Cancer-targeted, virally encoded, regulatable T cell (CATVERT) or NK cell (CATVERN) adaptors

Abstract

Recombinant polynucleotides and vectors are provided that contain an engineered (artificial) exon-intron-exon gene structure in a transgene that undergoes splicing when expressed in target cells.

Description

[0001] Government Support Statement

[0002] This invention was made with Government support under Grant No. NF170075 awarded by the Department of Defense. The Government may have certain rights in this invention.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 808,264, filed on February 20, 2019, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0005] The present invention relates to a method or vector for expressing a gene for treating a disease or condition. Background Art

[0006] Gene transfer as a disease treatment method (i.e., gene therapy) is now a reality. In 2017, the FDA approved Luxturna, an adeno-associated virus (AAV) designed to stably express a normal copy of the RPE65 gene in retinal cells, for the treatment of congenital retinal dystrophy. In 2019, the FDA approved Zolgensma, an AAV designed to express SMN1 in motor neurons for the treatment of spinal muscular atrophy. Ongoing clinical trials using AAV vectors to replace gene function in other diseases have also achieved very encouraging results, including a mini version of the dystrophin gene for Duchenne muscular dystrophy, and the factor VIII and IX genes for hemophilia A and B, respectively.

[0007] In all of the examples above, the goal is long-term gene expression to replace a missing or defective gene. However, there may be gene therapy applications where the transgene (which is expressed as a therapeutic agent in gene therapy) is expressed only for a short period of time, such as 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 ideal to minimize potential immune responses to Cas9 and minimize off-target gene mutations. Another application is situations 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.

[0008] Therefore, there is a need for a new method to activate transgene expression, allowing for short-term gene expression (weeks to months) using gene therapy platforms. An ideal system would place transgene expression under the control of a drug, so that drug administration activates gene expression and discontinuation of drug administration reverts to inactivation. In this scenario, if side effects occur or transgene expression is no longer desired, drug administration would not be necessary. The present invention addresses this need and provides related advantages. Summary of the Invention

[0009] The present invention provides a method or vector for expressing a therapeutic gene for a disease or condition, optionally under external control. In some cases, the disease or condition is cancer. In some cases, the vector expresses a polypeptide (referred to herein as "Dimert") that is used to bind immune executive cells to cancer cells, thereby triggering the killing of cancer cells.

[0010] The present invention provides a recombinant polynucleotide or vector comprising, or consisting essentially of, or consisting of the following sequences: (a) a first polynucleotide sequence comprising a first portion of an open reading frame encoding a first antibody or an antigen-binding fragment thereof; (b) a second polynucleotide sequence comprising a second portion of an open reading frame encoding the first antibody or an antigen-binding fragment thereof; (c) a third polynucleotide sequence encoding a second antibody or an 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, or consists essentially of, or consists of: a splice donor site, an upstream intron, an exon comprising one or more stop codon sequences in its respective reading frame, a downstream intron, and a splice acceptor site. In a further aspect, the gene regulatory polynucleotide sequence comprises one or more binding sequences for an antisense oligonucleotide. In a further aspect, the antisense oligonucleotide is a 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 NO. 24 (AATATGATCCAACAATAGAGGTAAATCTTG) or SEQ ID NO. 25 (GATCCAACAATAGAGGTAAATCTTGTTTTA). In another embodiment, the stop codon comprises an oligonucleotide from the following group: TAA, TAG, or TGA. In another aspect, the stop codon sequence comprises the polynucleotide sequence TAAxTAGxTGAxTAGxTAAxTGAx (SEQ ID NO. 1), wherein x is any nucleotide, or the stop codon sequence comprises the polynucleotide sequence TAATTAGTTGATTAGTTAATTGAT (SEQ ID NO. 2) or its equivalent. In another embodiment, the recombinant polynucleotide or vector encodes a bispecific or trispecific engager. In another embodiment, the bispecific cell engager is a bispecific engager. In another embodiment, the bispecific cell engager is a trispecific engager.

[0011] In a further embodiment, the first antibody or its antigen binding fragment specifically binds to the activation antigen on the immune effector cell, and the second antibody or its antigen binding fragment binds to a tumor antigen. On the other hand, the first antibody or its antigen binding fragment specifically binds to a tumor antigen, and the second antibody or its antigen binding fragment binds to the activation antigen on the immune effector cell. On the other hand, the recombinant polynucleotide or vector also includes a fourth polynucleotide sequence encoding a third antibody or its antigen binding fragment, wherein the third antibody or its antigen binding fragment binds to the activation antigen or tumor antigen on the immune effector cell. On the one hand, immune effector cells include dendritic cells, natural killer (" NK") cells, macrophages, T cells, B cells, neutrophils, eosinophils, basophils, mast cells or a combination thereof. On the one hand, immune effector cells are T cells. On the other hand, immune effector cells are NK cells. In another embodiment, the third antibody or its antigen binding fragment binds to the activation antigen on NK cells and induces an immune response mediated by NK cells.

[0012] 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.

[0013] In one aspect, the dimert (e.g., a 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 NKG2D. 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 CD19. In another embodiment, the first antigen-binding fragment binds to CD3 and the second antigen-binding fragment binds to GD2. In another embodiment, the first antigen-binding fragment binds to NKG2D and the second antigen-binding fragment binds to GD2.

[0014] In one embodiment, the dimer (e.g., a trispecific binder or antibody) comprises a first antigen-binding fragment, a second antigen-binding fragment, and a third antigen-binding fragment. In another embodiment, the trispecific binder or antibody comprises three antigen-binding fragments that bind to NKG2D, IL21R, and GD2, respectively. In a further aspect, the trispecific binder or antibody comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO. 11.

[0015] 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 NO. 3 and SEQ ID NO. 4, respectively.

[0016] SEQ ID NO.3: Amino acid sequence of IL-21

[0017] MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS

[0018] SEQ ID NO.4: cDNA sequence of IL-21

[0019] ATGAGAAGCAGCCCCGGCAACATGGAGAGAATCGTGATCTGCCTGATGGTGATCTTCCTGGGCACCCTGGTGCACAAGAGCAGCAGGGCCAGGACAGACACATGATCAGAATGAGACAGCTGATCGACATCGTGGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTCCTGCCCGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGAGCGCCTTCAGCTGCTTCCAGAAG GCCCAGCTGAAGAGCGCCAACACCGGCAACAACGAGAATCATCAACGTGAGCATCAAGAAGCTGAAGAGAAAGCCCCCCAGCACCAACGCCGGCAGAAGACAGAAGCACAGACTGACCTGCCCCAGCTGCGACAGCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGATTCAAGAGCCTGCTGCAGAAGATGATCCACCAGCACCTGAGCAGCAGAACCCACGGCAGCGAGGACAGC.

[0020] In one embodiment, the antigen-binding fragment that binds to NKG2D comprises MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, Rae-1α, Rae-1β, Rae-1γ, Rae-1δ, Rae-1ε, H60a, H60b, H60c, MULT1, or a fragment thereof. In one embodiment, the antigen-binding fragment that binds to NKG2D is MICA or a fragment thereof. The amino acid and cDNA sequences of MICA are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively.

[0021] SEQ ID NO.5: Amino acid sequence of MICA

[0022] EPHSLRYNLTVLSWDGSVQSGFLAEVHLDGQPFLRYDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETEEWTVPQSSRAQT LAMNVRNFLKEDAMKTKTHYHAMHADCLQELRRYLESSVVLRRTVPPMVNVTRSEASEGNITVTCRASSFYPRNIILTWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICRGEEQRFTCYMEHSGNHSTHPVPS

[0023] SEQ ID NO.6: MICA cDNA sequence

[0024]

[0025] 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 that comprises a methionine mutation instead of an 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 NO. 7 and SEQ ID NO. 8, respectively.

[0026] SEQ ID NO.7: Amino acid sequence of MUC-30

[0027] EPHSLRYNLTVLSWDGSVQSGFLAEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETEEWTMPQSSRAQT LAMNIRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPS

[0028] SEQ ID NO.8: cDNA sequence of MUC-30

[0029]

[0030]

[0031] In a further aspect, a precursor mRNA (pre-mRNA) is expressed which, when contacted with a morpholino oligonucleotide, encodes a trispecific antibody.

[0032] In one aspect, the antigen binding domain is a single chain variable region fragment or an antibody.

[0033] In a further aspect, the recombinant polynucleotide or vector include a polynucleotide sequence encoding a secretory peptide. On the other hand, the recombinant polynucleotide or vector further include a polynucleotide sequence encoding a dimerization domain. On the other hand, the recombinant polynucleotide or vector include 5' inverted terminal repeats (ITR) and 3' ITR. On the other hand, the vector includes sequence SEQ ID No.4, 6, 8, 12, 14, 16-23, 30-33 or 40-46. The non-limiting example of such vectors includes: recombinant viral vectors, which include a backbone vector selected from retroviral vectors, lentiviral vectors, murine leukemia virus ("MLV") vectors, Epstein-Barr virus ("EBV") vectors, adenoviral vectors, herpes virus ("HSV") vectors, adeno-associated virus ("AAV") vectors, AAV vectors or optional self-complementary AAV vectors.

[0034] The recombinant polynucleotide or vector can be contained within a host cell, such as a prokaryotic or eukaryotic cell.

[0035] Recombinant polynucleotides, vectors and cells can be included in compositions comprising vectors and / or host cells and carriers (e.g., pharmaceutically acceptable carriers). They can be formulated for various modes of administration and comprise an effective amount of polynucleotides, vectors and / or host cells that are effective for patients, diseases or conditions, vectors and modes of administration. On the one hand, the mode of administration is systemic or intravenous. On the other hand, topical administration is performed by direct injection. On the one hand, morpholino oligonucleotides are contacted with polynucleotides or carriers simultaneously or thereafter, for example, as systemic or topical injections. Alternatively, contact is made before the polynucleotides or carriers.

[0036] Recombinant polynucleotides or vectors can be used to treat a variety of diseases or conditions. In one aspect, a method for delivering a transgene is provided. The method comprises administering an effective amount of a recombinant polynucleotide or vector comprising a transgene to the cell, tissue, or patient to be treated. In one aspect, an effective amount of a morpholino oligonucleotide is administered to the cell, tissue, or patient to be treated. Non-limiting examples of providing and selecting transgenes according to the purpose of the method are provided. The cell or tissue can be a mammal, such as a human. In one aspect, the morpholino oligonucleotide is contacted with the vector simultaneously or thereafter. Alternatively, the vector is contacted before the vector. In another aspect, the vector is introduced into the cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof.

[0037] The present invention provides a method for 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 a further method, the method further comprises administering to the subject an effective amount of a morpholino oligonucleotide. In one aspect, an effective amount of an anticancer agent is administered to the subject. Non-limiting examples of anticancer agents include anticancer peptides, polypeptides, nucleic acid molecules, small molecules, viral particles, or a combination thereof. In another aspect, the vector is introduced into the cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof.

[0038] In one aspect of the disclosed method, the viral particle is an oncolytic HSV particle.

[0039] Another method provided herein is a method for producing a bispecific or trispecific antibody in a cell, the method 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 simultaneously with or after the vector. Alternatively, the morpholino oligonucleotide is contacted before the polynucleotide or vector. In one aspect, the morpholino oligonucleotide comprises a sequence at least 95% identical to SEQ ID NOs. 27 or 28. Non-limiting examples of bispecific antibodies comprise a polypeptide sequence at least 95% identical to SEQ ID NOs. 13 or 15. In one embodiment, the bispecific antibody is encoded by a polynucleotide sequence at least 95% identical to SEQ ID NOs. 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.

[0040] 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, bone cells, epithelial cells, muscle cells, neural cells, endocrine cells, melanocytes, blood cells, or a combination thereof.

[0041] Further provided are kits comprising one or more of the polynucleotides, vectors, cells, or compositions described herein, optionally with instructional materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 An overview of the strategy showing the general concept of splicing using a 3-exon gene structure is shown. Exons are labeled 1, 2, or 3, and introns are represented by lines. Different splicing modes are shown with lowercase letters (a, b, c), and the structure of the resulting RNA is shown based on which of these is used. Oligonucleotides that can interfere with the splice donor (1D, 2D) or acceptor (2A, 3A) sites are shown as short blue lines, and the expected expression of different possible RNA isoforms is shown as "+", depending on the site blocked by the oligonucleotide.

[0043] Figure 2 Shows Figure 1 Overview of the strategy for using splice type 1, in which exon 2 is normally included in the gene transcript. Transcripts containing all three exons spliced ​​together are expected to predominate, but if oligonucleotides are present that block the exon 2 splice acceptor or donor site, transcripts containing exon 1 fused to exon 3 will predominate.

[0044] Figure 3 An overview of strategy #1 for creating an engineered intron-exon STOP-intron in a transgene is presented. Transcripts retaining the intron will be nonfunctional because the intron will have a premature stop codon and / or will be out of frame. The "a+b" transcript will be nonfunctional due to the stop codon in exon 2. Only transcripts in which exon 2 is skipped (using splice c) are functional, which will have a lower baseline and be activated by an oligonucleotide blocking the exon 2 splice site.

[0045] Figure 4 Shows Figure 1 Diagram of transgene regulation for the indicated strategies.

[0046] Figure 5A second strategy was demonstrated to create an engineered intron-exon STOP-intron within the transgene, utilizing "splicing type 2," where exon 2 is not normally present in cancer cells but is present in normal cells. In this scenario, exon-skipping oligonucleotides activated transgene expression (exons 1+3) in normal cells, leaving it unchanged (or even higher) despite some off-target skipping of cellular genes, as a potential "additional" therapeutic effect.

[0047] Figure 6 A third strategy was demonstrated to create an engineered intron-exon STOP-intron within the transgene, utilizing "splicing type 3," in which exon 2 is normally present in certain cancer cells but absent in normal cells. In this scenario, high levels of active transgene (exons 1+3) are present in normal cells, and exon-skipping oligonucleotides activate expression in tumor cells.

[0048] Figure 7 Exemplary CD3xGD2-HDD AAV constructs are shown.

[0049] Figure 8 A cartoon depicting the experiments used to determine the structure and function of Dimert disclosed herein is shown.

[0050] Figure 9 Shown is SDS-PAGE of whole-cell lysates from transfected 293T cells. Figure 7 Three constructs #1101, #1040, and #1124 (which contain secreted peptides) retained less CD3xGD2-HDD Dimert in transfected 293T cells relative to construct #1104 and the control construct pcDNA3GFP.

[0051] Figure 10 Demonstrated that AAV vector-secreted CD3xGD2-HDD Dimert binds to and activates human T cells.

[0052] Figure 11 Secreted CD3xGD2-HDD Dimert is shown to activate human T cells.

[0053] Figure 12A It was demonstrated that the binding of CD3xGD2-HDD to T cells was dose-dependent.

[0054] Figure 12B Bar graphs showing median staining levels normalized to unstained controls are shown.

[0055] Figure 13Cartoon showing the binding assay for the anti-GD2 arm of CD3xGD2-HDD Dimert. This assay demonstrates that both GD2 and CD3 binding occur on a single molecule.

[0056] Figure 14 An exemplary CD3xGD2-HDD Dimert binding to CD3 and GD2 is shown.

[0057] Figure 15 A flow chart showing the assay to determine whether CD3xGD2-HDD induces killing of GD2+ target cells by T cells is shown.

[0058] Figure 16 demonstrated that secreted CD3xGD2-HDD induced T cell killing of neuroblastoma cells.

[0059] Figure 17 We demonstrated T cell-mediated cytotoxicity of CD3xGD2-HDD Dimert, which correlated with GD2 expression.

[0060] Figure 18A A diagram showing exemplary AAV constructs used to test CD19xCD3 Dimert expression is shown.

[0061] Figure 18B An exemplary CD19 TransJoin genome structure is shown. Elements of the AAV-encoded transgene are indicated in the figure.

[0062] Figure 18C A cartoon showing CD19 Dimert interacting with cancer cells and T cells. CD19 Dimert is produced by cells that contain the CD19 TransJoin. As shown in the figure, "Ca" represents cancer cells and "T" represents T cells.

[0063] Figure 19 Supernatants from cells transfected with an AAV CD19xCD3 construct containing a secretion sequence were shown to bind and activate T cells.

[0064] Figure 20 Supernatants from cells transfected with AAV vectors containing secreted peptides were shown to activate human T cells.

[0065] Figure 21 demonstrated that AAV-secreted CD19xCD3 specifically binds to CD19 but not CD45.

[0066] Figure 22A It was demonstrated that CD19xCD3 binds to T cells in a dose-dependent manner.

[0067] Figure 22BBar graphs showing median staining levels normalized to unstained controls are shown.

[0068] Figure 23A It was demonstrated that CD19xCD3 binds to B cells in a dose-dependent manner.

[0069] Figure 23B Bar graphs showing median staining levels normalized to unstained controls are shown.

[0070] Figure 24 We demonstrated that CD3 Dimert activated T cells via anti-CD28 co-stimulation better than anti-CD3 antibodies.

[0071] Figure 25 293T cells were shown to produce the highest transduction efficiency for AAV8 vectors.

[0072] Figure 26 We show that Dimert concentration in the supernatant of AAV8-infected cells depends on AAV dose and transduction efficiency.

[0073] Figure 27 demonstrated that a single intravenous injection of CD19xCD3 TransJoin selectively depletes B cells in humanized mice.

[0074] Figure 28 demonstrated that a single intravenous injection of CD19xCD3 TransJoin resulted in prolonged depletion of B cells in humanized mice.

[0075] Figure 29 Demonstrated that a single intravenous injection of CD19xCD3 TransJoin eliminates CD19+ lymphomas in humanized mice.

[0076] Figure 30 An overview of OncoSkip and TransSkip described in this article is presented.

[0077] Figure 31 We demonstrate that KRAS Oncoskip antisense morpholino induces exon skipping of endogenous KRAS in lung cancer cells.

[0078] Figure 32 An exemplary AAV vector map of the CD3xGD2-HDD TransSkip is shown, showing the reverse-engineered introns flanking the exons inserted into the CD3xGD2-HDD Dimert coding sequence.

[0079] Figure 33 An exemplary strategy for testing the activity of OncoSkip and TransSkip described herein is presented.

[0080] Figure 34 Antisense morpholinos that induce exon skipping of the CD3xGD2-HDD TransSkip transgene are shown.

[0081] Figure 35 We demonstrated that KRAS OncoSkip induced the secretory expression of CD3xGD2-HDD Dimert in cells transfected with the CD3xGD2-HDD TransSkip AAV vector.

[0082] Figure 36 We demonstrated that KRAS OncoSkip exon skipping of CD3xGD2-HDD TransSkip is an on-target effect.

[0083] Figure 37 We demonstrated that induction of CD3xGD2-HDD Dimert expression, as determined by T cell engagement, was an on-target effect.

[0084] Figure 38 AAV genome maps of exemplary TransSkip splice variants are shown to reduce baseline TransSkip "leakage" but maintain inducible exon skipping.

[0085] Figure 39 Demonstrated is the CD3xGD2-HDD TransSkip splice variant K3, which abolishes baseline but maintains inducible exon skipping.

[0086] Figure 40 We present the CD3xGD2-HDD TransSkip variant K3, which showed no baseline Dimert production but was more readily induced by KRAS OncoSkip than the other TransSkip variants tested.

[0087] Figure 41 We demonstrated that OncoSkip-mediated induction of Dimert expression from CD3xGD2-HDD TransSkip variants K3 and K5 was an on-target effect.

[0088] Figure 42 We show that OncSkip-induced secreted Dimert from an AAV CD3xGD2-HDD TransSkip vector plays a role in mediating T cell killing of neuroblastoma cells.

[0089] Figure 43 We show that transgene exon skipping in cells infected with the AV CD3xGD2-HDD TransSkip variant K3 can be induced by KRAS OncoSkip targeting.

[0090] Figure 44A AAV genome maps of exemplary CD19xCD3 TransSkip splice variants are shown.

[0091] Figure 44B An exemplary CD19 TransSkip genome structure is shown. Elements of the AAV-encoded transgene are indicated in the figure.

[0092] Figure 44C A cartoon showing the interaction of CD19 Dimer with cancer cells and T cells. CD19 Dimer is produced by cells that contain the CD19 TransSkip protein. As shown in the image, "Ca" represents cancer cells and "T" represents T cells.

[0093] Figure 45 We show that the OncoSkip morpholinos KTS1 and KTS2 induce on-target exon skipping of the CD19xCD3 TransSkip K1.

[0094] Figure 46 Demonstrated that KRAS OncoSkip induces secretory expression of CD19xCD3 Dimert in cells transfected with the CD19xCD3 TransSkip K1 AAV vector.

[0095] Figure 47 We show that the CD19xCD3 TransSkip splice variant K3 eliminates baseline but maintains inducible exon skipping.

[0096] Figure 48 We demonstrated that induction of CD19xCD3 Dimer expression, as determined by T cell engagement of the CD19xCD3-K3 TransSkip, was an on-target effect.

[0097] Figure 49 We demonstrated that induction of CD19xCD3 Dimer expression from CD19xCD3-K3 TransSkip is reproducible. DETAILED DESCRIPTION

[0098] The following will describe more fully embodiments according to the present invention. However, various aspects of the present invention may be embodied in different forms and should not be construed as limited to the embodiments described herein. Rather, these embodiments are provided to make the present invention thorough and complete and to fully convey the scope of the present invention to those skilled in the art. The terminology used in the description herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0099] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of this application and the related art, and should not be interpreted in an idealized or overly formal sense unless explicitly defined herein. Although not explicitly defined below, such terms should be interpreted according to their common meaning.

[0100] 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 present invention.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0101] 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.

[0102] Unless the context indicates otherwise, the various features of the invention described herein may be used in any combination. Furthermore, the present invention contemplates that in some embodiments, any feature or combination of features described herein may be excluded or omitted. For illustration, if the specification states that a compound includes components A, B, and C, it is specifically intended that any one or combination of A, B, or C may be omitted or disclaimed, individually or in any combination.

[0103] Unless expressly indicated otherwise, all specified embodiments, features, and terms are intended to include the stated embodiment, feature, or term and its biological equivalents.

[0104] All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximate and vary in increments of (+) or (-) 1.0 or 0.1, as appropriate, or by + / - 15%, or by 10%, or by 5%, or by 2%. It is understood that all numerical designations are preceded by the term "about," although not always explicitly stated. It is also understood that the reagents described herein are exemplary only and that equivalents of such reagents are known in the art, although not always explicitly stated.

[0105] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by identifying references or numerals. Full citations for publications identified by numerals can be found immediately preceding the claims. The disclosures of these publications, patents, and published patent specifications are hereby incorporated by reference in their entireties to more fully describe the state of the art to which this invention pertains.

[0106] definition

[0107] Unless otherwise indicated, the practice of the present technology will employ conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. For example, see Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols In Molecular Biology (FM Ausubel, et al. eds., (1987)); these series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988); Antibodies, a Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)).

[0108] As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0109] As used herein, the term "comprising" is intended to indicate that compositions and methods include the elements, but do not exclude other elements. As used herein, the transition phrase consisting essentially of (and grammatical variations) will be interpreted as including the materials or steps, as well as those that do not substantially affect the basic and novel features of the embodiments. Therefore, the term "consisting essentially of" as used herein should not be interpreted as being equivalent to "comprising". "Consisting of" should mean excluding trace elements that are more than other ingredients and the basic method steps for applying the compositions disclosed herein. Aspects defined by each of these transition terms are within the scope of the present invention.

[0110] As used herein, the term "about" when referring to a measurable value (eg, an amount or concentration, etc.) is intended to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the stated amount.

[0111] The terms "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 disclosed purpose.

[0112] 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 a combination when construed in the alternative ("or").

[0113] As used herein, the term "adeno-associated virus" or "AAV" refers to members of the class of viruses associated with that name and belonging to the genus Dependaviridae, family Parvoviridae. Multiple serotypes of this 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 VP3. 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 (a chimera obtained by shuffling eight different AAV wild types).

[0114] In some cases, an AAV serotype preferentially targets a tissue type. In some cases, the figure below illustrates typical tissue types and AAV serotypes that target each tissue type.

[0115] organize Serotype CNS AAV1, AAV2, AAV4, AAV5, AAV8, AAV9 heart AAV1, AAV8, AAV9 kidney AAV2 liver AAV7, AAV8, AAV9 lung AAV4, AAV5, AAV6, AAV9 pancreas AAV8 photoreceptor cells AAV2, AAV5, AAV8 RPE (retinal pigment epithelium) AAV1, AAV2, AAV4, AAV5, AAV8 skeletal muscle AAV1, AAV6, AAV7, AAV8, AAV9

[0116] As used herein, the term "cell" may refer to a prokaryotic or eukaryotic cell, optionally obtained from a subject or a commercial source.

[0117] "Eukaryotic cells" include all kingdoms of life except the anucleate kingdom. They can be easily distinguished by their membrane-bound nuclei. Animals, plants, fungi, and protists are eukaryotic organisms or organisms whose cells are organized into complex structures via an internal membrane and a 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 monkeys, cows, pigs, mice, rats, birds, reptiles, and humans, such as HEK293 cells and 293T cells.

[0118] "Prokaryotic cells," which generally lack a nucleus or any other membrane-bound organelles, are divided into two domains, bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in a ring called an episome. Bacterial cells are very small, roughly the size of animal mitochondria (about 1-2 μm in diameter and 10 μm long). Prokaryotic cells have three main shapes: rod-shaped, spherical, and spiral. Bacterial cells do not undergo the complex replication process like eukaryotic cells, but instead divide by binary fission. Examples include, but are not limited to, Bacillus, Escherichia coli, and Salmonella.

[0119] The term "encoding" as applied to a nucleic acid sequence refers to a polynucleotide that is described as "encoding" a polypeptide and that, 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 mRNA for the polypeptide and / or its fragments. The antisense strand is the complementary sequence of such a nucleic acid, from which the coding sequence can be deduced.

[0120] The terms "equivalent" or "bioequivalent" are used interchangeably in reference to specific molecules, organisms, or cellular materials, and mean materials with minimal homology while maintaining the desired structure or function. Non-limiting examples of equivalent polypeptides include polypeptides 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 a polypeptide sequence, or polypeptides encoded by a polynucleotide or its complementary sequence that hybridizes under highly stringent conditions to a polynucleotide encoding the polypeptide sequence, the polypeptide sequence having substantially the same or identical function as the reference polypeptide, and in one aspect encoding the reference polypeptide. Highly stringent conditions are described herein and are incorporated herein by reference. Alternatively, an equivalent thereof is a polypeptide encoded by a polynucleotide or its complementary sequence 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).

[0121] Non-limiting examples of equivalent polynucleotides include polynucleotides that have 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 to a reference polynucleotide, or at least 97% sequence identity, or at least 98% identity, or at least 99% identity. Equivalents also include polynucleotides or their complements that hybridize to a reference polynucleotide under highly stringent conditions.

[0122] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" to another sequence, meaning that, when aligned, that percentage of bases (or amino acids) is the same when comparing the two sequences. Comparisons and homology percentages or sequence identities can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. In certain 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; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort by = high score; databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. For more information about these programs, please visit the following website: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percent identity can be determined by combining them with clustalW (available at genome.jp / tools / clustalw / , last accessed January 13, 2017).

[0123] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a single 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 "nonhomologous" sequence is one that has less than 40% identity, or less than 25% identity, to one of the sequences of the invention.

[0124] "Homology" or "identity" or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions.

[0125] "Hybridization" refers to the reaction of one or more polynucleotides to form a complex that is stabilized by hydrogen bonds between the nucleotide residue bases. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogstein binding, or any other sequence-specific manner. The complex can include two chains that form a double-stranded structure, three or more chains that form a multi-chain complex, a single self-hybridizing chain, or any combination of these. A hybridization reaction can constitute a step in a broader process, such as the initiation of a PCR reaction, or the digestion of a polynucleotide by a ribozyme.

