Chimeric protein comprising membrane-bound IL-12 with a protease-cleavable linker

Abstract

The present disclosure provides chimeric proteins designed to more specifically target cell-based immunotherapy to tumors and the tumor microenvironment. The use of membrane-bound effector molecules reduces off-target side effects and toxicity of immunotherapy. The chimeric proteins of the present disclosure comprise an effector molecule, a transmembrane domain, and a protease-cleavable linker. The present disclosure also provides nucleic acid constructs expressing the chimeric proteins of the present disclosure. Pharmaceutical compositions comprising the chimeric proteins of the present disclosure are also provided.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy, created on October 19, 2023, is named 67000-1381_WO_SL.xml and is 35,788 bytes in size.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 383,943, filed November 16, 2022, which is incorporated herein in its entirety.

[0003] The present invention is in the field of targeted cell-based therapy for treating disease.The present invention relates to the preparation and use of membrane-cleavable chimeric protein-based therapy. [Background technology]

[0004] Conventional therapies for cancer treatment, such as chemotherapy and radiation therapy, have limited efficacy and are often associated with systemic toxicity and off-target adverse events. Cell-based therapies are designed to localize treatment to the tumor environment and avoid off-target adverse effects. One such cell-based therapy is chimeric antigen receptor (CAR) T-cell therapy. In CAR T-cell therapy, T cells are engineered to express synthetic receptors that redirect effector function to the tumor or tumor microenvironment (TME). Summary of the Invention

[0005] The present invention provides chimeric proteins designed to target cell-based immunotherapies more specifically to tumors and the TME. The use of membrane-bound effector molecules reduces off-target side effects and toxicity of immunotherapies. The chimeric proteins of the present invention are suitable for use in armored CAR T-cell therapy.

[0006] In certain aspects, the present invention provides a membrane-cleavable chimeric protein having, in N-terminal to C-terminal orientation, the formula: EL-TM or TM-LE During the ceremony, a) E comprises an effector molecule; b) L is a first peptide linker comprising a protease cleavage site and an amino acid sequence selected from the group consisting of GGGGSISSGLLSGRSDNHGGGGS (SEQ ID NO:3), GGGGSVPLSLYSGGGISSGLLSGRSDNHGGGGS (SEQ ID NO:4), GGGGSHPVGLLARGGGHPVGLLARGGSGRSAGGSGRSAGGGGS (SEQ ID NO:5), GGGGSHPVGLLARGGGGS (SEQ ID NO:6), and GGGGSLAQAVRSSGGGGS (SEQ ID NO:7); c) TM comprises a transmembrane protein; d) The EL-TM or TM-LE is constructed to be expressed as a single polypeptide.

[0007] In certain embodiments, effector molecule E comprises a cytokine or a functional fragment thereof. The cytokine is selected from the group consisting of IL-1-beta, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17A, IL-18, IL-21, IL-22, type I interferon, interferon-gamma, and tumor necrosis factor-alpha (TNF-alpha). In some embodiments, the cytokine is IL-12. In some embodiments, effector molecule E comprises the p40 and p35 subunits of IL-12 covalently linked by a second peptide linker. IL-12 molecules containing both the p40 and p35 subunits are often referred to as IL-12p70 or IL-12p70 fusion proteins.

[0008] In some embodiments, the transmembrane domain is derived from the group consisting of PDGFR-beta, CD8, CD28, CD3 zeta chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG03, 2B4, LNGFR, NKG2D, TNFR2, B7-1, and BTLA. In some embodiments, the transmembrane is linked to the cytoplasmic domain. In certain embodiments, the cytoplasmic domain is derived from PDGFR-beta, CD8, CD28, CD3 zeta chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, or BTLA.

[0009] In some embodiments, the chimeric protein comprises a signal peptide at the N-terminus.

[0010] In addition to the disclosed peptide sequences, the present disclosure also includes nucleic acids (e.g., mRNA, DNA, siRNA) that express all or a portion of the peptide sequences. In certain aspects, the present disclosure provides nucleic acid molecules that encode the chimeric proteins of the present disclosure. In some embodiments, the nucleic acid molecule comprises an expression cassette, the expression cassette comprising a promoter and an exogenous polynucleotide sequence that encodes the chimeric protein of the present disclosure.

[0011] In certain embodiments, the present disclosure provides an expression vector comprising a nucleic acid molecule encoding a chimeric protein of the present disclosure. In some embodiments, the expression vector is a viral vector.

[0012] In some aspects, the present disclosure provides a cell comprising a chimeric protein of the present disclosure, a nucleic acid encoding a chimeric protein of the present disclosure, or an expression vector comprising a chimeric protein of the present disclosure. In some embodiments, the cell is selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma-delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, bone marrow cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In some embodiments, the cell further comprises a protease capable of cleaving the protease cleavage site L. Advantageously, the protease is an endogenous protease.

[0013] In another aspect, the present disclosure provides a pharmaceutical composition comprising a chimeric protein of the present disclosure, a nucleic acid encoding a chimeric protein of the present disclosure, an expression vector comprising a chimeric protein of the present disclosure, or an isolated cell of the present disclosure, and a pharmaceutically acceptable excipient. In a preferred embodiment, the pharmaceutical composition is a lipid nanoparticle (LNP) pharmaceutical composition.

[0014] The present disclosure also provides a method of treating a disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition of the present disclosure. In certain embodiments, the disease is cancer. [Brief explanation of the drawings]

[0015] [Figure 1]

[0023] Figure 1 is a schematic diagram showing the protease-cleavable linker coding sequences tested. IL12p40 linked to IL12p35 forms bioactive IL-12p70 protein fused to various non-cleavable (NC, L1) or cleavable (L2-L5) substrates fused to the transmembrane domain of B7.1. In the presence of proteases, binding to their respective substrate sites and enzymatic cleavage releases IL-12p70 from the transmembrane linkage. [Figure 2] Figure 1 shows cell surface staining of CHO cells transfected overnight with 500 ng of mRNA constructs encoding F-luciferase, soluble IL-12p70, transmembrane-anchored IL-12p70 (TM_IL12, L1), or various transmembrane-anchored IL-12p70 constructs with protease-cleavable domains (L2, L3, L4, L4-short, L5). Cells were then treated with PBS or activated mouse MMP2 (1 μg / mL) for 180 minutes, at which point they were harvested for flow cytometric staining of surface IL12p40. [Figure 3] The concentration of soluble IL-12p70 released from CHO cells transfected overnight with 500 ng of mRNA constructs encoding soluble IL-12p70, transmembrane-anchored IL-12p70 (TM_IL12, L1), or various transmembrane-anchored IL-12p70 constructs with protease-cleavable domains (L2, L3, L4, L4-short, L5). Cells were then treated with activated mouse MMP2 (1 μg / mL) for 180 minutes, and supernatants were collected for detection of cleaved / soluble IL-12p70 by ELISA. [Figure 4] Figure 1 shows tumor growth over time, measured as tumor volume, in animals bearing B16F10 tumors. Animals were randomized into treatment groups when tumors reached a size of 100 mm, and were treated intratumorally with 10 μg of one of the mRNAs encoding soluble (dashed line), membrane-bound (dotted line), or protease-cleavable (solid line) membrane-bound IL-12p70 on days 0, 3, 6, and 9. Mean tumor growth over time was quantified. [Figure 5]Figure 5 is a bar graph showing the mean tumor volume in B16F10 tumor-bearing animals randomized at 100 mm and treated with 10 μg of each mRNA encoding soluble, membrane-bound, or protease-cleavable membrane-bound IL-12p70 on days 0, 3, 6, and 9. Figure 5 is a representative bar graph of the mean tumor size 11 days after treatment, before euthanasia of control F-luciferase-treated animals. [Figure 6] Kaplan-Meier survival curves are shown for B16F10 tumor-bearing animals treated intratumorally with 10 μg of mRNA encoding soluble (dashed line), membrane-bound (dotted line), or protease-cleavable membrane-bound (solid line) IL-12p70 on days 0, 3, 6, and 9. Animals were euthanized when tumors reached a size of >2000 mm. [Figure 7] Circulating IL-12p70 levels from B16F10 tumor-bearing animals (N=5 / group) bled 24 hours after treatment with 10 μg of LNP-mRNA encoding soluble, transmembrane-bound, or protease-cleaved IL-12p70 were measured. Serum was subjected to ELISA to quantify circulating IL-12p70 levels. [Figure 8] The mean tumor growth over time is shown as tumor volume in MC38 tumor-bearing animals randomized at 85 mm and treated with 10 μg of mRNA encoding soluble (dashed line), membrane-bound (dotted line), or protease-cleavable membrane-bound (solid) IL-12p70 on days 0, 3, 6, and 9. Mean tumor growth over time was quantified. [Figure 9] 1 shows the mean tumor volume in MC38 tumor-bearing animals randomized at 85 mm and treated with 10 μg of each mRNA encoding soluble, membrane-bound, or protease-cleavable membrane-bound IL-12p70 on days 0, 3, 6, and 9. A representative bar graph of the mean tumor size 13 days after treatment, before euthanasia of control F-luciferase-treated animals. [Figure 10]Kaplan-Meier survival curves are shown for MC38 tumor-bearing animals treated intratumorally with 10 μg of mRNA encoding soluble (dashed line), membrane-bound (dotted line), or protease-cleavable membrane-bound (solid line) IL-12p70 on days 0, 3, 6, and 9. Animals were euthanized when tumors reached a size of >2000 mm3. [Figure 11] Circulating IL-12p70 levels were measured in MC38 tumor-bearing animals (N=5 / group) that were bled 24 hours after treatment with 10 μg of LNP-mRNA encoding soluble, membrane-bound, or protease-cleaved membrane-bound IL-12p70. Serum was subjected to serum ELISA to quantify circulating IL-12p70 levels. DETAILED DESCRIPTION OF THE INVENTION

[0016] Cell-based therapies are of interest for the treatment of various diseases due to their potential to reduce off-target adverse effects. One such cell-based therapy is chimeric antigen receptor (CAR) T-cell therapy. T cells are engineered to express synthetic receptors that redirect effector function to the tumor or tumor microenvironment (TME). While this approach has been effective in treating hematological malignancies, its treatment of solid tumors has been less successful. The immunosuppressive solid tumor TME is a significant barrier, reducing the anticancer activity of endogenous tumor-resident immune cells, which allows tumor growth. Recently, efforts have been made to enhance CAR T-cell function in the TME by engineering cells to express other proteins alongside the CAR. Examples of this engineering include inducing CAR T cells to secrete cytokines, expressing cytokine receptors to regulate cytokine activity within the TME, or generating CAR T cells that secrete antibody-like proteins that target tumor antigens. These methods are known as "armored CAR T-cell therapy."

[0017] Interleukins have been widely studied in cancer treatment. However, interleukins can have both cancer-promoting and cancer-inhibiting effects. In addition, administration of interleukins is often associated with adverse effects, including lethality. For example, CAR T cells engineered to express interleukins have been shown to induce "cytokine release syndrome," resulting in febrile neutropenia, hypotension, acute vascular leak syndrome, and acute respiratory distress syndrome. Therefore, treatment with interleukins has proven difficult. Efforts are underway to specifically target interleukins to cancer cells.

[0018] The present disclosure provides chimeric proteins designed to target cell-based immunotherapies more specifically to tumors and the TME. The use of membrane-bound effector molecules reduces off-target side effects and toxicity of immunotherapies.

[0019] The present disclosure also provides nucleic acids that express the chimeric proteins of the present disclosure.

[0020] In some embodiments, the present disclosure provides an expression vector comprising either a chimeric protein of the present disclosure or a nucleic acid that expresses a chimeric protein of the present disclosure. Expression vectors include viral vectors.

[0021] Any host-vector system known to those skilled in the art can be used to express the protein-coding sequence. For example, a mammalian cell system infected with a viral vector containing the nucleic acid sequence can be used. Any method known to those skilled in the art can be used to insert a DNA fragment into a vector and construct an expression vector containing a chimeric gene containing appropriate transcriptional / translational control signals and the protein-coding sequence.

[0022] The chimeric protein, nucleic acid, or expression vector may be provided as a pharmaceutical composition. In a preferred embodiment, the pharmaceutical composition is provided in lipid nanoparticles.

[0023] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed.

[0024] Headings are for organizational purposes only and are not intended to limit the disclosure in any way.

