Fusion proteins and methods of use thereof

Abstract

The present application provides fusion proteins comprising an interleukin-15 (IL-15) sequence and an interleukin-15 receptor alpha (IL-15R alpha) sequence, the fusion proteins having an endoplasmic reticulum (ER) retention sequence and / or a myristicylation sequence.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of priority to U.S. Patent Application No. 63 / 467,123, filed May 17, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] sequence declaration

[0004] This application contains a sequence list that has been submitted electronically as an XML file named “51624-0073WO1_SL_ST26.XML”. The XML file, created on May 16, 2024, is 43,151 bytes in size. The material in the XML file is hereby incorporated in its entirety by reference. Technical Field

[0005] This disclosure relates to fusion proteins comprising an interleukin-15 (IL-15) sequence and an interleukin-15 receptor α (IL-15Rα) sequence, wherein the fusion proteins have endoplasmic reticulum (ER) preserved sequences and / or myristylated sequences. Background Technology

[0006] IL-15 is an important cytokine for immune cell function. IL-15 is essential for the survival, proliferation, and functional integrity of NK cells. It can enhance NK cell-mediated immunotherapy. IL-15 is secreted in very small amounts, but is efficiently delivered through trans-presentation via its unique receptor α (IL-15 Rα) on the surface of IL-15-producing cells, to interact with the receptor complex on target cells composed of IL-2Rβ and a common γ chain.

[0007] IL-15 can improve the persistence of natural killer (NK) cells. However, recombinant IL-15 has a short half-life, thus requiring continuous administration, which may lead to undesirable side effects. One strategy is to modify NK cells to express IL-15 to improve persistence. Preliminary in vivo results indicate that the use of wild-type IL-15 in membrane-bound or soluble forms in the context of genetically modified NK cells resulted in lethality in mice. Furthermore, these cells have proven difficult to culture and expand efficiently in subjects with cancer. There is a need to increase the proliferation and antitumor efficacy of these immune cells. Summary of the Invention

[0008] This disclosure relates to fusion proteins containing IL-15 and IL-15Rα sequences, which have endoplasmic reticulum (ER) preserved sequences and / or myristylated sequences.

[0009] In one respect, this disclosure relates to a fusion protein comprising (1) a signal peptide sequence; (2) an interleukin-15 (IL-15) sequence; and (3) an endoplasmic reticulum (ER) preserved sequence.

[0010] In some embodiments, the signal peptide sequence is a calreticulin signal peptide sequence.

[0011] In some embodiments, the calreticulin signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 3.

[0012] In some embodiments, the ER-retained sequence comprises the amino acid sequence shown in SEQ ID NO: 12 or 13, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 12 or 13.

[0013] In one respect, this disclosure relates to a fusion protein comprising (1) a cell membrane targeting sequence; and (2) an IL-15 sequence.

[0014] In some embodiments, the cell membrane targeting sequence is selected from the group consisting of: myristylation sequence, palmitylation sequence, and isopreneation sequence.

[0015] In some embodiments, the cell membrane targeting sequence is a myristylated sequence.

[0016] In some embodiments, the myristylated sequence comprises an amino acid sequence shown in any one of SEQ ID NO: 14-16, or an amino acid sequence having at least 90%, 95%, or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 14-16.

[0017] In some embodiments, the IL-15 sequence comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 6.

[0018] In some embodiments, the fusion protein further comprises an IL-15Rα sequence.

[0019] In some embodiments, the IL-15Rα sequence comprises the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11.

[0020] In some embodiments, the IL-15Rα sequence comprises or is composed of an IL-15Rα Sushi domain, wherein the IL-15Rα Sushi domain comprises or is composed of the following: the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 10.

[0021] In some embodiments, the fusion protein comprises a signal peptide sequence, a first adapter sequence, an IL-15 sequence, a second adapter sequence, an IL-15Rα sequence, and an ER-retained sequence from the N-terminus to the C-terminus.

[0022] In some embodiments, the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NO: 18-20, or an amino acid sequence having at least 90%, 95%, or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 18-20.

[0023] In some embodiments, the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 19.

[0024] In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, an optional signal peptide sequence, a myristylation sequence, an IL-15 sequence, a linker sequence, and an IL-15Rα sequence.

[0025] In some embodiments, the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NO: 21-24, or an amino acid sequence having at least 90%, 95%, or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 21-24.

[0026] In some embodiments, the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 24.

[0027] In some embodiments, the fusion protein is trapped inside the cell to reduce the amount of secreted IL-15 and thus reduce the toxicity of IL-15.

[0028] In one respect, this disclosure relates to a nucleic acid comprising one or more nucleic acid sequences encoding the fusion protein described herein or a portion thereof.

[0029] In some embodiments, the nucleic acid further comprises a second nucleic acid sequence encoding an engineered receptor, wherein the engineered receptor comprises an extracellular antigen-binding domain or a ligand-binding domain, and optionally an intracellular signal transduction domain.

[0030] In some embodiments, the engineered receptor nucleic acid sequence and the nucleic acid sequence encoding the fusion protein are separated by a third nucleic acid sequence encoding a cleavable adapter.

[0031] In some embodiments, the cleavable connector comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2.

[0032] In some embodiments, the engineered receptor is selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), T-cell antigen conjugates (TACs), or portions thereof.

[0033] In some embodiments, the engineered receptor is a CAR.

[0034] In some embodiments, the CAR includes an extracellular antigen-binding domain that specifically binds to an antigen, wherein the antigen is a tumor antigen selected from the group consisting of: CD19, CD20, CD22, CD30, CD33, CD38, BCMA, CS1, CD138, CD123 / IL3Rα, c-Met, gp100, MUC1, IGF-I receptor, EpCAM, EGFR / EGFRvIII, HER2, IGF1R, mesothelin, PSMA, WT1, ROR1, CEA, GD-2, NY-ESO-1, MAGE A3, DLL3, GPC3, guanylate cyclase 2C (GCC), micin 18.2, micin 6, glycolipid F77, PD-L1, and / or PD-L2.

[0035] In some embodiments, the tumor antigen is BCMA.

[0036] In some embodiments, the CAR comprises a first VHH antibody portion and a second VHH antibody portion. The first VHH antibody portion comprises CDR1 containing the amino acid sequence of SEQ ID NO: 29, CDR2 containing the amino acid sequence of SEQ ID NO: 30, and CDR3 containing the amino acid sequence of SEQ ID NO: 31. The second VHH antibody portion comprises CDR1 containing the amino acid sequence of SEQ ID NO: 32, CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO: 34.

[0037] In some embodiments, the CAR comprises a first VHH antibody portion and a second VHH antibody portion, the first VHH antibody portion comprising the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence of SEQ ID NO: 27, and the second VHH antibody portion comprising the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence of SEQ ID NO: 28.

[0038] In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0039] In one respect, this disclosure relates to a carrier containing the nucleic acid described herein.

[0040] In one respect, this disclosure relates to a cell that contains the fusion protein, nucleic acid, and / or vector described herein.

[0041] In some embodiments, cells express engineered receptors.

[0042] In some embodiments, the engineered receptor is selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), T-cell antigen conjugates (TACs), or portions thereof.

[0043] In some embodiments, engineered receptors specifically recognize tumor antigens.

[0044] In some embodiments, the cells are immune cells.

[0045] In some embodiments, the cells are selected from the group consisting of: T cells, αβT cells, γδT cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

[0046] In some embodiments, the cell is an NK cell.

[0047] In one respect, this disclosure relates to a method for generating the cells described herein, the method comprising introducing the vector described herein into the cells.