[0126] Examples of stringent hybridization conditions include: an incubation temperature of about 25°C to about 37°C; a hybridization buffer concentration of about 6× sodium citrate buffer (SSC) to about 10×SSC; a formamide concentration of about 0% to 25%; and a wash solution of about 4×SSC to about 8×SSC. Examples of moderately stringent hybridization conditions include: an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9×SSC to 2×SSC; a formamide concentration of about 30% to 50%; and a wash solution of about 5×SSC to about 2×SSC. Highly stringent hybridization refers to hybridization of the oligonucleotide to the target sequence without mismatches (or complete complementarity). Examples of high stringency conditions include: an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1×SSC to 0.1×SSC; a formamide concentration of about 55% to 75%; and a wash solution of about 1×SSC, 0.1×SSC, or deionized water. Typically, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2 or more wash steps, with wash incubation times of about 1, 2 or 15 minutes. SSC is a 0.15M NaCl and 15mM sodium citrate buffer. It should be understood that SSC equivalents using other buffer systems can be used.

[0127] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0128] "Gene" refers to a polynucleotide containing at least one open reading frame (ORF) that, after transcription and translation, encodes a specific polypeptide or protein. "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.

[0129] "Under transcriptional control" is a term well known in the art, meaning that the transcription of a polynucleotide sequence (usually a DNA sequence) is dependent upon its operative linkage to elements that aid in initiating or promoting transcription. "Operably linked" means that the polynucleotides are arranged in a manner that allows them to function in the cell. In one aspect, the invention provides a promoter that is operably linked to a downstream sequence.

[0130] The term "exon" refers to a nucleic acid sequence that contains a protein-coding sequence. A gene typically contains more than one exon, separated by introns.

[0131] As used herein, the term "intron" refers to a nucleic acid sequence flanked by a splice donor site at the 5' end and a splice acceptor site at the 3' end. In some embodiments, an intron is spliced ​​or removed from an RNA or mRNA sequence expressed from a vector in which the intron is present.

[0132] The term "splice donor site" is a nucleic acid sequence or domain at the 5' end of an intron. In one embodiment, the splice donor site marks the start of an intron and / or the boundary of an intron with the immediately preceding coding sequence (or exon).

[0133] 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, a splice acceptor site marks the beginning of an intron and its boundary with a subsequent coding sequence (exon). In another embodiment, a splice acceptor site comprises an intron branch point, which is the point to which the 5' end of an intron is connected during the splicing process. In some embodiments, the splice acceptor sequence and the intron branch site are placed adjacent to each other as a single unit. In some embodiments, the splice acceptor sequence and the intron branch site can be further separated by moving the branch site further to the 5' end of the splice acceptor sequence.

[0134] As used herein, the term "splice site" refers to a nucleic acid sequence or domain present at the 5' or 3' end of an intron as defined above.

[0135] The term "exon skipping" as used herein refers to modifying pre-mRNA splicing by targeting splice donor and / or acceptor sites within pre-mRNA using one or more complementary antisense oligonucleotides. By blocking the access of the spliceosome to one or more splice donor or acceptor sites, one or more complementary antisense oligonucleotides can prevent the splicing reaction, thereby resulting in the deletion of one or more exons from the fully processed mRNA. In one embodiment, exon skipping is achieved in the nucleus during the maturation of pre-mRNA. It includes masking key sequences involved in targeted exon splicing by using antisense oligonucleotides that are complementary to splice donor sequences within pre-mRNA.

[0136] The term "gene regulatory sequence" as used herein refers to a nucleic acid sequence that can control the transcription, splicing or modification of a gene, open reading frame or exon or intron. The gene regulatory sequence of the present invention may include a promoter, a binding site for an antisense oligonucleotide and / or an enhancer. Therefore, placing a gene under the regulatory control of a promoter or a regulatory element means positioning the gene so that the expression of the gene is controlled by the regulatory sequence. Therefore, in the construction of a promoter-gene combination, the promoter is preferably located upstream of the gene and the distance from the transcription start site is close to the distance between the promoter and the gene it controls in its natural environment. This change in distance is tolerable without losing 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. Compared to promoter elements, enhancers are considered to be independent of relative position and direction. In some embodiments, the gene regulatory sequence includes 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.

[0137] In some embodiments, the antisense oligonucleotides (ASOs) described herein comprise nucleotides from about 8 to about 50 in length. In some cases, the ASOs comprise nucleotides from about 8 to about 30, from about 8 to about 25, from about 8 to about 20, from about 8 to about 18, from about 8 to about 15, from about 10 to about 50, from about 10 to about 30, from about 10 to about 25, from about 10 to about 20, from about 10 to about 18, from about 10 to about 15, from about 12 to about 50, from about 12 to about 30, from about 12 to about 25, from about 12 to about 20, from about 12 to about 18, or from about 12 to about 15 in length. In some embodiments, the ASOs comprise nucleotides from about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 in length.

[0138] In some cases, the ASO comprises one or more modified nucleotides. In some cases, the ASO comprises about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100% modified nucleotides. In other cases, the 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 of the ribose moiety. In some cases, the modification includes 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, halogen, sulfur, thiol, thioether, thioester, amine (primary, secondary or tertiary), amide, ether, ester, alcohol 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 heterocycle (e.g., an imidazole group, a hydrazine group, or a hydroxylamine group), an isocyanate or a cyanate group, or a sulfur-containing group (e.g., a sulfoxide group, a sulfone group, a sulfide group, and a disulfide group). In some cases, 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 some cases, 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 lock or bridged ribose modification (e.g., locked nucleic acid or LNA), ethylene nucleic acid (ENA) (e.g., 2'-4'-ethylene bridged nucleic acid), a peptide nucleic acid, or a morpholinyl group. In some cases, the modified nucleotide further comprises one or more modified internucleotide bonds. Exemplary modified internucleotide linkages include, but are not limited to, phosphorothioates, phosphorodithioates, methylphosphonates, 5′-alkylenephosphonates, 5′-methylphosphonates, 3′-alkylenephosphonates, borazine fluorides, borate phosphates and selenates with 3′-5′ or 2′-5′ linkages, phosphotriesters, thioalkylphosphotriesters, hydrogen phosphonate linkages, alkylphosphonates, alkylthiophosphonates, arylthiophosphonates, phospho-selenites, phospho-selenites, phosphonates, phosphoamidates, 3′-alkylphosphoamidates, aminoalkylphosphoamidates, thiophosphoamidates, phospho-periodides, Hydrazide, thiophosphoranilide, phosphoranilide, ketone, sulfone, sulfonamide, carbonate, carbamate, methylenehydrazine nitrogen, methylenedimethylhydrazine nitrogen, methylal, thiomethylal, oxime, methyleneimino, thioamide, bond to ribose acetyl, aminoethylglycine, silicon or siloxane bond, alkyl or cycloalkyl bond with or without heteroatoms, for example, 1 to 10 saturated or unsaturated and / or substituted and / or containing heteroatoms, bond to morpholino structure, amide, polyamide wherein the base is directly or indirectly attached to the nitrogen of the backbone, and combinations thereof.

[0139] The term "morpholino" as used herein refers to a polymer molecule having a base backbone capable of forming hydrogen bonds with a polynucleotide. In some embodiments, the polymer on the morpholino group lacks a pentose backbone portion, more specifically, lacks a ribose backbone connected by a phosphodiester bond, which is a typical feature of nucleotides and nucleosides. In one embodiment, the morpholino oligonucleotide comprises a nitrogen ring. In another embodiment, the morpholino group is a stereo pure oligonucleotide (e.g., see wavelifesciences.com, last visited on January 25, 2019) or a derivative thereof. In another embodiment, the morpholino group comprises a sequence at least 95% identical to a stereo pure polynucleotide.

[0140] In another embodiment, the morpholino group 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, including a target base sequence complementary to a target region of a selected pre-treated mRNA or pre-mRNA, such as an intronic region of a pre-mRNA. In another embodiment, the morpholino antisense oligonucleotide promotes splicing of a target exon, which results in a transcript lacking the target exon.

[0141] In some cases, antisense oligonucleotides (ASOs) are referred to herein as OncoSkip. As used herein, the term "OncoSkip" refers to an ASO designed to induce skipping of a target exon during splicing of a target transgene, thereby inducing expression of the target transgene. In some cases, the target transgene encodes a polypeptide that binds to a surface polypeptide (e.g., a surface receptor) of a target cell. In some cases, the target cell is a tumor cell or an immune cell. In some cases, the target transgene is an oncogene. In this case, the use of OncoSkip induces skipping of the target exon during splicing, thereby inducing expression of the oncogene.

[0142] In some embodiments, the OncoSkip described herein comprises nucleotides from about 8 to about 50 in length. In some examples, the OncoSkip comprises nucleotides from about 8 to about 30, from about 8 to about 25, from about 8 to about 20, from about 8 to about 18, from about 8 to about 15, from about 10 to about 50, from about 10 to about 30, from about 10 to about 25, from about 10 to about 20, from about 10 to about 18, from about 10 to about 15, from about 12 to about 50, from about 12 to about 30, from about 12 to about 25, from about 12 to about 20, from about 12 to about 18, or from about 12 to about 15. In some embodiments, the OncoSkip comprises nucleotides from about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, or 50 in length.

[0143] In some embodiments, the OncoSkip comprises one or more modified nucleotides, e.g., about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% modified nucleotides. In some cases, 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 comprises one or more morpholino-modified nucleotides. In some cases, the OncoSkip comprises about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% morpholino-modified nucleotides. In some cases, an 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.

[0144] In some cases, OncoSkip works in conjunction with 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 transgenic encoded polypeptide. In some cases, the transgene is further encompassed by a construct comprising a polynucleotide encoding dimer. In some cases, in conjunction with OncoSkip, OncoSkip skips exons from the intron-exon-intron region during splicing to generate mRNA capable of expressing the transgenic encoded polypeptide. In the absence of OncoSkip, due to the presence of a stop codon in the intron-exon-intron region, transgenic expression from TransSkip is silenced.

[0145] As used herein, the term "dimert" refers to an engineered protein molecule comprising two or more single-chain variable fragments (scFv), 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, dimert targets two different cell types, such as 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 cells are effector immune cells. In some cases, the effector immune cells include effector T (T) cells. 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 cells. eff In some cases, dimert targets two different cancer cells, for example, two different cancer cells from the same cancer.

[0146] In some cases, two or more scFvs of dimert are linked by a linker. In some cases, the linker is a peptide linker that promotes the binding of each scFv to its respective target polypeptide. In some cases, the linker comprises a series of poly-Ala, poly-Gly or a combination thereof. In some cases, the poly-Ala linker, poly-Gly linker or a peptide linker comprising a combination of Ala and Gly is each independently about 2 residues to about 50 residues in length. In some cases, the poly-Ala linker, the poly-Gly linker, or the peptide linker comprising a combination of Ala and Gly is each independently about 2 residues to about 45 residues, about 4 residues to about 45 residues, about 5 residues to about 45 residues, about 8 residues to about 45 residues, about 10 residues to about 45 residues, about 15 residues to about 45 residues, about 20 residues to about 45 residues, about 30 residues to about 45 residues, about 2 residues to about 40 residues, about 4 residues to about 40 residues, about 5 residues to about 40 residues, about 8 residues to about 40 residues, about 10 residues to about 40 residues, or about 45 residues in length. In some embodiments, the present invention relates to a polynucleotide comprising a polynucleotide or a sequence of at least one amino acid sequence. The polynucleotide or the sequence of the polynucleotide is selected from the group consisting of: a polynucleotide, ... In certain instances, the poly-Ala linker, poly-Gly linker, or 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.

[0147] In some cases, the linker is a (Gly4Ser)n linker, wherein n is an integer from 1 to 10. In some cases, n is an integer from 1 to 6, from 1 to 4, or from 1 to 3. In some cases, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, 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 to about 20 amino acid residues in length. In some cases, the linker comprises one or more unnatural amino acids.

[0148] In some cases, the dimert further comprises an additional polypeptide. In some cases, the additional polypeptide enhances the affinity of dimert for target cells. In some cases, the additional polypeptide is a dimerization domain of human hepatocyte nuclear factor 1 alpha (HNF1α). In some cases, HNF1α 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 instances, HNF1α is encoded by a polynucleotide having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to ATGGTGAGCAAGCTGAGCCAGCTGCAGACCGAGCTGCTGGCCGCCCTGCTGGAGAGCGGCCTGAGCAAGGAGGCCCTGATCCAGGCCCTGGGCGAG (SEQ ID NO: 48). In some cases, the additional polypeptides do not induce any additional immunogenicity or toxicity.

[0149] In some embodiments, the additional polypeptide is connected to the rest of the dimer via a linker. In some cases, the linker comprises GSGGAP. As used herein, the GSGGAP peptide is also referred to herein as a spacer. In some cases, the linker comprises TPLGDTTHTSG. In some cases, the peptide TPLGDTTHTSG is from the hinge region of IgG3.

[0150] As used herein, the term "TransJoin" refers to a recombinant vector (e.g., a recombinant viral vector, such as an AAV vector) comprising a polypeptide encoding dimert as described herein but without an intron-exon-intron region (wherein the exon contains a stop codon). Thus, TransJoin differs from TransSkip in that TransJoin can express dimert without the need for OncoSkip.

[0151] In some embodiments, the TransJoin described herein is optimized for delivery of dimert to target cells or target tissues. In some cases, the TransJoin comprises a promoter and enhancer pair, or comprises a promoter and regulatory element pair optimized for delivery and / or expression to target cells or target tissues. In some cases, the TransJoin is optimized for constitutive expression of dimert over a period of time (e.g., using a promoter and enhancer pair, or a promoter and regulatory element pair). In some cases, the 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 some cases, the TransJoin comprises a promoter and regulatory element for enhanced expression, constitutive expression, stable expression, or a combination thereof, such as a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE).

[0152] In some embodiments, the TransJoin described herein comprises a promoter and enhancer pair, or a promoter and regulatory element pair optimized for balanced expression of dimert in target cells or target tissues. In some cases, balanced expression refers to an expression range where expression above this range induces toxicity, while expression below this range does not produce a therapeutic effect. In some cases, the promoter and enhancer, or the promoter and regulatory element (e.g., WPRE), act synergistically to balance the expression of dimert, thereby achieving a target range. In some cases, balanced expression includes a wide range, for example, providing a wide therapeutic window for dimert.

[0153] In some embodiments, the TransJoin described herein further comprises a secretion consensus sequence described below, which is optimized for the balanced expression of dimert in target cells or target tissues. In some cases, balanced expression refers to an expression range in which expression above this range induces toxicity, while expression below this range does not produce a therapeutic effect. In some cases, the secretion consensus sequence synergizes with a promoter, enhancer, regulatory element (e.g., WPRE), or a combination thereof to balance the expression of dimert, thereby achieving a target range. In some cases, balanced expression includes a wide range, for example, providing a wide therapeutic window for dimert.

[0154] As described above, the time period includes one day, two days, three days, four days, five days, seven days, twenty-one days, 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.

[0155] As used herein, the term "isolated" refers to a molecule, biological or cellular material that is substantially free of other material.

[0156] As used herein, the term "functional" may be applied to the modification of any molecule, biological product, or cellular material to achieve a particular specific effect.

[0157] 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-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases.

[0158] 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 can be constitutive, inducible, repressible, or tissue-specific. A "promoter" is a control sequence that 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 ROS sarcoma virus (RSV) LTR promoter (optionally with RSV enhancer), cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerate kinase (PGK) promoter, U6 promoter, or EF1α short form (EFS) promoter.

[0159] In some embodiments, the promoter is an EF1α short form (EFS) promoter. In certain instances, the EF1α short form (EFS) promoter comprises GTCCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGAATTCGCTAGCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGG (SEQ ID NO: 49), or an equivalent thereof.

[0160] Other non-limiting exemplary promoters with specific target specificity are provided below, including but not limited to cytomegalovirus (CMV), human polypeptide chain elongation factor (EF1a), SV40, phosphoglycerate kinase (PGK), such as 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, alpha-1-antitrypsin, spleen focus forming virus (SFFV) and chicken β-actin (CBA). Synthetic derived promoters can be used for ubiquitous or tissue-specific expression. In addition, virally derived promoters, some of which are described above, can be used in the methods disclosed herein, such as CMV, HIV, adenovirus and AAV promoters.

[0161] 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 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, SV40 / hAlb (InvivoGen); photoreceptor-specific promoters, such as human rhodopsin kinase (GRK1) and visual inhibitory protein (CAR); B cell-specific promoters, such as B29 (InvivoGen); hematopoietic cell-specific promoters, such as CD45 promoter and SV40 / CD45 from InvivoGen; myocyte-specific promoters, such as desmin promoter (InvivoGen); pancreatic acinar cell-specific promoters, such as elastase-1 promoter (InvivoGen); endothelial cell-specific promoters, such as Flt-1 promoter (InvivoGen); and neuron-specific promoters, such as SYN1 promoter (InvivoGen).

[0162] In some embodiments, the promoter is coupled to an enhancer to increase transcription efficiency. Non-limiting examples of enhancers include RSV enhancer, CMV enhancer, and α-fetoprotein MERII enhancer.

[0163] An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter / enhancer" is a polynucleotide that contains sequences that can provide both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. Enhancers / promoters may be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer / promoter is one that is naturally attached to a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one that has been juxtaposed to a gene by genetic manipulation (i.e., molecular biology techniques) such that transcription of the gene is controlled by the attached enhancer / promoter.

[0164] In some embodiments, the vector used herein (e.g., a viral vector, such as an AAV vector) 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 some cases, the exemplary WPRE comprises the nucleic acid sequence of SEQ ID NO: 50 or its equivalent. The sequence of SEQ ID NO: 50 is as follows:

[0165] (SEQ ID NO: 50).

[0166] The terms "protein," "peptide," and "polypeptide" are used interchangeably and in their broadest sense to refer to a compound composed of two or more subunits of amino acids, amino acid analogs, or peptidomimetics. The subunits can be linked by peptide bonds. In another aspect, the subunits can be linked by other bonds, such as esters, ethers, and the like. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that can comprise a protein or peptide sequence. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including glycine and its D and L optical isomers, amino acid analogs, and peptidomimetics.

[0167] As used herein, the term "vector" refers to a non-chromosomal nucleic acid containing a complete replicon so that it can be replicated when the vector is placed in a cell, for example, by a transformation process. A vector can be a viral vector or a non-viral vector. Viral vectors include retroviruses, adenoviruses, herpes viruses, baculoviruses, modified baculoviruses, pasteurian viruses, or other modified natural 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 (such as isomeric polylysine, limited length oligopeptides, and polyethyleneimine), in some cases contained in liposomes; and the use of ternary complexes comprising viruses 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.

[0168] "Viral vector" is defined as a recombinantly produced virus or viral particle comprising a polynucleotide that will be delivered to a host cell in vivo, in vitro or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenoviral vectors, alphaviral vectors, etc. Alphaviral vectors, such as vectors based on Semliki Forest virus and vectors based on Sindbis virus, have also been developed for gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5: 434-439 and Ying, et al. (1999) Nat. Med. 5 (7): 823-827. In some cases, 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 rh74. In some cases, the AAV rh74 comprises the vector sequence of GenBank No. LP899424.1 (accessed on February 7, 2020).

[0169] In certain embodiments, the carrying capacity of a viral vector (e.g., an AAV vector) is limited. For example, the carrying capacity of an AAV vector is limited to 4.7 kb. Therefore, the combination of dimert with a promoter, enhancer, and other regulatory elements needs to be within a capacity of 4.7 kb. In this case, the promoter used herein is selected based on its nucleic acid length so that dimert and other regulatory elements can be packaged into a viral vector (e.g., an AAV vector). In some cases, the promoter of this use is SFFV, EF1α, PGK, UbiC, CMV, CBA, or EFS. In some cases, the promoter is EFS.

[0170] In another embodiment, the promoter is an inducible promoter. In a specific embodiment, the promoter is an inducible tetracycline promoter. As described by Gossen & Bujard (1992; Tet-Off) and Gossen et al. (1995; Tet-On), the Tet-Off and Tet-On gene expression systems provide researchers with ready access to regulated, high-level gene expression systems. In the Tet-Off system, gene expression is turned on when tetracycline (Tc) or doxycycline (Dox; a Tc derivative) is removed from the culture 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 levels in the Tet system are very high and compare well to the maximum expression levels achieved with strong constitutive mammalian promoters, such as 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 nonspecific induction. In Escherichia coli, the Tet repressor protein (TetR) negatively regulates genes of the tetracycline resistance operon on the Tn10 transposable element. In the absence of Tc, TetR blocks transcription of these genes by binding to the tet operator sequence (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, also known as tetracycline activator protein), is encoded by the pTet-On regulatory plasmid.

[0171] 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 expression of the tetracycline activator protein.

[0172] Other inducible systems that can be used in the vectors, isolated cells, viral packaging systems, and methods described herein include ecdysone, estrogen, progesterone, chemical inducers of dimerization, and regulation by isopropyl-β-D1-thiogalactopyranoside (EPTG).

[0173] As used herein, the term "recombinant expression system" or "recombinant vector" refers to one or more genetic constructs for expressing certain genetic materials formed by recombination.

[0174] A "gene delivery vector" is defined as any molecule capable of carrying a polynucleotide for insertion into a host cell. Examples of gene delivery vectors include liposomes, micelles, biocompatible polymers, including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria or viruses, such as baculoviruses, adenoviruses, and retroviruses, phages, 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 can be used for gene therapy as well as simple protein expression.

[0175] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vectors. "Gene delivery," "gene transfer," "transduction," etc., as used herein, refer to the term for introducing exogenous polynucleotides (sometimes referred to as "transgenes") into host cells, regardless of the method for introducing. Such methods include a variety of well-known technologies, such as vector-mediated gene transfer (such as by viral infection / transfection or various other protein- or lipid-based gene delivery complexes) and technology (such as electroporation, "gene gun" delivery, and various other technologies for introducing polynucleotides) that promote the delivery of "naked" polynucleotides. The introduced polynucleotides can be stably or temporarily maintained in the host cell. Stable maintenance generally requires that the introduced polynucleotides include a replication origin that is compatible with the host cell, or are integrated into the replicon of the host cell, such as an extrachromosomal replicon (such as a plasmid) or a nuclear chromosome or a mitochondrial chromosome. Many known vectors are capable of mediating gene transfer to mammalian cells, as known in the art and described herein.

[0176] A "plasmid" is an extrachromosomal DNA molecule that is separate from chromosomal DNA and capable of replicating independently of it. In many cases, it is circular and double-stranded. Plasmids provide a mechanism for horizontal gene transfer within microbial populations and often provide a selective advantage under given environmental conditions. Plasmids may carry genes that confer resistance to naturally occurring antibiotics in competitive environmental niches, or they may produce proteins that act as toxins under similar circumstances.

[0177] The "plasmids" used in genetic engineering are called "plasmid vectors." Many commercially available plasmids are available for this purpose. The gene to be replicated is inserted into a copy of the plasmid, which contains the gene that makes the cell resistant to a specific antibiotic and a multiple cloning site (MCS or polylinker), a short region containing several commonly used restriction sites that allows DNA fragments to be easily inserted at this location. Another major use of plasmids is to produce large quantities of proteins. In this case, researchers grow bacteria that contain a plasmid carrying the gene of interest. Just as the bacterium produces the protein to confer antibiotic resistance, it can also be coaxed into producing large quantities of protein from the inserted gene.

[0178] In aspects where gene transfer is mediated by a DNA viral vector, such as adenovirus (Ad) or adeno-associated virus (AAV), a vector construct refers to a polynucleotide comprising the viral genome or portion thereof and a transgene. Adenovirus (Ad) is a relatively well-characterized group of homologous viruses that includes more than 50 serotypes. Ad does not require integration into the host cell genome. Recombinant Ad-derived vectors, particularly those that reduce the likelihood of 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 infective and specific and can integrate into the host cell genome. See, Wold and Toth (2013) Curr. Gene. Ther. 13(6):421-433, Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470, and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0179] Vectors comprising a promoter and a cloning site to which a polynucleotide can be operably linked are common knowledge in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). In order to optimize expression and / or in vitro transcription, it may be necessary to remove, add, or alter the 5′ and / or 3′ untranslated portion of the clone to eliminate additional, potential, inappropriate alternative translation start codons or other sequences that may interfere with or reduce expression, whether at the transcriptional or translational level. Alternatively, a consensus ribosome binding site can be inserted immediately 5′ of the start codon to enhance expression.

[0180] As used herein, the term "adapter," "adapter molecule," or "activation antigen" refers to a molecule secreted from a cell capable of activating an immune cell. In a specific embodiment, the adaptor activates specific immune effector cells according to the domain present in the adaptor. Illustrative examples of cells that secrete adaptors include, but are not limited to, T cells, NK cells, NKT cells, CAR T cells, mesenchymal stem cells (MSC), neural stem cells, hematopoietic stem cells, or a mixture thereof. In another embodiment, the immune effector cells include dendritic cells, natural killer ("NK") cells, macrophages, T cells, B cells, or a combination thereof.

[0181] The term "antigen recognition domain" or "antigen binding domain" refers to a portion of an adaptor molecule that recognizes an antigen. In particular embodiments, the antigen can be of any nature, including but not limited to proteins, carbohydrates, and / or synthetic molecules.

[0182] As used herein, the term "activation antigen" or "activation domain" refers to a portion of an adaptor 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 immunomodulatory signals include signals that inhibit cell proliferation, inhibit the secretion of immunosuppressive factors, or induce apoptosis.

[0183] As used herein, the term "innate immune cell" refers to an immune cell that is naturally present in the immune system. Illustrative examples include, but are not limited to, T cells, NK cells, NKT cells, B cells, and dendritic cells.

[0184] As used herein, the term "engineered immune cell" refers to an immune cell that has been genetically modified.

[0185] 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, tolerant T cells, chimeric B cells, and antigen-specific T cells.

[0186] As used herein, the term "antibody" refers not only to complete antibody molecules, but also to antibody molecule fragments that retain immunogen binding ability. Such fragments are common knowledge in the art and are frequently used both in vitro and in vivo. Therefore, as used herein, the term "antibody" refers not only to complete immunoglobulin molecules, but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments lacking the Fc fragment of a complete antibody are cleared from the circulation faster and may have less nonspecific tissue binding to the complete antibody (Wahl et al., J. Nucl. Med. 24: 316-325 (1983)). The antibodies of the present invention include all-natural antibodies, monoclonal antibodies, human antibodies, humanized antibodies, camelid antibodies, multispecific antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single-chain V region fragments (scFv), single-domain antibodies (e.g., nanobodies and single-domain camelid antibodies), VNAR fragments, bispecific T cell engagers (BiTE) antibodies, miniantibodies, disulfide-linked FVs (sdFv) and anti-idiotypic (anti-Id) antibodies, in vivo antibodies, fusion polypeptides, non-traditional antibodies and any of the above antigen-binding fragments. Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain antigen binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

[0187] In certain embodiments, antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V H ) and heavy chain constant (C H ) region. The heavy chain constant region consists of three domains: CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated here as V L ) and light chain constant C L The light chain constant region consists of a domain C L Composition. H and V L The regions can be further subdivided into regions of high variability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V LIt consists of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system. As used interchangeably herein, the terms "antigen binding portion", "antigen binding fragment" or "antigen binding region" refer to the region or portion of an antibody that binds to an antigen and confers antigen specificity to the antibody; a fragment of an antigen binding protein, for example, an antibody includes one or more antibody fragments (such as a peptide / HLA complex) that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be achieved by fragments of a full-length antibody. Examples of antigen binding portions within the term "antibody fragment" that include antibodies include: Fab fragments, L 、V H 、C L and CHI domains; (Fab)2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bond in the hinge region; H and CHI domains; the Fd fragment consists of the V L and V H Fv fragment composed of domains; dAb fragment (Ward et al., Nature 341: 544-546 (1989)), composed of V H domain composition; and isolated complementarity determining regions (CDRs).