[0025] Unless otherwise defined, 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. Unless otherwise stated, all patents, patent applications, published applications and publications, websites, and other published materials referenced throughout this disclosure are incorporated by reference in their entirety for all purposes. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, preferred methods are described.

[0026] Definition of Terms In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms "a," "an," and "the" are intended to include the plural as well, unless the context clearly indicates otherwise.

[0027] The term "and / or" refers to and includes all possible combinations of one or more of the associated listed items, and the lack of combinations when interpreted in the alternative ("or").

[0028] The term "or" refers to any one member of a particular list and also includes any combination of members of that list.

[0029] Unless the context clearly indicates otherwise, the singular articles "a," "an," and "the" include plural references. For example, the term "protein" or "at least one protein" can include a plurality of proteins, including mixtures thereof.

[0030] Unless otherwise specified, statistically significant means p<0.05.

[0031] As used herein, "treatment" refers to any delivery, administration, or application of a therapeutic agent to a disease or condition. Treatment can include curing a disease, inhibiting a disease, delaying or halting the onset of a disease, ameliorating one or more symptoms of a disease, or preventing the recurrence of one or more symptoms of a disease.

[0032] In this application, the use of "or" means "and / or" unless expressly stated otherwise. Also, if clear from the context in which it is used, "and" may be interpreted as "or," like a list of alternatives, all of which may not all be true at once or may not exist.

[0033] As used herein, the terms "comprises" and / or "comprising" specify the presence of specified features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof that are not specifically listed. For example, a composition that "comprises" or "includes" a protein may include the protein alone or in combination with other components. Furthermore, to the extent the terms "includes," "having," "has," "with," "composed," "comprised," or variations thereof, are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0034] The transitional phrase "consisting essentially of" means that the scope of the claim is to be construed to include the particular elements recited in the claim and any elements that do not materially affect the basic and novel characteristics of the claimed disclosure. Thus, the term "consisting essentially of" when used in the claims of this disclosure is not intended to be construed as the equivalent of "comprising."

[0035] When the terms "consist of," "consists of," or "consisting of" are used in the body of a claim, the claim term offset by "consist of," "consists of," and / or "consisting of" is limited to the elements recited immediately following "consist of," "consists of," and / or "consisting of," and not to the unrecited elements associated with that particular claim term. The term "combinations thereof," when included in the list of recited elements following "consist of," "consists of," and / or "consisting of," means combinations of only two or more of the recited elements.

[0036] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes examples when the event or circumstance occurs and examples when the event or circumstance does not occur.

[0037] As used herein, ranges and amounts may be expressed as "about" a particular value or range. "About" is intended to include the exact amount. Thus, "about 5 percent" also means "about 5 percent" and "5 percent." "About" means within typical experimental error for the intended use or purpose. Unless otherwise clear from the context, the term "about" encompasses values ​​within the standard error of measurement (e.g., SEM) of the indicated value.

[0038] When a range of numerical values ​​is recited, it is understood that it includes the endpoints, whether or not specifically recited, all values ​​within that range, and all narrower ranges within that range.

[0039] As used herein, an "effector molecule" is a molecule (e.g., a ligand) that binds to another molecule, e.g., a receptor, and modulates the biological activity of the molecule to which it binds. Effector molecules can, for example, modulate enzymatic activity, gene expression, or cell signaling. Effector molecules can be peptides, proteins (polypeptides), or nucleic acids such as DNA or RNA. Effector molecules that modulate immune responses are of particular interest to the present disclosure.

[0040] As used herein, "transmembrane protein" refers to a protein that traverses a distance through the cell wall lipid bilayer. A transmembrane protein has three regions. One region is outside the cell and is referred to as the "extracellular domain." Another region is within the lipid bilayer of the cell wall and is referred to as the "transmembrane domain," and for purposes of this disclosure, is commonly referred to as the "TM." The third region is inside the cell and is referred to as the "cytoplasmic domain," and for purposes of this disclosure, is commonly referred to as the "CD." When a TM is linked to a CD in a chimeric protein of the present disclosure, the construct is referred to as a "TMCD."

[0041] As used herein, "TME" refers to the tumor microenvironment.

[0042] The term "isolated," with respect to proteins, nucleic acids, and cells, includes proteins, nucleic acids, and cells that are relatively purified with respect to other cellular or biological components that may normally be present in situ, up to substantially pure preparations of proteins, nucleic acids, or cells. The term "isolated" also includes proteins and nucleic acids that have no naturally occurring counterpart, or proteins or nucleic acids that are chemically synthesized and thus are substantially uncontaminated by other proteins or nucleic acids. The term "isolated" also includes proteins, nucleic acids, or cells that have been separated or purified from most other cellular or biological components with which they are naturally associated (e.g., other cellular proteins, nucleic acids, or cellular or extracellular components).

[0043] The term "variant" refers to a nucleotide sequence that differs (e.g., by one nucleotide) from the most common sequence in a population, or a protein sequence that differs (e.g., by one amino acid) from the most common sequence in a population.

[0044] The term "fragment", when referring to a protein, means a protein that is shorter or has fewer amino acids than the full-length protein. The term "fragment", when referring to a nucleic acid, means a nucleic acid that is shorter or has fewer amino acids than the full-length nucleic acid. A fragment, for example, when referring to a protein fragment, can be an N-terminal fragment (i.e., removal of a portion of the C-terminus of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminus of the protein), or an internal fragment (i.e., removal of a portion of each of the N-terminus and C-terminus of the protein). A fragment, for example, when referring to a nucleic acid fragment, can be a 5' fragment (i.e., removal of a portion of the 3' terminus of the nucleic acid), a 3' fragment (i.e., removal of a portion of the 5' terminus of the nucleic acid), or an internal fragment (i.e., removal of a portion of each of the 5' terminus and 3' terminus of the nucleic acid).

[0045] "Sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to the residues of the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentages of sequence identity are used with respect to proteins, non-identical residue positions often differ by conservative amino acid substitutions, in which an amino acid residue is replaced with another amino acid residue that has similar chemical properties (e.g., charge or hydrophobicity) and therefore does not alter the functional properties of the molecule. When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of zero, conservative substitutions are given a score between zero and 1. Scoring of conservative substitutions is calculated, for example, as performed in the program PC / GENE (Intelligenetics, Mountain View, Calif.).

[0046] "Percentage of sequence identity" includes a value determined by comparing two optimally aligned sequences (maximum number of perfectly matched residues) over a comparison window, where the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) when compared to a reference sequence (not including additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes concatenated non-homologous sequences), the comparison window is the full length of the shorter of the two sequences being compared.

[0047] Unless otherwise specified, sequence identity / similarity values ​​include values ​​obtained using GAP version 10 using the following parameters: % identity and % similarity for nucleotide sequences using a GAP weight of 50 and a length weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using a GAP weight of 8 and a length weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. "Equivalent program" includes any sequence comparison program that produces alignments having identical nucleotide or amino acid residue matches and identical percent sequence identity for any two sequences of interest when compared to corresponding alignments produced by GAP version 10.

[0048] The term "conservative amino acid substitution" refers to the substitution of an amino acid normally present in a sequence with an amino acid of a different size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, or leucine for another non-polar residue. Similarly, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Furthermore, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another, are additional examples of conservative substitutions. Examples of non-conservative substitutions include substitutions of a non-polar (hydrophobic) amino acid residue, e.g., isoleucine, valine, leucine, alanine, or methionine, with a polar (hydrophilic) residue, e.g., cysteine, glutamine, glutamic acid, or lysine, and / or a polar residue with a non-polar residue.

[0049] A "homologous" sequence (e.g., a nucleic acid sequence) includes a sequence that is identical to or substantially similar to a known reference sequence, e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence. Homologous sequences can include, for example, orthologous and paralogous sequences. For example, homologous genes typically originate from a common ancestral DNA sequence through either speciation events (orthologous genes) or gene duplication events (paralogous genes). "Orthologous" genes include genes from different species that evolved from a common ancestral gene through speciation. Orthologs typically retain the same function during evolution. "Paralogous" genes include genes that are related by duplication within a genome. Paralogs can evolve new functions during evolution.

[0050] The term "in vitro" includes an artificial environment and processes or reactions that occur within an artificial environment (e.g., a test tube or an isolated cell or cell line). The term "in vivo" refers to the natural environment (e.g., a cell or organism or body) and processes or reactions that occur within a natural environment. The term "ex vivo" includes cells removed from an individual's body and processes or reactions that occur within such cells.

[0051] The term "reporter gene" refers to a nucleic acid having a sequence encoding a gene product (typically an enzyme) that is easily and quantitatively assayed when a construct containing the reporter gene sequence operably linked to an endogenous or heterologous promoter and / or enhancer element is introduced into a cell that contains (or can be made to contain) the factors necessary for activation of the promoter and / or enhancer element. Examples of reporter genes include, but are not limited to, the gene encoding beta-galactosidase (lacZ), the bacterial chloramphenicol acetyltransferase (cat) gene, the firefly luciferase gene, the gene encoding beta-glucuronidase (GUS), and genes encoding fluorescent proteins. "Reporter protein" refers to the protein encoded by the reporter gene.

[0052] As used herein, the term "fluorescent reporter protein" means a reporter protein that is detectable based on fluorescence, which can be either from the reporter protein directly, from the activity of the reporter protein on a fluorogenic substrate, or from a protein that has an affinity for binding to a fluorescently tagged compound. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, emerald, thistle green, monomeric thistle green, CopGFP, AceGFP, and ZsGreen1), yellow fluorescent proteins (e.g., YFP, eYFP, citrine, Venus, YPet, PhiYFP, and ZsYellow1), blue fluorescent proteins (e.g., BFP, eBFP, eBFP2, azurite, mKalamal, GFPuv, sapphire, and T-sapphire), cyan fluorescent proteins (e.g., CFP, eCFP, cerulean, CyPet, AmCyanl, and Acropora-Cyan), red fluorescent proteins (e.g., RFP, mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed), and the like. Examples of suitable fluorescent proteins include GFP (e.g., HcRed-monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRaspberry, mStrawberry, and Jred), orange fluorescent proteins (e.g., mOrange, mKO, Kusabira-Orange, Kusabira-Orange monomer, mTangerine, and tdTomato), and any other suitable fluorescent proteins whose presence within a cell can be detected by flow cytometry.

[0053] As used herein, "subject" means a living organism. Preferably, the subject is a mammal, such as a human, non-human primate, rodent, or companion animal, such as a dog, cat, cow, or pig.

[0054] Chimeric proteins The chimeric proteins of the present disclosure comprise an effector molecule, a protease-cleavable linker, and a transmembrane protein, preferably a transmembrane domain and / or a cytoplasmic domain of a transmembrane protein. The chimeric proteins of the present disclosure are suitable for treating various types of cancer.

[0055] The protein of the present disclosure is a membrane-cleavable chimeric protein having the following formula, in the N-terminal to C-terminal direction:

[0056] EL-TM or TM-LE E is an effector molecule, which is a moiety that induces a biological effect, such as, for example, inhibiting the growth of cancer cells, and / or inhibiting the metastasis or spread of cancer cells, and / or reducing the number of cancer cells, and / or reducing the volume of an existing tumor. Preferably, the effector molecule is an immunomodulatory molecule, such as a cytokine or portion thereof. Suitable effector molecules of the present disclosure are described in more detail below.

[0057] L is a first peptide linker. In some embodiments, L can comprise a protease-cleavable peptide. Preferably, L is cleavable by a protease expressed in cancer cells. L links the effector molecule E to the transmembrane protein TM. L is described in more detail below.

[0058] A TM is a transmembrane portion of a transmembrane protein that can be expressed in cells used in cell-based therapy. In some embodiments, the TM is linked to the cytoplasmic domain of the transmembrane protein and is referred to as a TMCD.

[0059] In the tumor or TME, a protease expressed on a cell (e.g., a cell expressing a chimeric protein of the present disclosure or a cancer cell) cleaves the linker L, thereby releasing the effector molecule from the cell membrane. Upon release, the effector molecule induces a desired biological effect on the cancer cell.

[0060] As used herein, "first linker" refers to a protease-cleavable linker "L."

[0061] As used herein, "second linker" refers to a peptide linker that covalently joins the alpha and beta subunits of the IL-12 family of cytokines.