[0048] In one respect, this disclosure relates to a method for treating a subject with cancer, the method comprising administering a therapeutically effective amount of the cells described herein to the subject in need.

[0049] In some embodiments, the subjects have breast cancer, lung cancer, pancreatic cancer, melanoma, oral cancer, mesothelioma, ovarian cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, skin cancer, multiple myeloma, lymphoma, epithelial tumor, soft tissue sarcoma, esophageal cancer, or CNS tumor.

[0050] In one aspect, this disclosure relates to a modified IL-15 (e.g., a modified IL-15 / IL-15R complex) comprising an ER-retained sequence and / or a myristylated sequence. The modified IL-15 provides an immune-stimulating / activating signal. This disclosure further relates to polynucleotides encoding the modified IL-15, modified cells expressing the modified IL-15, therapeutic uses of the modified IL-15, and pharmaceutical compositions comprising the modified IL-15.

[0051] In one aspect, this disclosure relates to modified IL-15 (e.g., modified IL-15 / IL-15R complexes) comprising endoplasmic reticulum (ER) preserved sequences and / or myristylated sequences, and methods of using them.

[0052] 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 invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of any conflict, this specification, including the definitions, shall prevail.

[0053] Other features and advantages of the invention will be apparent from the following detailed description and accompanying drawings, as well as from the claims. Attached Figure Description

[0054] Figure 1A-1B The levels of cell surface IL-15 and IL-15Rα in cells (transfected or modified) transfected with various modified IL-15 and CAR constructs are shown. On day 18, the expression of IL-15 and IL-15Rα designed with the selected constructs was tested using antibodies against IL-15 and IL-15Rα. Figure 1A The percentage of IL-15 and IL-15Rα positive NK cells is shown. Figure 1B The MFIs for IL-15 and IL-15Rα are shown.

[0055] Figure 2The percentage of CAR-positive cells in the transfected cells is shown. The CAR positivity rate of the selected constructs in the transfected cells was determined using the BCMA-FITC assay. All constructs showed good and comparable expression of the BCMA CAR.

[0056] Figures 3A-3B Cell proliferation and viability of the transfected cells were demonstrated. Cell proliferation of the selected constructs was assessed by cell counting, and viability was assessed by trypan blue staining. Construct 32 was used as a control. Cell cultures with equal numbers of cells were seeded in cytokine-free medium on day 0 and counted on day 9. Figure 3A The number of cells (millions) for each build design is shown. Figure 3B This demonstrates the dynamism of each building block design.

[0057] Figures 4A-4B Cell proliferation of transfected cells is shown. Cell proliferation of the selected constructs was tested using a dye dilution method. Construct 32 was used as a control. Cell trace violet-labeled cells were seeded in equal numbers in cytokine-free medium on day 0, and dye dilutions were evaluated on days 1 and 9. Figure 4A The reduction in MFI for each build design is shown. Figure 4B The multiplication factor for each construct design is shown.

[0058] Figures 5A-5B The percentage of phosphorylated STAT5 (pSTAT5) positive cells in the transfected cells is shown. The STAT5 activation activity of the selected construct was tested by measuring pSTAT5 using a pSTAT5 antibody. Figure 5A The percentage of pSTAT5-positive cells in cells transfected with different IL-15 constructs is shown. Figure 5B The MFI for each construct is shown. Simulation data were gated on total NK cells, while the remaining data were gated on CAR-positive cells.

[0059] Figure 6 The cytotoxicity of the transfected cells against tumor cells was demonstrated. The cytotoxicity of the selected construct was tested using a series of cytotoxicity assays against H929 cells, in which the transfected cells were repeatedly excited with tumor cells. Specifically, fresh trace violet-labeled H929 cells were added at the start of each round, and viable tumor cells were counted using flow cytometry after 24 hours.

[0060] Figure 7 illustrates the safety of the CAR constructs. The proliferation of the selected construct designs was tested in the absence of tumor antigens in cytokine-free medium. Cell cultures with equal numbers of cells were seeded into cytokine-free medium on day 0 and counted at different time points until day 49. Figure 7A The cell count (millions) is displayed. Figure 7B The viability of modified cells for each construct design is shown.

[0061] Figure 8A-8G The cytotoxicity of the transfected cells against tumor cells in vivo was demonstrated. The activity of the selected construct design against NCI-H929 cells in vivo was tested. To generate tumor xenografts, 2 million luciferase-labeled NCI-H929 tumor cells were intravenously injected into NCG mice. Twelve days after tumor implantation, 2 million IL-15-construct-armored CAR-NK cells or unNK cells were intravenously (iv) injected into the mice. Figure 8A This demonstrates the use of in vivo bioluminescence imaging (BLI) to monitor tumor progression weekly. Figure 8B Survival curves of mice treated with BCMA CAR-NK cells armored with different IL-15 constructs are shown. Figure 8C-8F These figures illustrate the persistence and pharmacokinetics of NK cells in peripheral blood at different time points. Figure 8C percentages and Figure 8E (absolute number) and CAR-positive NK cells ( Figure 8D percentages and Figure 8F The amplification of the absolute quantity in the middle. Figure 8G The changes in mouse weight are shown.

[0062] Figure 9 The selected sequences in this application are shown. Detailed Implementation

[0063] IL-15 is a key cytokine that exhibits pleiotropic effects on the development, proliferation, and activation of natural killer cells, as well as the proliferation and activation of CD8+ T cells, and is therefore considered one of the most promising molecules for anticancer immunotherapy.

[0064] IL-15 has attracted much attention due to its similarity to IL-2 in cytokine receptor biology. IL-15R is a heterotrimeric receptor composed of a unique IL-15Rα chain, a β subunit (CD122) shared with IL-2, and a common γ subunit (CD132) shared with several cytokines, suggesting that IL-2 and IL-15 have similar biological activities.

[0065] A unique aspect of IL-15 is its trans-presentation, in which IL-15-producing cells present the cytokine on their cell surface against the backdrop of the IL-15 receptor α (IL-15Rα) chain. The key role of IL-15Rα expressed by dendritic cells (DCs) in the trans-presentation of IL-15 to NK cells has been demonstrated. Binding of IL-15R on NK cells leads to autophosphorylation and activation of Janus kinases (JAK1 and JAK3), which induces at least three parallel signaling cascades: the Ras-Raf-MAPK pathway, the State of Signal Transduction and Activation of Transcription (STAT5), and the PI3K-AKT-mTOR pathway.

[0066] When IL-15 binds to the IL-15Rβ / γ complex, a conformational change leads to phosphorylation and activation of receptor-associated JAK1 and JAK3, followed by tyrosine phosphorylation of IL-15Rβ / γ itself. These phosphotyrosine residues provide binding sites for SH2-containing proteins, including STAT5 molecules, which are then phosphorylated, resulting in the formation and translocation of STAT5 dimers and / or tetramers to the nucleus to induce target gene expression. Sustained expression of one such STAT5 target gene, Mcl1, is required to maintain NK cell survival. Due to its very short half-life, the termination of IL-15 signaling leads to the rapid loss of MCL1 in NK cells, ultimately resulting in apoptosis.

[0067] Exogenous administration of IL-15 can be used to support the in vivo proliferation and antitumor activity of NK CAR cells, thereby overcoming the requirement to include IL-15 in the construct. However, when administered in combination with CAR, IL-15 is associated with significant toxicity even at a low dose of 0.5 μg / mouse every 2–3 days.

