereof.

Embodiment 25: the vector according to any one of embodiments 20-24, wherein the secretory consensus sequence further comprises one, two, three, four or more residues at the C-terminus of the sequence.

Embodiment 26: the vector of any one of embodiments 20-25, wherein the secretory consensus sequence further comprises one, two, three, four, or more Ala residues at the C-terminus of the sequence.

Embodiment 27: the vector of any one of embodiments 20-26, wherein the secretory consensus sequence further comprises one, two, or three Ala residues at the C-terminus of the sequence.

Embodiment 28: the vector of any one of embodiments 20-27, wherein the secretory consensus sequence further comprises two Ala residues at the C-terminus of the sequence.

Embodiment 29: the vector of embodiment 28, wherein the secretory consensus sequence comprises at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID No. 55, or consists of SEQ ID No. 55.

Embodiment 30: the vector of embodiment 28 or 29, wherein the secretory consensus sequence is encoded by a polynucleotide comprising SEQ ID NO: 56 or an equivalent thereof.

Embodiment 31: the vector of any one of embodiments 20-30, wherein the secretory consensus sequence further comprises an Ala residue at the C-terminus of the sequence.

Embodiment 32: the vector of embodiment 31, wherein said secretory consensus sequence comprises at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID No. 53, or consists of SEQ ID No. 53.

Embodiment 33: the vector of embodiment 31 or 32, wherein the secretory consensus sequence is encoded by a polynucleotide comprising SEQ ID NO: 54 or an equivalent thereof.

Embodiment 34: the vector according to any one of embodiments 1-33, wherein said secretory consensus sequence modulates expression and/or secretion of dimert.

Embodiment 35: the vector of any one of embodiments 1-34, wherein said secretory consensus sequence enhances expression and/or secretion of dimert.

Embodiment 36: the vector according to any one of embodiments 1-35, wherein said vector further comprises a polynucleotide sequence encoding a dimerization domain.

Embodiment 37: the vector of embodiment 36, wherein said dimerization domain comprises the dimerization domain of human hepatocyte nuclear factor 1 alpha (HNF 1 a).

Embodiment 38: the vector according to embodiment 37, wherein the dimerization domain of HNF1 a comprises a polypeptide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID No. 47.

Embodiment 39: the vector of any one of embodiments 1-38, wherein the vector further comprises a promoter.

Embodiment 40: the vector of embodiment 39, wherein said promoter is a constitutive promoter.

Embodiment 41: the vector of embodiment 39 or 40, wherein said promoter is a tissue specific promoter.

Embodiment 42: the vector of any one of embodiments 39-41, wherein said promoter comprises a Rous Sarcoma Virus (RSV) LTR promoter, a Cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a β -actin promoter, a phosphoglycerate kinase (PGK) promoter, a U6 promoter, an EF1 a short form (EFS) promoter, a phosphoglycerate kinase (PGK) promoter, an ubiquitin C (Ubic) promoter, an α -1-antitrypsin, a Spleen Focus Forming Virus (SFFV) promoter, or a chicken β -actin (CBA) promoter.

Embodiment 43: the vector of any one of embodiments 39-42, wherein said promoter is an EFS, optionally comprising SEQ ID NO 49 or an equivalent thereof.

Embodiment 44: the vector according to any one of embodiments 1-43, wherein said vector further comprises an enhancer.

Embodiment 45: the vector of embodiment 44, wherein the enhancer is a RSV enhancer, a CMV enhancer, and an alpha-fetoprotein MERII enhancer.

Embodiment 46: the vector according to any one of embodiments 1-45, wherein said vector further comprises one or more additional regulatory elements.

Embodiment 47: the vector according to any one of embodiments 1-46, wherein said vector comprises a regulatory element comprising a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE), optionally SEQ ID NO: 50, or an equivalent thereof.

Embodiment 48: the vector of any one of embodiments 1-47, wherein the vector comprises a 5 'Inverted Terminal Repeat (ITR) and a 3' ITR.