[0188] Antibodies and antibody fragments can be derived in whole or in part from mammals (e.g., humans, non-human primates, goats, guinea pigs, hamsters, horses, mice, rats, rabbits, and sheep) or non-mammalian antibody-producing animals (e.g., chickens, ducks, geese, snakes, and uromorphs). Antibodies and antibody fragments can be produced in vivo or in vitro, for example, from yeast or phage (e.g., as a single antibody or antibody fragment or as part of an antibody library).

[0189] In addition, although the two domains V L and V H are encoded by separate genes, but they can be joined using recombinant methods with synthetic linkers to make them into a single protein chain, where V L and V HThe regions pair to form monovalent molecules. These are called single-chain Fvs (scFvs); see, for example, Bird et al., Science 242:423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. 85:5879-5883 (1988). 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.

[0190] An "isolated antibody" or "isolated antigen binding protein" is an antibody that has been identified, separated and / or recovered from a component of its natural environment. A "synthetic antibody" or "recombinant antibody" is typically produced using recombinant or synthetic peptide technology known to those skilled in the art.

[0191] As used herein, the term "single-chain variable fragment" or "scFv" is a fragment of a polypeptide that is covalently linked to form a V H :V L Heterodimer immunoglobulin (e.g., mouse or human) heavy chain (V H ) and light chain (V L ) variable region fusion protein. Heavy chain (V H ) and light chain (V L ) are directly linked or linked via a peptide-encoded linker (e.g., about 10, 15, 20, 25 amino acids) that connects V H The N-terminal and V L C-terminus, or V H The C-terminus and V L The N-terminus of the molecule is cleaved. Linkers are typically rich in glycine to increase flexibility and serine or threonine to enhance solubility. This linker can connect the heavy and light chain variable regions of the extracellular antigen-binding domain.

[0192] Despite the removal of the constant region and the introduction of a linker, the scFv protein still retains the specificity of the original immunoglobulin. As described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85: 5879-5883 (1988)), a single-chain Fv polypeptide antibody can be obtained from a V-containing protein. H and V LSee also, U.S. Patent 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); Moosmayer et al., Ther Immunol 2(10):31-40(1995). Agonistic scFvs with stimulatory activity have been described (see, for example, Peter et al., J Biol Chem 25278(38):36740-7(2003); Xie et al., Nat Biotech 15(8):768-71(1997); Ledbetter et al., Crit Rev Immunol 17(5-6):427-55(1997); Ho et al., Bio Chim Biophys Acta 1638(3):257-66(2003)).

[0193] As used herein, "F(ab)" refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have an Fc portion, e.g., papain digestion of an antibody produces two F(ab) fragments and one Fc fragment (e.g., a heavy (H) chain constant region; the Fc region that does not bind to an antigen).

[0194] 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') (divalent) regions, wherein each (ab1) region comprises two independent amino acid chains, an H chain and a portion of a light (L) chain connected by an SS bond for antigen binding, wherein the remaining H chain portions are linked together. A single "F(ab')2" fragment can be split into two separate Fab' fragments.

[0195] As used herein, "CDR" is defined as the complementary determining region amino acid sequence of an antibody, which is the hypervariable region of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th USDepartment 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. CDRs provide most of the contact residues for the binding of an antibody to an antigen or epitope. In certain embodiments, the Kabat system is used to depict CDR regions (Kabat, EA, et al. Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 (1991)).

[0196] As used herein, the term "affinity" refers to a measure of binding strength. Without being bound by theory, affinity depends on the tightness of the stereochemical coordination between the antibody binding site and the antigenic determinant, 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 the antigen-antibody bond after the formation of a reversible complex (e.g., monovalent or multivalent). Methods known in the art for calculating the affinity of an antibody for an antigen include using binding experiments to calculate affinity. Antibody activity in functional assays (e.g., flow cytometry analysis) also reflects antibody affinity. Antibodies and affinities can be phenotypically characterized and compared by functional assays (e.g., flow cytometry analysis). Nucleic acid molecules that can be used in the subject matter of the present invention include any nucleic acid molecules encoding a polypeptide or a fragment thereof. In certain embodiments, nucleic acid molecules that can be used in the subject matter of the present invention include nucleic acid molecules encoding an antibody or its antigen-binding portion. Such nucleic acid molecules do not need to be 100% identical to the endogenous nucleic acid sequence, but will typically exhibit substantial consistency. Polynucleotides having "substantial homology" or "substantial identity" to endogenous sequences are generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. "Hybridization" refers to 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 Enzymol. 152: 399 (1987); Kimmel, R. M. Methods Enzymol. 152: 507 (1987)).

[0197] 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 used for comparison (e.g., a wild-type or native sequence). In some embodiments, the 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, the substantially homologous or substantially identical polypeptide comprises one or more non-natural amino acids or amino acid analogs, including D-amino acids and reverse transcriptase amino acids, in place of the homologous sequence.

[0198] 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 / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. In an exemplary method for determining the degree of identity, the BLAST program can be used, wherein e -3 and e -100 Probability scores between and indicate closely related sequences.

[0199] As used herein, the term "analog" refers to a structurally related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

[0200] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding properties of the currently disclosed engineered receptor (e.g., the extracellular antigen binding domain of the engineered receptor) comprising the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the human single-chain antibodies of the currently disclosed engineered 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 substitution refers to the replacement of 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, and histidine, negatively charged amino acids include aspartic acid and 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 can be substituted with other amino acid residues from the same group, and the altered antibody can be tested for retained function (i.e., the functions described in (c) to (1) above) using the functional assays 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.

[0201] As used herein, the term "ligand" refers to a molecule that binds to a receptor. Specifically, a ligand binds to a receptor on another cell, allowing for intercellular recognition and / or interaction.

[0202] As used herein, the term "costimulatory signal domain" or "costimulatory domain" refers to an engineered receptor portion comprising an intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for the effective activation and function of T lymphocytes when bound to an antigen. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2, and a ligand specifically binding CD83. Therefore, although the present invention provides exemplary costimulatory domains derived from CD28 and 4-1BB, it is expected that other costimulatory domains can be used for engineered receptors as described herein. One or more costimulatory signal domains can be included to enhance the efficacy and amplification of T cells expressing engineered receptors. Intracellular signals and costimulatory signal domains can be connected in series to the carboxyl terminus of the transmembrane domain in any order.

[0203] 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.

[0204] 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, the bispecific antibody comprises a polypeptide as described in Brinkmann et al., "The making of bispecific antibodies," MABS 9(2): 182-212 (2017). Figure 2 ; or Labrijn, et al., "Bispecific antibodies: a mechanistic review of the pipeline," Nature Reviews18:585-608 (2019) Figure 2 The disclosed antibody formats (or antibody structures).

[0205] In some embodiments, at least one of the antigen-binding regions of the bispecific or trispecific antibodies binds to an activating antigen on an immune effector cell. This can be understood as different targeted binding, but also includes binding to different epitopes within 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 (CD276), CD20, CD22, or a combination thereof. Non-limiting examples of bispecific antibodies include, but are not limited to, CD3xCD19, CD3xGD2, CD3xEphA2, and NKG2DxGD2 antibodies.

[0206] In one embodiment, the dimert comprises a bispecific antibody. In some cases, the dimert comprises a bispecific T cell engager ("BiTE") antibody, a bispecific killer cell engager ("BiKE") antibody, or other bispecific antibodies described herein, such as antibodies associated with different immune cells. In another embodiment, the dimert comprises a trispecific antibody. In some cases, the dimert comprises a trispecific T cell engager ("TriTE") antibody, a trispecific killer cell engager ("TriKE") antibody, or other trispecific antibodies described herein, such as antibodies associated with different immune cells.

[0207] As used herein, the term "bispecific T cell engager" or "BiTE" refers to a bispecific monoclonal antibody that includes a first antigen binding fragment that binds to a T cell receptor and a second antigen binding fragment that binds to a tumor cell through a tumor-specific molecule. As used herein, the term "trispecific T cell engager" ("TiTE" or "TriTE") refers to a trispecific monoclonal antibody that includes a first antigen binding fragment that binds to a T cell engager, a second antigen binding fragment that binds to a tumor cell through a tumor-specific molecule, and a third antigen binding fragment that binds to a T cell engager or a cytokine T cell activation domain. In one embodiment, the tumor-specific molecule comprises an antigen selected from adrenergic type A receptor 2 (EphA2), interleukin (IL) -13rα2, EGFR VIII, PSMA, EpCAM, GD2 or 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 (GM2), growth factor receptor, stromal antigen, vascular antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR1), mesothelin, CD38, CD123, human epidermal growth factor receptor 2 (HER2), B cell maturation antigen (BCMA), fibroblast activation protein (FAP) α or a combination thereof. Examples of BiTEs include, but are not limited to, Blinatumomab (MT103) and Solitomab (MT110).

[0208] 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 tumor cells 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 an 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 an NK cell engager or cytokine NK cell activation domain. The NKG2DxIL21RxGD2 antibody is an exemplary TriKE antibody. Examples of NK cell engager domains include, but are not limited to, binding to CD16, CD16, and NK cell activating domains. + CD2, CD16 + DNAM and CD16 +NKp46 ligand or molecule. Examples of cytokine NK activation domains include but are not limited to IL-15, IL-12, IL-18, IL-21 or other NK cell enhancing cytokines, chemokines and / or activating molecules. The NK binding domain may include any portion that binds to and / or activates NK cells and / or any portion that prevents NK cell inhibition. In certain embodiments, the NK binding domain may include an antibody that selectively binds to NK cell surface components. In other embodiments, the NK binding domain may include a ligand or small molecule that selectively binds to NK cell surface components.

[0209] In some embodiments, the NK binding domain can selectively bind to a receptor at least partially located on the surface of an NK cell. In certain embodiments, the NK binding domain can play a role in binding to NK cells, thereby making the space of NK and the selective binding of the targeting domain close. However, in certain embodiments, the NK binding domain can selectively bind to the receptor that activates NK cells and therefore also has an activation function. As described above, activation of the CD16 receptor can cause antibody-dependent cell-mediated cytotoxicity. Therefore, in certain embodiments, the NK binding domain may include at least a portion of an anti-CD16 receptor antibody that is effectively selectively bound to the CD16 receptor. In other embodiments, the NK binder cell domain can interrupt the mechanism of suppressing NK cells. In such embodiments, the NK binding domain may include, for example, anti-PD1 / PDL1, anti-NKG2A, anti-TIGIT, anti-killer immunoglobulin receptor (KIR) and / or any other inhibitory blocking domain.

[0210] In a particular embodiment, cells are genetically modified (including immune cells) with adapter molecules, and the molecules at least include an antigen recognition domain and an activation domain and an optional cytokine, a costimulatory domain and / or a domain that suppresses the negative regulatory molecules of T cell activation. The antigen recognition domain of the adapter molecule is combined with one or more molecules present in and / or on the target cell or secreted by the target cell. In particular aspects, the target cell is a cancer cell, at least including solid tumor cells. Once the adapter molecule is combined with the target molecule, they can activate the cells expressing the molecules identified by the activation domain. The adapter molecule can activate the cells that are genetically modified with the adapter molecule, or it can activate unmodified cells.

[0211] Depending on the desired effect, activation can produce positive or negative signals. Examples of positive signals include those that induce cell proliferation, cytokine secretion, or cytolytic activity. Examples of negative signals include those that inhibit T cell proliferation, suppress the secretion of immunosuppressive factors, or induce cell death.

[0212] In certain aspects, immune cells that secrete adaptor molecules are capable of redirecting resident (natural endogenous to a particular individual) immune cells to cancer cells.

[0213] Embodiments of the present 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 specific cancers), rather than simply delivering the adaptor molecule to the individual itself (in the absence of modified immune cell production). In the present invention, the individual receives modified immune cells that are allowed to produce adaptor molecules. In specific embodiments, the cells produce immunostimulatory cytokines; proliferate in an antigen-specific manner; kill appropriate target cells; redirect bystander immune cells (including at least T cells or NK cells) to cancer cells; upon activation, secrete adaptor molecules; and / or are effective against cancer in a localized or systemic manner. Figure 3 Examples of modified T cells or NK cells that secrete adaptor molecules are shown. Although a particular T cell or NK cell may produce an adaptor that targets the same cancer cell-specific antigen, the activation domain of the T cell or NK cell must be different because NK cells do not express CD3. Examples of NK cell activation domains include at least CD16, NKG2D, or NKp30.

[0214] Gene delivery vectors also include DNA / liposome complexes, micelles, and targeted viral protein DNA complexes. Liposomes containing targeting antibodies or fragments thereof can be used in the methods disclosed herein. In addition to delivering polynucleotides to cells or cell populations, proteins described herein can be directly introduced into cells or cell populations by non-limiting protein transfection techniques, or other non-limiting techniques can enhance the expression of proteins disclosed herein and / or promote culture conditions for their activity.

[0215] As used herein, the term "signal peptide" or "signal polypeptide" refers to an amino acid sequence that is typically present at the N-terminus of a newly synthesized secretory or membrane polypeptide or protein. Its function is to direct the polypeptide to a specific cellular location, for example, across the cell membrane, into the cell membrane, or into the cell 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.

[0216] As used herein, the term "viral capsid" or "capsid" refers to the protein coat or outer shell of a viral particle. The function of the capsid is to encapsidate, 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 encapsidation within a viral capsid.

[0217] As used herein, the term "helper" with respect to a virus or plasmid refers to a virus or plasmid that is used to provide the additional components required for replication and packaging of viral particles or recombinant viral particles, such as the modified AAV disclosed herein. The components encoded by the helper virus may include any genes required for virion assembly, encapsidation, genome replication and / or packaging. For example, the helper virus may encode an enzyme required for viral genome replication. Non-limiting examples of helper viruses and plasmids suitable for use in AAV constructs include pHELP (plasmid), adenovirus (virus), or herpes virus (virus).

[0218] As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA small virus that grows only in cells, where some of its functions are provided by a co-infected helper virus. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). It is entirely expected that the same principles described in these reviews will apply to other AAV serotypes characterized after the publication date of the reviews, as it is well known that the various serotypes are very closely related in structure and function, even at the genetic level. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, JR Pattison, ed.; and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, all AAV serotypes clearly display very similar replication properties mediated by homologous rep genes; they all carry three related capsid proteins, like those expressed in AAV2. The degree of relatedness is further demonstrated by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of similar self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs). The similar infection patterns also suggest that the replication functions of each serotype are under similar regulation.

[0219] 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 a vector encoding and expressing the rep and cap gene products.

[0220] "AAV virion" or "AAV viral particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and the encapsidated polynucleotide AAV vector. If the particle contains 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 generally referred to as an "AAV vector particle," or simply "AAV vector." Thus, the production of an AAV vector particle necessarily includes the production of an AAV vector, since such a vector is contained within the AAV vector particle.

[0221] In some embodiments, AAV is a replication-deficient parvovirus with a single-stranded DNA genome of approximately 4.7 kb in length, including two 145-nucleotide inverted terminal repeats (ITRs). AAV has multiple serotypes. The nucleotide sequences of the AAV serotype genomes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the complete genome of AAV-5 is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 45:555-564 (1983). al., J. Virol., 78: 6381-6388 (2004); AAV-10 genome is provided in Mol. Ther., 13 (1): 67-76 (2006); AAV-11 genome is provided in Virology, 330 (2): 375-383 (2004). U.S. Patent No. 9,434,928 provides the sequence of the AAVrh.74 genome, 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 host cell chromosome integration are contained in AAV ITRs. Three AAV promoters (named p5, p19, and p40 by relative map position) drive expression of two AAV internal open reading frames encoding rep and cap genes. Differential splicing of two rep promoters (p5 and p19) with a single AAV intron (at nucleotides 2107 and 2227) results in the production of four rep proteins (rep78, rep68, rep52, and rep40). Rep proteins have diverse enzymatic properties and are ultimately responsible for replicating the viral genome. The cap gene, expressed from the p40 promoter, encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation start sites are responsible for the production of the three related capsid proteins.A single consensus polyadenylation site is located in the AAV genome at map position 95. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0222] 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 noncytopathic, and natural infection in humans and other animals is asymptomatic. Furthermore, AAV infects many mammalian cells, making it possible to target many different tissues in vivo. Furthermore, AAV can transduce both slowly dividing and non-dividing cells and persist as a transcriptionally active nuclear episome (extrachromosomal element) throughout the life cycle of these cells. The AAV proviral genome is inserted as cloned DNA into a plasmid, making it possible to construct recombinant genomes. Furthermore, because the signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, part or all of the approximately 4.3 kb internal genome (encoding the replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA. To generate AAV vectors, the rep and cap proteins can be provided in trans. Another notable feature of AAV is that it is a very stable and robust virus. It readily tolerates the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making refrigeration of AAV less critical. AAV can even be freeze-dried. Finally, AAV-infected cells are not resistant to repeated infection.

[0223] Several studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8:659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93:14082-14087 (1996); and Xiao et al., J Virol, 70:8098-8108 (1996). See also Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). In addition, because muscle is highly vascularized, recombinant AAV transduction has resulted in the appearance of transgene products in the systemic circulation after intramuscular injection, as described by Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94:13921-13926 (1997). In addition, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers have the cytokines required for proper antibody glycosylation, folding, and secretion, indicating that muscle can stably express secretory protein therapeutics. The AAV DNA in the rAAV genome can be from any AAV serotype from which recombinant viruses can 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 generation of pseudotyped rAAVs is disclosed in WO 01 / 83692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also contemplated. See, for example, 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.

[0224] As used herein, the term "outside" referring to a viral capsid protein refers to a surface, domain, region or end of a capsid protein facing the outside in the viral capsid of an assembly. The term "inside" referring to a viral capsid protein refers to a surface, domain, region or end (amino terminal or carboxyl terminal) of a capsid protein facing the inside in the viral capsid of an assembly. When used for an assembled viral capsid, the term "inside" refers to the encapsidation space in the viral capsid and the inward surface of the capsid exposed to a closed space. The internal space is wrapped by viral capsid proteins and can include nucleic acids, such as viral genomes, viral proteins, proteins of hosts or packaging cells, and any other components or factors packaged or embedded during replication, virion assembly, encapsidation and / or packaging.

[0225] As used herein, the term "label" refers to a directly or indirectly detectable compound or composition, such as a polynucleotide or protein, such as an antibody, that is directly or indirectly coupled to a composition to be detected, to generate a "label" composition. The term also includes sequences coupled to polynucleotides that provide signals when the inserted sequence is expressed, such as green fluorescent protein (GFP). The label itself is detectable (such as radioisotope labeling or fluorescent labeling), or in the case of enzyme labeling, can catalyze the chemical alteration of a detectable substrate compound or composition. Labeling can be applicable to small-scale detection or is more suitable for high-throughput screening. Therefore, suitable labels include but are not limited to radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes and proteins, including enzymes. Labeling can be simply detected or quantified. The response of a simple detection generally includes a response that only confirms its presence, and the response of quantification generally includes a response with a quantifiable (such as, digitally reportable) value, such as intensity, polarization and / or other characteristics. In luminescent or fluorescent assays, the detectable response can be produced directly using a luminophore or fluorophore associated with the assay component that actually participates in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0226] Examples of luminescent markers that produce signals include, but are not limited to, bioluminescence and chemiluminescence. Detectable luminescent responses typically include changes or the appearance of luminescent signals. Suitable methods and luminophores for luminescent marker analysis components are known in the art, such as described in Haugland, 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.

[0227] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, red pigment, coumarin, methylcoumarin, pyrene, apple green, stilbene, Lucifer yellow, Cascade blue TM. and Texas red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

[0228] In another aspect, fluorescent labels are functionalized to facilitate covalent attachment to cellular components present in or on a cell or tissue surface, 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 the fluorescent label to a second molecule. The choice of fluorescent label functional group depends on the attachment site of the linker, reagent, label, or second label.

[0229] Attachment of the fluorescent label can be directly attached to the cellular component or compound, or can be attached through a linker. Suitable binding pairs for indirectly linking the fluorescent label to the intermediate include, but are not limited to, antigen / antibody, such as rhodamine / anti-rhodamine, biotin / avidin, and biotin / streptavidin.

[0230] The phrase "solid carrier / 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 many techniques known to those skilled in the art. In one technique, oligonucleotides are linked and arranged on a gene chip for determining DNA sequences by hybridization methods, such as those outlined in U.S. Patent Nos. 6,025,136 and 6,018,041. The polynucleotides of the present invention can be modified into probes, which can then be used to detect genetic sequences. For example, such techniques are described in U.S. Patent Nos. 5,968,740 and 5,858,659. Probes can also be attached or fixed to electrode surfaces for electrochemical detection of nucleic acid sequences, as described in Kayem et al. US Patent No., 952,172 and Kelley et al. (1999) Nucleic Acids Res. 27: 4830-4837.

[0231] A "composition" refers to a combination of an active polypeptide, polynucleotide, or antibody and another inert (eg, a detectable label) or active (eg, a gene delivery vehicle) compound or composition.

[0232] A "pharmaceutical composition" refers to a composition comprising an active polypeptide, polynucleotide or antibody in combination with a carrier (inert or active, such as a solid support) that makes the composition suitable for diagnostic or therapeutic use in vitro, in vivo or in vitro.

[0233] As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate-buffered saline, water, and emulsions, such as oil / water or water / oil emulsions, and 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 Publ. Co., Easton).

[0234] As used herein, the term "cancer" includes solid tumors and hematologic 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 gastric cancer. Exemplary hematologic 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, nodal marginal zone B-cell lymphoma, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B-lymphocytic 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 lymphomatoid granuloma. In some cases, the cancer is a metastatic cancer (e.g., a metastatic solid tumor or a metastatic hematologic malignancy). In some cases, the cancer is a relapsed or refractory cancer (e.g., a relapsed or refractory solid tumor or a relapsed or refractory hematologic malignancy).

[0235] In some embodiments, the tumor is characterized by upregulated expression of fibroblast activation protein (FAP) (the amino acid sequence of human FAP is disclosed in GenPept Accession Number 138593, obtained on February 10, 2020). FAP, also known as FAPα 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 some cases, cancers characterized by upregulated 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 kidney cancer. In some cases, FAP-positive cancers contain high levels of fibrosis. In some cases, this level is compared to an equivalent cancer in which FAP is not upregulated. In other cases, this level is compared to the fibrosis level of a normal subject.

[0236] In some cases, the dimert described herein binds to the extracellular portion of FAPα. In some cases, dimert binds to human FAPα (e.g., binds to the extracellular portion of FAPα having GenPept Accession Number 138593 or an equivalent thereof). In some cases, dimert binds to FAPα and an immune cell target, such as a cell surface polypeptide expressed on a T cell or NK cell. In some cases, dimert binds to FAPα and optionally binds to an immune cell target (e.g., a cell surface polypeptide expressed on a T cell or NK cell) for the treatment of FAP-positive cancer, optionally pancreatic cancer, and further optionally a FAP-positive cancer characterized by a high level of fibrosis.

[0237] In some embodiments, the cancer is characterized by expression and / or upregulation of B-cell maturation antigen (BCMA) (also known as tumor necrosis factor receptor superfamily member 17, TNFRSF17, and BCM), a cell surface receptor of the TNF receptor superfamily. In some cases, the amino acid sequence of human BCMA is disclosed in GenPept Accession Number BAB60895.1 (obtained on February 10, 2020). In some cases, the cancer characterized by BCMA expression and / or upregulation is myeloma (or multiple myeloma).

[0238] In some cases, dimert as described herein binds to the extracellular portion of BCMA. In some cases, dimert binds to human BCMA (e.g., binds to the extracellular portion of BCMA having GenPept Accession Number BAB60895.1, or its equivalent). In some cases, dimert binds to BCMA and immune cell targets, such as cell surface polypeptides expressed on T cells or NK cells. In some cases, dimert binds to BCMA and optionally binds to immune cell targets (e.g., cell surface polypeptides expressed on T cells or NK cells) to treat myeloma.

[0239] In certain embodiments, the feature of cancer is expression or the raising of epidermal growth factor receptor (EGFR) mutant (such as EGFR variant III (EGFRvIII)).EGFRvIII refers to the EGFR mutation comprising EGFR gene exon 2-7 deletion.In some cases, the cancer (EGFRvIII positive cancer) characterized by expressing or raising EGFRvIII includes but is 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.

[0240] In some cases, dimert as herein described is combined with the extracellular portion of EGFRvIII.In some cases, dimert is combined with the extracellular portion of people EGFRvIII.In some cases, dimert is combined with EGFRvIII and immune cell target, for example, the cell surface polypeptides expressed on T cell or NK cell.In some cases, dimert is combined with EGFRvIII, and optionally with immune cell target (for example, the cell surface polypeptides expressed on T cell or NK cell), is used to treat EGFRvIII positive cancer, optionally is glioblastoma.

[0241] In some embodiments, the cancer is characterized by upregulation of human epidermal growth factor receptor 2 (HER2) (also known as HER2 / neu, receptor tyrosine-protein kinase erbB-2, CD340, and ERBB2). In some cases, the amino acid sequence of human HER2 is disclosed in GenPept Accession Number NP-004439.2 (obtained on February 10, 2020). In some cases, HER2-positive cancers include, but are not limited to, breast cancer, ovarian cancer, gastric cancer, colorectal cancer, pancreatic cancer, and endometrial cancer.

[0242] In some cases, dimert as described herein is combined with the extracellular portion of HER2. In some cases, dimert is bound to the extracellular portion of human HER2 (e.g., comprising the amino acid sequence listed in GenPept Accession Number NP-004439.2 or its equivalent). In some cases, dimert combines HER2 and immune cell targets, such as cell surface polypeptides expressed on T cells or NK cells. In some cases, dimert combines HER2 and optionally combines immune cell targets (e.g., cell surface polypeptides expressed on T cells or NK cells) to treat HER2-positive cancers, optionally breast cancer.

[0243] In some embodiments, the cancer is characterized by upregulation of CD123 (also known as interleukin-3R or IL-3RA). In some cases, the amino acid sequence of human CD123 is disclosed in GenPept Accession Number NP_002174.1 (obtained on February 10, 2020). In some cases, CD123-positive cancers include, but are not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), blastic plasmacytoid dendritic cell neoplasm, and hairy cell leukemia.

[0244] In some cases, dimert as described herein is combined with the extracellular portion of CD123. In some cases, dimert is bound to the extracellular portion of human CD123 (for example, comprising the amino acid sequence listed in GenPept Accession Number NP_002174.1 or its equivalent). In some cases, dimert combines CD123 and immune cell targets, such as cell surface polypeptides expressed on T cells or NK cells. In some cases, dimert combines CD123 and optionally combines immune cell targets (for example, cell surface polypeptides expressed on T cells or NK cells) to treat CD123 positive cancers, optionally AML.

[0245] In some embodiments, the cancer is characterized by upregulation of CD38 (also known as ADP-ribosyl cyclase 1 or ADPRC 1). In some cases, the amino acid sequence of human CD38 is disclosed in GenPept Accession Number BAA18966.1 (accessed on February 10, 2020). In some cases, CD38-positive cancers include, but are not limited to, multiple myeloma, acute myeloid leukemia, prostate cancer, and lung cancer.