[0068] To reduce the toxicity of IL-15, it can be modified to trap IL-15 within the endoplasmic reticulum or beneath the plasma membrane, thereby reducing the amount of IL-15 secreted and thus decreasing its toxicity. This disclosure provides toxicity-reduced modified IL-15 or related fusion proteins containing the IL-15 sequence.

[0069] As used herein, the term "fusion protein" refers to a protein complex containing one or more polypeptides that have the desired function. A protein complex may contain or consist of a single polypeptide. A fusion protein may be a fusion polypeptide.

[0070] As used herein, the term "IL-15" refers to a polypeptide derived from wild-type IL-15 or a functional variant thereof. IL-15 can be wild-type IL-15 (e.g., human IL-15). IL-15 can have one or more mutations (e.g., insertion, deletion, or substitution). IL-15 can be human IL-15. The IL-15 sequence can be the complete IL-15 sequence or a portion of the complete IL-15 sequence.

[0071] As used herein, the term "IL-15Rα" refers to a polypeptide, a functional variant thereof, or a portion thereof derived from wild-type IL-15Rα. IL-15Rα can be wild-type IL-15Rα (e.g., human IL-15Rα). IL-15Rα can have one or more mutations (e.g., insertion, deletion, or substitution). IL-15Rα can be human IL-15Rα. The IL-15Rα sequence can be the complete sequence of IL-15Rα or a portion thereof. The IL-15Rα sequence can contain or consist of a sequence of the sushi domain of IL-15Rα.

[0072] As used herein, a “vector” is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An “expression vector” is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell in which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by operatively linking to regulatory elements such as promoters, enhancers, and / or poly-A tails, which are located within the vector or in the genome of the host cell at or near the integration site of the polynucleotide of interest, such that the polynucleotide of interest will be translated in the host cell in which the expression vector has been introduced.

[0073] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically transplant one or more antigens onto immune effector cells such as T cells or NK cells. Some CARs are also referred to as "artificial T cell receptors," "chimeric T cell receptors," or "chimeric immune receptors." CARs may contain an extracellular ligand-binding domain or an extracellular antigen-binding domain specific to one or more antigens (such as tumor antigens), a transmembrane region, and an intracellular signaling domain for T cell receptors and / or other receptors. "CAR-T cell" refers to a T cell expressing a CAR. "CAR-NK cell" refers to an NK cell expressing a CAR.

[0074] As used herein, the term "T cell receptor" or "TCR" refers to an endogenous or modified T cell receptor containing an extracellular antigen-binding domain that binds to a specific antigenic peptide bound to an MHC molecule. A TCR may contain TCRα and TCRβ polypeptide chains. A TCR may contain TCRγ and TCRδ polypeptide chains. A TCR can specifically bind to tumor antigens. "TCR-T" refers to T cells expressing a recombinant TCR. Expression of heterologous antigen receptors (such as heterologous TCRs or CARs) can alter the immunogenicity specificity of T cells, enabling them to recognize one or more tumor antigens present on the surface of cancer cells in an individual with cancer, or exhibiting improved recognition of one or more tumor antigens present on the surface of cancer cells in an individual with cancer.

[0075] As used herein, the term “cancer” refers to cells capable of autonomous growth. Examples of such cells include cells exhibiting an abnormal state or condition characterized by rapid proliferation of cell growth. The term implies the inclusion of cancerous growth, such as tumors; carcinogenic processes, metastatic tissues, and malignant transformations of cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. It also includes malignant tumors of various organ systems, such as the respiratory, cardiovascular, renal, reproductive, hematopoietic, nervous, hepatic, gastrointestinal, and endocrine systems; and adenocarcinomas, including most colon cancers, renal cell carcinomas, prostate and / or testicular tumors, non-small cell lung cancer, and small bowel cancer. “Naturally occurring” cancer includes any cancer that is not experimentally induced by implanting cancer cells into a subject, and includes, for example, spontaneously arising cancers, cancers caused by subject exposure to carcinogens, cancers caused by the insertion of transgenic oncogenes or the knockout of tumor suppressor genes, and cancers caused by infections (e.g., viral infections). The term “cancer” is generally accepted to refer to malignant tumors of epithelial or endocrine tissues. The term also includes carcinosarcoma, which comprises malignant tumors composed of both carcinomatous and sarcomatous tissue. "Adenocarcinoma" refers to cancer derived from glandular tissue or in which tumor cells form identifiable glandular structures. The term "sarcoma" is generally accepted as referring to a mesenchymal-derived malignant tumor. The term "hematopoietic neoplastic disorder" includes diseases involving proliferative / tumorous cells of hematopoietic origin. Hematopoietic neoplastic disorders can originate from myeloid, lymphoid, or erythroid lineages or their precursor cells.

[0076] As used herein, the term "subject" refers to an animal, human, or non-human being treated according to the methods disclosed herein. This disclosure envisions both veterinary and non-veterinary applications. Human subjects may be adults or adolescents (e.g., humans under the age of 18). In addition to humans, subjects include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Examples include, for instance, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other domesticated, farm, and zoo animals.

[0077] 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 pertains. This document describes the methods and materials used in this disclosure; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of any conflict, this specification, including the definitions, shall prevail.

[0078] Modified IL15 and related fusion proteins

[0079] This disclosure provides a modified IL-15 with reduced toxicity and an associated fusion protein containing the IL-15 sequence. The modified IL-15 and associated fusion protein can be trapped intracellularly, and the amount of secreted IL-15 is reduced, which can decrease toxicity. The modified IL-15 and associated fusion protein can lead to increased persistence of NK cells in vivo.

[0080] In one aspect, this disclosure provides modified IL-15 or related fusion proteins that can target certain locations within cells. The modified IL-15 or related fusion proteins may possess a calreticulin signal peptide sequence. The modified IL-15 or related fusion proteins may possess an endoplasmic reticulum (ER) preserved sequence. The modified IL-15 or related fusion proteins may possess a myristylated sequence. The modified IL-15 or related fusion proteins may possess an IL-15Rα sequence. The modified IL-15 or related fusion proteins may be co-expressed with engineered receptors (e.g., CARs or TCRs).

[0081] Modified IL-15 or related fusion proteins can enhance NK cell proliferation. Modified IL-15 or related fusion proteins can be used to improve the persistence and function of immune effector cells such as NK, T (αβ-T and δγ-T), Treg, or NKT cells. Modified IL-15 or related fusion proteins can be expressed in genetically modified immune cells such as CAR-T, CAR-NK, and CAR-NKT cells. Modified cells (e.g., modified CAR-NK cells) can be used to treat cancer and autoimmune diseases. In addition to immune cells, other cell types such as mesenchymal stem cells (MSCs) and hepatocytes can also be used to express modified IL-15 or related fusion proteins as described herein.

[0082] In one respect, this disclosure relates to various modified IL-15 or related fusion proteins. Recombinant IL-15 can be modified in various ways to alter its intracellular distribution and secretion levels. Modified IL-15 or related fusion proteins may contain a calreticulin signal peptide sequence that mediates membrane targeting of the ER. Modified IL-15 or related fusion proteins may contain an ER-retaining sequence that facilitates retention of the modified IL-15 or related fusion protein in the ER. Modified IL-15 or related fusion proteins may contain a myristylation sequence that facilitates retention of the modified IL-15 intracellularly. Modified IL-15 or related fusion proteins may contain a palmitoylation sequence that facilitates retention of the modified IL-15 intracellularly. Modified IL-15 or related fusion proteins may contain an isopreneation sequence that facilitates retention of the modified IL-15 intracellularly.