[0062] The terms "protein," "polypeptide," and "peptide," used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids and amino acids that are chemically or biochemically modified or derivatized. The terms also include modified polymers, such as polypeptides having modified peptide backbones. The term "domain" refers to any portion of a protein or polypeptide having a specific function or structure.

[0063] Proteins are said to have an "N-terminus" and a "C-terminus". The term "N-terminus" refers to the beginning of a protein or polypeptide, which ends with an amino acid having a free amine group (-NH2). The term "C-terminus" refers to the end of an amino acid chain (protein or polypeptide) which ends with a free carboxyl group (-COOH).

[0064] Effector molecules The effector molecule E of the present disclosure can include a cytokine or a functional portion thereof. Cytokines are a category of small proteins between about 5 and 20 kDa that are involved in cell signaling, and include, among others, chemokines, interferons (INFs), interleukins (ILs), and tumor necrosis factors (TNFs). Chemokines act as chemoattractants to guide cell migration and are classified into four subfamilies: CXC, CC, CX3C, and XC. Exemplary chemokines include those from the CC subfamily, e.g., CCL1, CCL2 (MCP-1), CCL3, CCL4, CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (or CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CC L27, and CCL28; the CXC subfamily, e.g., CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and CXCL17; the XC subfamily, e.g., XCL1 and XCL2; and the CX3C subfamily, e.g., CX3CL1.

[0065] The effector molecule can be a costimulatory domain. In some cases, the costimulatory domain enhances antigen-specific cytotoxicity. In some cases, the costimulatory domain further enhances cytokine production. In some embodiments, the costimulatory domain comprises CD27, CD28, CD70, CD80, CD83, CD86, CD134 (OX-40), CD134L (OK-40L), CD137 (41BB), CD137L (41BBL), or CD224.

[0066] In some embodiments, the effector molecule is an immune checkpoint inhibitor polypeptide that inhibits negative regulators of T cell activation. Immune checkpoint inhibitors bind to immune checkpoint molecules, a group of molecules on the cell surface of CD4 and CD8 T cells. Exemplary immune checkpoint molecules include, but are not limited to, programmed cell death ligand 1 (PDL1, also known as B7-H1, CD274), programmed cell death 1 (PD-1), PD-L2 (B7-DC, CD273), LAG3, TIM3, 2B4, A2aR, B7H1, 87H3, B7H4, BTLA, CD2, CD16, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, inducible T cell costimulatory receptor (ICOS), KIR, LAIR, LIGHT, macrophage receptor with collagen structure (MARCO), OX-40, phosphatidylserine (PS), SLAM, TIGHT, VISTA, and VTCN1. In some embodiments, the immune checkpoint inhibitor inhibits one or more of PDL1, PD-1, CTLA-4, PD-L2, LAG3, TIM3, 2B4, A2aR, B7H1, B7H3, B7H4, BTLA, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD160, CD226, CD276, DR3, GAL9, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS, KIR, LAIR1, LIGHT, MARCO, OX-40, PS, SLAM, TIGHT, VISTA, and VTCN1.

[0067] Suitable cytokines for use as effector molecules in the present disclosure include, but are not limited to, IL-1-beta, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17A, IL-18, IL-21, IL-22, type I interferons (e.g., IFN-alpha, IFN-beta), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha).

[0068] Cytokines are produced by several types of cells, including endothelial cells, fibroblasts, and stromal cells, as well as some immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells. Their actions are mediated by cell surface receptors. Immune cell-mediated cytokine activity is of interest for certain diseases, such as cancer. However, the use of cytokines such as interleukins has been limited by their toxic effects, as discussed above.

[0069] IFN-alpha immunocytokines have been shown to exert antitumor effects mediated by the activation of immune system cells. IFN-alpha has been used to treat hematological malignancies and solid tumors. IFN-alpha has direct proapoptotic / antiproliferative activity on tumor cells. Notably, high doses of IFN-alpha are antiangiogenic, thereby exerting antitumor activity by affecting tumor vasculature and reducing blood flow to tumors. However, in cancer-bearing subjects, systemic treatment with IFN-alpha is associated with toxicity. Attempts have been made to reduce toxicity by fusion to apolipoprotein A-1, which incorporates the cytokine into high-density lipoproteins and improves the pharmacokinetics and antitumor activity of IFN-alpha. Another method that can attenuate toxicity is through the use of AcTakines (active on-target cytokines). AcTakines technology is based on the fusion of a mutant cytokine that exhibits reduced affinity for its receptor to a cell-specific targeting domain. IFN-alpha fused to a single-domain antibody targeting Clec9A, a molecule expressed on dendritic cells (DCs) specialized for cross-priming, also exhibits potent antitumor effects.

[0070] IL-15 is primarily produced by activated myeloid cells as a membrane-bound heterodimer associated with the receptor IL-15R-alpha that is transexpressed by IL-2 / IL-15R-beta-expressing NK cells and T cells. IL-15 is essential for the ontogeny of NK cells and CD8+ T cells and also induces proliferation, cytotoxicity, and release of other cytokines, such as IFN-gamma, from these cells. IL-15 does not stimulate Treg cells.

[0071] IL-10 is released by innate and adaptive immune cells and fine-tunes the activity of proinflammatory cytokines. IL-10 is considered an immunosuppressive cytokine because it can reduce the antigen-presenting activity of DCs and inhibit the cytotoxic and cytokine-releasing functions performed by T and NK lymphocytes. IL-10 activity can also be context-dependent. For example, in chronic infections and cancer, autocrine IL-10 activity on CD8+ T lymphocytes may be important for inhibiting antigen-induced CD8+ T cell apoptosis, thereby prolonging the effector activity of these cytotoxic lymphocytes.

[0072] IL-2 may be an important cytokine in promoting the expansion of natural killer (NK) and T lymphocytes. Improvements in the pharmacokinetic profile of IL-2 can be achieved by covalently linking IL-2 to moieties that increase its half-life in circulation (e.g., the Fc domain of an immunoglobulin or polyethylene glycol (PEG)) or by chimerizing it with an antibody that targets the cytokine to the TME. Improvements in the pharmacodynamic properties of IL-2 can be achieved by using techniques that reduce binding to the high-affinity IL-2 receptor while maintaining binding to the intermediate-affinity IL-2 receptor, increasing the amount of cytokine available to stimulate NK and T cells.

[0073] IL-12 can have significant antitumor activity by inducing antitumor immune responses, however, administration of IL-12 in vivo can also be associated with significant systemic toxicity.

[0074] The IL-12 family of cytokines includes IL-2, IL-23, IL-27, IL-35, and IL-39, as well as heterodimeric cytokines including IL-12. The biologically active heterodimeric form of IL-12 contains two subunits, p35 (alpha subunit) and p40 (beta subunit), covalently linked by a disulfide bond (second linker). The IL-12 heterodimer is a 70 kDa protein and can be referred to as IL-12p70. IL-12 is primarily produced by activated antigen-presenting cells (such as DCs, macrophages, monocytes, and B cells). IL-12 production is a tightly regulated process, primarily at the transcriptional level. In addition to its cytotoxic activity, IL-12 is antiangiogenic, reducing the vasculature of cancer cells. The antiangiogenic activity may be due to the effect of IL-12-activated recruited kinases, which upon dimerization and nuclear translocation ultimately lead to IFN-gamma production. In clinical trials, systemic IL-12 can be toxic. The use of local IL-12 delivery and immunostimulatory monoclonal antibodies can produce synergistic results.

[0075] Protease cleavage site In the present disclosure, the effector molecule E is connected to a transmembrane protein or a portion thereof via a first peptide linker "L". The peptide cleavable linker is cleavable by a protease expressed in vivo in isolated cells or cancer cells. Advantageously, the peptide protease cleavable linker L (first linker) is a sequence that is cleaved by a protease expressed in cancer cells or the TME. For example, the linker L may be cleavable by a matrix metalloprotease such as MMP2.

[0076] In certain embodiments, the linker L comprises one or more protease-binding substrate sequences selected from ISSGLLSGRSDNH (SEQ ID NO: 8), VPLSLYSGGGISSGLLSGRSDNH (SEQ ID NO: 9), HPVGLLAR (SEQ ID NO: 10), SGRSA (SEQ ID NO: 11), and LAQAVRSS (SEQ ID NO: 12).

[0077] In the example described herein, the non-cleavable Gly-Ser peptide linker is G4Sx3™ (SEQ ID NO: 1). Linker L1 is a longer non-cleavable Gly-Ser type linker, G4Sx4 (SEQ ID NO: 2). Gly-Ser type linkers are commonly used as peptide linkers in fusion proteins (see Klein et al. (2014). Design and characterization of structured protein linkers with different flexibilities. Protein Engineering, Design & Selection, vol. 27 no. 10 pp. 325-330). The Gly-Ser linker is also described in linkers L2, L3, L4, L4s, and L5, which are protease-cleavable linkers containing a protease substrate sequence inserted between the Gly-Ser peptide sequences.

[0078] L2, [ka] is a peptide linker cleavable by the proteases urokinase plasminogen activator (uPA), matriptase (also known as MT-SP1), and matrix metalloproteinases (MMPs). The bolded and underlined amino acids in this sequence (and in other sequences) are the protease-cleavable peptide portions of the linker. The sequence LSGRSDNH (SEQ ID NO: 13) is cleavable by uPA and / or matriptase (see, e.g., US2017 / 0204139, WO2022 / 178753, WO2022 / 178751, WO2019 / 173382). The sequence ISSGLLSS (SEQ ID NO: 14) is cleavable by MMPs (see, e.g., WO2019 / 173382, US2017 / 0204139).

[0079] L3, [ka] is a peptide linker containing an MMP9+MMP / uPA / matriptase (matriptase is also known as MT-SP1) cleavable sequence. The sequence VPLSLYSG (SEQ ID NO: 16) is cleavable by MMPs, particularly MMP2 and MMP9 (see, e.g., WO2021 / 016599, WO2021 / 016640, WO2020 / 069398). The sequence LSGRSDNH (SEQ ID NO: 13) is cleavable by uPA and / or matriptase (see, e.g., US2017 / 0204139, WO2022 / 178753, WO2022 / 178751, WO2019 / 173382). The sequence ISSGLLSS (SEQ ID NO: 14) is cleavable by MMPs (see, e.g., WO2019 / 173382, US2017 / 0204139).

[0080] L4, [ka] is a cell surface TNF prodrug cleavable linker, surface MMP2 + uPA. The sequence HPVGLLAR (SEQ ID NO: 10) is a substrate for MMP2 (see, e.g., WO2021 / 016640). L4 has two repeats of HPVGLLAR. The sequence SGRSA (SEQ ID NO: 11) is cleavable by uPA and / or matriptase (see, e.g., WO2022 / 178753, WO2022 / 178751, WO2021 / 149697, WO2021 / 016599, WO2021 / 016640, WO2020 / 069398, US2020 / 0207846).

[0081] L4s has only one repeat of HPVGLLAR (SEQ ID NO: 10) and is cleavable by MMP2.

[0082] L5, [ka] contains an ADAM / TACE / CD156q (i.e., TNF-alpha converting enzyme) cleavage site. The sequence LAQAVRSS (SEQ ID NO: 12) is a substrate for ADAM / TACE / CD156q.

[0083] In a preferred embodiment, the protease cleavage site linker L is selected from the peptide sequences of Table A, where the bolded sequences are protease-binding substrates. [Table 1]

[0084] Proteases Of particular interest are proteases that are preferentially expressed by tumor cells or whose expression is dysregulated in cancer cells and the TME.

[0085] In some embodiments, the isolated cells expressing the chimeric protein of the present disclosure comprise an endogenous or exogenous protease that cleaves the protease-cleavable linker.

[0086] Metalloproteinases, a family of Zn2+-binding protease homologs, are associated with many cancer-related functions. The most important metalloproteinases active in the tumor microenvironment are matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAM), and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS).

[0087] Increased MMP activity has been detected in nearly all types of cancer. MMPs target a wide range of extracellular matrix (ECM) proteins, thus contributing to cancer initiation, progression, invasive growth, and cancer cell spread. The proteolytic action of MMPs on ECM scaffolding proteins alters the composition, structure, and function of the ECM. While MMPs can be soluble (e.g., MMP-1, -2, -7, -8, -9, 10, -11, -13, -26), other MMPs are membrane-bound and anchored to the cell surface by GPI anchors (e.g., MMP-14, -15, -16, -17, -24, and -25).