[0246] In some cases, dimert as described herein binds to the extracellular portion of CD38. In some cases, dimert binds to the extracellular portion of human CD38 (e.g., comprising the amino acid sequence listed in GenPept Accession Number BAA18966.1 or its equivalent). In some cases, dimert binds to CD38 and immune cell targets, such as cell surface polypeptides expressed on T cells or NK cells. In some cases, dimert binds to CD38 and optionally binds to immune cell targets (e.g., cell surface polypeptides expressed on T cells or NK cells) to treat CD38-positive cancers, optionally AML.

[0247] In some embodiments, the cancer is characterized by upregulation of mesothelin (also known as MSLN). In some cases, the amino acid sequence of human mesothelin is disclosed in GenPept Accession Number AAV87530.1 (obtained on February 10, 2020). In some cases, mesothelin-positive cancers include, but are not limited to, mesothelioma, pancreatic cancer, ovarian cancer, endometrial cancer, bile duct cancer, gastric cancer, lung adenocarcinoma, and childhood acute myeloid leukemia.

[0248] In some cases, dimert as described herein binds to the extracellular portion of mesothelin. In some cases, dimert binds to the extracellular portion of human mesothelin (e.g., comprising the amino acid sequence listed in GenPept Accession Number AAV87530.1 or its equivalent). In some cases, dimert binds to mesothelin and immune cell targets, such as cell surface polypeptides expressed on T cells or NK cells. In some cases, dimert binds to mesothelin and optionally binds to immune cell targets (e.g., cell surface polypeptides expressed on T cells or NK cells) to treat mesothelin-positive cancers, optionally mesothelioma.

[0249] In some embodiments, the cancer is characterized by upregulation of interleukin-13 receptor alpha (IL13Rα). In some cases, IL13Rα comprises IL13Rα1 (IL13Rα1) and IL13Rα2 (IL13Rα2). In some cases, the amino acid sequence of human IL13Rα1 is disclosed in GenPept Accession Number P78552.1 (obtained on February 10, 2020). In some cases, the amino acid sequence of human IL13Rα2 is disclosed in GenPept Accession Number Q14627.1 (obtained on February 10, 2020). In some cases, IL13Rα-positive cancers include, but are not limited to, brain cancer (such as glioblastoma) and renal cell carcinoma (RCC).

[0250] In some cases, dimert as described herein binds to the extracellular portion of IL13Rα. In some cases, dimert binds to the extracellular portion of human IL13Rα (e.g., comprising the amino acid sequence listed in GenPept Accession Number P78552.1, Q14627.1, or its equivalent). In some cases, dimert binds to IL13Rα and immune cell targets, such as cell surface polypeptides expressed on T cells or NK cells. In some cases, dimert binds to IL13Rα and optionally binds to immune cell targets (e.g., cell surface polypeptides expressed on T cells or NK cells) to treat IL13Rα-positive cancers, optionally brain cancer.

[0251] In some embodiments, the cancer is characterized by upregulation of B7-H3 (also known as CD276, an immune checkpoint member). In some cases, the amino acid sequence of human B7-H3 is disclosed in GenPept Accession Number CAE47548.1 (obtained on February 10, 2020). In some cases, 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, kidney cancer, gastric cancer, ovarian cancer, melanoma, and thyroid cancer.

[0252] In some cases, dimert described herein binds to the extracellular portion of B7-H3. In some cases, dimert binds to the extracellular portion of human B7-H3 (e.g., comprising the amino acid sequence listed in GenPept Accession Number CAE47548.1 or its equivalent). In some cases, dimert binds to B7-H3 and T cell or NK cell targets, such as cell surface polypeptides expressed on T cells or NK cells. In some cases, dimert binds to B7-H3 and optionally binds to T cell or NK cell targets (e.g., cell surface polypeptides expressed on T cells or NK cells) to treat B7-H3 positive cancers, optionally lung cancer (e.g., non-small cell lung cancer), breast cancer, prostate cancer, renal cell carcinoma, brain cancer, pancreatic cancer, kidney cancer, gastric cancer, ovarian cancer, melanoma or thyroid cancer.

[0253] In some embodiments, the cancer is characterized by upregulation of receptor tyrosine kinase-like orphan receptor 1 (ROR1) (also known as neurotrophic tyrosine kinase, receptor-related 1, or NTRKR1). In some cases, the amino acid sequence of human ROR1 is disclosed in GenPept Accession Number NP_005003 (obtained on February 10, 2020). In some cases, ROR1-positive cancers include, but are not limited to, breast cancer, lung cancer, gastric cancer, ovarian cancer, chronic lymphocytic leukemia (CLL), and acute lymphoblastic leukemia (ALL).

[0254] In some cases, dimert as described herein binds to the extracellular portion of ROR1. In some cases, dimert binds to the extracellular portion of human ROR1 (e.g., comprising the amino acid sequence listed in GenPept Accession Number NP_005003 or its equivalent). In some cases, dimert binds to ROR1 and immune cell targets, such as cell surface polypeptides expressed on T cells or NK cells. In some cases, dimert binds to ROR1 and optionally binds to immune cell targets (e.g., cell surface polypeptides expressed on T cells or NK cells) to treat ROR1-positive cancers, optionally breast cancer, lung cancer, gastric cancer, ovarian cancer, CLL or ALL.

[0255] In some embodiments, the cancer is characterized by upregulation of adrenergic type A receptor 2 (EphA2) (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 Accession Number NP_004422.2 (accessed on February 10, 2020). In some cases, EphA2-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.

[0256] In some cases, the dimert described herein binds to the extracellular portion of EphA2. In some cases, dimert binds to the extracellular portion of human EphA2 (e.g., comprising the amino acid sequence listed in GenPept Accession Number NP_004422.2 or its equivalent). In some cases, dimert binds to EphA2 and an immune cell target, such as a cell surface polypeptide expressed on a T cell or NK cell. In some cases, dimert binds to EphA2 and optionally binds to an immune cell target (e.g., a cell surface polypeptide expressed on a T cell or NK cell) to treat an EphA2-positive cancer, optionally 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, or liver cancer.

[0257] 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-lymphocytic leukemia; hairy cell leukemia. In some cases, B-cell leukemia, B-cell lymphoma, or a combination thereof is characterized by high expression of CD20 and / or CD22 on B cells. In some cases, the dimert described herein binds to CD20 or CD22. 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 a cancer cell or a cell surface polypeptide expressed on a T cell or NK cell. 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.

[0258] As used herein, "first-line treatment" includes primary treatment for a subject, optionally a subject with cancer. In some cases, the cancer is a primary cancer. In other cases, the cancer is a metastatic or recurrent cancer. In some cases, the first-line treatment includes chemotherapy. In other cases, the first-line treatment includes radiation therapy. A skilled artisan will readily appreciate that different first-line treatments may be appropriate for different types of cancer.

[0259] As used herein, second-line therapy includes treatment used after primary or first-line therapy has stopped. Third-line therapy, fourth-line therapy, or fifth-line therapy include subsequent treatments. As indicated by the naming convention, third-line therapy includes a course of treatment during which primary and second-line therapy have stopped.

[0260] The "subject" of diagnosis or treatment is a cell or animal, such as a mammal or human. Subjects are not limited to a particular species and include non-human animals being diagnosed or treated, as well as animals affected by infection or animal models, for example, monkeys, rodents (such as rats, mice, chinchillas), canines (such as dogs), lagomorphs (such as rabbits), livestock, sports animals, and pets. Human patients are also included in the term.

[0261] As used herein, the term "tissue" refers to the tissue of a living or dead organism, or any tissue derived from or designed to simulate a living or dead organism. 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 can be interconnected) or the tissue that makes up an organism's organ or body part or region. The tissue may include a homogenous cell material or may be a composite structure, such as a composite structure found in a body region including the chest, which may, for example, include lung tissue, bone 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, bile duct, duodenum, abdominal aorta, iliac vein, heart and intestine, including any combination thereof.

[0262] 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 to a biological molecule of interest (e.g., a polypeptide) but does not substantially recognize and bind to other molecules in a sample (e.g., a biological sample containing or expressing a tumor antigen).

[0263] As used herein, "treating" or "treating" a disease in a subject means (1) preventing the onset of symptoms or disease in a subject who is susceptible or not yet showing symptoms of the disease; (2) inhibiting the disease or arresting its development; 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 the purposes of the present technology, beneficial or desired results may include one or more of, but are not limited to: alleviation or amelioration of one or more symptoms; reduction in the extent of a condition (including a disease); stabilization (i.e., not worsening) of the condition (including a disease); delay or slowing of a condition (including a disease); progression, improvement, or remission of a condition (including a disease); and remission (whether partial or complete), whether detectable or not. In one aspect, the term "treating" does not include prevention.

[0264] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired effect. In the case of therapeutic or preventive applications, the effective amount will depend on the type and severity of the disease in question and the characteristics of the individual subject, such as general health, age, sex, body weight and tolerance to the pharmaceutical composition. In the context of gene therapy, in some embodiments, the effective amount is an amount sufficient to cause partial or complete functional restoration of the gene lacking in the subject. In other embodiments, the effective amount of the recombinant polynucleotide, vector or AAV viral particle is an amount sufficient to cause gene expression in the subject. In some embodiments, the effective amount is the amount required to increase the galactose metabolism of a subject in need. Those skilled in the art will be able to determine the appropriate amount based on these and other factors.

[0265] In some embodiments, the effective amount will depend on the size and nature of the application. It will also depend on the nature and sensitivity of the target object and the method of use. Those skilled in the art will be able to determine the effective amount based on these and other considerations. Depending on the embodiment, the effective amount may include one or more administrations of the composition.

[0266] As used herein, the term "administration" or "administration" refers to the delivery of a substance to a subject (such as an animal or human). Throughout the course of treatment, administration may be a single administration, continuous administration, or intermittent administration. Methods for determining the most effective mode of administration and dosage are well known to those skilled in the art and will vary with the composition used for treatment, the purpose of treatment, and the age, health, or sex of the subject being treated. Single or multiple administrations can be selected by the treating physician for dosage levels and patterns, and for pets and animals, by the treating veterinarian for dosage levels and patterns. Formulations and methods for administration of appropriate dosages are known in the art. The route of administration can be determined, and methods for determining the most effective route of administration are well known to those skilled in the art and will vary with the composition used for treatment, the purpose of treatment, the health status or disease stage of the subject being treated, and the target cells or tissues. Non-limiting examples of routes of administration include intravenous injection, intraarterial injection, intramuscular injection, intracardiac injection, intrathecal injection, subventricular injection, epidural injection, intracerebral injection, intracerebroventricular injection, subretinal injection, intravitreal injection, intraarticular injection, intraocular injection, intraperitoneal injection, intrauterine injection, intradermal injection, subcutaneous injection, transdermal injection, submucosal injection, and inhalation.

[0267] Mode for Carrying Out the Invention

[0268] Cancer is the second leading cause of death worldwide, accounting for an estimated 9.6 million deaths in 2018. Numerous different types of cancer therapies are in clinical development and on the market, including immunotherapy, hormone 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 enable targeted delivery of payloads to the tumor microenvironment of interest. However, these oncolytic viruses express their payloads within tumor cells and induce a cell-killing effect, limiting payload expression to a maximum of a few days and typically to a few hours. Therefore, to achieve sustained and long-lasting therapeutic effects, multiple dosing is required, which can increase toxicity and lead to adverse immune responses. Adoptive cell therapies, such as chimeric antigen receptor (CAR) T cell therapy, offer personalized 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, seizures, and loss of balance; and neutropenia and anemia. In addition, the preparation of CAR-T cells requires several weeks of culture and expansion before administration, which is particularly important for patients with advanced cancer.

[0269] In certain embodiments, disclosed herein is a method for delivering a therapeutic transgene (e.g., dimert disclosed herein) using a gene therapy vector. In some embodiments, the gene therapy vector provides stable, sustained expression of a therapeutic transgene (e.g., dimert). In other embodiments, the gene therapy vector provides constitutive expression. In further embodiments, the gene therapy vector provides regulated expression. In some cases, the gene therapy vector is transduced in normal cells (e.g., in organ cells such as hepatocytes or muscle cells). In some cases, a single administration of the gene therapy vector is sufficient to induce stable, sustained expression of a therapeutic transgene (e.g., dimert). In other cases, the gene therapy vector provides sustained, long-term expression of a therapeutic transgene (e.g., dimert), thereby providing long-term pressure on cancer cells.

[0270] In certain embodiments, disclosed herein is a method of delivering a therapeutic transgene (e.g., dimert disclosed herein) using a TransJoin. In some cases, a TransJoin provides constitutive expression of a therapeutic transgene (e.g., dimert). In some cases, a TransJoin provides sustained, stable, long-term expression of a therapeutic transgene (such as dimert). 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, a TransJoin is transduced in normal cells (e.g., in organ cells such as hepatocytes or muscle cells). In some cases, a single administration of a TransJoin is sufficient to induce stable, sustained expression of a therapeutic transgene (e.g., dimert). In other cases, a TransJoin provides sustained, long-term expression of a therapeutic transgene (e.g., dimert), thereby providing long-term pressure on cancer cells.

[0271] In certain embodiments, disclosed herein is a novel method for activating transgene expression (e.g., dimer disclosed herein), which can be used as a gene therapy platform for modulating 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 utilizes 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. In another aspect, when the drug or agent is absent or discontinued, gene expression returns to its original state prior to administration. For example, when a drug or agent activates gene expression, if there are side effects or if transgene expression is no longer desired, discontinuation of the drug can inactivate gene expression, thereby minimizing side effects, if any. In some cases, TransSkip is used for transduction in normal cells (e.g., in organ cells such as hepatocytes 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 regulated but sustained expression of a therapeutic transgene (e.g., dimert), thereby providing long-term pressure on cancer cells.

[0272] Exon skipping is a technique used to treat genetic diseases in which defects occur in short regions of certain genes. DNA mutations result in damaged proteins, either because the wrong amino acid ends up in the protein, because they produce stop mutations that result in a truncated protein, or because they alter the reading frame that produces both. Because mammalian genes are typically encoded in exons, meaning they are divided into multiple gene segments (exons) that are spliced ​​together during mRNA processing, the DNA mutations (changes or deletions in base pairs) that cause many diseases are contained within a single exon. If that exon can be skipped and not included in the final spliced ​​mRNA, the mutated region will not be included in the final protein. While the protein will be shorter and may be missing some parts, it will still be "in frame" and may retain some of its functions.

[0273] For example, Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are the most common childhood muscular dystrophies and are caused by genetic defects in the DMD gene that encodes dystrophin, a muscle protein required for the interaction of the cytoskeleton and the extracellular matrix to maintain the stability of muscle fibers during contraction. DMD mutations in the dystrophin gene are characterized by frameshift insertions or deletions or nonsense point mutations, resulting in the absence of functional dystrophin. BMD mutations typically leave the reading frame intact, allowing the synthesis of partially functional dystrophin. Exon skipping-based therapies result in the conversion of out-of-frame mutations in DMD patients to in-frame mutations that encode a partially functional dystrophin protein. In 2016, the FDA approved the first exon-skipping drug, eteplirsen. TM Sarepta Therapeutics is being developed for Duchenne muscular dystrophy, a disease characterized by a mutation in exon 51 of the dystrophin gene. When the drug (a short, modified piece of DNA, also called an oligonucleotide) is administered, exon 51 is “skipped,” restoring a near-full-length, more functional protein. Similar technology is being developed to skip other dystrophin exons and exons in other disease-causing genes.

[0274] In contrast to exon skipping, the present invention utilizes transgene activation in gene therapy applications. Most, if not all, gene therapy approaches currently utilize complementary (cDNA) gene sequences; that is, the genetic sequence of the transgene is solely the coding sequence (exons only) and does not include any intervening (intronic) sequences. Therefore, the gene does not undergo any RNA splicing.

[0275] The present invention and technology utilize the intervention sequence containing splicing donor and acceptor sites and RNA spliceosome binding sites to force transgene to carry out normal exon splicing. Therefore, a "reverse engineered" (artificial) exon-intron-exon gene structure in transgene is provided, which undergoes splicing when expressed in target cells. However, splicing is regulated according to the principle of exon skipping. On the one hand, the exon containing the intentional insertion of a stop codon in the middle of the gene regulates gene expression, that is, only when the exon is skipped, the artificial structure will express a normal functional transgene. 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 (BiKE), a trispecific T cell engager (TriTE) or a trispecific NK cell engager (TriKE).

[0276] On the other hand, the degree and type of splicing vary depending on the cell type, as many genes often undergo alternative splicing, which sometimes varies 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 cells and cancer cells). This technology can exploit these characteristics to achieve different transgene control than in normal cells and cancer cells. In addition, antibodies encoded by functional transgenes, such as bispecific or trispecific antibodies, can be used to treat cancer. Therefore, in one aspect, the regulation of transgene splicing in the present invention provides a method for treating cancer.

[0277] Construct Examples

[0278] In certain embodiments, provided herein is a polynucleotide or vector comprising the following sequence, or consisting essentially of the following sequence, or consisting of the following sequence: (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 some cases, 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 the second example, the first target cell can be an immune cell, and the second target cell can be a tumor cell. In the 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 cell type different from 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, eg, associated with the same genetic defect or optionally of the same tissue type.

[0279] In some embodiments, the first polypeptide is a first antibody or a binding fragment thereof, and the second polypeptide is a second antibody or a binding fragment thereof. In some cases, provided herein is a polynucleotide or vector comprising the following sequences, or consisting essentially of the following sequences, or consisting of the following sequences: (a) a first polynucleotide sequence comprising a first portion of an open reading frame encoding a first antibody or an antigen-binding fragment thereof; (b) a second polynucleotide sequence comprising a second portion of an open reading frame encoding a first antibody or an antigen-binding fragment thereof; (c) a third polynucleotide sequence comprising a gene regulatory polynucleotide sequence encoding a second antibody or an 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 polynucleotides, their complementary sequences, and / or vectors 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 and the second target cell can be a second immune cell, and the first immune cell is a cell type different from the second immune cell. In a fourth example, the first target cell is a first cancer cell and 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, for example, 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, and the two epitopes are present on the same antigen. In some cases, the first antibody and the second antibody have different amino acid sequences. On the one hand, the polynucleotide is contained in a gene expression vector, non-limiting examples of which include plasmids, DNA viral vectors, or gene delivery vectors.

[0280] In some embodiments, the present invention also discloses a vector for gene therapy, comprising: a first polynucleotide sequence encoding a first antibody or an antigen-binding fragment thereof; and a second polynucleotide sequence encoding a second antibody or an antigen-binding fragment thereof. Complementary sequences of the polynucleotides are also provided. In one aspect, the polynucleotides, their complementary sequences, 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 and the second target cell can be a second immune cell, and the first immune cell is a different cell type from the second immune cell. In a fourth example, the first target cell is a first cancer cell and 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, for example, 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, and the two epitopes are present on the same antigen. In some cases, the first antibody and the second antibody have different amino acid sequences.

[0281] On the other hand, 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. On the other hand, the gene regulatory polynucleotide sequence includes a binding sequence for one or more antisense oligonucleotides. On the other hand, the antisense oligonucleotide is a morpholino. In another aspect, the binding sequence of the morpholino oligonucleotide includes a polynucleotide sequence at least 95% identical to SEQ ID NO: 24 (AATATGATCCAACAATAGAGGTAAATCTTG) or SEQ ID NO. 25 (GATCCAACAATAGAGGTAAATCTTGTTTTA), or has at least 96%, or at least 97%, or 98%, or at least 99% identity thereto. In one embodiment, the morpholino oligonucleotide comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO. 27 (CAAGATTTACCTCTATTGTTGGATCATATT) or SEQ ID NO. 28 (TAAAACAAGATTTACCTCTATTGTTGGATC), or at least 96%, or at least 97%, or 98%, or at least 99% identical to each of them. Splice donor sites and splice acceptor sites are well known in the art. One of ordinary skill in the art will recognize the sequences of splice donor sites and splice acceptor sites, such as consensus sequences. An exemplary splice site consensus sequence for the U2 intron may include the 5' splice site MAG- GT RAGT, where M is A or C; R is A or G, the underlined nucleotides indicate that "GT" is invariant; the dash "-" indicates the splice site. The 3' splice site of the U2 intron can be C AG -G, where 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:

[0282] p53 exon 10 5' splice site (donor): CAG-gtgagt, where the dash "-" indicates the splice site;

[0283] Brd2 exon 3 5'ss: AAG-gtgagt, where the dash “-” indicates the splice site;

[0284] BRCA1 exon 22 5' splice site: CAG-gtaagt, where the dash "-" indicates the splice site;

[0285] SMN1 exon 1 5'ss: CAG-gtgagg, where the dash "-" indicates the splice site;

[0286] BRD2 3′ splice site acceptor intron 1 (lower case) / exon 2 (upper case): cccatctttacag-GCTCCC, where the dash “-” indicates the splice site;

[0287] BCL-X 3' splice acceptor intron 2 (lower case) / exon 3 (upper case): tctctccctgcag-GATACT, where the dash “-” indicates the splice site;

[0288] Fibronectin 3' splice acceptor intron 28 (lower case) / exon 29 (upper case): ctttttcatacag-GAGGAA, where the dash "-" indicates the splice site; and

[0289] Survivin 3' splice acceptor intron 2 (lower case) / exon 3 (upper case): tctttatttccagGCAAAG, where the dash "-" indicates the splice site.

[0290] In some embodiments, the splice site consensus sequence is obtained from / / science.umd.edu / labs / mount / RNAinfo / matrices.html.

[0291] In some embodiments, the stop codon comprises an oligonucleotide comprising TAA, TAG, or TGA. In another aspect, the stop codon sequence comprises a polynucleotide sequence of TAAxTAGxTGAxTAGxTAAxTGAx (SEQ ID NO. 1), wherein x is any nucleotide, or the stop codon sequence comprises a polynucleotide sequence of TAATTAGTTGATTAGTTAATTGAT (SEQ ID NO. 2). In a further aspect, the gene regulatory polynucleotide comprises a polynucleotide sequence that is at least 95% identical to SEQ ID NO. 2, or a polynucleotide sequence that is at least 96%, or at least 97%, or 98%, or at least 99% identical to SEQ ID NO. 2.

[0292] In a further embodiment, the first antibody or its antigen binding fragment specifically binds to the activation antigen on the immune effector cell, and the second antibody or its antigen binding fragment binds to the tumor antigen. On the other hand, the first antibody or its antigen binding fragment specifically binds to the tumor antigen, and the second antibody or its antigen binding fragment binds to the activation antigen on the immune effector cell. On the other hand, the carrier also includes a fourth polynucleotide sequence encoding a third antibody or its antigen binding fragment, wherein the third antibody or its antigen binding fragment binds to the activation antigen or tumor antigen on the immune effector cell. On the one hand, the immune effector cell includes dendritic cells, natural killer (" NK") cells, macrophages, T cells, B cells or combinations thereof. Non-limiting examples of immune effector cells include T cells or NK cells.

[0293] 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.

[0294] Non-limiting examples of target antigens on antigen presenting cells include, but are not limited to, B7-H3 (CD276).

[0295] Non-limiting examples of target antigens on B cells include, but are not limited to, CD20 and CD22.

[0296] Non-limiting examples of tumor antigens include one or more of the following: adrenergic type A receptor 2 (EphA2), interleukin (IL) -13rα2, 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 antigens, surface glycoproteins, gangliosides (GM2), growth factor receptors, matrix antigens, vascular antigens, receptor tyrosine kinase-like orphan receptor 1 (ROR1), mesothelin, CD38, CD123, human epidermal growth factor receptor 2 (HER2), B cell maturation antigen (BCMA), fibroblast activation protein (FAP) α or a combination thereof. Other examples can be found in the art, see, for example, incorporated herein by reference. In another aspect, the recombinant vector expresses a precursor mRNA encoding dimert as described herein. In some cases, when the precursor mRNA is contacted with a morpholino oligonucleotide, dimert is a bispecific antibody or a trispecific antibody. Non-limiting examples of dimert are: bispecific T cell engagers (BiTE) or bispecific NK cell engagers (BiKE); trispecific antibodies include trispecific T cell engagers (TriTE) or trispecific NK cell engagers (TriKE). On the one hand, trispecific antibodies include a first antibody or its antigen-binding fragment and a second antibody or its antigen-binding fragment.

[0297] In one aspect, the dimert (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, CD19; lymphocyte function-associated antigen 1 (LFA1); CD45; interleukin 21 receptor (IL21R); natural killer group 2, member D (NKG2D); natural cytotoxicity receptor (NCR), such as NKp44, NKp46, or NKp30; or DNAX accessory molecule-1 (DNAM or DNAM-1; also known as CD226, or platelet and T cell activation antigen 1 (PTA1)). In another embodiment, the polypeptide sequence encodes an antigen-binding fragment for CD3, CD19, GD2, or NKG2D. 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 CD19. In another embodiment, the first antigen-binding fragment binds to CD3 and the second antigen-binding fragment binds to GD2. In another embodiment, the first antigen-binding fragment binds to NKG2D and the second antigen-binding fragment binds to GD2.

[0298] In one embodiment, the trispecific binder or antibody comprises a first antigen-binding fragment, a second antigen-binding fragment, and a third antigen-binding fragment. In another embodiment, the trispecific binder or antibody comprises three antigen-binding fragments that bind to NKG2D, IL21R, and GD2, respectively. In another aspect, the trispecific binder or antibody has a polypeptide sequence that is at least 95% identical to SEQ ID NO. 11, or has a sequence identity of at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO. 11.

[0299] 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 NO.3 and SEQ ID NO.4, respectively. In one embodiment, the antigen-binding fragment that binds to NKG2D comprises MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, Rae-1α, Rae-1β, Rae-1γ, Rae-1δ, Rae-1ε, H60a, H60b, H60c, MULT1, or a fragment thereof. In one embodiment, the antigen-binding fragment that binds to NKG2D is MICA (SEQ ID NO.5) or a fragment thereof, or an equivalent thereof.

[0300] 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 described in SEQ ID NO:5). In another embodiment, the MICA mutant is a sequence variant of MUC-30 (SEQ ID NO.7) that comprises a methionine mutation instead of an alanine at position 129 of the wild-type MICA sequence (MICA-129Met). An equivalent of the MICA mutant (MICA-129Met) retains the methionine mutation at position 129 of wild-type MICA. In another embodiment, the antigen-binding fragments of the bispecific or trispecific binder or antibody are separated by a linker sequence. One embodiment of the linker sequence comprises, consists essentially of, or consists of the sequence: GGGGSGGGGSGGGGS (SEQ ID NO.9) or an equivalent thereof. The linker is encoded by the polynucleotide sequence GGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGCGGCAGCGGCGGCGGCAGC (SEQ ID NO. 10) or its equivalent. 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-129Met) or their respective fragments or equivalents. In one embodiment, the two antigen-binding fragments separated by the linker sequence are GD2 and MICA (e.g., wild-type MICA or a MICA mutant such as MUC-30 or MICA-129Met) or their respective fragments or equivalents. In one embodiment, the two antigen-binding fragments separated by the linker sequence are IL21 and GD2, or their respective fragments or equivalents. In another embodiment, the linker is inserted between the antigen-binding fragments of any of the following trispecific binders:

[0301] IL21-MICA129-GD2

[0302] MICA129-IL21-GD2

[0303] GD2-IL21-MICA129

[0304] GD2-MICA129-IL21

[0305] IL21-MICA / V129M-GD2-HDD

[0306] MICA / V12M-IL21-GD2-HDD

[0307] GD2-IL21-MICA129-HDDGD2-MICA129-IL21-HDD.

[0308] In another embodiment, the dimert (e.g., bispecific or trispecific adaptor) comprises a secretion consensus sequence (also referred to herein as sec0A). In some cases, the secretion consensus sequence (sec0A) 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 some cases, sec0A is encoded by a polynucleotide comprising ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCC (SEQ ID NO: 52) or an equivalent thereof.