[0083] The modified IL-15 or related fusion proteins described herein may have the structures and / or sequences described in Tables 1 and 2.

[0084] Modified IL-15 or related fusion proteins may contain an IL-15 sequence. The IL-15 sequence may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 6, or may consist of an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 6. The IL-15 sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations (e.g., compared to SEQ ID NO: 6 or the entire wild-type IL-15). These mutations may be amino acid insertions, deletions or substitutions. Insertions, deletions and substitutions may be within the IL-15 sequence and / or at one or both ends of the IL-15 sequence.

[0085] Modified IL-15 or related fusion proteins may contain an IL-15 propeptide sequence. The IL-15 propeptide sequence may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 5, or may consist of an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 5. The IL-15 precursor sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations (e.g., compared to SEQ ID NO: 5). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the IL-15 precursor sequence and / or at one or both ends of the IL-15 precursor sequence.

[0086] The modified IL-15 or related fusion protein may contain a signal peptide sequence. The signal peptide sequence may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or may consist of at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4. The signal peptide sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations (e.g., compared to SEQ ID NO: 3 or SEQ ID NO: 4). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the signal peptide sequence and / or at one or both ends of the signal peptide sequence.

[0087] The modified IL-15 or related fusion protein may contain an ER-reserved sequence. The ER-reserved sequence may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13, or may consist of at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 13. The ER-retained sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations (e.g., compared to SEQ ID NO: 12 or SEQ ID NO: 13). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the ER-retained sequence and / or at one or both ends of the ER-retained sequence.

[0088] Modified IL-15 or related fusion proteins may contain an IL-15Rα sequence. The IL-15Rα sequence may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 11, or may consist of an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 11. The IL-15Rα sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations (e.g., compared to SEQ ID NO: 11). These mutations may be amino acid insertions, deletions or substitutions. Insertions, deletions and substitutions may be within the IL-15Rα sequence and / or at one or both ends of the IL-15Rα sequence.

[0089] The IL-15Rα Sushi domain is the region where trans-presented IL-15 binds with a high affinity to IL-15Rβ / γ. Modified IL-15 or related fusion proteins may contain an IL-15Rα sequence, wherein the IL-15Rα sequence contains or is composed of an IL-15Rα Sushi domain sequence. The IL-15Rα Sushi domain sequence may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 10, or may be composed of at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of SEQ ID NO: 10. The IL-15Rα Sushi domain sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., compared to SEQ ID NO: 10). These mutations can be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions can occur within the IL-15Rα Sushi domain sequence and / or at one or both ends of the IL-15Rα Sushi domain sequence.

[0090] The modified IL-15 or related fusion protein may contain a myristoylated sequence. The myristoylated sequence may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to any of SEQ ID NO: 14-16. The myristoylated sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., compared to any of SEQ ID NO: 14-16). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may occur within the myristoylated sequence and / or at one or both ends of the myristoylated sequence.

[0091] The modified IL-15 or related fusion protein may contain an adapter sequence. The adapter sequence may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 7-9, or may consist of an amino acid sequence that is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence of any one of SEQ ID NO: 7-9. The adapter sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations (e.g., compared to any one of SEQ ID NO: 7-9). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the adapter sequence and / or at one or both ends of the adapter sequence.

[0092] The modified IL-15 or related fusion protein may comprise or consist of the following from the N-terminus to the C-terminus: a signal peptide sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4), a linker sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 7-9), and an IL-15 sequence (e.g., a sequence identical to SEQ ID NO: 4). 6. At least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences to any one of SEQ ID NO: 7-9, a connector sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 7-9), and an ER-reserved sequence (e.g., a sequence identical to SEQ ID NO: 12 or SEQ ID NO: 10). 13. The modified IL-15 or related fusion protein may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 17. The modified IL-15 or related fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., compared to SEQ ID NO: 17). These mutations may be amino acid insertions, deletions, or substitutions. Insertion, deletion, and substitution can occur within the modified IL-15 or related fusion protein, and / or at one or both ends of the modified IL-15 or related fusion protein.

[0093] The modified IL-15 or related fusion protein may comprise or consist of the following from the N-terminus to the C-terminus: a signal peptide sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4), an IL-15 sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6), and an adapter sequence (e.g., a sequence identical to SEQ ID NO: 4). The sequence is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 10 or SEQ ID NO: 11), the IL-15Rα sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 12 or SEQ ID NO: 13), and the ER-reserved sequence (e.g., a sequence identical to any one of SEQ ID NO: 12). 13. The modified IL-15 or related fusion protein may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 18. The modified IL-15 or related fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations (e.g., compared to SEQ ID NO: 18). These mutations may be amino acid insertions, deletions, or substitutions. Insertion, deletion, and substitution can occur within the modified IL-15 or related fusion protein, and / or at one or both ends of the modified IL-15 or related fusion protein.

[0094] The modified IL-15 or related fusion protein may comprise or consist of the following from the N-terminus to the C-terminus: a signal peptide (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4), a linker sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 7-9), and an IL-15 sequence (e.g., a sequence identical to SEQ ID NO: 4). 6. At least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences to any one of SEQ ID NO: 7-9; 6. Connector sequences (e.g., sequences at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences to any one of SEQ ID NO: 7-9); IL-15Rα sequences (e.g., sequences identical to any one of SEQ ID NO: 10 or SEQ ID NO: 10). 11. At least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences) and ER-reserved sequences (e.g., sequences that are at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12 or SEQ ID NO: 13). The modified IL-15 or related fusion protein may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO: 19.The modified IL-15 or related fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations (e.g., compared to SEQ ID NO: 19). These mutations may be amino acid insertions, deletions or substitutions. Insertions, deletions and substitutions may be within the modified IL-15 or related fusion protein, and / or at one or both ends of the modified IL-15 or related fusion protein.

[0095] The modified IL-15 or related fusion protein may comprise or consist of the following from the N-terminus to the C-terminus: a signal peptide (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4), a linker sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 7-9), and an IL-15 sequence (e.g., a sequence identical to SEQ ID NO: 4). 6. At least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences to any one of SEQ ID NO: 7-9; 6. Connector sequences (e.g., sequences at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences to any one of SEQ ID NO: 7-9); IL-15Rα sequences (e.g., sequences identical to any one of SEQ ID NO: 10 or SEQ ID NO: 10). 11. At least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences) and ER-reserved sequences (e.g., sequences that are at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12 or SEQ ID NO: 13). The modified IL-15 or related fusion protein may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO: 20.The modified IL-15 or related fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations (e.g., compared to SEQ ID NO: 20). These mutations may be amino acid insertions, deletions or substitutions. Insertions, deletions and substitutions may be within the modified IL-15 or related fusion protein and / or at one or both ends of the modified IL-15 or related fusion protein.

[0096] The modified IL-15 or related fusion protein may comprise or consist of the following from the N-terminus to the C-terminus: a signal peptide (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4), a myristylated sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 14-16), and an IL-15 sequence (e.g., a sequence identical to SEQ ID NO: 3 or SEQ ID NO: 4). 6. At least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences to any one of SEQ ID NO: 7-9, a connector sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 7-9), and an IL-15Rα sequence (e.g., a sequence identical to any one of SEQ ID NO: 10 or SEQ ID NO: 10). 11. The modified IL-15 or related fusion protein may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 21. The modified IL-15 or related fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations (e.g., compared to SEQ ID NO: 21). These mutations may be amino acid insertions, deletions, or substitutions. Insertion, deletion, and substitution can occur within the modified IL-15 or related fusion protein, and / or at one or both ends of the modified IL-15 or related fusion protein.