Embodiment 49: the vector according to any one of embodiments 1-48, wherein said vector comprises the sequence set forth in SEQ ID NOs 4, 6, 8, 12, 14, 16-23, 30-33, or 40-46.

Embodiment 50: a composition comprising a carrier (vector) according to any one of embodiments 1-49 and a carrier (carrier), optionally a pharmaceutically acceptable carrier.

Embodiment 51: the composition of embodiment 50, wherein the composition is formulated for systemic administration.

Embodiment 52: the composition of embodiment 50, wherein the composition is formulated for topical administration.

Embodiment 53: the composition according to any one of embodiments 50-52, wherein the composition is formulated for parenteral administration.

Embodiment 54: a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the vector of any one of embodiments 1-49 or the pharmaceutical composition of any one of embodiments 50-53, wherein the vector expresses a therapeutic anti-cancer antibody or antigen-binding fragment thereof.

Embodiment 55: the method of embodiment 54, further comprising administering to the subject an anti-cancer agent.

Embodiment 56: the method of embodiment 55, wherein the anti-cancer agent comprises an agent selected from a peptide, a polypeptide, a nucleic acid molecule, a small molecule, a viral particle, or a combination thereof.

Embodiment 57: the method of embodiment 56, wherein the viral particle is an oncolytic HSV particle.

Embodiment 58: the method according to any one of embodiments 54-57, wherein the subject is a mammal.

Embodiment 59: the method according to any one of embodiments 54-58, wherein the subject is a human.

Embodiment 60: a method of producing a bispecific or trispecific antibody in a cell comprising contacting the cell with the vector of any one of embodiments 1-49.

Embodiment 61: the method of embodiment 60, wherein said contacting comprises transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof.

Embodiment 62: the method of embodiment 60 or 61, wherein the cells comprise fibroblasts, skeletal cells, epithelial cells, muscle cells, neural cells, endocrine cells, melanocytes, blood cells, or a combination thereof.

Embodiment 63: the method according to any one of embodiments 60-62, wherein the bispecific antibody comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO 13 or 15.

Embodiment 64: the method according to any one of embodiments 60-62, wherein the trispecific antibody comprises a polypeptide sequence at least 95% identical to SEQ ID NO 11.

Embodiment 65: the method of any one of embodiments 60-62, wherein the bispecific antibody is encoded by a polynucleotide sequence that is at least 95% identical to SEQ ID NO 14, 16, 22, 23, 30-33, or 40-46.

Embodiment 66: the method according to any one of embodiments 60-62, wherein said trispecific antibody is encoded by a polynucleotide sequence at least 95% identical to SEQ ID NO 12.

Embodiment 67: a kit comprising the vector of any one of embodiments 1-49 or the pharmaceutical composition of any one of embodiments 50-53.

Embodiment 68: the kit of embodiment 67, further comprising instructional materials.

Equivalents of the formula

It should be understood that while the disclosure has been described in conjunction with the above-described embodiments, the foregoing description and examples are intended to illustrate, but not limit the scope of the disclosure. Other aspects, advantages, and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All nucleotide sequences provided herein are presented in a 5 'to 3' orientation.

The embodiments illustratively described herein suitably may be practiced in the absence of any element, limitation, or restriction which is not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like are to be construed broadly and not restrictively. Furthermore, the terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention, in the use of such terms and expressions, of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure.

Thus, it should be understood that although the present disclosure has been specifically disclosed by particular embodiments and optional features, those skilled in the art may resort to modifications, improvements, and variations of the examples disclosed herein, and that such modifications, improvements, and variations are considered to be within the scope of the present disclosure. The materials, methods, and examples provided herein are representative of particular embodiments, are exemplary, and are not intended to limit the scope of the present disclosure.

The scope of the present disclosure has been described broadly and generally herein. Each of the narrower species and subgeneric groups that fall within the generic disclosure also form part of the disclosure. This includes the generic description with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Further, where features or aspects of the disclosure are described in terms of markush groups, those skilled in the art will recognize that embodiments of the disclosure may also be described thereby in terms of any individual member or subgroup of members of the markush group.