[0309] 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 some cases, one, two, three, four or more residues are Ala residues, Gly residues, Val residues, Ile residues or combinations thereof. In some cases, one, two, three, four or more residues are Ala residues, Gly residues, Val residues or combinations thereof. In some cases, one, two, three, four or more residues are Ala residues, Gly residues or combinations 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.

[0310] In some cases, the secretory consensus sequence further comprises one, two or three residues with an aliphatic side chain (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 combinations thereof. In some cases, one, two or three residues are Ala residues, Gly residues, Val residues or combinations thereof. In some cases, one, two or three residues are Ala residues, Gly residues or combinations 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.

[0311] In some cases, the secretion consensus sequence further comprises one or two residues with an aliphatic side chain (e.g., Ala, Met, Ile, Val or Leu). In some cases, one or two residues are Ala residues, Gly residues, Val residues, Ile residues or a combination thereof. In some cases, one or two residues are Ala residues, Gly residues, Val residues or a combination thereof. In some cases, one or two residues are Ala residues, Gly residues, Val residues or a combination thereof. In some cases, the secretion consensus sequence further comprises one or two Ala residues at the C-terminus of the sequence. In some cases, the secretion consensus sequence further comprises one or two Gly residues at the C-terminus of the sequence. In some cases, the secretion consensus sequence further comprises one or two Val residues at the C-terminus of the sequence. In some cases, the secretion consensus sequence further comprises one or two Ile residues at the C-terminus of the sequence.

[0312] In some cases, the secretory consensus sequence further comprises a residue with 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.

[0313] In some cases, the secretory consensus sequence further comprises one or two Ala residues at the C-terminus of the sequence, and such sequences are referred to as secrecon1A (or sec1A) with one Ala at the C-terminus, while secrecon2A (or sec2A) has two Ala residues at the C-terminus. In some cases, sec1A has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to MWWRLWWLLLLLLLLWPMVWAA (SEQ ID NO: 53). In some cases, sec1A is encoded by a polynucleotide comprising ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCCGCC (SEQ ID NO: 54) or an equivalent thereof. In some cases, sec2A has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to MWWRLWWLLLLLLLLWPMVWAAA (SEQ ID NO: 55). In some cases, sec2A is encoded by a polynucleotide comprising ATGTGGTGGAGACTGTGGTGGCTGCTGCTGCTGCTGCTGCTGCTGTGGCCCATGGTGTGGGCCGCCGCC (SEQ ID NO: 56) or an equivalent thereof.

[0314] In some embodiments, the secretion consensus sequence regulates the expression and / or secretion of dimert. In some cases, the secretion consensus sequence (e.g., sec1A or sec2A) enhances the expression and / or secretion of dimert. In some cases, the secretion consensus sequence (e.g., sec1A or sec2A) regulates (e.g., enhances) the expression of dimert 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 cases, the secretion consensus sequence (e.g., sec1A or sec2A) regulates (e.g., enhances) the secretion of dimert 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.

[0315] In some embodiments, a dimert (e.g., a trispecific adapter) described herein comprises a secretion consensus sequence (e.g., sec0A, sec1A, or sec2A), an IL21 sequence (e.g., SEQ ID NO: 3), a MICA sequence (e.g., a wild-type sequence, MUC-30, or MICA-129Met), an anti-GD2 peptide sequence, or an equivalent of one or more thereof. In some cases, a dimert (e.g., a trispecific adapter) comprises a secretion consensus sequence (e.g., sec0A, sec1A, or sec2A), an IL21 sequence (e.g., SEQ ID NO: 3), a MICA sequence (e.g., a wild-type sequence, MUC-30, or MICA-129Met), an anti-GD2 peptide sequence, or an equivalent of one or more thereof. In one embodiment ... the polypeptide sequence of SEQ ID NO: 11.

[0316] SEQ ID NO.11

[0317] MWWRLWWLLLLLLLLWPMVWAARSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDSGGGGSGGGGSGGGGSEPHSLRYNLTVLSWDGSVQSGFLAEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETEEWTMPQSSRAQTLAMNIRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGGGGSGGGGSGGGGSQVQLQQSGPELVKPGASVKISCKTSGYKFTEYTMHWVKQSHGKCLEWIGGINPNNGGTNYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCARDTTVPYAYWGQGTTVTVSSGGGGSGGGGSGGGGSDIELTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASSVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPLTFGCGTKLEIKRASTKGP or its equivalents.

[0318] In one embodiment, dimert (e.g., a trispecific adapter) is encoded by the sequence of SEQ ID NO.12.

[0319] SEQ ID NO.12

[0320]

[0321] In one embodiment, a dimert (e.g., a bispecific adaptor) comprises a secrecon1A sequence (sec1A), a CD3 sequence, and an anti-GD2 peptide sequence, or equivalents of one or more thereof. In one embodiment, the bispecific adaptor comprises the polypeptide sequence of Sequence ID NO. 13, arranged as sec1A-anti-CD3-linker-anti-GD2-HDD. The sec1A portion is underlined. The anti-CD3 portion is in bold. The anti-GD2 portion is underlined and italicized. The gray shaded area represents the HDD portion, which comprises the HDD peptide in bold, the HDD peptide upstream hinge region in lowercase, and the HDD peptide downstream spacer region in lowercase.

[0322] SEQ ID NO.13

[0323] or its equivalent.

[0324] In another embodiment, the dimert (eg, bispecific adaptor) is encoded by the sequence of SEQ ID NO. 14.

[0325] SEQ ID NO.14

[0326]

[0327] In another embodiment, a dimer (e.g., a bispecific adapter) comprises a secrecon1A sequence, an anti-CD19 sequence, and an anti-CD3 sequence arranged as sec1A-anti-CD19-linker-anti-CD3. The bispecific adapter comprises the polypeptide sequence of SEQ ID NO. 15. The sec1A portion is underlined.

[0328] SEQ ID NO.15

[0329] MWWRLWWLLLLLLLLWPMVWAA or its equivalent.

[0330] In another embodiment, the dimer (e.g., bispecific adapter) comprises a secrecon1A sequence, an anti-CD19 sequence, and an anti-CD3 sequence, arranged as sec1A-anti-CD19-linker-anti-CD3, or an equivalent of one or more thereof. In one aspect, it is encoded by the polynucleotide sequence of SEQ ID NO. 16.

[0331] SEQ ID NO.16

[0332]

[0333] In some embodiments, dimert comprises a secretion signal called secreconAA (sec2A). In some cases, dimert comprises secreconAA in tandem with an amino acid sequence from Blinatumomab (targeting CD3 and CD19). In some cases, secreconAA-BlinatumAb (or sec2A-CD19xCD3) comprises the polypeptide sequence of SEQ ID NO: 29, wherein the secreconAA portion is underlined.

[0334] SEQ ID NO:29

[0335] MWWRLWWLLLLLLLLWPMVWAAA DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQ VQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGS DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGS GGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKHHHHHH or its equivalent.

[0336] In some embodiments, the dimert comprising sec2A-CD19xCD3 is encoded by the polynucleotide sequence of SEQ ID NO:30.

[0337] SEQ ID NO:30

[0338]

[0339] In some embodiments, dimert comprises the sequence of secreconA (sec1A), CD19, and CD3 arranged in sec1A-CD19xCD3, or an equivalent of one or more thereof. In one aspect, it is encoded by the polynucleotide sequence of SEQ ID NO: 31.

[0340] SEQ ID NO:31

[0341]

[0342] In some embodiments, dimert comprises a secrecon (sec0A) sequence, CD19, and CD3 arranged in sec0A-CD19xCD3, or an equivalent of one or more thereof. In one aspect, it is encoded by the polynucleotide sequence of SEQ ID NO: 32.

[0343] SEQ ID NO:32

[0344]

[0345] In some embodiments, dimert comprises CD19 and CD3 sequences arranged as CD19xCD3 without the secreton sequence, or an equivalent of one or both. In one aspect, it is encoded by the polynucleotide sequence of SEQ ID NO: 33.

[0346] SEQ ID NO:33

[0347]

[0348] In one aspect, the bispecific T cell engager comprises a polypeptide sequence that is at least 95% identical to any one of SEQ ID NOs. 12 and 13, or at least 96%, or at least 97%, or 98%, or at least 99% identical. In one embodiment, the 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 comprises a polypeptide sequence that is at least 95% identical to any one of SEQ ID NOs. 12 and 13, or at least 96%, or at least 97%, or 98%, or at least 99% identical to any one of SEQ ID NOs. 11.

[0349] In a further aspect, the recombinant polynucleotide expresses a pre-mRNA that, when contacted with a morpholino oligonucleotide, encodes a trispecific antibody.

[0350] In one aspect, the antigen binding domain is a single chain variable fragment of an antibody.

[0351] In a further aspect, the recombinant polynucleotide or vector further comprises a polynucleotide sequence encoding a secretory peptide. On the other hand, the vector further comprises a polynucleotide sequence encoding a dimerization domain. On the other hand, the vector includes 5' inverted terminal repeats (ITRs) and 3' ITRs. On the other hand, the vector has a sequence or polynucleotide with at least 95% or at least 96% or at least 97% or any 98% or at least 99% identity to SEQ ID No. 4, 6, 8, 12, 15, 16, 30-33 or its equivalent. The non-limiting examples of this vector 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 polynucleotides optionally detectably labeled.

[0352] The recombinant polynucleotide vector can be contained in a host cell, such as a prokaryotic cell or a eukaryotic cell. By culturing the cell containing the polynucleotide under conditions that allow the polynucleotide to replicate and optionally express the polynucleotide, the cell can be used to recombinantly express or replicate the polynucleotide. The polynucleotide and / or expression product are optionally isolated from the cell culture.

[0353] The present invention also provides a viral packaging system comprising: a vector as described above, wherein the backbone is derived from a plasmid or virus; a packaging plasmid; and an envelope plasmid. The packaging plasmid contains nucleosides, capsid proteins, and matrix proteins. Examples of packaging plasmids are also described in the patent literature, for example, U.S. Patent 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.

[0354] The present invention also provides suitable packaging cell lines. In one aspect, the packaging cell line is a 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.

[0355] The present invention also provides a method for preparing AAV particles, comprising, consisting essentially of, or consisting of the following steps: transducing a packaging cell line with a viral system as described above under conditions suitable for packaging viral vectors. 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 present invention further provides such isolated particles.

[0356] The present invention further provides an isolated AAV viral particle produced by this method.The viral particle comprises, consists essentially of, or consists of a polynucleotide as described herein.

[0357] host cells

[0358] Further provided is an isolated cell or cell population comprising, consisting essentially of, or consisting of an isolated polynucleotide, a viral particle, a vector, and a packaging system, as described above and incorporated herein by reference. In one aspect, the isolated cell is a packaging cell line.

[0359] Also provided is an isolated cell or cell population comprising, consisting essentially of, or consisting of a polynucleotide sequence as described herein.

[0360] The isolated cells described herein can be any cell of the following species: mouse, rat, rabbit, monkey, cow, sheep, pig, canine, feline, farm animal, sports animal, pet, horse and primate, especially human cells.

[0361] Carrier and cell can be included in the composition comprising carrier and / or host cell and carrier (for example pharmaceutically acceptable carrier).They can be prepared for various modes of administration, and comprise effective amount of carrier and / or host cell effective to patient, disease or illness, carrier and mode of administration.On the one hand, mode of administration is systemic or intravenous injection.On the other hand, local administration is carried out by direct injection.On the one hand, morpholino oligonucleotide is contacted with carrier simultaneously or contacts after carrier.Or, contact morpholino oligonucleotide before carrier.

[0362] How to use

[0363] Polynucleotides and vectors can be used to treat a variety of diseases or conditions. On the one hand, a method for delivering a transgenic is provided. The method includes administering an effective amount of a polynucleotide or vector comprising a transgenic to cells, tissues or patients to be treated. On the one hand, an effective amount of antisense oligonucleotides (e.g., morpholino oligonucleotides) is administered to cells, tissues or patients to be treated. Non-limiting examples of transgenics are provided, and are selected according to the purpose of the method. Cells or tissues can be mammals, such as humans. On the one hand, antisense oligonucleotides (e.g., morpholino oligonucleotides) are contacted with the vector simultaneously or after the vector. Alternatively, contact is made before the vector. On the other hand, the vector is introduced into the cell by transfection, infection, transformation, electroporation, injection, microinjection or a combination thereof.

[0364] Also provided herein is a method for treating cancer in a subject in need thereof. The method comprises, or consists essentially of, or consists of the following steps: administering an effective amount of a recombinant viral vector or cell as described herein to the subject. On the other hand, the method also includes administering an effective amount of an antisense oligonucleotide (e.g., morpholino oligonucleotide) to the subject. On the one hand, an effective amount of an anticancer agent is administered to the subject. Non-limiting examples of anticancer agents include anticancer peptides, polypeptides, nucleic acid molecules, small molecules, viral particles, or a combination thereof. On the other hand, the vector is introduced into the cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof. The therapy can be implemented as a first-line, second-line, third-line, fourth-line, or fifth-line treatment. The therapy can be an adjuvant therapy or can be combined with other cancer therapies.

[0365] In one aspect of the disclosed method, the viral particle is an oncolytic HSV particle.

[0366] Application

[0367] The administration of the recombinant polynucleotides and / or vectors (e.g., AAV), viral particles or compositions of the present invention can be carried out in a single dose, continuously or intermittently throughout the course of treatment. Administration can be by any suitable mode of administration, including but not limited to: intravenous, intraarterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intraventricular, 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 polynucleotides or vectors or compositions are administered by intramuscular injection or intravenous injection. In another embodiment, the recombinant polynucleotides or vectors or compositions are administered systemically. In another embodiment, the recombinant polynucleotides or vectors or compositions are administered parenterally by injection, infusion or implantation.

[0368] Methods for 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 treatment, and the subject being treated. The dosage level and pattern can be selected by the treating physician for single or multiple administrations. It is worth noting that dosage may be affected by the route of administration. Formulations and methods of administration for appropriate dosages are known in the art. Non-limiting examples of such appropriate dosages may be as low as 1E+9 vector genomes to as high as 1E+17 vector genomes per administration.

[0369] In some embodiments of the methods described herein, the number of viral particles (e.g., AAV) administered to a subject is in the range of about 10 9 to about 10 17 In a specific embodiment, about 10 10 to about 10 16 , about 10 10 to about 10 15 , about 10 10 to about 10 12 , about 10 11 to about 10 13 , about 10 11 to about 10 12 , about 10 11 to about 10 14 , about 10 11 to about 10 15 , about 10 11 to about 10 16 , about 5x10 11 to about 5x10 12 , or about 10 12 to about 10 13 In some cases, about 10 11 to about 10 12 In some cases, about 1013 to about 10 15 In some cases, about 10 9 to about 10 12 In some cases, about 10 9 to about 10 11 In some cases, the amount of viral particles administered is based on the subject's body weight. One skilled in the art will understand how to adjust the amount of viral particles delivered so that the total amount delivered to the subject is in the range of about 10 9 to about 10 17 , optionally about 10 10 to about 10 16 , about 10 10 to about 10 15 , about 10 10 to about 10 12 , about 10 11 to about 10 13 , about 10 11 to about 10 12 , about 10 11 to about 10 14 , about 10 11 to about 10 15 , about 10 11 to about 10 16 , about 5x10 11 to about 5x10 12 , or about 10 12 to about 10 13 In some cases, the subject is a pediatric subject (e.g., a subject under 18 years of age). In some cases, about 10 9 to about 10 12 , about 10 10 to about 10 12 , about 10 11 to about 10 12 , or about 10 9 to about 10 10 Virus particles.

[0370] In another aspect, the viral particles and compositions of the invention can be administered in conjunction with other therapies, eg, approved treatments for cancer and its related diseases or conditions.

[0371] Successful treatment and / or repair is determined when one or more of the following is detected: alleviation or improvement of one or more diseases, disorders, or conditions of the subject; reduction in the extent of the subject's disease, disorder, or condition, stabilization of the disease, disorder, or condition (i.e., no worsening); delay or slowing of the progression of the subject's disease, disorder, or condition; and improvement or alleviation of the subject's disease, disorder, or condition. In some embodiments, the success of 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 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.

[0372] Reagent test kit

[0373] 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 present invention comprise one or more of the following: modified viral capsid proteins, isolated polynucleotides, vectors, host cells, recombinant viral particles, recombinant expression systems, modified AAVs, modified cells, isolated tissues, compositions, or pharmaceutical compositions as described herein.

[0374] In certain embodiments, the test kit further includes instructions for use. Specifically, such test kits may include one or more reagents as described herein, and instructions for describing the intended application and proper use of these reagents. For example, in one embodiment, the test kit may include one or more components and / or separation and mixing samples for mixing the test kit and applying to the experimenter. In certain embodiments, the reagents in the test kit are pharmaceutical preparations and dosages suitable for specific applications and methods of administration. The test kit for research purposes may include the composition of the appropriate concentration or quantity for carrying out various experiments.

[0375] The design of the kit can facilitate the use of the methods described herein and can take a variety of forms. Where applicable, each composition of the kit can 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., to an active form), such as by adding a suitable solvent or other species (e.g., water or cell culture medium), which may or may not be provided with the kit. In some embodiments, the composition may be provided in a preservation solution (e.g., a cryogenic preservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde, and (Stem Cell Technologies, Vancouver, Canada) In some embodiments, the storage solution comprises an amount of a metalloproteinase inhibitor.

[0376] As used herein, "instructions" can define an element of instructions and / or promotion, and generally refers to written instructions for or associated with the packaging of the method, recombinant vector, or composition. Instructions can also include any oral or electronic instructions provided in any manner so that the user will clearly understand that the instructions will be associated with the kit, for example, audio-visual (e.g., videotape, DVD, etc.), Internet and / or web-based communication, etc. In some embodiments, the written instructions are in a format prescribed by a governmental agency that regulates the manufacture, use, or sale of drugs or biological products, and the instructions can also reflect approval of manufacture, use, or sale by an animal regulatory agency.

[0377] In some embodiments, the test kit comprises any one or more components described herein in one or more containers. Thus, in some embodiments, the test kit may include containers for holding reagents described herein. These reagents may be in the form of liquids, gels, or solids (powders). The preparations may be aseptically prepared, packaged in syringes, and transported refrigerated. Alternatively, they may be placed in bottles or other containers for storage. A second container may be equipped with other sterile prepared reagents. Alternatively, the test kit may include premixed active agents and transported in syringes, bottles, test tubes, or other containers. The test kit may have one or more or all components required for administering the agent to the subject, such as syringes, topical application devices, or IV needles and bags.

[0378] The treatments described herein can be combined with appropriate diagnostic techniques to identify and select patients for such treatment.

[0379] Production method

[0380] In an additional method provided herein, a method for producing a bispecific antibody or trispecific antibody in a cell comprises 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 NO. 27 (CAAGATTTACCTCTATTGTTGGATCATATT) or SEQ ID NO. 28 (TAAAACAAGATTTACCTCTATTGTTGGATC), or an equivalent thereof. In one aspect, the morpholino oligonucleotide is contacted simultaneously with the vector or after the vector. Alternatively, the morpholino oligonucleotide is contacted before the vector. Non-limiting examples of the bispecific antibody have a polypeptide sequence that is at least 95% identical to any one of SEQ ID NOs: 13 and 15, or 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 that is at least 95% identical to SEQ ID NO: 11, or has a sequence that is at least 96%, or at least 97%, or at least 98%, or at least 99% identical to SEQ ID NO: 11.

[0381] 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, bone cells, epithelial cells, muscle cells, neural cells, endocrine cells, melanocytes, blood cells, or a combination thereof.

[0382] Further provided are kits comprising one or more vectors, cells, or compositions as described herein, optionally with instructional documentation or materials.

[0383] Specific implementation plan

[0384] There are several approaches to regulate gene expression through the principle of exon skipping.

[0385] On the one hand, at least three exons are required to skip an exon in this strategy. For a three-exon structure, there are two splice donor and two splice acceptor sites, e.g. Figure 1 The first donor site is spliced ​​to the first acceptor site ( Figure 1 "a" in ), or, spliced ​​to a second acceptor site ( Figure 1 The second donor site can only be spliced ​​to the second acceptor site ( Figure 1 Thus, this three-exon / two-intron structure can give rise to five possible RNA structures, depending on whether splicing occurs, whether only one splicing event occurs (a, b, or c), or whether two splicing events occur (a+b).

[0386] Sequence-specific oligonucleotides ("oligonucleotides") are constructed that specifically interfere with each splicing event, usually at the splice donor or acceptor site, or at a binding site for a spliceosomal component (sometimes in the middle of an intron). For the purposes of this example and as Figure 1 As shown, each oligonucleotide was classified as 1-donor (1D), 2-acceptor (1A), 2-donor (2D), or 3-acceptor (3D) based on whether it interferes with the donor or the acceptor.

[0387] For example, due to alternative splicing or interference with oligonucleotides, exon 2 of a gene is skipped in cancer cells but retained in normal cells. In this embodiment, the normal gene containing the skipped exon is not expressed in cancer cells, but expresses protein in normal cells. The intron sequences on both sides of exon 2 in this structure can be used to reproduce the same splicing pattern in the transgene. Therefore, the construct containing the transgene in the typical intron-exon-intron structure spliced ​​in normal cells and tumor cells maintains relatively efficient splicing and produces high levels of fully spliced ​​transcripts (exons 1+2+3, using splicing a+b). In addition, when the oligonucleotide blocks the splicing of intron a or intron b (or both), exon 2 is skipped to generate a transcript that fuses exon 1 to exon 3. Therefore, Figure 1 The relative expected abundance of the “theoretically possible” transcripts shown in Figure 2 was modified to show potentially enriched transcripts.

[0388] In one embodiment, based on the splicing concept described above, oligonucleotides and "exon skipping" technology are used to convert the transgene from a transcript comprising exon 1, exon 2, and exon 6 to a transcript comprising exon 1 and exon 3. Thus, the construct or vector produces a functional polypeptide encoded by a transcript fused to exon 1 and exon 3. In one embodiment, the stop codon is designed to be operably linked to exon 2 so that transcription stops at the stop codon before exon 3 is translated, in the absence of oligonucleotide interference to skip exon 2. A premature stop codon results in a non-functional transcript comprising 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 (exons 1+2+3) is switched to the functional transcript (exons 1+2), as shown in FIG. Figure 3 This is shown because ribosomes stop translation of the transcript after the stop codon that includes exon 3.

[0389] The construction of such a transgene is relatively simple, but an intron-exon STOP-intron structure is selected that is normally "spliced" appropriately. The intron-exon STOP-intron structure can be engineered using sequences flanking the normally spliced ​​exons and cloned into specific sites within the transgene, thereby forming splice donor and acceptor consensus sequences in the flanking bases. Exons should be carefully selected because exons derived from sequences in the normal gene will also be skipped. For example, the intron-exon-intron structure could be derived 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 against cancer, exploitation of oncogene intron-exon-intron boundaries could be considered, potentially resulting in a defect in the oncogene. This strategy would exploit the non-targeted skipping of cellular genes as a potential "additional" therapeutic effect.

[0390] Some exons are differentially regulated in cancer cells compared to normal cells. For example, splice type 2 exons are "spliced ​​in" in normal cells but are excluded from the same gene in some cancer cells. In this case, if the intron-exon-intron boundaries of splice type 2 exons are used in the construction (see Figure 4 ), then the baseline and skipped expression of the transgene will be different in normal and cancer cells, e.g. Figure 5 Because the engineered exon STOP is not normally present in cancer cells, the transgene will be activated in these cells but not in normal cells ( Figure 5 This exon skipping can be used to selectively express a transgene in cancer cells, but not in normal cells even without exon skipping. This may be desirable, for example, for toxin genes or prodrug enzymes. Using oligonucleotides to skip exon 2 will activate the transgene in normal cells and may increase expression in cancer cells if baseline skipping in these cells is less than 100%.

[0391] In contrast, splice type 3 exons are excluded in normal cells but are "spliced ​​in" in certain cancer cells. Therefore, regulated splicing using this exon will produce the opposite results to splice type 2, such as Figure 6 shown.

[0392] In summary, the following strategies have been disclosed to achieve the goal of regulating transgene expression in the context of gene therapy:

[0393] Strategy #1: Activation of transgene expression relies on administration of exon-skipping oligonucleotides. This effect is seen in both normal and cancer cells.

[0394] Strategy #2: Activation of transgene expression, without any drugs or external additives, is typically high in certain cancer cells (and only in certain cancers) but low in normal cells. This application can be used for cancer-selective expression in cancers with appropriately regulated splicing. Administration of exon-skipping oligonucleotides will activate expression in normal cells in addition to specific cancer cells.

[0395] Strategy #3: Activation of transgene expression is high in normal cells but low in certain cancer cells. Administration of exon-skipping oligonucleotides can activate expression in these cancer cells.

[0396] Although the principles of controllable gene expression can be applied to any gene therapy application, gene therapy for cancer treatment is a particular application. In this embodiment, a therapeutic drug expressed and secreted in normal cells can be activated when needed by administering appropriate exon skipping oligonucleotides.

[0397] For these purposes, the applicants designed an intron-exon STOP-intron cassette from exon 1 of the KRAS gene as an example of a strategy #1 construct to be inserted into a transgene of interest to achieve regulatable gene expression. Oligonucleotides that induce exon skipping are selected for therapeutic applications.

[0398] On the one hand, the applicant took advantage of the structure of the KRAS gene, which is one of the most commonly mutated genes in cancer. KRAS is not an enzyme and does not have an obvious drug binding pocket on its surface, so it cannot be used as a drug target. Therefore, to the best of the applicant's knowledge, this skipping technology is the first method to target KRAS in cancer. For these purposes, the applicant's construct utilizes the first exon of KRAS in the 5' untranslated region, and the ATG start codon for protein translation is located within the second exon. (Therefore, the second exon is usually referred to as exon 1 and the first exon is called exon 0). Oligonucleotides that induce exon skipping of exons containing ATG will cause transcripts to be unable to be translated normally due to the lack of the ATG start codon.

[0399] KRAS-based intron-exon STOP-intron

[0400] According to the Genbank sequence (NCBI reference sequence: NG_007524.1, last accessed on February 20, 2019), the exons of KRAS are located at:

[0401] 4990..5170,10526..10647,28509..28687,30148..30307,46010..51132, while cDNA was added at: 10537..10647,28509..28687,30148..30307,46010..46126.

[0402] The first ATG of the normal KRAS gene is located at position 10537.

[0403] The exon to be skipped is located between 10526-10647 (122 bp).

[0404] This construct contains a pseudo-version with a stop (STOP) codon in each reading frame.

[0405] In one embodiment, for the KRAS1 exon, the shaded and underlined nucleotides (including the ATG of KRAS) are replaced by a stop codon:

[0406]

[0407] Exemplary stop codons are: TAA, TAG and TGA. In one aspect, all three reading frames are: TAAxTAGxTGA, where x is any nucleotide.

[0408] To design all three stop codons twice in a row, a stop codon can be inserted. In addition, the 11 base pair repeat can be avoided by using the sequence TAAxTAGxTGAxTAGxTAAxTGAx (24 bp) (SEQ ID No: 1), where x is any nucleotide. A specific example is: TAATTAGCTGAGTAGATAAGTGAT (SEQ ID No: 2). Therefore, an example containing the exon Kras1 STOP (stop codon is underlined) is: GCCTGCTGAAA TAATTAGCTGAGTAGAT AAGTGAT GTTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAG (SEQ ID NO. 18).

[0409] The normal upstream intron is 5171-10525 (5355 bp) of the NCBI reference sequence NG_007524.1: “gtacg…ataag”, where the rest of the upstream intron is shown as “…”.