[0097] The modified IL-15 or related fusion protein may contain or consist of the following from the N-terminus to the C-terminus: a myristylated sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 14-16), an IL-15 sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6), and a linker sequence (e.g., a sequence identical to any one of SEQ ID NO: 14-16). The sequence of any one of 7-9 is at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 10 or SEQ ID NO: 11. The modified IL-15 or related fusion protein may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to that of SEQ ID NO: 22. The modified IL-15 or related fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations (e.g., compared to SEQ ID NO: 22). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may occur within the modified IL-15 or related fusion protein and / or at one or both ends of the modified IL-15 or related fusion protein.

[0098] The modified IL-15 or related fusion protein may comprise or consist of the following from the N-terminus to the C-terminus: a signal peptide (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4), a myristylated sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 14-16), and an IL-15 sequence (e.g., a sequence identical to SEQ ID NO: 3 or SEQ ID NO: 4). 6. At least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences to any one of SEQ ID NO: 7-9, a connector sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 7-9), and an IL-15Rα sequence (e.g., a sequence identical to any one of SEQ ID NO: 10 or SEQ ID NO: 10). 11. The modified IL-15 or related fusion protein may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 23. The modified IL-15 or related fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations (e.g., compared to SEQ ID NO: 23). These mutations may be amino acid insertions, deletions, or substitutions. Insertion, deletion, and substitution can occur within the modified IL-15 or related fusion protein, and / or at one or both ends of the modified IL-15 or related fusion protein.

[0099] The modified IL-15 or related fusion protein may comprise or consist of the following from the N-terminus to the C-terminus: a signal peptide (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4), a myristylated sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 14-16), and an IL-15 sequence (e.g., a sequence identical to SEQ ID NO: 3 or SEQ ID NO: 4). 6. At least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences to any one of SEQ ID NO: 7-9, a connector sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 7-9), and an IL-15Rα sequence (e.g., a sequence identical to any one of SEQ ID NO: 10 or SEQ ID NO: 10). 11. The modified IL-15 or related fusion protein may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 24. The modified IL-15 or related fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations (e.g., compared to SEQ ID NO: 24). These mutations may be amino acid insertions, deletions, or substitutions. Insertion, deletion, and substitution can occur within the modified IL-15 or related fusion protein, and / or at one or both ends of the modified IL-15 or related fusion protein.

[0100] The modified IL-15 or related fusion protein may contain or consist of the following from the N-terminus to the C-terminus: an IL-15 sequence (e.g., at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 6). The modified IL-15 or related fusion protein may contain an amino acid sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 25. The modified IL-15 or related fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations (e.g., compared to SEQ ID NO: 25). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the modified IL-15 or related fusion protein, and / or at one or both ends of the modified IL-15 or related fusion protein.

[0101] The modified IL-15 or related fusion protein may comprise or consist of the following from the N-terminus to the C-terminus: a signal peptide (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4), an IL-15 propeptide sequence (e.g., a sequence at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5), and an IL-15 sequence (e.g., a sequence identical to SEQ ID NO: 4). 6. The modified IL-15 or related fusion protein may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to the amino acid sequence of SEQ ID NO: 26. The modified IL-15 or related fusion protein may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., compared to SEQ ID NO: 26). These mutations may be amino acid insertions, deletions, or substitutions. Insertion, deletion, and substitution can occur within the modified IL-15 or related fusion protein, and / or at one or both ends of the modified IL-15 or related fusion protein.

[0102] Modified IL-15 or related fusion proteins can be captured in the ER of cells. Modified IL-15 or related fusion proteins can be captured within cells (e.g., the cytoplasm).

[0103] NK cell therapy

[0104] Chimeric antigen receptors (CARs) that specifically redirect autologous T cells to lymphomas have yielded promising clinical results. Nevertheless, CAR-modified T cells still have several limitations. Generating autologous products for each individual subject is logistically cumbersome and limits widespread clinical use. The manufacture of CAR T cells typically takes several weeks, which is impractical for subjects with rapidly progressing disease. Furthermore, generating clinically relevant doses of CAR T cells is not always possible for subjects who have received substantial prior therapy and often have lymphopenia. Previously collected allogeneic products can overcome these limitations; however, allogeneic T cells (even if HLA-matched) carry the risk of graft-versus-host disease (GVHD) mediated by their native αβ T cell receptors.

[0105] Natural killer (NK) cells offer an attractive alternative to T cells for CAR engineering. NK cells do not induce GVHD, thus providing an opportunity to produce off-the-shelf products for immediate clinical use. Furthermore, because engineered NK cells should retain their full array of native receptors, they have the potential to induce cytotoxicity through mechanisms other than those determined by CAR specificity, which in principle could reduce the risk of relapse mediated by loss of CAR-target antigens, as reported for CAR-T cell therapies.

[0106] Functional NK cells can be derived from several sources. Autologous NK cells can be reproducibly generated in vitro. Umbilical cord blood (CB) is an readily available source of allogeneic NK cells, offering significant advantages. CB is available as a ready-to-use cryopreservation product, an advantage supported by methods for generating large quantities of highly functional NK cells ex vivo from frozen CB units. The generation of CAR-transduced NK cells from frozen CB units stored in large global CB banks promises extensive scalability, which is not replicable in individual adult donors requiring screening and leukocyte separation. However, a major drawback of NK cells is their lack of persistence after adoptive transfer in the absence of cytokine support. Finally, CAR-engineered NK cells may also cause potentially serious toxicities such as cytokine release syndrome (CRS) or extratumor / on-target toxicity, as reported in CAR-T cells.

[0107] Mature NK cells exhibit short lifespans and poor in vivo persistence in both humans and mice. While recent data support the existence of long-lived memory NK cells in mice (and possibly in humans), the lack of reliable and stable biomarkers (or sets of biomarkers) to define memory NK cells hinders their selection for immunotherapy. This poses a significant limitation to their use in adoptive therapy, as the in vivo persistence of effector cells is crucial for sustained clinical responses. IL-15 is a cytokine that drives NK cell proliferation and persistence. Ectopic production of IL-15 leads to more robust NK cell activation with enhanced in vivo proliferation, persistence, and antitumor activity compared to CAR-transduced NK cells lacking IL-15. Although CAR-transduced NK cells lacking IL-15 can mediate antitumor responses, the effects are transient, further highlighting the importance of the in vivo persistence of CAR-expressing NK cells for effective and durable antitumor immunity.

[0108] In cancer patients, NK cells frequently exhibit impaired function. Therefore, a major strategy in immunotherapy aims to enhance NK cell-mediated antitumor activity in vivo. One approach is based on in vivo administration of cytokines that determine NK cell activation, differentiation, and expansion, such as IL-2 and IL-15. IL-2 administration was approved in the 1990s for treating patients with metastatic RCC and melanoma. Two major obstacles in IL-2-based therapies are dose-related toxicity (primarily vascular leakage) and the induction of T-reg cell activation and expansion, leading to suppression of NK cell function. Recently, IL-2 variants with lower affinity for the IL-2Rα subunit (highly expressed by Treg cells) have been engineered. Furthermore, pegylated IL-2 (also known as NKTR-214), which binds to CD122 (IL-2Rβ) expressed by both T cells and NK cells, preferentially enhances these cells and their antitumor responses.