[0088] ADAMs are transmembrane proteases involved in cell proliferation, adhesion, and migration. They play a key role in the complexities of proteolysis in tumors. ADAMs generally target the extracellular domains of both type II and type III transmembrane proteins, contributing to the cleavage of cell adhesion molecules, the shedding of cell surface receptors, and, interestingly, for the purposes of this disclosure, the maturation of cytokines and chemokines. ADAM-10 and ADAM-17 (also known as tumor necrosis-alpha converting enzyme, TACE) have been shown to be primarily involved in cancer. ADAM-10 processes and activates epidermal growth factor receptor (EGFR) ligands, cleaving E-cadherin (suggesting an effect on signal transduction) and cleaving CD44 adhesion molecules from the cell surface. ADAM-10 expression has been shown to correlate with the invasive growth of some cancers. ADAM-17 contributes to the release of soluble TNF-alpha. As discussed above, TNF-alpha may have anti-angiogenic effects. ADAM-17 can also activate IL-6 / ERK signaling, release EGF receptors, and shed adhesion molecules such as CD44.

[0089] ADAMTS are primarily involved in the degradation of structural ECM proteins. Different ADAMTS may be involved in cancer initiation and progression and can promote tumor development, although some ADAMTS may also have tumor-suppressing effects.

[0090] Other proteases that may play a role in cancer include serine proteases, cysteine ​​proteases, aspartic acid proteases, and threonine proteases. For example, urokinase-type plasminogen activator (uPA) is a trypsin-like protease that catalyzes the activation of urokinase-type plasminogen activator receptor (uPAR) and cleaves several components of the extracellular matrix (ECM). Matriptase is a membrane-anchored serine protease involved in the serine protease growth factor signaling axis and has been investigated as a potential diagnostic marker for cancer.

[0091] N-terminal signal peptide In some embodiments, the chimeric proteins disclosed herein further comprise a signal peptide at the N-terminus of the chimeric protein. The signal peptide directs newly synthesized proteins destined for secretion to membrane localization and the appropriate protein processing pathway. The signal peptide may comprise a native signal peptide, i.e., a signal peptide native to the effector molecule. Alternatively, the signal peptide may comprise a non-native signal peptide. Suitable non-native signal peptides include IL-12, IL-2, optimized IL-2, trypsinogen-2, Gaussia luciferase, CD5, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL-6, IL-8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GRO alpha, CXCL12, IL-21, CD8, NKG2D TNFR2, and GMCSF.

[0092] Transmembrane proteins Transmembrane proteins are integral membrane proteins that traverse the entire cell wall lipid bilayer. They have three regions: an extracellular domain, a transmembrane domain (TM), and an intracellular domain (CD). Structurally, there are several types of transmembrane proteins: (1) single-pass alpha-helical or bitopic membrane proteins that traverse the membrane only once; (2) polytopic transmembrane alpha-helical proteins (e.g., seven-transmembrane proteins such as G protein-coupled receptors) that traverse the membrane more than once, and polytopic beta-sheet proteins. Transmembrane proteins are also classified based on their topology, i.e., the location of their N- and C-termini on different sides of the lipid bilayer, e.g., type I, type II, type III, and type IV single-pass molecules. Type I transmembrane proteins are single-pass transmembrane proteins with an extracellular (intraluminal) N-terminus and a cytoplasmic C-terminus relative to the cell. Type II transmembrane proteins are single-pass transmembrane proteins with an extracellular (intraluminal) C-terminus and a cytoplasmic N-terminus relative to the cell.

[0093] The transmembrane proteins (TM) of the present disclosure are preferably transmembrane domain portions of transmembrane proteins. In certain embodiments, the TM is derived from PDGFR-beta, CD8, CD28, CD3 zeta chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, and BTLA. In some embodiments, the TM is further linked to a cytoplasmic domain to obtain a TMCD construct. The cytoplasmic domain is derived from PDGFR-beta, CD8, CD28, CD3 zeta chain, CD4, 4-1BB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, and BTLA. In certain embodiments, either the TM or CD, or both the TM and CD, are derived from the B7-1 transmembrane protein.

[0094] PDGFR-beta (platelet-derived growth factor receptor-beta) is a type I transmembrane protein. PDGFR-beta is expressed in several tumors, and its expression correlates with tumor growth, invasiveness, drug resistance, and poor clinical outcome.

[0095] CD8 (cluster of differentiation 8) is a dimeric type I transmembrane protein expressed on cytotoxic T cells and natural killer (NK) cells. CD8+ cytotoxic T cells have been shown to be useful in anti-cancer therapy.

[0096] CD28 is a type I transmembrane protein expressed on most naive CD4 and CD8 T cells. CD28 is a costimulatory molecule on T cells. CD28 has been studied in cancer immunotherapy.

[0097] The CD3 zeta chain (also known as CD247) is part of the T cell receptor (TCR) CD3 complex. The CD3 zeta chain is a transmembrane protein in the TCR complex.

[0098] CD4 is a monomeric transmembrane protein found on immune cells, including T helper cells, monocytes, macrophages, and dendritic cells. It is a co-receptor for the TCR.

[0099] 4-1BB is a glycosylated type I membrane protein. 4-1BB is not expressed on naive T cells, but is upregulated after T cell binding to MHC:peptide on antigen-presenting cells. 41-BBL is a binding partner of 4-1BB and is a type II membrane protein of the tumor necrosis factor superfamily.

[0100] OX40 is a type I transmembrane glycoprotein that is expressed on several types of cells, including CD4+ and CD8+ T cells.

[0101] ICOS (also known as CD278) is a type I transmembrane glycoprotein. ICOS belongs to the CD28 family of costimulatory immune receptors and is expressed on antigen-presenting cells.

[0102] CTLA-4 (cytotoxic T lymphocyte-associated protein 4) is a type I transmembrane protein expressed on activated T cells. Blocking CTLA-4 can be a treatment for cancer.

[0103] PD-1 (programmed cell death protein 1; also known as CD279) is a type I transmembrane protein expressed on T cells and B cells. Blockade of PD-1 may be a treatment for cancer.

[0104] LAG-3 (lymphocyte activation gene 3; also known as CD223) is a type I transmembrane protein. It is expressed as a dimer or oligomer on activated CD4+ and CD8+ T cells, NK cells, B cells, regulatory T cells, Tr1 cells, natural killer cells, and plasmacytoid dendritic cells. It is upregulated on exhausted T cells in cancer.

[0105] 2B4 is a type I transmembrane protein expressed on natural killer (NK) cells and may play a role in tumorigenesis.

[0106] LNGFR (low affinity nerve growth factor receptor; also known as p75 neurotrophin receptor (p75NTR)) is a type I transmembrane protein. LNGFR may be a marker for cancer stem cells. The use of LNGFR as a spacer in CAR T cells is also of interest.

[0107] NKG2D (natural killer cell 2D receptor) is expressed on NK cells and T cells. NKG2D is a type II transmembrane glycoprotein. NKG2D binds to ligands expressed on tumor cells.

[0108] EpoR (erythropoietin receptor) is a member of the cytokine type I transmembrane receptor family. Splice variants of EpoR have been detected in tumors.

[0109] TNFR2 (tumor necrosis factor receptor type II) is expressed on several types of tumor cells and immune cells, such as regulatory T cells and myeloid-derived suppressor cells, and is enriched in the TME. TNFR2 is a type I transmembrane protein.

[0110] B7-1 is a type I transmembrane protein. It is a ligand for the CD28 receptor family and is expressed on activated antigen-presenting cells, T lymphocytes, and tumor cells. B7-1 is involved in mediating the dynamic interactions between cancer cells and the host immune system.

[0111] BTLA (B and T lymphocyte attenuator) is an immunomodulatory receptor expressed on B cells, T cells, and all mature lymphoid cells. BTLA is a type I transmembrane protein.

[0112] Isolated cells In certain embodiments, the chimeric proteins of the present disclosure are expressed in isolated cells. Suitable isolated cells include T cells, CD8+ T cells, CD4+ T cells, gamma-delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK)15 cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, bone marrow cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In some aspects, the isolated cells are natural killer (NK)20 cells.

[0113] Isolated cells also include tumor cells. Suitable tumor cells include bladder tumor cells, brain tumor cells, breast tumor cells, cervical tumor cells, colon tumor cells, esophageal tumor cells, glioma cells, kidney tumor cells, liver tumor cells, lung tumor cells, melanoma cells, ovarian tumor cells, pancreatic tumor cells, prostate tumor cells, skin tumor cells, thyroid tumor cells, and uterine tumor cells.

[0114] In some embodiments, when the chimeric protein of the present disclosure is expressed in a cell, the effector molecule E is linked to the cell membrane. The transmembrane domain TM is integral to the cell membrane, and the effector molecule E is attached to TM via the protease cleavage site L.

[0115] In some embodiments, when a chimeric protein of the present disclosure is expressed in a cell that expresses a protease capable of cleaving the protease cleavage site L, the effector molecule E is released from the cell membrane.

[0116] In some embodiments, the protease expressed on the cell membrane is endogenous to the cell. In other embodiments, the cell has been engineered to express a protease that is exogenous to the cell. Preferably, the protease is one expressed by cancer cells.

[0117] Advantageously, the cell comprises a chimeric protein of the present disclosure, a nucleic acid molecule of the present disclosure (described below), or an expression vector of the present disclosure (described below).

[0118] As used herein, "stable expression" of a transfected or transduced gene in a host cell refers to the integration of the gene in the genome of the host cell, such that the transfected genetic material can be expressed.

[0119] nucleic acid The present disclosure also includes recombinant nucleic acids encoding the chimeric proteins of the present disclosure. As used herein, "recombinant" refers to a modification of a nucleic acid or amino acid sequence that results in a product not found in nature. When referred to in reference to a nucleic acid construct, the term refers to a molecule composed of nucleic acid sequences linked together or produced by means of molecular biological techniques. When referred to in reference to a protein or polypeptide, the term "recombinant" refers to a protein or polypeptide molecule expressed using a recombinant nucleic acid construct made by molecular biological techniques. A recombinant nucleic acid construct may contain nucleotide sequences that are not naturally ligated or that are ligated or engineered to be ligated to nucleic acid sequences that are ligated at a different location in nature. Thus, referring to a nucleic acid construct as "recombinant" indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e., by human intervention. A recombinant nucleic acid construct may be introduced into a host cell by any suitable means, for example, as described herein or known in the art. Such a recombinant nucleic acid construct may contain sequences derived from the same host cell species or a different host cell species that have been isolated and reintroduced into cells of the host species. The recombinant nucleic acid construct sequence may be integrated ("stably integrated") into the host cell genome, e.g., the genome of an oncolytic virus, as a result of the original transformation of the host cell or as a result of subsequent recombination and / or repair events.

[0120] A nucleic acid may be an RNA sequence. As used herein, "RNA" refers to a molecule containing one or more ribonucleotide residues. A "ribonucleotide" is a nucleotide having a hydroxyl group at the 2' position of a beta-D-ribofuranose moiety. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA), essentially pure RNA, synthetic RNA, and recombinantly produced RNA. The term "RNA" also refers to modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. In other embodiments, a nucleic acid may be a DNA sequence.

[0121] The terms "nucleic acid" and "polynucleotide," used interchangeably herein, include polymeric forms of nucleotides of any length, containing ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. These include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers that contain purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0122] The present disclosure also provides nucleic acid molecules comprising expression vectors (expression constructs, expression cassettes), wherein the expression cassette comprises a promoter and an exogenous polynucleotide sequence encoding a chimeric protein. In some embodiments, the expression cassette can comprise a polynucleotide encoding a reporter gene, such as mCherry or enhanced green fluorescent protein (EGFP).

[0123] A "promoter" is a regulatory region of DNA that typically contains a TATA box that can direct RNA polymerase II to begin RNA synthesis at the appropriate transcription start site for a particular polynucleotide sequence. A promoter may further contain other regions that affect the rate of transcription initiation. The promoter sequences disclosed herein regulate the transcription of an operably linked polynucleotide. The promoter may be active in one or more cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, one-cell stage embryos, differentiated cells, or combinations thereof). The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO2013 / 176772, the entire contents of which are incorporated herein by reference. "Promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. This sequence may be the core promoter sequence, or it may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.