[0410] The normal downstream intron is 10648-28508 (17861 bp) of the NCBI reference sequence NG_007524.1: “gtaaa…ctcag”, wherein the remaining portion of the downstream intron is shown as “…”.

[0411] 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:

[0412] GTACGGAGCGGACCACCCCTCCTGGGCCCCTGCCCGGGTCCCGACCCTCTTTGCCGGCGCCGGGCGGGGCCGGCGGAGTATTTGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTATTATAAG (SEQ ID NO. 19).

[0413] In one embodiment, the downstream intron sequence is as follows:

[0414] GTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGACCATTTTGAGTTGTATATAACACCTTTTTTGAAGTAAAAGGTGCACTGTAATAATCCAGACTGTGTTTCTCCCTTCTCAG (SEQ ID NO. 20).

[0415] Therefore, in one embodiment, the following sequence is an intron-exon Kras1 STOP-intron sequence (the sequence without highlighting is the upstream intron, the gray sequence is the intron containing the STOP sequence, and the underlined sequence is the downstream intron):

[0416]

[0417] On the one hand, a series of oligonucleotides that span the junction of the exon-downstream intron boundary can be added because they have the ability to cause exon skipping. Exon skipping oligonucleotides are usually 20-30 base pairs long and antisense to DNA.

[0418] In one embodiment, the morpholino binding site is a Kras1 derived intron-exon STOP-intron morpholino binding site (SEQ ID NO. 26). The sequence of the K1ExonStopIntron 3' junction sequence is shown below (exons are marked in grey and introns are underlined):

[0419]

[0420] Applicants created and tested two morpholino binding sites, designated KTS1 (SEQ ID NO. 24) and KTS2 (SEQ ID NO. 25). The KTS1 morpholino sequence comprised the sequence of SEQ ID NO. 27, and the KTS2 morpholino sequence comprised the sequence of SEQ ID NO. 28. Applicants used the reversed KTS2 morpholino sequence (SEQ ID NO. 29) as a negative control.

[0421] SEQ ID NO. 24—KTS1 (KRAS TransSkip 1) (exons are marked in gray, introns are underlined)

[0422]

[0423] SEQ ID NO. 27—KTS1 morpholino

[0424] CAAGATTTACCTCTATTGTTGGATCATATT

[0425] SEQ ID NO.25—KTS2(KRAS TransSkip 2)

[0426]

[0427] SEQ ID NO. 28—KTS2 morpholino

[0428] TAAAACAAGATTTACCTCTATTGTTGGATC

[0429] SEQ ID NO. 29—KTS2 morpholino reverse sequence

[0430] CTAGGTTGTTATTCTCATTTAGAACAAAAT.

[0431] In some cases, the splice donor site includes A / C AG***GT A / G AGT (SEQ ID NO: 34), where "***" indicates an exon-intron boundary and the site for insertion of an intron-exon STOP-intron sequence.

[0432] In certain instances, the splice acceptor comprises (Py)XCAG***G / T (SEQ ID NO: 35), where "***" indicates an intron-exon boundary.

[0433] In some cases, exemplary splice donor sites for insertion into 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), where "-" indicates the insertion site.

[0434] CATAAVERT constructs

[0435] To create cancer-targeted AAV-expressing regulated T( Ca ncer T Argeted AAV e xpressed, r egulated T In one embodiment, a vector comprising a regulated CATAAVERT (CATAAVERT) linker (also known as "TransSkip") inserts an 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, a vector comprising a regulated CATAAVERT expresses CD3 and GD2.

[0436] In another embodiment, the vector comprises a sequence encoding the secrecon-AA-CD3xGD2-HDD dimert. For this construct, the secrecon-AA-CD3xGD2-HDD dimert sequence comprises all potential sites (highlighted in grey) for insertion into the intron-exon Kras1 stop-intron to generate a consensus splice donor / acceptor (the insertion sequence is located after the third of one or more 5 bp) and is provided below:

[0437]

[0438] On the one hand, it is inserted at the most upstream site to minimize the length of the CD3xGD2 dimer that is translated before the stop codon (the underlined sequence is the splice site that remains in the mRNA when the insert is spliced, the double underlined sequence is the splice donor site at the beginning of the upstream insert sequence, the gray sequence is the exon containing the STOP sequence, and the bold sequence is the downstream intron). In some cases, this sequence is called the CD3xGD2 K1 dimer:

[0439]

[0440] In some cases, the upstream splicing factor binding site is modified (the modified region is italicized and bold). The underlined sequence is the splice junction that remains in the mRNA when the insert is spliced, the double-underlined sequence is the splice donor site at the beginning of the upstream intron sequence, the gray sequence is the exon containing the STOP sequence, and the bold sequence is the downstream intron. In some cases, this sequence is called CD3xGD2 K2 dimert:

[0441]

[0442]

[0443] In some cases, the CD3xGD2 dimerT construct comprises one or more additional modifications in the polynucleotide sequence. In some cases, the CD3xGD2 dimerT construct is sec2A-CD3xGD2-HDD-K3, and the sequence of sec2A-CD3xGD2-HDD-K3 is shown in SEQ ID NO:41. The underlined sequence is the splice junction retained in the mRNA when the intron is spliced, the double-underlined sequence is the splice donor site at the beginning of the upstream insert sequence, the gray sequence is the exon containing the STOP sequence, and the bold sequence is the downstream intron.

[0444] SEQ ID NO:41

[0445]

[0446]

[0447] In some cases, the CD3xGD2 dimert construct is sec2A-CD3xGD2-HDD-K4, and the sequence of sec2A-CD3xGD2-HDD-K4 is shown in SEQ ID NO: 42. The underlined sequence is the splice junction retained in the mRNA during intron splicing, the double-underlined sequence is the splice donor site at the beginning of the upstream intron sequence, the gray sequence is the exon containing the STOP sequence, and the bold sequence is the downstream intron.

[0448] SEQ ID NO:42

[0449]

[0450]

[0451] In some cases, the CD3xGD2 dimert construct is sec2A-CD3xGD2-HDD-K5, and the sequence of sec2A-CD3xGD2-HDD-K5 is shown in SEQ ID NO: 43. The underlined sequence is the splice junction retained in the mRNA during intron splicing, the double-underlined sequence is the splice donor site at the beginning of the upstream intron sequence, the gray sequence is the exon containing the termination sequence, and the bold sequence is the downstream intron.

[0452] SEQ ID NO:43

[0453]

[0454] In some embodiments, the dimert described herein comprises a CD3 sequence, a CD19 sequence, and an optional secreton sequence. In some cases, the dimert comprises the CD3xCD19 construct as described in SEQ ID NO: 44, highlighting all potential sites for insertion of the intron-exon-Kras1stop-intron sequence to create a consensus splice donor / acceptor site. Each potential insertion site is highlighted in gray.

[0455] SEQ ID NO:44

[0456]

[0457] In some cases, the insertion is made at the most upstream site. In some cases, the dimer construct 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, the underlined sequences are splice junctions retained in the mRNA during intron splicing, the italicized and bold sequences are upstream intron sequences, the gray sequences are exons containing STOP sequences, and the bold sequences are downstream introns.

[0458] SEQ ID NO:45

[0459]

[0460]

[0461] In some cases, the CD3xCD19 construct is sec1A-CD3xCD19-K3, the sequence of which is shown in SEQ ID NO: 46. As shown below, the underlined sequence is the splice junction retained in the mRNA when the intron is spliced, the italicized and bold regions are the modified regions of the upstream intron binding site, the gray sequence is the exon containing the STOP sequence, and the bold sequence is the downstream intron.

[0462] SEQ ID NO:46

[0463]

[0464]

[0465] Example

[0466] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0467] Example 1 - Generation of CD3xGD2-HDD Dimer using an exemplary CD3xGD2-HDD TransJoin

[00105] Mapping of exemplary AAV constructs used to test CD3xGD2-HDD expression.

[0468] As a proof of principle, a previously described bispecific molecule targeting human CD3 on T cells and the disialoganglioside GD2 on neuroblastoma and other cancer cell types was expressed. This bispecific protein has the amino acid sequences of the heavy and light chain variable regions of human CD3 derived from clone OKT3 (using a single-chain variable fragment format, scFv), fused via a short linker (L) to a scFv construct for GD2 derived from clone 5F11, connected as a dimer by the HNF1a dimerization domain (HDD), as reported by Ahmed et al., OncoImmunology, 4:4, e989776, DOI: 10.4161 / 2162402X.2014.989776. The optimal human DNA coding sequence for CD3xGD2-HDD was reverse engineered using the vectorbuilder.com codon optimization tool (http: / / en.vectorbuilder.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 sequence derived from AAV2. Based on the finding that alanine enhances protein secretion (Güler-Gane et al., PLoS ONE 11(5):e0155340.doi:10.1371 / journal.pone.0155340), three other versions were prepared containing a consensus secretion signal domain ("secrecon", based on Barash et al., Biochem Biophys Res Commun. 2002; 294:835–842) located downstream of the ATG start site and ending with 0, 1, or 2 alanines. The proteins derived from these constructs are referred to as heterodimeric scFvs or Dimerts. Figure 7 Four exemplary CD3xGD2-HDD dimert constructs are illustrated.

[0469] Tests to determine the structure and function of Dimert.

[0470] 293T cells were transduced with AAV expression vectors (or controls) and the supernatants were collected and stored. The supernatants were tested for the presence of proteins of the correct size on electrophoresis (SDS-PAGE), binding to CD3 by a binding competition assay using flow cytometry, activation of T cells by flow cytometry, and killing of tumor cells when co-incubated with T cells. Figure 8 , which shows a cartoon illustration of the assays used to determine the structure and function of the dimerts disclosed herein.

[0471] The three constructs containing secreted peptides showed less CD3xGD2-HDD Dimert retention in cells.

[0472] 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 50 μg of total protein per lane, and the gel was stained with Ponceau S. The results showed that there was more protein using construct #1104 that lacked the secretion sequence, while all three constructs with the secretion sequence showed less protein. Controls included cells alone or cells transfected with a control plasmid (pcDNA3-GFP). KDa, kilodaltons; Sec, secretion domain; GFP, green fluorescent protein; MW, molecular weight. See Figure 9 .

[0473] Only supernatants from cells transfected with the AAV CD3xGD2-HDD construct containing the secretion sequence bound to and activated T cells.

[0474] The supernatants were tested for binding and activation of human T cells. Binding was determined by competition with fluorescently labeled anti-CD3 antibodies pre-bound to T (Jurkat) cells. The stained cells showed 77.6%, 79.24% and 78.7% (Q2+Q3) CD3 positivity in the control group (DMEM, control, GFP), and 77.7% in the AAV vector #1104 lacking the secretory peptide. In contrast, binding of the fluorescently labeled anti-CD3 antibody was reduced to 2.15%, 3.67% and 3.99% (Q2+Q3) by the supernatant of cells transfected with each vector containing the secretory peptide. In addition, the cells were co-stained with anti-CD69, a marker of T cell activation. Both the control group and AAV vector #1104 showed less than 13.4% CD69 positivity (Q1+Q2), while the three different AAV vectors containing the secretory peptide showed 58.87-68.5% CD69 positivity. See Figure 10 .

[0475] Method details: Jurkat 2e5 cells / well in 400ul RPMI+10% FBS were seeded in a 24-well plate, and then 100ul supernatant from 293T transfected cells (20% of the total culture) was added to each well. After incubation for 4 hours, the Jurkat cells were centrifuged, washed once with PBS, and then stained with PE-anti-hCD69 (1:100) and PerCP-anti-hCD3e (1:300#OKT3) on ice for 30 minutes. After washing with FACS buffer, each sample was fixed in 1% PFA and analyzed by flow cytometry. DMEM was the culture medium added to Jurkat alone, the control was the supernatant added from untransfected 293T cells, and GFP was the supernatant added from 293T cells transfected with an AAV plasmid expressing GFP.

[0476] Only supernatants from cells transfected with AAV vectors containing the secreted peptide activated human T cells.

[0477] Human T (Jurkat) cells were incubated with supernatants from 293T cells transfected with various AAV vectors, stained with PE-conjugated antibodies for CD69, and examined under a fluorescence microscope. Figure 11 , where the left panel is phase contrast and the right panel is fluorescence. Neither the EGFP vector control nor the vector #1104 lacking the secreted peptide showed positive staining, while the other three vectors showed high levels of staining (red). Method details: Jurkat 2e5 cells / well in 400ul RPMI+10% FBS were seeded into a 24-well plate, and then 100ul supernatant from 293T transfected cells (20% of the total culture) was added to each well. After incubation for 48 hours, the Jurkat cells were centrifuged, washed once with PBS, and then stained with phycoerythrin (PE)-anti-hCD69 (1:100) on ice for 30 minutes. After washing with FACS buffer, each sample was fixed in 1% PFA for 5 minutes. Scale bar: 100um.

[0478] The binding of CD3xGD2-HDD to T cells was dose-dependent.

[0479] Different amounts of supernatant from 293T cells transfected with different AAV plasmids containing secreted peptides were incubated and tested for competition with Peridinin Chlorophyll Protein Complex (PerCP)-conjugated anti-hCD3 on Jurkat T cells. Figure 12A , where the left panel shows the FACS plots and the right panel shows the median staining level. The gray shades in the left panel are unstained, and the dark gray line in each panel indicates the maximum number of cells stained without the addition of supernatant. Figure 12BShown are bar graphs of median staining levels normalized to unstained controls.

[0480] Method Details: Jurkat 2e5 cells / well in 400ul RPMI + 10% FBS were seeded into 24-well plates, and 10ul, 25ul, 50ul, or 100ul of supernatant from 293T transfected cells (2-20% of the total culture) were added to each well. After a 24-hour incubation, Jurkat cells were centrifuged, washed once with PBS, and then stained with PerCP-anti-hCD3e (1:300 #OKT3) on ice for 30 minutes. After washing with FACS buffer, each sample was fixed in 1% PFA for 5 minutes and analyzed by flow cytometry. Sec = secreted peptide, A = alanine.

[0481] Binding of the anti-GD2 arm of the CD3xGD2-HDD Dimer was assayed and confirmed that both GD2 and CD3 binding occurred on a single molecule.

[0482] Binding of CD3xGD2-HDD to GD2 is measured indirectly by first incubating supernatant from 293T transfected cells with GD2-positive or GD2-negative cells and then repeating the CD3 T cell binding competition assay. Proteins that bind to GD2 should be taken up by GD2-positive cells but not by GD2-negative cells, resulting in a loss of competition for T cell binding. This assay is designed to confirm that GD2 binding is coupled to CD3 binding, in other words, that a single molecule is bispecific. See Figure 13 , is a cartoon illustration of the binding assay.

[0483] CD3xGD2-HDD combines CD3 and GD2.

[0484] like Figure 13 Supernatants from 293T cells transfected with the #1101CD3xGD2-HDD AAV vector were collected and preincubated with GD2-positive SK-N-Be(2) or GD2-negative Raji cells as indicated. After rotation and washing, binding competition assays were performed on CD3 T (Jurkat) cells.

[0485] Figure 14Figure 3 illustrates an exemplary CD3xGD2-HDD Dimert that binds both CD3 and GD2. The gray shading in the top panel with the black outline is unstained Jurkat T cells, and the dark gray line is Jurkat T cells fully stained for CD3 without the addition of supernatant. Sample Jurkat_cd3e shows complete competition for CD3 binding using supernatant without preincubation, almost overlapping with supernatant preincubated with GD2-negative Raji cells. In contrast, sample Jurkat_cd3e sknbe2 shows loss of competition when the supernatant is preincubated with GD2-positive SK-N-Be(2) neuroblastoma cells, confirming that the same molecule binds CD3 and GD2.

[0486] Test to determine whether CD3xGD2-HDD induces T cell killing of GD2+ target cells.

[0487] Figure 15 Flowchart illustrating the assay to determine whether CD3xGD2-HDD induces killing of G2+ target cells by T cells. Figure 15 As shown, GD2+ neuroblastoma (SK-N-Be(2)) cells were seeded into the wells, followed by primary human T cells (purchased from StemExpress) and supernatants from 293T cells transfected with AAV vectors. Cell viability was assessed using CellTiter-Glo from Promega (Madison, WI).

[0488] Secreted CD3xGD2-HDD induces T cell killing of neuroblastoma cells.

[0489] use Figure 15 In the assay shown in , the cytotoxicity of human T cells plus supernatants (from 293T cells transfected with various AAV vector plasmids) was tested on GD2+SK-N-Be(2) neuroblastoma cells. A T cell to target cell ratio of 10:1 was used. Cell viability was determined after 48 hours of co-culture. There was no significant cytotoxicity using supernatants from cells transfected with a construct expressing an irrelevant dimert (CD19xCD3) or a vector lacking a secreted peptide (#1104) compared to supernatants from untransfected cells ("no dimert"). Figure 16 In contrast, supernatants from cells transfected with either vector containing the secreted peptide induced statistically significant cytotoxicity (p<0.001), killing 25-30% of the cells ( Figure 16 ).

[0490] T cell-mediated cytotoxicity of CD3xGD2-HDD Dimert is associated with GD2 expression.

[0491] A panel of neuroblastoma cell lines was tested for sensitivity to human T cell killing, in combination with supernatant from AAV vector #1101 ( Figure 17 and their GD2 expression was measured by flow cytometry ( Figure 17 (lower panel, shaded curve is isotype control). CHP-134 cells showed the greatest cytotoxicity and the highest GD2 expression.

[0492] Example 2 - Generation of CD19xCD3 Dimers Using an Exemplary CD19xCD3 TransJoin

[0493] Map of exemplary AAV constructs used to test CD19xCD3 Dimert expression.

[0494] The FDA-approved protein therapeutic, called blinatumomab, is a so-called bispecific T-cell engager (BiTE) that targets human CD19 on B cells / B-cell malignancies and human CD3 on T cells. The publicly available amino acid sequence of blinatumomab (http: / / www.drugbank.ca / drugs / DB09052) was used and the vectorbuilder.com codon optimization tool (http: / / en.vectorbuilder.com / tool / codon-optimization.html) was further used to reverse engineer the optimal human DNA coding sequence for CD19xCD3. The resulting DNA sequence, which began with the ATG start codon, was synthesized and cloned into an adenovirus-associated virus expression cassette downstream of the chicken-actin-b-globin promoter (CAGp) with inverted terminal repeats derived from AAV2. Based on the finding that alanine enhances protein secretion (Güler-Gane et al., PLoS ONE 11(5):e0155340.doi:10.1371 / journal.pone.0155340), three other versions were prepared containing a consensus secretion signal domain ("secrecon," based on Barash et al., Biochem Biophys Res Commun. 2002; 294:835–842) located downstream of the ATG start site and ending with 0, 1, or 2 alanines. Proteins derived from these constructs are referred to as heterodimeric scFvs or Dimerts. Figure 18A and Figure 18B Five exemplary CD19xCD3 constructs are described.

[0495] Figure 18CCartoon illustration of the interaction of CD19 dimer with cancer cells and T cells. CD19 dimer is produced by cells containing the CD19 TransJoin. When administered intravenously to a subject, for example, an AAV TransJoin (e.g., the AAV CD19 TransJoin shown in this figure) enters normal cells (e.g., liver or muscle) and expresses its encoded polypeptide. The secretory signal peptide is cleaved during the secretion process, leaving the active dimer, which binds to cancer cells (Ca) at one end and to T immune cells at the other.

[0496] Only supernatants from cells transfected with the AAV CD19xCD2 construct containing the secretion sequence bound to and activated T cells.

[0497] The supernatant was tested for binding and activation of human T cells. Binding was determined by competition with fluorescently labeled anti-CD3 antibodies pre-bound to human T (Jurkat) cells. The stained cells showed 77.6%, 79.24% and 78.7% (Q2+Q3) CD3 positivity in the control group (DMEM, control, GFP), and 79.4% in the vector #1323 lacking the secretory peptide. In contrast, the binding of the fluorescently labeled anti-CD3 antibody was reduced to 23.88%, 18.59% and 30.76% (Q2+Q3) by the supernatant of cells transfected with each vector containing the secretory peptide. In addition, the cells were co-stained with anti-CD69, a marker of T cell activation. Both the control group and #1323 showed less than 13.38% CD69 positivity (Q1+Q2), while the three different vectors containing the secretory peptide showed 63.9%, 67.3% and 66.6% CD69 positivity ( Figure 19 ).

[0498] Method details: Jurkat 2e5 cells / well in 400ul RPMI+10% FBS were seeded in a 24-well plate, and then 100ul supernatant from 293T transfected cells (20% of the total culture) was added to each well. After incubation for 48 hours, the Jurkat cells were centrifuged, washed once with PBS, and then stained with PE-anti-hCD69 (1:100) and PerCP-anti-hCD3e (1:300#OKT3) on ice for 30 minutes. After washing with FACS buffer, each sample was fixed in 1% PFA and analyzed by flow cytometry. DMEM was the culture medium added to Jurkat alone, the control was the supernatant added from untransfected 293T cells, and GFP was the supernatant added from 293T cells transfected with an AAV plasmid expressing GFP.

[0499] Only supernatants from cells transfected with AAV vectors containing the secreted peptide activated human T cells.

[0500] Human T (Jurkat) cells were incubated with supernatants of 293T cells transfected with various AAV vectors, stained with PE-labeled antibodies for CD69, and examined under a fluorescence microscope. Figure 20 The left panel is phase contrast, and the right panel is fluorescence. Neither the EGFP vector control nor the AAV vector #1323 lacking the secretory peptide showed positive staining, while the other three vectors showed high levels of staining (red).

[0501] Method Details: Jurkat 2e5 cells / well in 400 μl of RPMI + 10% FBS were seeded in a 24-well plate, and 100 μl of supernatant from 293T transfected cells (20% of the total culture) was added to each well. After a 48-hour incubation, Jurkat cells were centrifuged, washed once with PBS, and then stained with phycoerythrin (PE)-anti-hCD69 (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 μm.

[0502] AAV-secreted CD19xCD3 specifically binds to CD19 but not CD45.

[0503] A binding competition assay was used to determine whether supernatants from AAV vector-transfected 293T cells would interfere with the staining of Epstein-Barr virus (EBV)-transformed human B cells by two different antibodies, one staining the B cell marker CD19 and the other staining the pan-leukocyte marker CD45.

[0504] See Figure 21 , where cells in the three control groups (DMEM, control, GFP) showed 93%, 93.1%, and 93.2% CD19 (Q1+Q2) positive staining, respectively, while the supernatant of cells transfected with the AAV vector #1323 lacking the secretory peptide did not compete for staining and showed 93.2% CD19 positivity. In contrast, each AAV vector containing a secretory peptide competed out the CD19 signal to 33.33%, 31.07%, and 35.88%. Note that the cutoff value was set so that unstained cells showed 14.58% CD19 positivity, indicating that the supernatant from cells transfected with these three AAV vectors competed down to 2-fold above the background. In contrast, none of the vectors competed for CD45 staining, with 78.89%, 79.14%, and 78.32% positivity in the three controls (Q2+Q3), 82.9% in the vector lacking the secreted peptide, and 80.5%, 78.5%, and 79.4% positivity using supernatants from cells transfected with the other three vectors.

[0505] Method details: B cells were transformed with EBV designated NB122R (Gene Ther. 2013 Jul; 20(7):761-9. doi:10.1038 / gt.2012.93) at 2e5 cells / well in 400ul of RPMI + 10% FBS and seeded in 24-well plates. 100ul of 293T transfected cell supernatant (2-20% of the total culture) was added to each well. After incubation for 24 hours, the cells were centrifuged, washed once with PBS, and then stained with PEcy7-anti-hCD45 (1:100) and APC-anti-hCD19 (1:300) on ice for 30 minutes. After washing with FACS buffer, each sample was fixed in 1% PFA for 5 minutes before flow cytometric analysis. DMEM was the culture medium added to Jurkat cells alone, the control was the supernatant added from untransfected 293T cells, and GFP was the supernatant added from 293T cells transfected with an AAV plasmid expressing GFP.

[0506] Binding of CD19xCD3 to T cells was dose-dependent and superior to the vector containing a single alanine downstream of the secretory peptide consensus sequence compared to other vectors tested.

[0507] Different amounts of supernatant from 293T cells transfected with different AAV plasmids containing secreted peptides were incubated and tested for competition with Peridinin Chlorophyll Protein Complex (PerCP)-conjugated anti-hCD3 on Jurkat T cells. Figure 22A , where the left panel shows the FACS plots and the right panel shows the median staining level. The gray shadows in each left panel are unstained cells, and the dark gray line in each left panel is the maximum stained cells without the addition of supernatant. Figure 22B Bar graphs showing median staining levels normalized to unstained controls are shown. Vector #1325, which has a single alanine downstream of the secretory peptide consensus sequence, exhibited the greatest competition compared to the other vectors tested and was selected for further experiments.

[0508] Method Details: Jurkat 2e5 cells / well in 400ul RPMI + 10% FBS were seeded into 24-well plates, and 10ul, 25ul, 50ul, or 100ul of supernatant from 293T transfected cells (2-20% of the total culture) were added to each well. After a 24-hour incubation, Jurkat cells were centrifuged, washed once with PBS, and then stained with PerCP-anti-hCD3e (1:300 #OKT3) on ice for 30 minutes. After washing with FACS buffer, each sample was fixed in 1% PFA for 5 minutes and analyzed by flow cytometry. MFI = mean fluorescence intensity, Sec = secreted peptide, A = alanine.

[0509] Binding of CD19xCD3 to B cells was dose-dependent and superior to the vector containing a single alanine downstream of the secretory peptide consensus sequence compared to other vectors tested.

[0510] Different amounts of supernatant from 293T cells transfected with different AAV plasmids containing secreted peptides were incubated and tested for competition with allophycocyanin (APC)-conjugated anti-hCD19 on human B cells. Figure 23A , where the left panels show FACS plots and the right panels show median staining levels. The gray shades in each left panel are unstained, and the dark gray line in each left panel is the maximum number of stained cells without added supernatant. Figure 23B Bar graphs showing median staining levels normalized to unstained controls are shown. Vector #1325, which has a single alanine at the end of the secreted peptide, showed the strongest competition, with Figure 22A and Figure 22B The T cell binding results were consistent with those shown in and were selected for further experiments.

[0511] Method Details: NB122R human B cells in 400 μl RPMI + 10% FBS were seeded at 2e5 cells / well in 24-well plates, and 10 μl, 25 μl, 50 μl, or 100 μl of supernatant from 293T transfected cells (2-20% of the total culture) was added to each well. After a 24-hour incubation, cells were centrifuged, washed once with PBS, and then stained with APC-anti-hCD19 (1:300) on ice for 30 minutes. After washing with FACS buffer, each sample was fixed in 1% PFA for 5 minutes and analyzed by flow cytometry. MFI = mean fluorescence intensity, Sec = secreted peptide, A = alanine.

[0512] CD3 Dimert activates T cells via anti-CD28 co-stimulation better than anti-CD3 antibodies.

[0513] Human T (Jurkat) cells were incubated with anti-CD28 antibodies and supernatants from 293T cells transfected with AAV vectors ( Figure 24 Left panel) or increasing concentrations of anti-CD3 antibody ( Figure 24 The cells were harvested and the mRNA was expressed in IL-2 ( Figure 24 ) and IL-8 ( Figure 24Quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed on mRNA of CD3xGD2-HDD (lower panel) and expression was calculated relative to the housekeeping mRNA GAPDH. Only supernatants from cells transfected with AAV vector constructs containing a secretion domain showed stimulation of either gene above that of the control (gfp, 293t) and above that of vector constructs lacking a secretion domain (#1104 for CD3xGD2-HDD and #1323 for CD19xCD3).

[0514] 293T cells produced the highest transduction efficiency for AAV8.