[0109] Signal peptide (SP)

[0110] Cleavable endoplasmic reticulum (ER) signal peptides (SPs) and other uncleavable signal sequences target the ER for approximately one-quarter of the human proteome. These short peptides, mostly located at the N-terminus of proteins, exhibit high diversity. For most ER-targeting proteins, the interaction between the signal sequence and various ER targeting and translocation mechanisms, such as signal recognition particles (SRPs), the protein conduction channel Sec61, and the signal peptidase complex (SPC), determines the protein's target location and provides translocation fidelity.

[0111] The secretion pathway is a protein transport highway utilized by more than a quarter of the human proteome. Soluble secretory proteins such as antibodies and protein hormones depend on this pathway. This pathway also delivers transmembrane proteins (TMPs) to the endoplasmic reticulum (ER), its downstream organelles such as the Golgi apparatus, and the plasma membrane.

[0112] All secreted proteins are translated by cytoplasmic ribosomes and must first be targeted to the ER membrane in a co-translational or post-translational manner early in their life, before being transported across (or inserted into) the ER membrane. A complex network of cytoplasmic and ER membrane-resident macromolecules facilitates and assists ER targeting and translocation.

[0113] SP is characterized by a trigonal structure: (i) a normally positively charged N-terminal “n-region” facing the cytosol; (ii) a short hydrophobic core—usually between 7 and 15 amino acids, but no longer than 18–20 amino acids—called the “h-region”; and (iii) a polar luminal C-terminal “c-region” containing easily broken bonds and which must be occupied by short hydrophobic residues at positions -1 and -3 relative to the cleavage site. Initially, SP is inserted into the ER membrane with the N-terminus facing the cytosol (Nin) and the mature sequence facing the organelle lumen (Cout).

[0114] This disclosure provides a signal peptide sequence. The signal peptide sequence may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to those of SEQ ID NO: 3 or SEQ ID NO: 4. The signal peptide sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., compared to SEQ ID NO: 3 or SEQ ID NO: 4). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may occur within the signal peptide sequence and / or at one or both ends of the signal peptide sequence.

[0115] Modified IL-15 or related fusion proteins may contain signal peptide sequences.

[0116] Calreticulin (CALR) is a 46 kDa Ca2+-binding protein and molecular chaperone in the ER lumen. This protein contains an N-terminal cleavable signal peptide sequence that directs it to the ER.

[0117] The signal peptide sequence may be a calreticulin signal peptide (CALRSP) sequence. The calreticulin signal peptide sequence may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to the amino acid sequence of SEQ ID NO: 3. The signal peptide sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., compared to SEQ ID NO: 3). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may occur within the signal peptide sequence and / or at one or both ends of the signal peptide sequence.

[0118] Modified IL-15 or related fusion proteins may contain a calreticulin signal peptide sequence. Compared to other signal peptides, the combination of the calreticulin signal peptide sequence with the ER retention sequence can help to more effectively retain modified IL-15 or related fusion proteins in the ER.

[0119] ER preserved sequence

[0120] After co-translation into the ER lumen or ER membrane, most proteins are transported downstream via the Golgi apparatus along the secretory pathway, while a few protein types are retained in the ER. Peptide retention in the ER is either signal-independent or dependent on specific retention signals encoded by the peptide's primary sequence. The first class, newly synthesized peptides that cannot reach their final conformation, are retained in the ER, where such quality control typically leads to their degradation. The second class, ER-resident proteins, escape the bulkflow of secretion due to the presence of specific N-terminal or C-terminal signals that interact with the integrated membrane or soluble receptors. ER retention of soluble proteins is mediated by KDEL, HDEL, or related sequences.

[0121] In order to be retained in the ER and thus diverted from the overall flow of secretory proteins, ER-resident proteins typically require specific retention or recovery signals. By comparing the polypeptide sequences of many soluble proteins residing in the ER lumen, a common tetrapeptide H / KDEL has been identified at their C-terminus. Furthermore, the C-terminal tetrapeptide KDEL has been shown to act as an ER retention signal in animal cells. Numerous laboratories have demonstrated that adding KDEL to the C-terminus of various secretory proteins results in the retention of these proteins in the ER of animal cells, or at least significantly delays their downstream transport in the secretory pathway.

[0122] This disclosure provides an ER-reserved sequence. The ER-reserved sequence may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to that of SEQ ID NO: 12 or SEQ ID NO: 13. The ER-reserved sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., compared to SEQ ID NO: 12 or SEQ ID NO: 13). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the ER-reserved sequence and / or at one or both ends of the ER-reserved sequence.

[0123] Modified IL-15 or related fusion proteins may contain ER-retaining sequences, which help retain the modified IL-15 or related fusion proteins in the ER. Modified IL-15 or related fusion proteins may contain calreticulin signal peptide sequences and KDEL, which helps retain the modified IL-15 or related fusion proteins in the ER more effectively compared to other signal peptides and ER-retaining sequences.

[0124] Membrane targeting sequence

[0125] Membrane-targeting sequences facilitate the transport of fusion proteins to the cell surface membrane, and some or other sequences can encode the binding of the fusion protein to the cell surface membrane. Such sequences include, but are not limited to, myristylation targeting sequences, palmitoylation targeting sequences, and isopreneation sequences (i.e., farnesylation, geranylation-geranylation, CAAX box).

[0126] Modified IL-15 or related fusion proteins may contain cell membrane targeting sequences that help retain the modified IL-15 or related fusion proteins within the cell. Cell membrane targeting sequences may be selected from the group consisting of: myristylation sequences, palmitoylation sequences, and isopreneation sequences.

[0127] Myristylated sequence

[0128] Covalent linkage between fatty acids and proteins is now a widely accepted form of protein modification. Many fatty acid-acylated proteins play key roles in regulating cellular structure and function. The two most common forms of protein fatty acylation are modification with myristate (a 14-carbon saturated fatty acid) and palmitate (a 16-carbon saturated fatty acid). The enzymatic nature of the myristoylation reaction is well-known. Proteins destined to become myristoylated begin with the sequence Met-Gly. The initiating methionine is removed by methionine aminopeptidase via co-translation, and the myristate is linked to Gly-2 via an amide bond. N-Myristoylation is catalyzed by N-myristyltransferase.

[0129] Myristylation involves labeling the N-terminal α-group of cysteine ​​or glycine residues via an amide bond, or directly or indirectly labeling lysine and cysteine ​​side chains via glycerothioesters and ester bonds. Before transfer to proteins, myristate esters must be activated to myristoyl-CoA in eukaryotes or to derivatives such as phosphatidylethanolamine in bacteria. Myristate esters typically act as molecular anchors, allowing labeled proteins to be targeted to membranes and travel across the internal membrane network in eukaryotes.

[0130] N-Myristyltransferase (NMT) is a 50-60 kDa monomeric enzyme that catalyzes the transfer of myristate from myristoyl-CoA to suitable peptide and protein substrates. To date, nearly a dozen NMTs from fungal and mammalian sources have been identified. Studies of NMT from the yeast *Saccharomyces cerevisiae* have shown that the catalytic cycle exhibits the following BiBi reaction mechanism: (1) myristoyl-CoA binds to NMT; (2) the peptide substrate binds to NMT; (3) myristate is transferred to the N-terminal glycine of the peptide; (4) CoA is released from the enzyme; and (5) the myristoyl peptide is released. In cells, N-myristoylation is a co-translational process that occurs while the nascent polypeptide chain is still attached to the ribosome.