[0124] As used herein, a "constitutive promoter" is one that is active in all tissues or in a specific tissue at all developmental stages. Examples of constitutive promoters include, but are not limited to, the cytomegalovirus immediate-early promoter (CMV), the simian virus (SV40) promoter, the adenovirus major late (MLP) promoter, the Rous sarcoma virus (RSV) promoter, the elongation factor-alpha (EF1a) promoter, the ubiquitin promoter, the actin promoter, the tubulin promoter, the immunoglobulin promoter, functional fragments thereof, or combinations thereof. Other suitable promoters are known to those skilled in the art.

[0125] As used herein, an "inducible promoter" is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when the promoter's corresponding inducer is present in the cell. Examples of inducible promoters include, for example, chemically regulated promoters and physically regulated promoters. Examples of inducible promoters include, but are not limited to, those that are inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. Chemically regulated promoters include, for example, alcohol-regulated promoters (e.g., the alcohol dehydrogenase (alcA) gene promoter), tetracycline-regulated promoters (e.g., a tetracycline-responsive promoter, a tetracycline operator sequence (tetO), a tet-On promoter, or a tet-Off promoter), steroid-regulated promoters (e.g., a rat glucocorticoid receptor promoter, an estrogen receptor promoter, or an ecdysone receptor promoter), or metal-regulated promoters (e.g., a metalloprotein promoter). Physically regulated promoters include, for example, temperature-regulated promoters (eg, heat shock promoters) and light-regulated promoters (eg, light-inducible or light-repressible promoters).

[0126] In some embodiments, the promoter can be tissue-specific, such as a neuronal, glial, muscle, cardiac, renal, bone, endothelial, or immune cell-specific promoter (e.g., a B cell or T cell promoter).

[0127] As used herein, a "tissue-specific promoter" is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes a gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter. Tissue-specific promoters include, but are not limited to, CAG, SYN1, CMV, NSE, CBA, PDGF, SV40, RSV, LTR, SV40, dihydrofolate reductase promoter, beta-actin promoter, PGK, EF1 alpha, GRK, MT, MMTV, TY, RU486, RHO, RHOK, CBA, chimeric CMV-CBA, MLP, RSV, ubiquitin promoter, actin promoter, tubulin promoter, immunoglobulin promoter, functional fragments thereof, and the like.

[0128] The tissue-specific promoter can be, for example, a neuron-specific promoter, a glial-specific promoter, a muscle cell-specific promoter, a cardiac cell-specific promoter, a kidney cell-specific promoter, a bone cell-specific promoter, an endothelial cell-specific promoter, or an immune cell-specific promoter (e.g., a B cell promoter or a T cell promoter).

[0129] In a preferred embodiment of the present disclosure, the promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-specific promoter, and a synthetic promoter.

[0130] The term "wild-type" includes entities having structure and / or activity as found in a normal state or context (as opposed to mutant, diseased, altered, etc.). Wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0131] The term "endogenous sequence" refers to a nucleic acid sequence that occurs naturally within a cell or eukaryote (eg, an animal, a non-human animal, a mammal, or a non-human mammal).

[0132] An "exogenous" molecule or sequence includes a molecule or sequence that is not normally present in a cell in that form. Normal presence includes presence with respect to a particular developmental stage and environmental conditions of the cell. An exogenous molecule or sequence can include, for example, a mutated version of a corresponding endogenous sequence in a cell, e.g., a humanized version of an endogenous sequence, or can include a sequence that corresponds to an endogenous sequence within a cell but in a different form (i.e., not within a chromosome). In contrast, an endogenous molecule or sequence includes a molecule or sequence that is normally present in that form, in a particular cell, at a particular developmental stage, and under particular environmental conditions.

[0133] The term "heterologous," when used in the context of a nucleic acid or a protein, indicates that the nucleic acid or protein comprises at least two segments that do not naturally occur together within the same molecule. For example, the term "heterologous," when used with reference to a segment of a nucleic acid or a segment of a protein, indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other (e.g., linked together) in nature. As an example, a "heterologous" region of a nucleic acid vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with that other molecule in nature. For example, a heterologous region of a nucleic acid vector can include a coding sequence adjacent to a sequence not found in association with the coding sequence in nature. Similarly, a "heterologous" region of a protein is a segment of amino acids within or attached to another peptide molecule that is not found in association with that other peptide molecule in nature (e.g., a fusion protein or a tagged protein). Similarly, a nucleic acid or protein can include a heterologous tag or a heterologous secretion or localization sequence.

[0134] The term "locus" refers to the specific location of a gene (or key sequence), DNA sequence, polypeptide coding sequence, or location on a chromosome in the genome of an organism. For example, a "Ttr locus" can refer to the specific location of the Ttr gene, Ttr DNA sequence, TTR coding sequence, or Ttr location on a chromosome in the genome of an organism in which such a sequence is identified to reside. A "Ttr locus" can include regulatory elements of the Ttr gene, including, for example, an enhancer, promoter, 5' and / or 3' untranslated regions (UTRs), or a combination thereof.

[0135] The term "gene" refers to a DNA sequence in a chromosome that encodes a product (e.g., an RNA product and / or a polypeptide product) and includes coding regions interrupted by non-coding introns and sequences located adjacent to the coding region at both the 5' and 3' ends, such that a gene corresponds to a full-length mRNA (including 5' and 3' untranslated sequences). The term "gene" also includes other non-coding sequences, including regulatory sequences (e.g., promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequences, and matrix attachment regions. These sequences may be close to (e.g., within 10 kb) or distant from the coding region of a gene, and they influence the level or rate of transcription and translation of the gene.

[0136] The term "allele" refers to variant forms of a gene. Some genes have different forms located at the same position, or locus, on a chromosome. Diploid organisms have two alleles at each locus. Each pair of alleles represents a genotype at a particular locus. A genotype is described as homozygous if there are two identical alleles at a particular locus, or heterozygous if the two alleles are different.

[0137] A "promoter" is a regulatory region of DNA that typically contains a TATA box that can direct RNA polymerase II to begin RNA synthesis at the appropriate transcription start site for a particular polynucleotide sequence. A promoter may further contain other regions that affect the rate of transcription initiation. The promoter sequences disclosed herein regulate transcription of an operably linked polynucleotide. A promoter may be active in one or more cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, one-cell stage embryos, differentiated cells, or combinations thereof). A promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO2013 / 176772, the entire contents of which are incorporated herein by reference.

[0138] The chimeric proteins disclosed herein may contain one or more transcription activation domains. Transcription activation domains include regions of naturally occurring transcription factors that, in combination with a DNA binding domain, can activate transcription from a promoter by contacting the transcription machinery directly or through other proteins such as coactivators. Transcription activation domains also include functional fragments or variants of such regions of transcription factors, as well as engineered transcription activation domains that are derived from naturally occurring transcription activation domains or artificially created or synthesized to activate transcription of a target gene. A functional fragment is a fragment that can activate transcription of a target gene when operably linked to a suitable DNA binding domain. A functional variant is a variant that can activate transcription of a target gene when operably linked to a suitable DNA binding domain.

[0139] Specific transcription activation domains for use in the chimeric proteins disclosed herein include the VP64 transcription activation domain or functional fragments or variants thereof. VP64 is a tetrameric repeat of the minimal activation domain from the herpes simplex virus VP16 activation domain. Other examples of transcription activation domains include the herpes simplex virus VP16 transactivation domain, VP64 (four tandem repeats of herpes simplex virus VP16), NF-kBp65 (NF-kB transactivation subunit p65) activation domain, MyoD1 transactivation domain, HSF1 transactivation domain (transactivation domain from human heat shock factor 1), RTA (Epstein-Barr virus R transactivator activation domain), SETT / 9 transactivation domain, p53 activation domain 1, p53 activation domain 2, CREB (cAMP response element binding protein) activation domain, E2A activation domain, NFAT (nuclear factor of activated T cells) activation domain, and functional fragments and variants thereof. See, e.g., US2016 / 0298125, US2016 / 0281072, and WO2016 / 049258, each of which is incorporated by reference in its entirety for all purposes. Other examples of transcription activation domains include Gcn4, MLL, Rtg3, Gln3, Oaf1, Pip2, Pdr1, Pdr3, Pho4, Leu3, and functional fragments and variants thereof. See, e.g., US2016 / 0298125, which is incorporated by reference in its entirety for all purposes. Still other examples of transcription activation domains include Sp1, Vax, GATA4, and functional fragments and variants thereof. See, e.g., WO2016 / 149484, which is incorporated by reference in its entirety for all purposes. Other examples include activation domains from Oct1, Oct-2A, AP-2, CTF1, P300, CBP, PCAF, SRC1, PvALF, ERF-2, OsGAI, HALF-1, C1, AP1, ARF-5, ARF-6, ARF-7, ARF-8, CPRF1, CPRF4, MYC-RP / GP, and TRAB1PC4, and functional fragments and variants thereof.See, e.g., US2016 / 0237456, EP3045537, and WO2011 / 146121, each of which is incorporated by reference in its entirety for all purposes. Additional suitable transcription activation domains are also readily known. See, e.g., WO2011 / 146121, which is incorporated by reference in its entirety for all purposes.

[0140] vector In some embodiments, a chimeric protein of the present disclosure, or a nucleic acid expressing a chimeric protein of the present disclosure, is expressed in the isolated cell via transduction with a vector.

[0141] As used herein, a "vector" refers to a composition of matter that contains an isolated nucleic acid encoding a chimeric protein of the present disclosure and can be used to deliver the isolated nucleic acid to the interior of a cell. Vectors include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. The term "vector" includes autonomously replicating plasmids or viruses. "Vector" may also include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, lipid nanoparticles, and non-lipid nanoparticles. In a preferred embodiment, the vector is a viral vector. Viruses that can be engineered for use as viral vectors include, but are not limited to, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAVs), alphaviruses, flaviviruses, rhabdoviruses, paramyxoviruses, picornaviruses, poxviruses, and herpesviruses. Among these, oncolytic viral vectors are preferred, including, but not limited to, adenovirus, herpesvirus, alphavirus, flavivirus, rhabdovirus, paramyxovirus, and cytomegalovirus. In some embodiments, the recombinant oncolytic virus can be an oncolytic RNA virus such as vesicular stomatitis virus (VSV), Maraba virus, Newcastle disease virus, poliovirus, measles virus, or reovirus. In some embodiments, the recombinant oncolytic virus can be an oncolytic DNA virus such as vaccinia virus (VV), herpes simplex virus (HSV), or adenovirus. In some embodiments, the recombinant oncolytic virus can be VSV-CXCL12, VV-CXCL12, VSV-CXCL13, VSV-M51R-VacE3L, VV-CXCL13, VSV-CXCL10, or VV-CXCL10. Preferably, the viral vector is an oncolytic virus.

[0142] Adeno-associated virus (AAV) is a small, replication-deficient parvovirus. AAV is approximately 20-24 nm long and has a density of approximately 1.40-1.41 g / cc. AAV contains a single-stranded, linear genomic DNA molecule approximately 4.7 kb in length. The single-stranded AAV genomic DNA can be either positive- or negative-sense. AAV contains two open reading frames, Rep and Cap, flanked by two 145-base inverted terminal repeats (ITRs). AAV contains a single intron. Cis-acting sequences that direct viral DNA replication (Rep), encapsidation / packaging, and integration into host cell chromosomes are contained within the ITRs. Three AAV promoters, p5, p19, and p40 (named after their relative map positions), drive expression of two AAV internal open reading frames encoding the rep and cap genes. p5 and p19 are rep promoters. Combined with alternative splicing of a single AAV intron, two rep promoters drive the rep gene to produce four rep proteins (rep78, rep68, rep52, and rep40). The rep proteins possess multiple enzymatic properties involved in viral genome replication. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. A single polyadenylation site is located at map position 95 of the AAV genome. Muzyczka has reviewed the life cycle and genetics of AAV (Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992)).

[0143] AAV infection is noncytopathic in cultured cells. Natural infection in humans and other animals is silent and asymptomatic (does not cause disease). AAV infects many mammalian cell types, potentially targeting many different tissues in vivo. In addition to dividing cells, AAV can transduce slowly dividing and nondividing cells and persist essentially for the lifespan of those cells as transcriptionally active nuclear episomes (i.e., extrachromosomal elements). The AAV proviral genome is infectious as cloned DNA in plasmids, enabling the construction of recombinant genomes. Furthermore, because signals directing AAV replication, genome encapsidation, and integration are all contained within the ITRs of the AAV genome, part or all of the approximately 4.3 kb genome encoding the replication and structural capsid proteins (rep-cap) can be replaced with heterologous DNA, such as a gene cassette containing a promoter, DNA of interest, and a polyadenylation signal. The rep and cap proteins can be provided in trans.