[0515] Four different cell lines were infected with two different concentrations of AAV8 expressing green fluorescent protein (GFP) at a multiplicity of infection (MOI) of 1e4 or 1e5 genome copies (gc) in medium containing 2% fetal bovine serum. GFP signal was assessed by fluorescence microscopy 48 hours after viral infection. Figure 25 AML-12: normal mouse liver cells; 293T: human embryonic kidney cells transformed by SV40-T antigen; H441-CRM: human lung cancer cells with KRAS mutation; SK-N-Be(2): human neuroblastoma cells with NMYC amplification.

[0516] The concentration of Dimert in the supernatant of AAV8-infected cells depends on the AAV dose and transduction efficiency.

[0517] T(Jurkat) binding assays were performed on supernatants from 293T and H441 cells transfected with TransJoin vector or AAV-GFP control. Figure 26 , where the gray shading in the panels represents unstained control cells and the dark gray line in each panel represents T cells fully stained with anti-CD3 antibody. Although CD19xCD3 Dimert appears to be much more effective than CD3xGD2-HDD Dimert in competing for binding, this effect is dose-dependent (the curve shifts to the left with increasing MOI) and was not observed in a cell line with poor AAV8 transduction efficiency (H441 cells). MOI, multiplicity of infection.

[0518] A single intravenous injection of CD19xCD3 TransJoin selectively depletes B cells in humanized mice.

[0519] Immunodeficient mice (NSG-SGM3, Jackson Labs) were purchased that had been irradiated and intravenously injected with human CD34+ hematopoietic stem cells. By 12 weeks, the mice had engrafted human blood cells, including T and B lymphocytes, as demonstrated by flow cytometry of peripheral blood. A single injection of CD19xCD3 TransJoin (AAV8-Sec1A-CAG-193, vector #1325 packaged into an AAV8 capsid) was administered to the mice at various doses, and blood collected at various time points for human lymphocytes was analyzed by flow cytometry. Figure 27 While low doses (5e9 and 5e10 vector genomes (vg) per kilogram (kg) of body weight) had no effect, higher doses (5e11 and 5e12 vg / kg) were sufficient to deplete circulating B cells without affecting CD4+ or CD8+ T cells.

[0520] A single intravenous injection of CD19xCD3 TransJoin results in prolonged B cell depletion in humanized mice.

[0521] Humanized mice were treated with a single dose of CD19xCD3 AAV8 TransJoin, and lymphocyte subsets were subsequently measured in the blood. Figure 28 As shown for individual mice, prolonged and selective B cell depletion was observed in all analyzed mice as long as these mice were alive.

[0522] A single intravenous injection of CD19xCD3 TransJoin eliminates CD19+ lymphomas in humanized mice.

[0523] Human CD19+ Raji cells (from a patient with Burkitt's lymphoma) were implanted into the flanks of two humanized mice and allowed to grow to more than 250 mm. 3 The size of the tumor was observed in the control AAV virus AAVGFP injected into the tail vein of one animal, and the CD19xCD3 AAV8 TransJoin injected into the tail vein of the second animal. The tumors in the control mice eventually grew rapidly, requiring the animals to be euthanized. The tumors in the TransJoin-treated mice grew slowly and eventually shrank almost completely. Figure 29 The animals needed to be sacrificed due to symptoms of graft-versus-host disease, a known outcome of humanized mice, but the experiment demonstrated proof of principle that a single TransJoin injection could treat cancer.

[0524] Example 3 - Use of OncoSkip and TransSkip to Simultaneously Target Oncogene Expression and Activate Therapeutic Transgene Expression

[0525] This article describes an overview of OncoSkip and TransSkip.

[0526] OncoSkip is an antisense morpholino that can induce exon skipping in oncogenes. In some embodiments, OncoSkip is designed as an antisense morpholino that induces exon skipping in oncogenes. Figure 30 For proof of principle, KRAS (exons 1, 2, and 3, represented by Figure 30 Black-gold-grey on the left) and tested a morpholino designed to skip KRAS exon 2 ( Figure 30 The exon is skipped, which is shown as the light grey bar on top of the oncogene in the figure) and contains an ATG start site, so the normal expression of the oncogene is reduced. A derivative of the same exon to be skipped is then created, but mutated to contain multiple stop codons in each reading frame as well as flanking intronic sequences. The new intron-exon (STOP)-intron is then inserted into the transgene coding sequence at a site that recreates the donor and acceptor splice sites, allowing it to be spliced ​​into the transgene mRNA and interrupt the normal coding sequence. In the presence of an antisense morpholino (the light grey bar on top of the oncogene), the newly inserted exon is skipped and the coding sequence is reconnected to generate a functional product. The class of AAV vectors containing genes interrupted by intron-exon-intron sequences is called TransSkip viruses, while morpholinos designed to downregulate oncogenes are called OncoSkip.

[0527] KRAS OncoSkip antisense morpholino induces exon skipping of endogenous KRAS in lung cancer cells.

[0528] Lung cancer cell lines A549 and H441 (not shown) were incubated with KTS1 and KTS2 OncoSkip antisense morpholinos, as well as a reverse control morpholino for KTS2. Both KTS1 and KTS2 bind to the 3'-prime end of the KRAS exon 2 exon-intron junction and are designed to induce skipping of exon 2 because it contains an ATG start site. Endogenous KRAS mRNA was then analyzed for the presence of exon 2 by reverse transcriptase RT-PCR using primers present in exon 1 (forward) and exon 2 (reverse). Primers in exon 4 were used as a control for total KRAS mRNA. A dose-dependent reduction in transcripts containing exon 2 ("exon 1+2") was observed with both OncoSkip morpholinos. See Figure 31 .

[0529] AAV vector map of the CD3xGD2-HDD TransSkip vector shows reverse-engineered introns flanking the exons inserted into the CD3xGD2-HDD Dimert coding sequence.

[0530] A 3-priming sequence of a human intron (Ki1) upstream of KRAS exon 2 (a derivative of KRAS exon 2 containing mutations to multiple stop codons (STOP) in all three reading frames) and a 5-priming sequence of a human intron (Ki) downstream of KRAS exon 2 were synthesized and cloned into the gene sequence encoding GD2 Dimert, located within specific sequences of the duplicated splice donor and acceptor sites. Figure 32 When the new STOP exon is spliced ​​into the transcript, no functional Dimert is produced. When cells are exposed to an antisense morpholino that binds to the 3-initiation exon-intron junction, the STOP exon is skipped and full-length Dimert mRNA is expressed. Selecting the KRAS sequence such that an antisense morpholino designed to cause exon 2 skipping simultaneously alters mRNA splicing of the TransSkip transgene, resulting in expression of full-length Dimert in transduced cells and reducing or eliminating native KRAS expression in cancer cells because exon 2 contains the KRAS ATG start codon.

[0531] Exemplary strategies for testing the activity of OncoSkip and TransSkip.

[0532] like Figure 33 As shown, cell pellets and supernatants were collected from 293T cells transduced with the AAV Dimert vector. 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 was spliced ​​into the mRNA. Supernatants were analyzed for Dimert expression by T cell binding and killing assays.

[0533] Antisense morpholinos induce exon skipping of the CD3xGD2-HDD TransSkip transgene.

[0534] 293T cells were transfected with CD3xGD2-HDD Dimert plasmid #1042. Cells were harvested 48 hours after transfection for total RNA isolation. Approximately 1 μg of RNA was used for RT-PCR. Figure 34, wherein lane #9 shows the full-length transgenic RNA without splicing between primers, using DNA plasmid as template. Lanes 7 and 8 and the control lane do not have antisense morpholinos and show that most transcripts include internal exons (striped rectangles). Some transcripts do not include exons (minimal bands), which indicates that the construct has some "leakage" of activated transcripts. Adding any morpholino (KTS1, KTS2) reduces the proportion of inactivated, exon-containing transcripts (219bp) in a dose-dependent manner and increases activated transcripts (97bp) that do not include exons.

[0535] KRAS OncoSkip induces secretory expression of CD3xGD2-HDD Dimert in cells transfected with the CD3xGD2-HDD TransSkip AAV vector.

[0536] Supernatants were collected from transfected 293T cells and tested for T cell binding (interference with fluorescently labeled anti-CD3 antibodies). Figure 35 As shown, the gray line in the top panel is an unstained T (Jurkat) cell, and the dark gray line in the top panel is a T cell fully stained with anti-CD3 antibody. As a positive control, supernatants from cells transfected with the constitutively expressed CD3xGD2-HDD TransJoin #1011 almost completely abolished anti-CD3 staining. Supernatants from cells transfected with CD3xGD2-HDD TransSkip #1042 showed moderate competition, consistent with some "leakage" of exon-skipped Dimert mRNA, which was further induced by OncoSkip morpholinos (KTS1, KTS2).

[0537] KRAS OncoSkip's exon skipping of CD3xGD2-HDD TransSkip is an on-target effect.

[0538] Exon skipping induced by the targeted antisense morpholino KTS2 was compared with that of a morpholino containing the same bases but in reverse sequence ( Figure 36 KTS2 induced exon skipping to almost 100% of the transcript (97 bp), whereas the reverse control morpholino had no effect compared to the Endoporter only (carrier for the morpholino) control.

[0539] Induction of CD3xGD2-HDD Dimert expression as determined by T cell engagement is an on-target effect.

[0540] Supernatants from 293T cells transfected with CD3xGD2-HDD TransJoin (positive control) and CD3xGD2-HDD TransSkip, incubated without or with different antisense morpholinos, were collected and assayed for human T (Jurkat) cell binding by flow cytometry (competition of fluorescently labeled anti-CD3 antibody binding). Figure 37 , where the gray line in the top panel is unstained T cells and the dark gray line in the top panel is fully stained T cells. The line indicating #1101CD3xGD2-HDDTransJoin is almost completely competitive with the signal from the constitutively expressed GD3 TransJoin. As before, supernatants from cells transfected with CD3xGD2-HDD TransSkip showed slight competition at baseline (#1042CD3xGD2-HDD TransSkip, no morpholino), which was not altered by the control morpholino (CD3xGD2-HDD TransSkip + "KTS2-Reverse Control"), but was induced to compete with the on-target "OncoSkip" morpholino KTS2 (CD3xGD2-HDD TransSkip + KTS2).

[0541] AAV genome maps of exemplary TransSkip splice variants were created and tested to reduce baseline TransSkip "leakiness" but maintain inducible exon skipping.

[0542] TransSkip splice variants were designed to reduce baseline exon skipping observed with the CD3xGD2-HDD TransSkip. A woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) downstream of the coding sequence was used to increase transgene expression. Due to AAV packaging size limitations, a shorter promoter was required to contain the WPRE sequence, so a new series of vectors were created whose promoters were derived from a shortened form of the human eukaryotic translation elongation factor 1α1 promoter (EFSp). These variants were created by altering specific base pairs in the U2 auxiliary factor (U2AF) binding site located in the polypyrimidine track at the 3-prime end of the first intron (Ki1-5). See Figure 38 .

[0543] The CD3xGD2-HDD TransSkip splice variant K3 abolished baseline but remained inducible exon skipping.

[0544] 293T cells were transfected with five different new CD3xGD2-HDD TransSkip constructs without and with the KTS2 antisense "OncoSkip" morpholino and compared to an "OncoSkip-only" control with the morpholino vector but without the morpholino. mRNA transcripts were analyzed by reverse transcriptase RT-PCR and revealed distinct phenotypes. Figure 39 As shown, variant K4 exhibited similar (or even slightly more) baseline exon skipping levels as K1 and could induce skipping even more than K1. Variants K2, K3, and K5 showed virtually no baseline skipping. Of these three, variant K3 was the most induced to skip exons.

[0545] The CD3xGD2-HDD TransSkip variant K3 showed no baseline Dimert production but was most readily induced by KRASOncoSkip.

[0546] Using a T (Jurkat) cell binding assay (far right), a panel of CD3xGD2-HDD TransJoin splice variants was tested for secreted CD3xGD2-HDD Metarho expression without or with the KRAS KTS2 OncoSkip antisense morpholino. Figure 40 As shown, the gray line in the right panel is an unstained T cell, the dark gray is a fully stained T cell, the blue is the constitutively expressed CD3xGD2-HDD TransJoin, and the green is the corresponding CD3xGD2-HDD TransSkip without OncoSkip ("endotransporter only"). The sequence shows the polypyrimidine U2AF binding site, where the base pair variants are 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 in which the exon is skipped. Variants K1 and K4 show some detectable baseline exon skipping and labeled baseline Dimert expression (green, far right) by RT-PCR, while variants K2, K3 and K5 show no baseline exon skipping by RT-PCR and no Dimert expression by binding competition (green, far right). All three variants showed a small amount of inducible exon skipping by RT-PCR expression in the presence of the KRAS antisense morpholino OncoSkip KTS2, and varying degrees of dose-dependent Dimer expression in the presence of KRAS OncoSkip (light purple and dark purple represent lower and higher concentrations, respectively). The K3 variant appeared to be the most inducible, so K3 was selected as the lead construct for further study.

[0547] OncoSkip-mediated induction of Dimert expression by CD3xGD2-HDD TransSkip variants K3 and K5 is an on-target effect.

[0548] RT-PCR and T(Jurkat) binding assays were repeated using CD3xGD2-HDD TransSkip variants K2, K3, and K5, and a "reverse KTS2" antisense morpholino was included as a control. Figure 41 , reverse KTS2 contains the same nucleotide bases as KTS2, but in reverse order. Reverse KTS2 failed to induce exon skipping in K3 and K5 (lower bands on the gel), while the correct KTS2 sequence induced exon skipping in K3 and K5 (not induced in either K2 variant). In the T cell binding assay of Dimert expression (rightmost panel), gray is unstained T cells, dark gray is fully stained T cells, blue is constitutively expressed CD3xGD2-HDD TransJoin, purple is KRAS OncoSkip KTS2 (light colors are lower doses, dark colors are higher doses), and yellow is the reverse KTS2 control. Dimert protein detection was consistent with RT-PCR; none of the morpholinos induced Dimert expression from the K2 variant, and only KTS2 OncoSkip, not the reverse control, induced Dimert expression in the K3 and K5 variants.

[0549] OncoSkip-induced secretion of Dimert from the AAV CD3xGD2-HDD TransSkip vector plays a role in mediating T cell killing of neuroblastoma cells.

[0550] The cytotoxicity of human T cells plus supernatants from 293T cells transfected with various AAV CD3xGD2-HDD TransSkip variants against GDS2+SK-N-Be(2) neuroblastoma cells was tested in the absence or presence of the KRAS OncoSkip KTS2. Figure 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 supernatant from untransfected cells. CD3xGD2-HDD TransSkip variants K1 and K4 showed cytotoxicity in the absence of KRAS OncoSkip KTS2, consistent with baseline exon skipping, and cytotoxicity could be further induced using K4. CD3xGD2-HDD TransSkip variant K2 did not exhibit baseline or inducible cytotoxicity, consistent with its known efficient splicing and exon inclusion. In contrast, CD3xGD2-HDD TransSkip variants K3 and K5 did not exhibit baseline, but did exhibit inducible cytotoxicity, with the K3 variant being dose-dependent.

[0551] Transgene exon skipping in cells infected with the AAV CD3xGD2-HDD TransSkip variant K3 can be induced by KRAS OncoSkip targeting.

[0552] Exon skipping was examined in the context of AAV viral infection to determine if it reflected what has been seen in other studies when transfected with AAV plasmids. CD3xGD2-HDD TransSkip 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 in the absence ("endotransporter only") or presence of the antisense morpholino KRAS OncoSkip KTS2 or a reverse control ("InvtKTS2 control"). Figure 43 As shown and across vector plasmids, the AAV GD2 K1 variant exhibited exon skipping at baseline, which was further induced by the KRAS OncoSkip KTS2, but not by the control. In contrast, no baseline exon skipping was observed with the K3 variant, but induction was observed with the OncoSkip KTS2 (alone or in complex with the vector "Vivo-KTS2"). In contrast, it was not induced by the control morpholino.

[0553] AAV genome map of the CD19xCD3 TransSkip splice variant.

[0554] Based on the results of the CD3xGD2-HDD TransSkip study, in which variant K3 showed lower baseline, but still inducible Dimert expression compared to K1, K1 and K3 intronic variants were created, inserting the CD19xCD3 Dimert transgene. As with the GD2 series, a WPRE downstream of the coding sequence was used to increase transgene expression, necessitating the use of a shorter promoter, EFSp. Ki, derived from a partial KRAS intronic sequence. STOP, derived from exon 2 of KRAS, was mutated to include a stop codon in all three reading frames. Figure 44A and Figure 44B , is an illustrative example of a CD19xCD3 TransSkip construct.

[0555] Figure 44C Cartoon illustrating the interaction of CD19 dimerT with cancer cells and T cells. CD19 dimerT is produced by cells containing CD19 TransSkip. When administered to a subject (e.g., intravenously), an AAV TransSkip (e.g., the AAV CD19 TransSkip shown in this figure) enters normal cells, such as liver or muscle, but does not express the polypeptide due to the insertion of introns and exons containing a stop codon into the normal coding sequence of the transgene. In the presence of antisense polynucleotides that induce exon skipping (e.g., morpholino antisense oligonucleotides), the mRNA transcript processed by the antisense oligonucleotide contains 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 the secretion process, leaving an active dimerT that binds cancer cells (CA) at one end and T immune cells at the other end.

[0556] Both oncoSkip morpholinos KTS1 and KTS2 induce on-target exon skipping of the CD19xCD3 transSkip K1.

[0557] like Figure 45 As shown, 293T cells were transfected with AAV vectors, including CD19xCD3 TransJoin (positive control, #1325) and CD19xCD3 TransSkip K1 (#1098), without co-incubation (lane #4) or with a control morpholino (lane #3) or two different KRAS OncoSkip morpholinos (lanes 1 and 2). CD19xCD3 TransSkip K1 showed some baseline exon skipping (lower "activation" bands in lanes 3 and 4), consistent with our findings with the parallel construct CD3xGD2-HDDTransSkip. These results demonstrate that the engineered TransSkip design works similarly with a variety of different transgenes.

[0558] KRAS OncoSkip induces secretory expression of CD19xCD3 Dimert in cells transfected with the CD19xCD3 TransSkip K1 AAV vector.

[0559] Supernatants were collected from transfected 293T cells and tested for T cell binding (interference with fluorescently labeled anti-CD3 antibodies). Figure 46 As shown, the gray line in the top panel is an unstained T (Jurkat) cell, and the dark gray line in the top panel is a T cell fully stained with anti-CD3 antibody. As a positive control, supernatant from cells transfected with the constitutively expressed CD19xCD3 TransJoin #1325 competed for anti-CD3 staining (#1325). Supernatants from cells transfected with CD19xCD3 TransSkip #1098 in the presence of a morpholino control showed some baseline competition (KTS2 reverse control), consistent with "leakage" of exon-skipped Dimert mRNA, and were further induced by OncoSkip morpholinos (KTS1; KTS2) to the level achieved using the constitutive CD19xCD3 TransJoin.

[0560] The CD19xCD3 TransSkip splice variant K3 abolished baseline but remained inducible for exon skipping.

[0561] like Figure 47 As shown, 293T cells were transfected with K1 and K3 CD19xCD3 TransSkip constructs in the absence and presence of KTS2 antisense "OncoSkip" morpholino or control morpholino ("InvtKTS2 control") and compared to an "endotransporter only" control with morpholino vector but no morpholino. 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 skipping ("endotransporter only") or skipping by the morpholino control. Skipping was observed with the K3 (#1168) variant using KTS2 OncoSkip, which was confirmed by analysis of protein expression by T cell binding assay.

[0562] Induction of CD19xCD3 Dimer expression determined by T cell engagement of CD19xCD3 TransSkip K3 is an on-target effect.

[0563] Supernatants were collected from 293T cells transfected with CD3xGD2-HDD TransJoin (positive control) and CD19xCD3 TransSkip without or with different antisense morpholinos and assayed for human T (Jurkat) cell binding by flow cytometry (competition with fluorescently labeled anti-CD3 antibody binding). Figure 48 As shown, in each top panel, gray represents unstained T cells, dark gray represents fully stained T cells, pink represents fully stained T cells in the presence of supernatant from untransfected 293T cells, and blue represents the signal competing with supernatant from cells transfected with the constitutively expressed CD19xCD3 TransJoin #1073. Supernatant from CD19xCD3 TransSkip K1 #11166 exhibited high expression comparable to the positive control CD19xCD3 TransJoin both at baseline and with the control morpholino, indicating that this construct is not suitable for controlling gene expression. In contrast to the experience with CD3xGD2-HDD TransSkip, the CD19xCD3 TransSkip variant K3 #1168 exhibited no baseline expression (green) or expression induced by the control morpholino (light and dark orange, "IntCtl"), but had dose-dependent expression with the KRAS OncoSkip morpholino KTS2 (light and dark purple).

[0564] Induction of CD19xCD3 Dimer expression from CD19xCD3 TransSkip K3 is reproducible.

[0565] The exon skipping assay and T cell binding assay were repeated to confirm the absence of leakiness and inducibility of the K3 CD19xCD3 TransSkip relative to the K1 CD19xCD3 TransSkip ( Figure 49 ). See also Figure 48 .

[0566] Implementation Plan

[0567] Embodiment 1: A vector for gene therapy, comprising: a first polynucleotide sequence encoding a first antibody or an antigen-binding fragment thereof; and a second polynucleotide sequence encoding a second antibody or an antigen-binding fragment thereof.

[0568] Embodiment 2: The vector according to embodiment 1, wherein the vector is a recombinant vector.

[0569] Embodiment 3: The vector according to embodiment 1 or 2, wherein the vector is a viral vector.

[0570] Embodiment 4: The vector according to any one of Embodiments 1-3, wherein the viral vector is a retroviral vector.

[0571] Embodiment 5: The vector of any one of Embodiments 1-4, wherein the viral vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a lentiviral vector, a murine leukemia virus ("MLV") vector, an Epstein-Barr virus ("EBV") vector, or a herpes virus ("HSV") vector.

[0572] 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.

[0573] Embodiment 7: The vector of embodiment 6, wherein the AAV vector is AAV rh74 (GenBank Accession No. LP899424.1).

[0574] 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 activating antigen on an immune effector cell, and the second antibody or antigen-binding fragment thereof binds to a tumor antigen.

[0575] 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 activation antigen on an immune effector cell.

[0576] Embodiment 10: The vector according to any one of Embodiments 1-9, further comprising a third polynucleotide sequence encoding a third antibody or an antigen-binding fragment thereof, wherein the third antibody or antigen-binding fragment thereof binds to an activation antigen or a tumor antigen on an immune effector cell.

[0577] Embodiment 11: The vector of any one of Embodiments 8-10, wherein the immune effector cells comprise dendritic cells, natural killer ("NK") cells, macrophages, T cells, or B cells.

[0578] Embodiment 12: The vector according to any one of Embodiments 8-11, wherein the immune effector cell is a T cell or a NK cell.

[0579] Embodiment 13: The vector of any one of Embodiments 8-12, wherein the activating antigens on the immune effector cells comprise CD3, CD2, CD4, CD8, CD19, LFA1, CD45, NKG2D, NKp44, NKp46, NKp30, DNAM, B7-H3, CD20, CD22, or a combination thereof.

[0580] Embodiment 14: The vector according to any one of embodiments 8-13, wherein the tumor antigen comprises one or more of the following: ephrin type A receptor 2 (EphA2), interleukin (IL)-13rα2, 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 (GM2), growth factor receptor, stromal antigen, vascular antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR1), mesothelin, CD38, CD123, human epidermal growth factor receptor 2 (HER2), B cell maturation antigen (BCMA), fibroblast activation protein (FAP) alpha, or a combination thereof.

[0581] Embodiment 15: The vector according to any one of Embodiments 1-14, wherein the first antibody or antigen-binding fragment thereof forms a dimer with the second antibody or antigen-binding fragment thereof.

[0582] Embodiment 16: The vector of embodiment 15, wherein the dimert is a bispecific antibody.

[0583] Embodiment 17: The vector of embodiment 15, wherein the dimert is a trispecific antibody.

[0584] Embodiment 18: The vector of embodiment 15 or 16, wherein the bispecific antibody comprises a polypeptide sequence that is at least 95% identical to any one of SEQ ID NO: 13 or 15.

[0585] Embodiment 19: The vector of embodiment 15 or 17, wherein the trispecific antibody comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:11.

[0586] Embodiment 20: The vector according to any one of Embodiments 1-19, wherein the vector further comprises a polynucleotide sequence encoding a secretory peptide.

[0587] Embodiment 21: The vector of Embodiment 20, wherein the secretory peptide comprises a secretory consensus sequence.

[0588] 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.

[0589] Embodiment 23: The vector of embodiment 20 or 21, wherein the secretion consensus sequence consists of SEQ ID NO: 51.

[0590] Embodiment 24: The vector of any one of Embodiments 20-23, wherein the secretion consensus sequence is encoded by a polynucleotide comprising SEQ ID NO: 52 or an equivalent thereof.

[0591] Embodiment 25: The vector of 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.

[0592] 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.

[0593] 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.

[0594] 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.

[0595] 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.

[0596] 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.

[0597] 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.

[0598] Embodiment 32: The vector of embodiment 31, wherein the 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.

[0599] 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.

[0600] Embodiment 34: The vector of any one of Embodiments 1-33, wherein the secretion consensus sequence regulates the expression and / or secretion of dimert.

[0601] Embodiment 35: The vector of any one of Embodiments 1-34, wherein the secretion consensus sequence enhances expression and / or secretion of dimert.

[0602] Embodiment 36: The vector of any one of Embodiments 1-35, wherein the vector further comprises a polynucleotide sequence encoding a dimerization domain.

[0603] Embodiment 37: The vector of Embodiment 36, wherein the dimerization domain comprises the dimerization domain of human hepatocyte nuclear factor 1α (HNF1α).

[0604] Embodiment 38: The vector of embodiment 37, wherein the dimerization domain of HNF1α comprises a polypeptide sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:47.

[0605] Embodiment 39: The vector of any one of Embodiments 1-38, wherein the vector further comprises a promoter.

[0606] Embodiment 40: The vector of Embodiment 39, wherein the promoter is a constitutive promoter.

[0607] Embodiment 41: The vector of embodiment 39 or 40, wherein the promoter is a tissue-specific promoter.

[0608] Embodiment 42: The vector of any one of embodiments 39-41, wherein the 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 EF1α short form (EFS) promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin C (UbiC) promoter, α-1-antitrypsin, a spleen focus forming virus (SFFV) promoter, or a chicken β-actin (CBA) promoter.

[0609] Embodiment 43: The vector of any one of Embodiments 39-42, wherein the promoter is EFS, optionally comprising SEQ ID NO: 49 or an equivalent thereof.

[0610] Embodiment 44: The vector of any one of Embodiments 1-43, wherein the vector further comprises an enhancer.

[0611] Embodiment 45: The vector of Embodiment 44, wherein the enhancer is an RSV enhancer, a CMV enhancer, and an alpha-fetoprotein MERII enhancer.

[0612] Embodiment 46: The vector of any one of Embodiments 1-45, wherein the vector further comprises one or more additional regulatory elements.

[0613] Embodiment 47: The vector of any one of Embodiments 1-46, wherein the 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.

[0614] 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.

[0615] Embodiment 49: The vector of any one of Embodiments 1-48, wherein the vector comprises the sequence set forth in SEQ ID NO: 4, 6, 8, 12, 14, 16-23, 30-33, or 40-46.

[0616] Embodiment 50: A composition comprising the vector of any one of embodiments 1-49 and a carrier, wherein the carrier is optionally a pharmaceutically acceptable carrier.

[0617] Embodiment 51: The composition of Embodiment 50, wherein the composition is formulated for systemic administration.