[0131] The common sequence for NMT protein substrates is: Met-Gly-XXX-Ser / Thr-. The initiating Met is removed by methionine aminopeptidase during translation, and Gly-2 is replaced by the N-terminal amino acid. The requirement for Gly at the N-terminus is absolute; no other amino acid can substitute. However, not all proteins with an N-terminal glycine are N-myristoylated, and their ability to be recognized by NMT depends on the downstream amino acid sequence. Typically, serine or threonine is preferably at position 6, and lysine or arginine is preferably at positions 7 and / or 8.

[0132] This disclosure provides a myristoylated sequence. The myristoylated sequence may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to any of SEQ ID NO: 14-16. The myristoylated sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., compared to any of SEQ ID NO: 14-16). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the myristoylated sequence and / or at one or both ends of the myristoylated sequence.

[0133] For some proteins, the myristic ester moiety not only provides membrane binding but also membrane targeting functions. For example, when the N-terminal sequence of Src or Gag is attached to a soluble protein, the chimera exhibits specific localization to the plasma membrane. Conversely, mutations at the N-myristication site in yeast Gpa 1p protein or HIV-1 Gag redirect the protein to the intracellular membrane, implying that myristic ester is involved in plasma membrane targeting. In Mason-Pfizer monkey virus, the virion core assembles in the cytosol, and myristication of Gag is essential for intracellular transport to the plasma membrane.

[0134] Modified IL-15 or related fusion proteins may contain myristylated sequences that bind to the cell surface membrane, which helps retain the modified IL-15 or related fusion proteins inside the cell.

[0135] Palmitylation sequence

[0136] Palmitoylation is the covalent linking of fatty acids such as palmitic acid to cysteine ​​(and less commonly, serine and threonine) residues in proteins that are typically membrane proteins. The precise function of palmitoylation depends on the specific protein being modified. Palmitoylation enhances the hydrophobicity of proteins and facilitates their membrane association.

[0137] Modified IL-15 or related fusion proteins may contain palmitoylated sequences that bind to the cell surface membrane, which helps retain the modified IL-15 or related fusion proteins inside the cell.

[0138] Isoprendylation sequence

[0139] Isoprenelation is the covalent linkage of a lipid consisting of three (farnesyl) or four (geranyl-geranyl) isoprene units to a free thiol on the cysteine ​​side chain at or near the C-terminus of a protein. Protein isoprenelation leads to increased hydrophobicity, which typically results in increased affinity for membranes.

[0140] Modified IL-15 or related fusion proteins may contain isoprene-encapsulated sequences that bind to the cell surface membrane, which helps retain the modified IL-15 or related fusion proteins within the cell.

[0141] Engineered receptors (e.g., CAR and TCR)

[0142] This disclosure provides cells (e.g., immune cells) containing engineered receptors and modified IL-15 or related fusion proteins. The engineered receptor may include an extracellular ligand-binding domain or an extracellular antigen-binding domain, and optionally an intracellular signaling domain. Exemplary engineered receptors include, but are not limited to, CAR, engineered TCR, and TAC receptors. The engineered receptor may include an extracellular domain, a transmembrane region, and an intracellular signaling domain, the extracellular domain including an antigen-binding domain that specifically binds to an antigen (e.g., a tumor antigen). The intracellular signaling domain may include a primary intracellular signaling domain and / or a co-stimulatory signaling domain. The intracellular signaling domain may include the intracellular signaling domain of a TCR co-receptor. The engineered receptor may be encoded by a heteropolynucleotide operatively linked to a promoter, such as a constitutive or inducible promoter.

[0143] Engineered receptors may include one or more specific binding domains that target at least one tumor antigen, and one or more intracellular effector domains, such as one or more primary intracellular signaling domains and / or co-stimulatory signaling domains.

[0144] Engineered receptors can be chimeric antigen receptors (CARs). Many chimeric antigen receptors are known in the art and can be adapted for modified cells containing the modified IL-15 or related fusion proteins described herein. CARs can also be constructed to be specific for any cell surface marker by using, for example, antigen-binding fragments of antibody molecules or variable domains of antibodies.

[0145] The CAR disclosed herein comprises an extracellular domain, a transmembrane region, and an intracellular signaling domain, the extracellular domain including at least one antigen-binding domain that specifically binds at least one tumor antigen. The intracellular signaling domain can generate signals that promote immune effector functions in CAR-containing cells (e.g., CAR-T cells). "Immune effector function or immune effector response" refers to a function or response, such as that of immune effector cells, that enhances or promotes the immune attack of target cells. For example, an immune effector function or response can refer to the property of T or NK cells to promote the killing or inhibition of target cell growth or proliferation. Examples of immune effector functions, such as in CAR-T cells, include cytolytic activities (such as antibody-dependent cytotoxicity, or ADCC) and helper activities (such as cytokine secretion). CARs may have an intracellular signaling domain that has attenuated immune effector functions. Compared to CARs having full-length and wild-type CD3ζ and optionally one or more co-stimulatory signaling domains, this CAR may have an intracellular signaling domain having no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less of an immune effector function (such as cell lysis against target cells). The intracellular signaling domain can generate signals that promote the proliferation and / or survival of CAR-containing cells. The CAR may contain one or more intracellular signaling domains selected from CD28, CD137, CD3, CD27, CD40, ICOS, GITR, and OX40. The signaling domain of a naturally occurring molecule may comprise the entire intracellular (or cytoplasmic) portion of the molecule or a fragment or derivative thereof, or the entire native intracellular signaling domain.

[0146] The intracellular signaling domains of CARs can include primary intracellular signaling domains. A "primary intracellular signaling domain" refers to an intracellular signaling sequence that acts in a stimulatory manner to induce the function of immune effectors. Primary intracellular signaling domains can contain signaling motifs known as immune receptor tyrosine-based activation motifs or ITAMs. Primary intracellular signaling domains can contain functional signaling domains of proteins selected from the following groups: CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγRIIa, DAP10, and DAP12. Primary intracellular signal transduction domains may contain nonfunctional or attenuated signal transduction domains of proteins selected from the group consisting of: CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγR IIa, DAP10, and DAP12. Nonfunctional or attenuated signal transduction domains may be point-mutated, insertion- or deletion-mutated signal transduction domains that attenuate or eliminate one or more immune effector functions, such as cytolytic or accessory activities, including antibody-dependent cytotoxicity (ADCC). CARs may contain a nonfunctional or attenuated CD3ζ (i.e., CD3ζ or CD3z) signal transduction domain. Intracellular signal transduction domains may lack primary intracellular signal transduction domains. Compared to CARs with the same construct but with wild-type primary intracellular signaling domains, the weakened primary intracellular signaling domains can induce immune effector functions (such as cell lysis against target cells) of no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less.

[0147] The intracellular signaling domain of a CAR may contain one or more (such as any one of 1, 2, 3 or more) co-stimulatory signaling domains. A “co-stimulatory signaling domain” can be the intracellular portion of a co-stimulatory molecule. The term “co-stimulatory molecule” refers to a homologous binding partner on an immune cell (such as a T cell) that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response of the immune cell, such as, but not limited to, proliferation and survival. Co-stimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that contribute to an efficient immune response. Co-stimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activated NK cell receptors. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Other examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, L... FA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and ligands that specifically bind to CD83.