[0144] Several AAV serotypes have been identified, each with different tropism (cell types they infect). Serotype AAV1 is tropic for the following tissues: CNS, heart, retinal pigment epithelium (RPE), and skeletal muscle. Serotype AAV2 is tropic for the following tissues: CNS, kidney, photoreceptor cells, and RPE. Serotype AAV4 is tropic for the following tissues: CNS, lung, and RPE. Serotype AAV5 is tropic for the following tissues: CNS, lung, photoreceptor cells, and RPE. Serotype AAV6 is tropic for the following tissues: lung and skeletal muscle. Serotype AAV7 is tropic for the following tissues: liver and skeletal muscle. Serotype AAV8 is tropic for the following tissues: CNS, heart, liver, pancreas, photoreceptor cells, RPE, and skeletal muscle. Serotype AAV9 exhibits tropism for the following tissues: CNS, heart, liver, lung, and skeletal muscle. The tropism of AAV viruses may be related to variability in the amino acid sequence of the capsid protein, which can bind to different functional receptors present on different cell types.

[0145] For example, it was recently shown that inclusion of the human rhodopsin kinase (hGRK1) promoter in an AAV5 vector results in rod- and cone-specific expression in the primate retina (Boye, et al., Human Gene Therapy, 23:1101-1115 (October 2012) (DOI: 10.1089 / hum.2012.125)).

[0146] Recently, it has also been shown that AAV virions with altered capsid proteins can confer greater tissue-specific infectivity. For example, AAV6 containing a variant capsid protein exhibits increased infectivity of retinal cells compared to wild-type AAV capsid proteins (US Pat. No. 8,663,624). A variant capsid protein containing a peptide insertion between two adjacent amino acids corresponding to amino acids 570 and 611 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype, results in increased infectivity of retinal cells compared to wild-type AAV (US Pat. No. 9,193,956).

[0147] Lentiviruses are a genus of retroviruses that cause chronic and fatal diseases characterized by long latency periods in humans and other mammalian species. The most well-known lentivirus is the human immunodeficiency virus (HIV), which causes AIDS. Lentiviruses also infect apes, cattle, goats, horses, cats, and sheep. Recently, lentiviruses have been discovered in monkeys, lemurs, Malaysian flying lemurs (not a true lemur or primate), rabbits, and ferrets. Lentiviruses and their hosts are distributed worldwide. Lentiviruses are one of the most efficient methods of gene delivery because they can integrate significant amounts of viral cDNA into the DNA of host cells and efficiently infect non-dividing cells. Lentiviruses can become endogenous (ERVs) and integrate their genome into the host germline genome, allowing the virus to be subsequently inherited by the host's offspring.

[0148] Lentiviruses are primarily used as research tools to introduce gene products into in vitro systems or animal models. Conversely, lentiviruses can also be used to stably overexpress certain genes, thus allowing researchers to investigate the effects of increased gene expression in model systems.

[0149] Another common application is the use of lentiviruses to introduce new genes into human or animal cells. For example, a mouse model of hemophilia can be corrected by expressing wild-type platelet factor VIII, the gene mutated in human hemophilia. Lentiviral infection offers advantages over other gene therapy methods, including highly efficient infection of dividing and non-dividing cells, long-term stable expression of the transgene, and low immunogenicity. Lentiviruses have also been successfully used to transduce diabetic mice with a gene encoding PDGF (platelet-derived growth factor), a therapy being investigated for use in humans. Finally, lentiviruses have also been used to induce immune responses against tumor antigens. These treatments, like most current gene therapy experiments, are promising but have yet to be established as safe and effective in controlled human studies. Gammaretroviral and lentiviral vectors have been used in over 300 clinical trials to date, addressing treatment options for a variety of diseases.

[0150] The Indiana strain of vesicular stomatitis virus is the prototype strain of the vesicular stomatitis virus genus, which belongs to the Rhabdoviridae family, and the term VSV refers to this strain. Other major serotypes include Cocal, the New Jersey strain of vesicular stomatitis virus, Chandipura, Maraba, and Piry viruses. VSV is enveloped and has a negative-sense RNA genome. The single-stranded negative-sense RNA genome of VSV encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M; involved in the formation of the viral core and anchoring of glycoproteins to the viral membrane, allowing the formation of glycoprotein homotrimers), glycoprotein (G; controlling receptor recognition, cell entry, and viral fusion), and viral polymerase. The glycoprotein is the primary target of the primary humoral response because it controls receptor recognition, cell entry, and viral fusion. The glycoprotein is designated VSV.G.

[0151] Most viral vectors based on enveloped viruses are pseudotyped, meaning that their envelope is not encoded by their genome but rather derived from a related virus. VSV.G is one of the most widely used viral envelopes due to its broad tropism (its primary receptor, LDLR, is widely expressed in most tissues), thermal stability, and physical stability. For example, lentiviral vectors pseudotyped with VSV.G are used in gene therapy applications.

[0152] VSV has been used as a vector for vaccine therapy. However, there are currently two problems with this approach. VSV.G generates a strong neutralizing antibody response, making it impossible to re-administer vaccine vectors expressing VSV.G. Also, VSV is a neurotropic virus and may have off-target neurotoxicity-related side effects.

[0153] VSV may be an oncolytic virus. The lack of antiviral mechanisms in cancer cells allows oncolytic viruses to preferentially infect and replicate in cancer cells, spread to other tumor tissues, and kill them. VSV's tumor selectivity is the result of its sensitivity to type I interferon-dependent cellular immune responses. VSV infection in healthy cells is reduced by a robust IFN-1 response, but in cancer cells, parts of this immune pathway are missing or impaired. Therefore, VSV preferentially replicates in cancer tissues.

[0154] Viral vectors may comprise an expression vector, expression construct, or expression cassette. The terms "expression vector" or "expression construct" or "expression cassette" refer to a recombinant nucleic acid containing a desired coding sequence operably linked to appropriate nucleic acid sequences necessary for expression of the operably linked coding sequence in a particular host cell or organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), a ribosome binding site, and other sequences. Eukaryotic cells are known to generally utilize promoters, enhancers, and termination and polyadenylation signals, although some elements may be deleted and others added without sacrificing the necessary expression.

[0155] The expression cassettes disclosed herein can also include other components. Such expression cassettes can further include a 3' splicing sequence at the 5' end of the expression cassette and / or a second polyadenylation signal following the coding sequence. The term 3' splicing sequence refers to a nucleic acid sequence at the 3' intron / exon boundary that can be recognized and bound by the splicing machinery. The expression cassette can further include a selection cassette containing, for example, a coding sequence for a drug resistance protein.

[0156] Examples of suitable selection markers include neomycin phosphotransferase (neo.sup.r), hygromycin B phosphotransferase (hyg.sup.r), puromycin-N-acetyltransferase (puro.sup.r), blasticidin S deaminase (bsr.sup.r), xanthine / guanine phosphoribosyltransferase (gpt), and herpes simplex virus thymidine kinase (HSV-k). Optionally, the selection cassette may be flanked by recombinase recognition sites for a site-specific recombinase. When the expression cassette further comprises recombinase recognition sites adjacent to a polyadenylation signal upstream of the coding sequence as described above, the selection cassette may be flanked by the same recombinase recognition sites or by different sets of recombinase recognition sites recognized by different recombinases.

[0157] The expression cassette can also include nucleic acids encoding one or more reporter proteins, such as a fluorescent protein (e.g., green fluorescent protein). Any suitable reporter protein can be used. For example, a fluorescent reporter protein can be used, or a non-fluorescent reporter protein can be used. Examples of fluorescent reporter proteins are provided elsewhere herein. Non-fluorescent reporter proteins include reporter proteins that can be used in histochemical or bioluminescence assays, such as beta-galactosidase, luciferase (e.g., Renilla luciferase, firefly luciferase, and NanoLuc luciferase), and beta-glucuronidase. The expression cassette can include a reporter protein that can be detected in a flow cytometry assay (e.g., a fluorescent reporter protein such as green fluorescent protein) and / or a reporter protein that can be detected in a histochemical assay (e.g., a beta-galactosidase protein). One example of such a histochemical assay is the histochemical visualization of in situ beta-galactosidase expression by hydrolysis of X-Gal (5-bromo-4-chloro-3-indoyl-bD-galactopyranoside), which produces a blue precipitate, or using fluorogenic substrates such as beta-methylumbelliferyl galactoside (MUG) and fluorescein digalactoside (FDG).

[0158] The expression cassettes described herein can be in any form. For example, the expression cassette can be within a vector or plasmid. The expression cassette can be operably linked to a promoter in an expression construct that can direct protein or RNA expression (e.g., upon removal of an upstream polyadenylation signal). Alternatively, the expression cassette can be within a targeting vector. For example, the targeting vector can include homology arms flanking the expression cassette, which are suitable for directing recombination with a desired target genomic locus to facilitate genomic integration and / or replacement of endogenous sequences.

[0159] Pharmaceutical Compositions The present disclosure provides a pharmaceutical composition comprising a chimeric protein of the present disclosure, a nucleic acid encoding a chimeric protein of the present disclosure, an expression vector comprising a chimeric protein of the present disclosure, or an isolated cell of the present disclosure, and a pharmaceutically acceptable excipient, including, but not limited to, a water-insoluble diluent, a water-soluble diluent, a disintegrant, a binder, a wetting agent, a solubilizer, a glidant, a lubricant, and a granulation solvent.

[0160] lipid nanoparticles In a preferred embodiment, the pharmaceutical composition of the present disclosure is a lipid nanoparticle (LNP) pharmaceutical composition. LNP formulations are described in WO2017 / 173054. Lipid nanoparticles ("LNPs") are an example of a vector according to the present disclosure. LNPs are particles containing multiple lipid molecules physically bound to each other by intermolecular forces. These include microspheres (including unilamellar and multilamellar vesicles, e.g., liposomes), the dispersed phase in an emulsion, micelles, or the internal phase in a suspension. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery. Formulations containing cationic lipids are useful for delivering polyanions such as nucleic acids. Other lipids that can be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, helper lipids that enhance transfection, and stealth lipids that increase the time the nanoparticles can persist in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids can be found in WO2016 / 010840A1 and WO2017 / 173054A1, which are incorporated herein by reference in their entirety for all purposes. Exemplary lipid nanoparticles can include cationic lipids and one or more other components. In one example, the other components can include a helper lipid such as cholesterol. In another example, the other components can include a helper lipid such as cholesterol and a neutral lipid such as DSPC. In another example, the other components can include a helper lipid such as cholesterol, an optional neutral lipid such as DSPC, and a stealth lipid such as S010, S024, S027, S031, or S033.

[0161] LNPs can include one or more or all of the following: (i) lipids for encapsulation and endosomal escape, (ii) neutral lipids for stabilization, (iii) helper lipids for stabilization, and (iv) stealth lipids. See, e.g., Finn et al. (2018) Cell Rep. 22(9):2227-2235 and WO2017 / 173054A1, which are incorporated by reference in their entirety for all purposes. In a preferred embodiment, the LNPs include a nucleic acid molecule encoding a chimeric protein of the present disclosure.

[0162] The lipid for encapsulation and endosomal escape can be a cationic lipid. The lipid can also be a biodegradable lipid, such as a biodegradable ionizable lipid. One example of a suitable lipid is lipid A or LPO1, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy-)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl-)oxy)methyl))propyl(9Z,12Z)-octadeca-9,12-dienoate. See, e.g., Finn et al. (2018) Cell Rep. 22(9):2227-2235 and WO2017 / 173054A1, which are incorporated by reference in their entirety for all purposes. Another example of a suitable lipid is lipid B, which is ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate), also known as ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate). Another example of a suitable lipid is lipid C, which is 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1-,3-diyl(9Z,9'Z,12Z,127)-bis(octadeca-9,12-dienoate). Another example of a suitable lipid is lipid D, which is 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl 3-octylundecanoate. Other suitable lipids include heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (also known as [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butanoate or Dlin-MC3-DMA (MC3)).