[0618] Embodiment 52: The composition of embodiment 50, wherein the composition is formulated for topical administration.

[0619] Embodiment 53: The composition of any one of Embodiments 50-52, wherein the composition is formulated for parenteral administration.

[0620] 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.

[0621] Embodiment 55: The method of Embodiment 54, further comprising administering an anti-cancer agent to the subject.

[0622] 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.

[0623] Embodiment 57: The method of embodiment 56, wherein the viral particle is an oncolytic HSV particle.

[0624] Embodiment 58: The method of any one of Embodiments 54-57, wherein the subject is a mammal.

[0625] Embodiment 59: The method of any one of Embodiments 54-58, wherein the subject is a human.

[0626] Embodiment 60: A method of producing a bispecific antibody or trispecific antibody in a cell, comprising contacting the cell with the vector of any one of embodiments 1-49.

[0627] Embodiment 61: The method of Embodiment 60, wherein the contacting comprises transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof.

[0628] 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 of 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.

[0629] Embodiment 64: The method of any one of Embodiments 60-62, wherein the trispecific antibody comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 11.

[0630] 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.

[0631] Embodiment 66: The method of any one of Embodiments 60-62, wherein the trispecific antibody is encoded by a polynucleotide sequence that is at least 95% identical to SEQ ID NO: 12.

[0632] 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.

[0633] Embodiment 68: The kit of embodiment 67, further comprising instructional materials.

[0634] Equivalent

[0635] It should be understood that although the present disclosure has been described in conjunction with the above embodiments, the foregoing description and examples are intended to illustrate rather than limit the scope of the present disclosure. Other aspects, advantages and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which the present disclosure belongs.

[0636] 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 5' to 3' orientation.

[0637] The embodiments illustratively described herein may suitably be practiced in the absence of any elements, limitations or restrictions not specifically disclosed herein. Thus, for example, the terms "comprising," "containing," "including," and the like are to be interpreted broadly and not restrictively. Furthermore, the terms and expressions used herein have been used as terms of description and not of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalents of the features shown and described or portions thereof, but it is to be understood that various modifications may be made within the scope of the present disclosure.

[0638] Therefore, it should be understood that although the present disclosure has been specifically disclosed through specific embodiments and optional features, those skilled in the art may resort to modifications, improvements and variations of the embodiments disclosed herein, and 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 specific embodiments and are exemplary and are not intended to limit the scope of the present disclosure.

[0639] The scope of the present disclosure has been described broadly and generically herein. Each narrower species and subgeneric grouping falling within the generic disclosure also forms part of this disclosure. This includes the generic description with a proviso or negative limitation removing any subject matter from the genus, whether or not the excised material is specifically cited herein.

[0640] In addition, 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 are also thereby described in terms of any individual member or subgroup of members of the Markush group. Sequence Listing <110> Children's National Hospital Research Institute <120> Cancer-targeted, virally encoded, regulatable T cell (CATVERT) or NK cell (CATVERN) adaptors <130> 106887-7610 <140> PCT / US2020 / 018806 <141> 2020-02-19 <150> 62 / 808,264 <151> 2019-02-20 <160> 71 <170> PatentIn version 3.5 <210> 1 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (4)..(4) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (8)..(8) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (12)..(12) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (16)..(16) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (20)..(20) <223> a, c, t, g, unknown or other <220> <221> modified_base <222> (24)..(24) <223> a, c, t, g, unknown or other <400> 1 taantagntg antagntaan tgan 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 2 taattagttg attagttaat tgat 24 <210> 3 <211> 162 <212> PRT <213> Unknown <220> <223> Description of Unknown: IL-21 sequence <400> 3 Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met 1 5 10 15 Val Ile Phe Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln 20 25 30 Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln 35 40 45 Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro 50 55 60 Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln 65 70 75 80 Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile 85 90 95 Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala 100 105 110 Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr 115 120 125 Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu 130 135 140 Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu 145 150 155 160 Asp Ser <210> 4 <211> 486 <212> DNA <213> Unknown <220> <223> Description of Unknown: IL-21 sequence <400> 4 atgagaagca gccccggcaa catggagaga atcgtgatct gcctgatggt gatcttcctg 60 ggcaccctgg tgcacaagag cagcagccag ggccaggaca gacacatgat cagaatgaga 120 cagctgatcg acatcgtgga ccagctgaag aactacgtga acgacctggt gcccgagttc 180 ctgcccgccc ccgaggacgt ggagaccaac tgcgagtgga gcgccttcag ctgcttccag 240 aaggcccagc tgaagagcgc caacaccggc aaacgaga gaatcatcaa cgtgagcatc 300 aagaagctga agagaaagcc ccccagcacc aacgccggca gaagacagaa gcacagactg 360 acctgcccca gctgcgacag ctacgagaag aagcccccca aggagttcct ggagagattc 420 aagagcctgc tgcagaagat gatccaccag cacctgagca gcagaaccca cggcagcgag 480 gacagc 486 <210> 5 <211> 274 <212> PRT <213> Unknown <220> <223> Description of Unknown: MICA sequence <400> 5 Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp Gly 1 5 10 15 Ser Val Gln Ser Gly Phe Leu Ala Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Tyr Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Glu Glu Trp Thr 115 120 125 Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met 145 150 155 160 His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Glu Ser Ser Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu 180 185 190 Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe Tyr 195 200 205 Pro Arg Asn Ile Ile Leu Thr Trp Arg Gln Asp Gly Val Ser Leu Ser 210 215 220 His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr 225 230 235 240 Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Arg Gly Glu Glu Gln Arg 245 250 255 Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val 260 265 270 Pro Ser <210> 6 <211> 822 <212> DNA <213> Unknown <220> <223> Description of Unknown: MICA sequence <400> 6 gagccccaca gtcttcgtta taacctcacg gtgctgtcct gggatggatc tgtgcagtca 60 gggttcttg ctgaggtaca tctggatggt cagcccttcc tgcgctatga caggcagaaa 120 tgcagggcaa agccccaggg acagtgggca gaagatgtcc tgggaaataa gacatgggac 180 agagagacca gggacttgac agggaacgga aaggacctca ggatgaccct ggctcatatc 240 aaggaccaga aagaaggctt gcattccctc caggagatta gggtctgtga gatccatgaa 300 gacaacagca ccaggagctc ccagcatttc tactacgatg gggagctctt cctctcccaa 360 aacctggaga ctgaggaatg gacagtgccc cagtcctcca gagctcagac cttggccatg 420 aacgtcagga atttcttgaa ggaagatgcc atgaagacca agacacacta tcacgctatg 480 catgcagact gcctgcagga actacggcga tatctagaat ccagcgtagt cctgaggaga 540 acagtgcccc ccatggtgaa tgtcacccgc agcgaggcct cagagggca catcaccgtg 600 acatgcaggg cttccagctt ctatccccgg atatcatac tgacctggcg tcaggatggg 660 gtatctttga gccacgacac ccagcagtgg ggggatgtcc tgctgatgg gatggaacc 720 taccagacct gggtggccac caggattgc cgaggagagg agcagaggtt cacctgctac 780 atggacaca gcgggaatca cagcactcac cctgtgccct ct 822 <210> 7 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 7 Glu Pro is found in the Arg Tyr Asn and in the Thr Val and in the Asp Gly 1 5 10 15 Ser Val Gln Ser Gly Phe Leu Ala Glu Val His Leu Asp Gly Gln Pro 20 25 30 Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly Gln 35 40 45 Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr Arg 50 55 60 Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His Ile 65 70 75 80 Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val Cys 85 90 95 Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr Tyr 100 105 110 Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Glu Glu Trp Thr 115 120 125 Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Ile Arg Asn 130 135 140 Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala Met 145 150 155 160 His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly Val 165 170 175 Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser Glu 180 185 190 Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe Tyr 195 200 205 Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu Ser 210 215 220 His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly Thr 225 230 235 240 Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln Arg 245 250 255 Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro Val 260 265 270 Pro Ser <210> 8 <211> 822 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 8 gagccccaca gtcttcgtta taacctcacg gtgctgtcct gggatggatc tgtgcagtca 60 gggtttctcg ctgaggtaca tctggatggt cagcccttcc tgcgctgtga caggcagaaa 120 tgcagggcaa agccccaggg acagtgggca gaagatgtcc tgggaaataa gacatgggac 180 agagagacca gggacttgac agggaacgga aaggacctca ggatgaccct ggctcatatc 240 aaggaccaga aagaaggctt gcattccctc caggagatta gggtctgtga gatccatgaa 300 gacaacagca ccaggagctc ccagcatttc tactacgatg gggagctctt cctctcccaa 360 aacctggaga ctgaggaatg gacaatgccc cagtcctcca gagctcagac cttggccatg 420 aacatcagga atttcttgaa ggaagatgcc atgaagacca agacacacta tcacgctatg 480 catgcagact gcctgcagga actacggcga tatctaaaat ccggcgtagt cctgaggaga 540 acagtgcccc ccatggtgaa tgtcacccgc agcgaggcct cagagggcaa cattaccgtg 600 acatgcaggg cttctggctt ctatccctgg aatatcacac tgagctggcg tcaggatggg 660 gtatctttga gccacgacac ccagcagtgg ggggatgtcc tgcctgatgg gaatggaacc 720 taccagacct gggtggccac caggatttgc caaggagagg agcagaggtt cacctgctac 780 atggaacaca gcgggaatca cagcactcac cctgtgccct ct 822 <210> 9 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 9 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 10 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 10 ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagc 45 <210> 11 <211> 732 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 11 Met Trp Trp Arg Leu Trp Trp Leu Leu Leu Leu Leu Leu Leu Leu Trp 1 5 10 15 Pro Met Val Trp Ala Ala Arg Ser Ser Pro Gly Asn Met Glu Arg Ile 20 25 30 Val Ile Cys Leu Met Val Ile Phe Leu Gly Thr Leu Val His Lys Ser 35 40 45 Ser Ser Gln Gly Gln Asp Arg His Met Ile Arg Met Arg Gln Leu Ile 50 55 60 Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu 65 70 75 80 Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala 85 90 95 Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn 100 105 110 Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro 115 120 125 Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro 130 135 140 Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg 145 150 155 160 Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His Leu Ser Ser Arg 165 170 175 Thr His Gly Ser Glu Asp Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 180 185 190 Ser Gly Gly Gly Gly Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr 195 200 205 Val Leu Ser Trp Asp Gly Ser Val Gln Ser Gly Phe Leu Ala Glu Val 210 215 220 His Leu Asp Gly Gln Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg 225 230 235 240 Ala Lys Pro Gln Gly Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr 245 250 255 Trp Asp Arg Glu Thr Arg Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg 260 265 270 Met Thr Leu Ala His Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu 275 280 285 Gln Glu Ile Arg Val Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser 290 295 300 Ser Gln His Phe Tyr Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu 305 310 315 320 Glu Thr Glu Glu Trp Thr Met Pro Gln Ser Ser Arg Ala Gln Thr Leu 325 330 335 Ala Met Asn Ile Arg Asn Phe Leu Lys Glu Asp Ala Met Lys Thr Lys 340 345 350 Thr His Tyr His Ala Met His Ala Asp Cys Leu Gln Glu Leu Arg Arg 355 360 365 Tyr Leu Lys Ser Gly Val Val Leu Arg Arg Thr Val Pro Pro Met Val 370 375 380 Asn Val Thr Arg Ser Glu Ala Ser Glu Gly Asn Ile Thr Val Thr Cys 385 390 395 400 Arg Ala Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln 405 410 415 Asp Gly Val Ser Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu 420 425 430 Pro Asp Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys 435 440 445 Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Asn 450 455 460 His Ser Thr His Pro Val Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly 465 470 475 480 Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Pro 485 490 495 Glu Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser 500 505 510 Gly Tyr Lys Phe Thr Glu Tyr Thr Met His Trp Val Lys Gln Ser His 515 520 525 Gly Lys Cys Leu Glu Trp Ile Gly Gly Ile Asn Pro Asn Asn Gly Gly 530 535 540 Thr Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp 545 550 555 560 Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu 565 570 575 Asp Ser Ala Val Tyr Tyr Cys Ala Arg Asp Thr Thr Val Pro Tyr Ala 580 585 590 Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly 595 600 605 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr 610 615 620 Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met 625 630 635 640 Thr Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met His Trp Tyr Gln Gln 645 650 655 Lys Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu 660 665 670 Ala Ser Ser Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser 675 680 685 Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr 690,695,700 Tyr Cys His Gln Arg Ser Ser Tyr Pro Leu Thr Phe Gly Cys Gly Thr 705,710,715,720 Lys Leu Glu Ile Lys Arg Ala Ser Thr Lys Gly Pro 725 730 <210> 12 <211> 2199 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polynucleotide <400> 12 atgtggtgga gactgtggtg gctgctgctg ctgctgctgc tgctgtggcc catggtgtgg 60 gccgccagaa gcagccccgg caacatggag agaatcgtga tctgcctgat ggtgatctttc 120 ctgggcaccc tggtgcacaa gagcagcagc cagggccagg acagacacat gatcagaatg 180 agacagctga tcgacatcgt ggaccagctg aagaactacg tgaacgacct ggtgcccgag 240 ttcctgcccg cccccgagga cgtggagacc aactgcgagt ggagcgcctt cagctgcttc 300 cagaaggccc agctgaagag cgccaacacc ggcaacaacg agagaatcat caacgtgagc 360 atcaagaagc tgagagaaa gccccccagc accaacgccg gcagagaca gaagcacaga 420 ctgacctgcc ccagctgcga cagctacgag aagaagcccc ccaggagtt cctggagaga 480 ttcaagagcc tgctgcagaa gatgatccac cagcacctga gcagcagaac ccacggcagc 540 gaggacagcg gcggcggcgg gcggcggcg gcggcggcgg cagcgagcccc 600 cacagtcttc gttataacct cacggtgctg tcctgggatg gatctgtgca gtcaggttt 660 ctcgctgagg tacatctgga tggtcagccc ttcctgcgct gtgacaggca gaaatgcagg 720 gcaaagcccc agggacagtg gtcctgggaa atagacatg ggacagagag 780 accagggact tgacaggga cggaaggac ctcaggatga ccctggctca tatchcaggac 840 cagaaagaag gcttgcattc cctccaggag attaggtct gtgagatcca tgaagacaac 900 agcaccagga gctcccagca tttctactac gatggggagc tctcctc ccaaaacctg 960 gagactgagg aatggacaat gcccagtcc tccagagctc agaccttggc catgaacatc 1020 aggaatttct tgaaggaga tgccatgaag accagacac actacacgc tatgcatgca 1080 gactgcctgc aggaactcg gcgatatcta aaatccggcg tagtcctgag gagaacagtg ccccccatgg tgaatgtcac ccgcagcgag gcctcagagg gcaacattac cgtgacatgc agggcttctg gcttctatcc ctggaatatc acactgagct ggcgtcagga tggggtatct 1260 ttgagccacg acacccagca gtggggggat gtcctgcctg atgggaatgg aacctaccag 1320 acctgggtgg ccaccaggat ttgccaagga gaggagcaga ggttcacctg ctacatggaa cacagcggga atcacagcac tcaccctgtg ccctctggcg gcggcggcag cggcggcggc ggcagcggcg gcggcggcag ccaggtgcag ctgcagcag gcggccccga gctggtgaag cccggcgcca gcgtgaagat cagctgcaag accagcggct acaagttcac cgagtacacc atgcactggg tgaagcagag ccacggcaag tgcctggagt ggatcggcgg catcaacccc 1620 aacaacggcg gcaccaacta caaccagaag ttcaagggca aggccaccct gaccgtggac aagagcagca gcaccgccta catggagctg aagagcctga ccagcgagga cagcgccgtg tactactgcg ccagagacac caccgtgccc tacgcctact ggggccaggg caccaccgtg accgtgagca gcggcggcgg cggcagcggc ggcggcggca gcggcggcgg cggcagcgac 1860 atcgagctga cccagagccc cgccatcatg agcgccagcc ccggcgagaa ggtgaccatg 1920 acctgcagcg ccagcagcag catcagctac atgcactggt accagcagaa gcccggcacc 1980 agccccaaga gatggatcta cgacaccagc aagctggcca gcagcgtgcc cgccagattc 2040 agcggcagcg gcagcggcac cagctacagc ctgaccatca gcagcatgga ggccgaggac 2100 gccgccacct actactgcca ccagagaagc agctaccccc tgaccttcgg ctgcggcacc 2160 aagctggaga tcaagagagc cagcaccaag ggcccctag 2199 <210> 13 <211> 572 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 13 But Too Too Arg Slow Too Too Slow Slow Slow Slow Slow Slow Slow Too 1 5 10 15 Pro Met Val Trp Ala Ala Gln Val Gln Leu Gln Gln Ser Gly Pro Glu 20 25 30 Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Thr Ser Gly 35 40 45 Tyr Lys Phe Thr Glu Tyr Thr Met His Trp Val Lys Gln Ser His Gly 50 55 60 Lys Cys Leu Glu Trp Ile Gly Gly Ile Asn Pro Asn Asn Gly Gly Thr 65 70 75 80 Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys 85 90 95 Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser Glu Asp 100 105 110 Ser Ala Val Tyr Tyr Cys Ala Arg Asp Thr Thr Val Pro Tyr Ala Tyr 115 120 125 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln 145 150 155 160 Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr 165 170 175 Cys Ser Ala Ser Ser Ser Ile Ser Tyr Met His Trp Tyr Gln Gln Lys 180 185 190 Pro Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala 195 200 205 Ser Ser Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr 210 215 220 Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr 225 230 235 240 Cys His Gln Arg Ser Ser Tyr Pro Leu Thr Phe Gly Cys Gly Thr Lys 245 250 255 Leu Glu Ile Lys Arg Ala Ser Thr Lys Gly Pro Gly Gly Gly Gly Ser 260 265 270 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln 275 280 285 Ser Gly Gly Gly Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys 290 295 300 Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Arg 305 310 315 320 Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser 325 330 335 Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile 340 345 350 Ser Arg Asp Asn Ser Lys Asn Thr Ala Phe Leu Gln Met Asp Ser Leu 355 360 365 Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys Ala Arg Tyr Tyr Asp Asp 370 375 380 His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Pro Val Thr Val Ser ...

Claims

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; as well as (d) a gene regulatory polynucleotide sequence located between the first polynucleotide and the second polynucleotide and positioned to create a consensus splice donor / acceptor site, wherein the gene regulatory polynucleotide sequence comprises a splice donor site, an upstream intron, an exon including more than one stop codon sequence in its respective reading frame, a downstream intron, and a splice acceptor site, and wherein the gene regulatory polynucleotide sequence further comprises a binding sequence for an antisense oligonucleotide, and wherein the gene regulatory polynucleotide sequence comprises: GTACGGAGCGGACCACCCCTCCTGGGCCCCTGCCCGGGTCCCGACCCTCTTTGCCGGCGCCGGGCGGGGCCGGCGGAGTATTTGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCCTTCTTTTTTTTTAAGGCCTGCTGAAATAATTAGCTGAGTAGATAAGTGATGTTGGAGCTGGTGGCGTAGGCAAGAG TGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGACCATTTTGAGTTGTATATAACACCTTTTTTGAAGTAAAAGGTGCACTGTAATAATCCAGACTGTGTTTCTCCCTTCTCAG or GTACGGAGCGGACCACCCTCCTGGGCCCCTGCCCGGGTCCCGACCCTCTTTGCCGGCGCCGGGCGGGGCCGGCGGAGTATTTGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCATTATTTTTTTTTTCAGGCCTGCTGAAATAATTAGCTGAGTAGATAAGTGATGTTGGAGCTGGTGGCGTAGGCAAGAG TGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGACCATTTTGAGTTGTATATAACACCTTTTTTGAAGTAAAAGGTGCACTGTAATAATCCAGACTGTGTTTCTCCCTTCTCAG.

2. The polynucleotide or vector of claim 1, wherein the antisense oligonucleotide is a morpholino oligonucleotide.

3. The polynucleotide or vector according to claim 2, wherein the binding sequence for morpholino oligonucleotide comprises the polynucleotide sequence of SEQ ID NO. 24 or 25.

4. The polynucleotide or vector of claim 2, wherein the morpholino oligonucleotide comprises the polynucleotide sequence of SEQ ID NO: 27 or 28.

5. The polynucleotide or vector of claim 1, wherein the stop codon comprises: oligonucleotides in the group of TAA, TAG or TGA; A polynucleotide sequence of TAAxTAGxTGAxTAGxTAAxTGAx, wherein x is any nucleotide; or The polynucleotide sequence of TAATTAGTTGATTAGTTAATTGAT.

6. The polynucleotide or vector according to claim 1, wherein the gene regulatory polynucleotide sequence comprises: GTACGGAGCGGACCACCCCTCCTGGGCCCCTGCCCGGGTCCCGACCCTCTTTGCCGGCGCCGGGCGGGGCCGGCGGAGTATTTGATAGTGTATTAACCTTATGTGTGACATGTTCTAATATAGTCACATTTTCCTTCTTTTTTTTTAAGGCCTGCTGAAATAATTAGCTGAGTAGATAAGTGATGTTGGAGCTGGTGGCGTAGGCAAGAG TGCCTTGACGATACAGCTAATTCAGAATCATTTTGTGGACGAATATGATCCAACAATAGAGGTAAATCTTGTTTTAATATGCATATTACTGGTGCAGGACCATTCTTTGATACAGATAAAGGTTTCTCTGACCATTTTGAGTTGTATATAACACCTTTTTTGAAGTAAAAGGTGCACTGTAATAATCCAGACTGTGTTTCTCCCTTCTCAG.

7. The polynucleotide or vector of claim 1, wherein the first polypeptide is a first antibody or an antigen-binding fragment thereof, and the second polypeptide is a second antibody or an antigen-binding fragment thereof.

8. The polynucleotide or vector of claim 7, wherein the first antibody or antigen-binding fragment thereof specifically binds to an activating antigen on an immune effector cell, and the second antibody or antigen-binding fragment thereof binds to a tumor antigen.

9. The polynucleotide or vector of claim 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 activating antigen on an immune effector cell.

10. The polynucleotide or vector of claim 7, 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 activation antigen or a tumor antigen on an immune effector cell.

11. The polynucleotide or vector of any one of claims 8-10, wherein the immune effector cells comprise dendritic cells, natural killer ("NK") cells, macrophages, T cells, B cells, or a combination thereof.

12. The polynucleotide or vector of any one of claims 8-10, wherein the immune effector cell is a T cell or a NK cell.

13. The polynucleotide or vector of any one of claims 8-10, wherein the activating antigens on the immune effector cells comprise CD3, CD2, CD4, CD8, CD19, LFA1, CD45, NKG2D, NKp44, NKp46, NKp30, DNAM, B7-H3, CD20, CD22, or a combination thereof.

14. The polynucleotide or vector of any one of claims 8-10, wherein the tumor antigen comprises one or more of the following: ephrin type A receptor 2, interleukin (IL)-13rα2, 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 (GM2), growth factor receptor, stromal antigen, vascular antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR1), mesothelin, CD38, CD123, human epidermal growth factor receptor 2 (HER2), B-cell maturation antigen (BCMA), fibroblast activation protein (FAP) alpha, or a combination thereof.

15. The polynucleotide or vector according to any one of claims 7 to 10, wherein the vector is a recombinant viral vector and is capable of expressing a precursor mRNA, which, when contacted with an antisense oligonucleotide, encodes a polypeptide for binding immune executive cells to cancer cells, thereby triggering the killing of cancer cells.

16. The polynucleotide or vector according to claim 15, wherein the polypeptide encoded by the pre-mRNA is a bispecific antibody.

17. The polynucleotide or vector of claim 15, wherein the polypeptide encoded by the pre-mRNA is a trispecific antibody.

18. The polynucleotide or vector of claim 16, wherein the bispecific antibody comprises the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof.

19. The polynucleotide or vector of claim 17, wherein the trispecific antibody comprises the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof.

20. The polynucleotide or vector according to claim 16, wherein the bispecific antibody comprises the polypeptide sequence of SEQ ID NO. 13 or 15.

21. The polynucleotide or vector of claim 10, wherein the vector expresses a pre-mRNA that encodes a trispecific antibody when the pre-mRNA is contacted with an antisense oligonucleotide.

22. The polynucleotide or vector of claim 21, 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.

23. The polynucleotide or vector of claim 21, wherein the trispecific antibody comprises the polypeptide sequence of SEQ ID NO.

11.

24. The polynucleotide or vector of any one of claims 7-10, wherein the first antibody and the second antibody are each independently a single chain variable fragment.

25. The polynucleotide or vector according to any one of claims 1-10, further comprising a polynucleotide sequence encoding a secretory peptide.

26. The polynucleotide or vector of any one of claims 1-10, further comprising a polynucleotide sequence encoding a dimerization domain.

27. The polynucleotide or vector of any one of claims 1-10, further comprising a 5' inverted terminal repeat and a 3' inverted terminal repeat.

28. The polynucleotide or vector of any one of claims 1-10, wherein the vector comprises the sequence set forth in SEQ ID NO: 4, 6, 8, 12, 14, 16-23, 30-33, or 40-46.

29. The polynucleotide or vector of any one of claims 1-10, wherein the vector is a recombinant viral vector comprising a backbone vector selected from a retroviral vector, a lentiviral vector, a murine leukemia virus vector, an Epstein-Barr virus vector, an adenoviral vector, a herpes virus ("HSV") vector, or an adeno-associated virus ("AAV") vector.

30. The polynucleotide or vector of any one of claims 1-10, wherein the vector is an AAV vector.

31. A composition comprising: the polynucleotide or vector of any one of claims 1-30; and a pharmaceutically acceptable carrier.

32. Use of the polynucleotide or vector of any one of claims 8 to 14 in the preparation of a medicament for treating cancer in a subject in need thereof, wherein the polynucleotide or vector expresses a therapeutic anti-cancer antibody or antigen-binding fragment thereof.

33. The use according to claim 32, wherein the medicament is formulated for administration together with an antisense oligonucleotide.

34. The use according to claim 32, wherein the medicament is formulated for administration together with an anticancer agent.

35. The use according to claim 34, wherein the anticancer agent comprises an agent selected from a polypeptide, a nucleic acid molecule, a small molecule, a viral particle or a combination thereof.

36. The use according to claim 35, wherein the viral particle is an oncolytic HSV particle.

37. The use according to any one of claims 32 to 36, wherein the subject is a mammal.

38. The use according to any one of claims 32 to 36, wherein the subject is a human.

39. An in vitro method for producing a bispecific antibody or a trispecific antibody in a cell, comprising contacting a cell comprising the vector of any one of claims 1-30 with an effective amount of an antisense oligonucleotide.

40. The method of claim 39, wherein the antisense oligonucleotide is a morpholino oligonucleotide, and wherein the morpholino oligonucleotide comprises a stereopure polynucleotide.

41. The method of claim 39, wherein the vector is introduced into the cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof.

42. The method of claim 39, wherein the cells comprise fibroblasts, bone cells, epithelial cells, muscle cells, neural cells, endocrine cells, melanocytes, blood cells, or a combination thereof.

43. The method of any one of claims 39-42, wherein the bispecific antibody comprises the polypeptide sequence of SEQ ID NO. 13 or 15.

44. The method of any one of claims 39-42, wherein the trispecific antibody comprises the polypeptide sequence of SEQ ID NO.

11.

45. The method of any one of claims 39-42, wherein the bispecific antibody is encoded by a polynucleotide sequence of SEQ ID NO. 14, 16, 22, 23, 30-33, or 40-46.

46. ​​The method of any one of claims 39-42, wherein the trispecific antibody is encoded by the polynucleotide sequence of SEQ ID NO.

12.

47. A kit comprising the polynucleotide or vector of any one of claims 1-30 or the composition of claim 31.

48. The kit of claim 47, further comprising instructional documentation.

Images