[0163] Some such lipids suitable for use in the LNPs described herein are biodegradable in vivo. For example, LNPs comprising such lipids include those in which at least 75% of the lipid is cleared from plasma within 8, 10, 12, 24, or 48 hours, or within 3, 4, 5, 6, 7, or 10 days. As another example, at least 50% of the LNP is cleared from plasma within 8, 10, 12, 24, or 48 hours, or within 3, 4, 5, 6, 7, or 10 days.

[0164] Such lipids may be ionizable depending on the pH of the medium in which they are contained. For example, in a slightly acidic medium, the lipids may be protonated and thus positively charged. Conversely, in a slightly basic medium, such as blood, which has a pH of approximately 7.35, the lipids may not be protonated and therefore may not be charged. In some embodiments, the lipids may be protonated at a pH of at least about 9, 9.5, or 10. The ability of such lipids to carry a charge is related to their inherent pKa. For example, the lipids may independently have a pKa ranging from about 5.8 to about 6.2.

[0165] Neutral lipids function to stabilize and improve processing of LNPs. Examples of suitable neutral lipids include various neutral, uncharged, or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-diarachidonoyl-s n-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyllysophosphatidylcholine phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and combinations thereof. For example, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE).

[0166] Helper lipids include lipids that enhance transfection. The mechanism by which helper lipids enhance transfection may include enhancing particle stability. In certain cases, helper lipids can enhance membrane fusion. Helper lipids include steroids, sterols, and alkylresorcinols. Suitable examples of helper lipids include cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In one example, the helper lipid can be cholesterol or cholesterol hemisuccinate.

[0167] Stealth lipids include lipids that change the length of time that nanoparticles can exist in vivo. Stealth lipids can, for example, aid in the formulation process by reducing particle aggregation and controlling particle size. Stealth lipids can adjust the pharmacokinetic properties of LNPs. Suitable stealth lipids include lipids that have a hydrophilic head group linked to the lipid moiety.

[0168] The hydrophilic head group of the stealth lipid can comprise a polymer moiety selected from, for example, polymers based on PEG (often poly(ethylene oxide), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyamino acids, and poly-N-(2-hydroxypropyl) methacrylamide). The term PEG refers to polyethylene glycol or other polyalkylene ether polymers. In certain LNP formulations, the PEG is PEG-2K, also known as PEG2000, and has an average molecular weight of about 2,000 daltons. See, e.g., WO2017 / 173054A1, incorporated herein by reference in its entirety for all purposes.

[0169] The lipid portion of the stealth lipid can be derived from a diacylglycerol or diacylglycamide, including, for example, those containing a dialkylglycerol or dialkylglycamide group having an alkyl chain length independently containing from about C4 to about C40 saturated or unsaturated carbon atoms, where the chain can contain one or more functional groups, such as, for example, an amide or ester. The dialkylglycerol or dialkylglycamide group can further contain one or more substituted alkyl groups.

[0170] Exemplary stealth lipids include PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl- -[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol))-2000] (PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol))-2000] (PEG2k-DSPE), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In one specific example, the stealth lipid can be PEG2k-DMG.

[0171] LNPs may contain different molar ratios of the component lipids in the formulation. The mol% of the CCD lipid may be, for example, about 30 mol% to about 60 mol%, about 35 mol% to about 55 mol%, about 40 mol% to about 50 mol%, about 42 mol% to about 47 mol%, or about 45%. The mol% of the helper lipid may be, for example, about 30 mol% to about 60 mol%, about 35 mol% to about 55 mol%, about 40 mol% to about 50 mol%, about 41 mol% to about 46 mol%, or about 44 mol%. The mol% of the neutral lipid may be, for example, about 1 mol% to about 20 mol%, about 5 mol% to about 15 mol%, about 7 mol% to about 12 mol%, or about 9 mol%. The mol % of the stealth lipid may be, for example, about 1 mol % to about 10 mol %, about 1 mol % to about 5 mol %, about 1 mol % to about 3 mol %, about 2 mol %, or about 1 mol %.

[0172] LNPs can have different ratios between the positively charged amine groups of the biodegradable lipids (N) and the negatively charged phosphate groups of the encapsulated nucleic acid (P). This can be mathematically represented by the equation N / P. For example, the N / P ratio can be about 0.5 to about 100, about 1 to about 50, about 1 to about 25, about 1 to about 10, about 1 to about 7, about 3 to about 5, about 4 to about 5, about 4, about 4.5, or about 5. The N / P ratio can also be about 4 to about 7, or about 4.5 to about 6. In specific examples, the N / P ratio can be 4.5 or 6.

[0173] A specific example of a suitable LNP has a nitrogen-to-phosphate (N / P) ratio of 4.5 and comprises a biodegradable cationic lipid, cholesterol, DSPC, and PEG2k-DMG in a molar ratio of 45:44:9:2. The biodegradable cationic lipid can be (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy-)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also referred to as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl-)oxy)methyl))propyl(9Z,12Z)-octadeca-9,12-dienoate. See, e.g., Finn et al. (2018) Cell Rep. 22(9):2227-2235, incorporated herein by reference in its entirety for all purposes. Another specific example of a suitable LNP comprises Dlin-MC3-DMA (MC3), cholesterol, DSPC, and PEG-DMG in a molar ratio of 50:38.5:10:1.5.

[0174] Another specific example of a suitable LNP has a nitrogen-to-phosphate (N / P) ratio of 6 and comprises a biodegradable cationic lipid, cholesterol, DSPC, and PEG2k-DMG in a molar ratio of 50:38:9:3. The biodegradable cationic lipid can be (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy-)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also referred to as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl-)oxy)methyl))propyl(9Z,12Z)-octadeca-9,12-dienoate.

[0175] Another specific example of a suitable LNP has a nitrogen-to-phosphate (N / P) ratio of 3 and may include a cationic lipid, a structural lipid, cholesterol (e.g., cholesterol (ovine) (Avanti 700000)), and PEG2k-DMG (e.g., PEG-DMG 2000 (NOF America-STJNBRIGHT.RTM.GM-020(DMG-PEG)) in a ratio of 50:10:38.5:1.5 or a ratio of 47:10:42:1. The structural lipid may be, for example, DSPC (e.g., DSPC (Avanti 850365)), SOPC, DOPC, or DOPE. The cationic / ionizable lipid may be, for example, Dlin-MC3-DMA (e.g., Dlin-MC3-DMA (Biofine International)).

[0176] Another specific example of a suitable LNP contains Dlin-MC3-DMA, DSPC, cholesterol, and PEG lipid in a ratio of 45:9:44:2. Another specific example of a suitable LNP contains Dlin-MC3-DMA, DOPE, cholesterol, and PEG lipid or PEG-DMG in a ratio of 50:10:39:1. Another specific example of a suitable LNP contains Dlin-MC3-DMA, DSPC, cholesterol, and PEG2k-DMG in a ratio of 55:10:32.5:2.5. Another specific example of a suitable LNP contains Dlin-MC3-DMA, DSPC, cholesterol, and PEG-DMG in a ratio of 50:10:38.5:1.5. Another specific example of a suitable LNP contains Dlin-MC3-DMA, DSPC, cholesterol, and PEG-DMG in a ratio of 50:10:38.5:1.5.

[0177] Upon administration to a subject, the effector molecule is cleaved and the concentrated (retained) bioactive effector (e.g., IL-12) is released within the TME.

[0178] The present disclosure also provides a method of treating a disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition of the present disclosure. In certain embodiments, the disease is cancer. The pharmaceutical composition may be administered intratumorally, locally within the TME, or systemically. [Example]

[0179] mRNA constructs encoding membrane-bound IL-12p70 and membrane-bound IL-12p70 with a protease-cleavable linker were validated using a Chinese hamster ovary (CHO) transfection system. The CHO transfection system was treated with either vehicle control or matrix metalloproteinase 2 (MMP2) to detect cleavage of IL-12p70 from its membrane-bound localization to its soluble form. Following validation, the antitumor efficacy of localized delivery of lipid nanoparticles (LNPs) encapsulating these mRNA constructs encoding various IL-12p70 sequences (soluble, membrane-bound, or membrane-bound with a protease-cleavable substrate) was determined. For these syngeneic tumor growth studies, two relevant in vivo models (B16F10 melanoma and MC38 colon carcinoma) were utilized, in which tumor cells were injected subcutaneously into the flanks of C57BL / 6 mice purchased from Jackson Laboratories. For in vivo studies, mice were divided equally according to tumor size (calculated as follows: (L 2 × W) / 2 (where L is the smallest size) measured by calipers. [Table 2]

[0180] Example 1. In vitro validation of mRNA constructs In vitro study 1 aimed to validate various mRNA constructs, as listed in Table 1. This study involved transfecting specific wells with 500 ng of mRNA encoding F-luciferase (negative control), membrane-bound IL-12p70 (TM, L1), or membrane-bound IL-12p70 using protease-cleavable substrates (L2, L3, L4s, L5), as indicated in Table 1. The bolded sequences represent the protease-bound substrates. Sixteen hours after transfection, these corresponding wells were treated with phosphate-buffered saline (PBS) or 1 μg / mL of the activated protease, MMP2. MMP2 was activated by pretreating recombinant MMP2 with 4-aminophenylmercuric acetate for 1 hour at 37°C. Cells were treated for 180 minutes, then gently detached with a pipette and stained for cell surface expression of IL-12 using an antibody detecting IL-12p40 conjugated to PerCP-Cy5.5 (Biolegend, catalog no. 505212, clone C15.6). Enzymatic detachment was avoided to prevent interference / degradation of surface protein expression. Samples were acquired on a Becton Dickinson LSR Fortessa Flow Cytometer. Similarly, supernatants containing soluble IL-12p70 (positive control) or mCherry (negative control) were collected at 90 and 180 minutes and subjected to ELISA quantitation of IL-12p70.

[0181] This study demonstrated that mRNA constructs encoding IL-12p70-TM sequences with different protease-cleavable linker substrates resulted in cleavage upon MMP2 treatment. This example illustrates the various IL-12p70 transmembrane or IL-12p70 transmembrane linkers linked to the protease-cleavable substrate mRNA sequences utilized in this report (Figure 1). Using a Chinese hamster ovary cell (CHO) mRNA transfection system, these constructs were validated in two different assays using exogenous addition of MMP2, a potent protease with broad specificity. Using flow cytometry analysis after 180 minutes of MMP2 treatment, the transmembrane (TM and L1) sequences showed no effect on surface IL-12p40 staining. However, sequences with cleavable linker substrates (L2, L3, L4, L4s, and L5) all showed a significant reduction in cell surface staining of IL-12p40, suggesting efficient cleavage (Figures 2A-2C). In parallel, we utilized enzyme-linked immunosorbent assay (ELISA) to detect the release of cleaved (and therefore soluble) IL-12p70 and observed a time-dependent release of IL-12p70, confirming the expression and validity of the protease-cleavable linker sequence within each construct (Fig. 3 ).

[0182] Example 2. In vivo study of the antitumor activity of the chimeric protein in mice injected with B16F10 melanoma tumor cells. In vivo study 2 included nine groups with 7–8 mice per group. On day 0, mice were anesthetized by isoflurane inhalation and then injected with 3.5 × 10 mice in a suspension of 50 μL of medium. 5 B16F10 melanoma tumor cells were injected subcutaneously into the right flank. On day 10, mice were cultured at an average of 100 mm 3Mice were randomized equally into nine groups according to tumor size and received intratumoral injections of 10 μL of LNP-mRNA in PBS encoding F-luciferase, soluble IL-12p70 (sIL-12), membrane-bound IL-12p70 (TM, L1), or membrane-bound IL-12p70, along with protease-cleavable substrates (L1, L2, L3, L4s, L5). Mice were treated every three days for a total of four intratumoral LNP-mRNA injections. LNPs utilizing fixed concentrations of ionizable lipids, cholesterol, structural lipids, and polyethylene glycol (PEG) were used. The experimental dosing and treatment protocol for mouse groups is shown in Table 2.

[0183] Intratumoral delivery of an LNP-mRNA construct encoding membrane-bound IL-12p70 containing a protease-cleavable linker substrate sequence resulted in greater antitumor efficacy than membrane-bound stable forms of IL-12p70 in a B16F10 tumor model. This example demonstrates the potent antitumor efficacy of intratumoral delivery of LNP-mRNA encoding membrane-bound IL-12p70 with a protease-cleavable linker sequence in an aggressive B16F10 melanoma tumor model. Soluble IL-12p70 mRNA, which demonstrated robust antitumor efficacy in previous studies, was used as a benchmark for comparison between the antitumor efficacy of the tested constructs. Similarly, membrane-bound IL-12p70, which demonstrated modest efficacy in previous studies, was also used to benchmark the antitumor efficacy of the tested constructs.

[0184] C57BL / 6 strain background mice (Jackson Laboratories) were transfected with B16F10 cells (3.5 × 10 5 (cells / mouse) were implanted subcutaneously. Tumors were measured using a caliper, and tumor volume was calculated using the formula (L 2 × W) / 2, where L is the minimum size. 3Mice were randomized equally into nine treatment groups when the tumor size reached 10 days, which was day 10. Mice received intratumoral injections of 10 μg of LNP-mRNA encoding F-luciferase on days 10, 13, 16, and 19, with either control, soluble IL-12p70, membrane-bound IL-12p70 (IL-12p70_TM), or a linker length control (L1) or various sequences found in Figure 1 describing the linker between IL-12p70 and the transmembrane signal peptide with a protease-substrate sequence for cleavage to soluble IL-12p70. Tumor volume was monitored twice weekly by caliper measurement until the end of the study on day 45. Mean tumor volume over time for each group indicates that all treatment groups produced strong antitumor efficacy compared to the F-luciferase control. As expected, soluble IL-12p70 had the greatest overall antitumor efficacy and survival rate (Figures 4, 5, and 6). Interestingly, most sequences with protease-cleavable linkers had stronger efficacy compared to the membrane-bound sequences, TM, and L1 (Figures 4, 5, and 6). Specifically, linkers L4, L4-short, and L3 had the strongest overall efficacy. Furthermore, serum collected from animals 24 hours after the first dose revealed that, although no circulating IL-12p70 was found within the transmembrane-bound sequences, all protease-cleavable sequences resulted in elevated levels of circulating IL-12p70, suggesting that these proteins were indeed cleaved in vivo (Figure 7). [Table 3]

[0185] As shown in Table 2, for in vivo study 1 using the B16F10 subcutaneous tumor model, mice treated with sIL-12p70 had robust antitumor efficacy. Interestingly, all tested constructs carrying a protease-cleavable linker between IL-12p70 and the transmembrane domain (L2, L3, L4, L4s, L5) had better antitumor efficacy compared to the modest efficacy seen with membrane-bound IL-12p70 (TM, L1).

[0186] Example 3. In vivo study of the antitumor activity of chimeric proteins in mice injected with MC38 colon tumor cells In vivo study 3 included nine groups with 6–7 mice per group. On day 0, mice were anesthetized by isoflurane inhalation and then administrated with 3 × 10 mice in a suspension of 50 μL of medium. 5 MC38 colon tumor cells were injected subcutaneously into the right flank. On day 11, mice were cultured at an average size of 85 mm 3 Mice were randomized equally into nine groups according to tumor size and received intratumoral injections of 10 μL of LNP-mRNA in PBS encoding F-luciferase, soluble IL-12p70 (sIL12), membrane-bound IL-12p70 (TM, L1), or membrane-bound IL-12p70, along with protease-cleavable substrates (L1, L2, L3, L4s, L5). Mice were treated every three days for a total of four intratumoral injections of LNP-mRNA. LNPs utilizing fixed concentrations of ionizable lipids, cholesterol, structural lipids, and polyethylene glycol (PEG) were used. The experimental dosing and treatment protocol for mouse groups is shown in Table 3.

[0187] Intratumoral delivery of an LNP-mRNA construct encoding membrane-bound IL-12p70 containing a protease linker substrate sequence resulted in greater antitumor efficacy than membrane-bound stable forms of IL-12p70 in an MC38 tumor model. This example demonstrates the potent antitumor efficacy of intratumoral delivery of LNP-mRNA encoding membrane-bound IL-12p70 with a protease-cleavable linker sequence in an aggressive MC38 colon tumor model. Soluble IL-12p70 mRNA, which demonstrated robust antitumor efficacy in previous studies, was used as a benchmark for comparison between the antitumor efficacy of the tested constructs. Similarly, membrane-bound IL-12p70, which demonstrated modest efficacy in previous studies, was also used to benchmark the antitumor efficacy of the tested constructs.

[0188] C57BL / 6 strain background mice (Jackson Laboratories) were transfected with MC38 cells (3 × 10 5(cells / mouse) were implanted subcutaneously. Tumors were measured using a caliper, and tumor volume was calculated using the formula (L 2 × W) / 2, where L is the minimum size. 3 Mice were randomized equally into nine treatment groups when the tumor size reached 11 days. Mice received intratumoral injections of 10 μg of LNP-mRNA encoding F-luciferase on days 11, 14, 17, and 20. These injections included control, soluble IL-12p70, membrane-bound IL-12p70, or various sequences (see Figure 1) describing the linker length between IL-12p70 and the transmembrane signal peptide, either as a control (L1) or a linker with a protease-substrate sequence for cleavage to soluble IL-12p70. Tumor volume was monitored twice weekly by caliper measurement until the end of the study on day 45. Similar to in vivo study 1, soluble IL-12p70 had the greatest overall antitumor efficacy and survival rate (Figures 8, 9, and 10). Interestingly, most sequences with protease-cleavable linkers had stronger efficacy compared to the membrane-bound sequences, TM, and L1 (Figures 8, 9, and 10). Specifically, linkers L4-short, L4, L5, and L2 (in that order) had the best overall trend in antitumor efficacy, resulting in 3, 2, 1, and 1 tumor-free mice, respectively. Furthermore, serum collected from animals 24 hours after the first dose revealed that, while no circulating IL-12p70 was found within the membrane-bound sequences, all protease-cleavable sequences resulted in elevated levels of circulating IL-12p70, suggesting that these proteins were indeed cleaved in vivo (Figure 11). [Table 4]

[0189] For in vivo study 2 using the MC38 subcutaneous tumor model, mice treated with sIL-12p70 had robust antitumor efficacy, as shown in Table 3. Interestingly, all tested constructs carrying a protease-cleavable linker between IL-12p70 and the transmembrane domain (L2, L3, L4, L4s, L5) had better antitumor efficacy compared to the modest efficacy seen with membrane-bound IL-12p70 (TM, L1).

[0190] Although the present disclosure has been described in detail, including preferred embodiments thereof, it will be appreciated that those skilled in the art, upon consideration of the present disclosure, may make modifications and / or improvements thereto within the scope and spirit of the present disclosure. Sequences of IL-12p70 constructs with or without cleavable peptide linkers IL-12p70 (SEQ ID NO: 17) IL-12p70_TM (membrane-bound) (SEQ ID NO: 18) IL-12p70-L1-TM (SEQ ID NO: 19) IL-12p70-L2-TM (SEQ ID NO: 20) IL-12p70-L3-TM (SEQ ID NO: 21) IL-12p70-L4-TM (SEQ ID NO: 22) IL-12p70-L4s-TM (SEQ ID NO: 23) IL-12p70-L5-TM (SEQ ID NO: 24) Table 5-1 Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 5-6 Table 5-7 Table 5-8 Table 5-9

Claims

1. 1. A membrane-cleavable chimeric protein having, in N-terminal to C-terminal direction, the formula: E-L-TM or TM-L-E During the ceremony, a) E comprises an effector molecule; b) L is a first linker comprising a protease cleavage site and an amino acid sequence selected from the group consisting of GGGGSISSGLLLSGRSDNHGGGGGS (SEQ ID NO: 3), GGGGSVPLSLYSGGGISSGLLSGRSDNHGGGGGS (SEQ ID NO: 4), GGGGSHPVGLLARGGGHPVGLLARGGSGRSAGGSGRSAGGGGGS (SEQ ID NO: 5), GGGGSHPVGLLARGGGGS (SEQ ID NO: 6), and GGGGSLAQAVRSSGGGGGS (SEQ ID NO: 7), and L is a cleavable linker; c) the TM comprises a transmembrane protein; d) A membrane-cleavable chimeric protein in which EL-TM or TM-LE is constructed to be expressed as a single polypeptide.

2. The membrane-cleavable chimeric protein of claim 1 , wherein the membrane-cleavable chimeric protein further comprises a signal peptide at the N-terminus.

3. The membrane-cleavable chimeric protein of claim 1 or 2, wherein the effector molecule comprises a cytokine or a functional fragment thereof.

4. 4. The membrane-cleavable chimeric protein of claim 3, wherein the cytokine is selected from the group consisting of IL-1-beta, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-17A, IL-18, IL-21, IL-22, type I interferon, interferon-gamma, and TNF-alpha.

5. The membrane-cleavable chimeric protein of claim 4, wherein the cytokine is IL-12.

6. The membrane-cleavable chimeric protein of any one of claims 1 to 5, wherein the effector molecule comprises the p40 and p35 subunits of IL-12 linked by a second linker.

7. 7. The membrane-cleavable chimeric protein of any one of claims 1 to 6, wherein the transmembrane domain is derived from the group consisting of PDGFR-beta, CD8, CD28, CD3 zeta chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, and BTLA.

8. The membrane-cleavable chimeric protein according to any one of claims 1 to 7, wherein the transmembrane domain is derived from B7-1.

9. The membrane-cleavable chimeric protein according to any one of claims 1 to 8, wherein the transmembrane domain is linked to a cytoplasmic domain.

10. 10. The membrane-cleavable chimeric protein of claim 9, wherein the cytoplasmic domain is derived from the group consisting of PDGFR-beta, CD8, CD28, CD3 zeta chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, and BTLA.

11. The membrane-cleavable chimeric protein of claim 9 or 10, wherein the cytoplasmic domain is derived from B7-1.

12. The membrane-cleavable chimeric protein according to any one of claims 1 to 11, wherein the effector molecule is cleaved from the chimeric protein upon cleavage of the cleavable linker L.

13. The membrane-cleavable chimeric protein according to any one of claims 1 to 12, wherein the cleavable linker L is preferentially cleaved in cancer cells.

14. a) when expressed in a cell, the effector molecule is linked to the plasma membrane of the cell; and / or b) the effector molecule is released from the cell membrane when expressed in a cell expressing a protease capable of cleaving the protease cleavage site; and / or c) The membrane-cleavable chimeric protein according to any one of claims 1 to 13, wherein the protease expressed on the cell membrane is endogenous to the cell.

15. A nucleic acid molecule encoding the membrane-cleavable chimeric protein of any one of claims 1 to 14.

16. A nucleic acid molecule comprising an expression cassette, wherein the expression cassette comprises a promoter and an exogenous polynucleotide sequence encoding the membrane-cleavable chimeric protein of any one of claims 1 to 14.

17. 17. The nucleic acid molecule of claim 16, wherein the promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, a tissue-specific promoter, and a synthetic promoter.

18. An expression vector comprising the nucleic acid molecule of any one of claims 15 to 17.

19. The expression vector of claim 18 , wherein the vector is a viral vector.

20. A cell comprising the chimeric protein of any one of claims 1 to 14, the nucleic acid molecule of any one of claims 15 to 17, or the expression vector of claim 18 or 19.

21. 21. The cell of claim 20, wherein the cell is selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma-delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

22. 22. The cell of claim 20 or 21, wherein the cell further comprises a protease capable of cleaving the protease cleavage site.

23. The cell of claim 22 , wherein the protease is an endogenous protease.

24. The cell of claim 20 , wherein the membrane-cleavable chimeric protein is expressed in the cell and the effector molecule is linked to the cell membrane of the cell.

25. 25. The cell of claim 24, wherein the cell expresses a protease that cleaves the protease cleavage site and releases the effector molecule from the cell membrane.

26. A pharmaceutical composition comprising the membrane-cleavable chimeric protein of any one of claims 1 to 14, the nucleic acid of any one of claims 15 to 17, the expression vector of claim 18 or 19, or the cell of any one of claims 20 to 25, and a pharmaceutically acceptable excipient.

27. 27. The pharmaceutical composition of claim 26, wherein the pharmaceutical composition is a lipid nanoparticle pharmaceutical composition.

28. 30. A method of treating a disease in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 26 or 27.

29. 29. The method of claim 28, wherein the disease is cancer.

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