[0148] CARs may contain a single costimulatory signaling domain. CARs may contain two or more costimulatory signaling domains. Intracellular signaling domains may contain a functional primary intracellular signaling domain and one or more costimulatory signaling domains. CARs may lack a functional primary intracellular signaling domain (such as CD3ζ). CARs may contain an intracellular signaling domain consisting of one or more costimulatory signaling domains or substantially consisting of one or more costimulatory signaling domains. CARs may contain an intracellular signaling domain consisting of a non-functional or attenuated primary intracellular signaling domain (such as mutant CD3ζ) and one or more costimulatory signaling domains, or substantially consisting of a non-functional or attenuated primary intracellular signaling domain (such as mutant CD3ζ) and one or more costimulatory signaling domains. After the antigen-binding domain binds to a tumor antigen, the costimulatory signaling domains of the CAR can transduce signals to enhance the proliferation, survival, and differentiation of CAR-equipped modified immune cells (such as T cells) and inhibit activation-induced cell death. One or more co-stimulatory signal transduction domains can be derived from one or more molecules selected from the group consisting of: CD27, CD28, 4-1BB (i.e., CD137), OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0149] The intracellular signal transduction domain of a CAR may include a co-stimulatory signal transduction domain derived from CD28. The intracellular signal transduction domain may include both the intracellular signal transduction domain of CD3ζ and the co-stimulatory signal transduction domain of CD28. The intracellular signal transduction domain in the chimeric receptor of this application may include a co-stimulatory signal transduction domain derived from 4-1BB (i.e., CD137). The intracellular signal transduction domain may include both the intracellular signal transduction domain of CD3ζ and the co-stimulatory signal transduction domain of 4-1BB.

[0150] The intracellular signal transduction domains of CARs may include a co-stimulatory signal transduction domain of CD28 and a co-stimulatory signal transduction domain of 4-1BB. The intracellular signal transduction domains may also include the intracellular signal transduction domain of CD3ζ, the co-stimulatory signal transduction domain of CD28, and the co-stimulatory signal transduction domain of 4-1BB. Alternatively, the intracellular signal transduction domains may comprise a polypeptide containing, from N-terminus to C-terminus, the co-stimulatory signal transduction domain of CD28, the co-stimulatory signal transduction domain of 4-1BB, and the intracellular signal transduction domain of CD3ζ.

[0151] The antigen-binding domain of a CAR can be an antibody or antibody fragment, such as scFv, Fv, Fab, (Fab')2, single-domain antibody (sdAb), or V. H The H domain. The antigen-binding domain of a CAR may contain an extracellular portion of a ligand or receptor that specifically binds to a tumor antigen. CARs can be monospecific, bispecific, or multispecific. The antigen-binding domain of a CAR can specifically bind to a single tumor antigen. The antigen-binding domain of a CAR can bind to two or more tumor antigens.

[0152] Tumor antigens can be selected from the following groups: CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, CD20, CD22, CD30, CD33, CD38, CEA, CS1, CD138, CD123 / IL3Rα, c-Met, gp100, MUC1, IGF-I receptor, EpCAM, CEA, EGFR (such as EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, phosphatidylinositol proteoglycan 3 (GPC3), guanylate cyclase 2C (GCC), DLL3, dentin 18.2, dentin 6, glycolipid F77, PD-L1, PD-L2, and other clinically significant tumor antigens, as well as combinations thereof. Tumor antigens can be derived from intracellular proteins of tumor cells. Tumor antigens can be expressed on the surface of tumor cells. Many TCRs specific to tumor antigens, including tumor-associated antigens, have been described, such as the NY-ESO-1 cancer-testis antigen, the p53 tumor suppressor antigen, and TCRs targeting tumor antigens in melanoma (e.g., MARTI, gp 100), leukemia (e.g., WT1, minor histocompatibility antigen), and breast cancer (e.g., HER2, NY-BR1).

[0153] Many CARs targeting different tumor antigens have been widely disclosed in the art, such as CD19 CARs or BCMACARs. The extracellular antigen-binding domain of a CD19 CAR may be or contain a CD19-binding fragment (e.g., FMC63, SJ25C1, or those disclosed in various patents such as WO 2022 / 012683). BCMACARs have also been well described, with related patents including but not limited to WO 2016 / 014789, WO 2016 / 014565, WO 2013 / 154760, WO 2018 / 028647, and WO2021 / 121228.

[0154] The extracellular antigen-binding domain of a BCMA (B cell maturation antigen) CAR may be or may contain a BCMA-binding fragment. The BCMA-binding fragment may bind to one or more epitopes on BCMA. A BCMA CAR may be a bivalent CAR containing two anti-BCMA sdAbs targeting different BCMA epitopes.

[0155] The transmembrane region of a CAR may contain transmembrane regions selected from the following: α, β, or ζ chains of the T cell receptor; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154; KIRDS2, OX40, CD2, CD27; LFA-1 (CD11a, CD18); ICOS (CD278); 4-1BB (CD137); GITR; CD40; BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 (KLRF1); CD160; CD19; IL-2Rβ; IL-2Rγ; IL-7R. a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITG AX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT Transmembrane regions of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C. Transmembrane regions of CARs can be CD4, CD3, CD8α, or CD28 transmembrane regions. CAR transmembrane regions may include CD8α transmembrane regions.

[0156] Extracellular domains can connect to transmembrane regions via hinge regions. Hinge regions can contain the hinge regions of CD8α.

[0157] CARs can contain signal peptides (SPs), such as the CD8α signal peptide.

[0158] CARs can be BCMA CARs. Various antigen-binding domain sequences can be used as antigen-binding domains for CARs. A BCMA CAR can contain, from the N-terminus to the C-terminus, an anti-BCMA antibody, a CD8α hinge region, a CD8α transmembrane domain, a 4-1BB co-stimulatory signal transduction domain, and a CD3ζ intracellular signal transduction domain.

[0159] The BCMA CAR can be composed of the following from the N-terminus to the C-terminus: CD8α signal peptide, anti-BCMA binding protein sequence, CD8α hinge region, CD8α transmembrane region, 4-1BB co-stimulatory signal transduction domain and CD3ζ intracellular signal transduction domain. The BCMA CAR may comprise a first VHH antibody portion and a second VHH antibody portion. The first VHH antibody portion comprises: CDR1, which comprises the amino acid sequence of SEQ ID NO: 29 or a variant thereof comprising up to about 3 amino acid substitutions; CDR2, which comprises the amino acid sequence of SEQ ID NO: 30 or a variant thereof comprising up to about 3 amino acid substitutions; and CDR3, which comprises the amino acid sequence of SEQ ID NO: 31 or a variant thereof comprising up to about 3 amino acid substitutions. The second VHH antibody portion comprises: CDR1, which comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof comprising up to about 3 amino acid substitutions; CDR2, which comprises the amino acid sequence of SEQ ID NO: 33 or a variant thereof comprising up to about 3 amino acid substitutions; and CDR3, which comprises the amino acid sequence of SEQ ID NO: 34 or a variant thereof comprising up to about 3 amino acid substitutions. The CDR sequences may be determined according to a well-known numbering system. In some embodiments, the CDRs are numbered according to Kabat.

[0160] The BCMA CAR may comprise a first VHH antibody portion and a second VHH antibody portion. The first VHH antibody portion comprises the amino acid sequence of SEQ ID NO: 27 (i.e., BCMA 269A37948 VHH) or an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 27. The second VHH antibody portion comprises the amino acid sequence of SEQ ID NO: 28 (i.e., BCMA 269AS34822 VHH) or an amino acid sequence having at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 28.

[0161] The BCMA CAR may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequence to that of SEQ ID NO: 1. The BCMA CAR may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., compared to SEQ ID NO: 1). These mutations may be amino acid insertions, deletions, or substitutions. Insertions, deletions, and substitutions may be within the BCMA CAR and / or at one or both ends of the BCMA CAR. The CAR may specifically bind to BCMA-positive tumor cells (e.g., H929 cells).

[0162] The BCMA CAR can be co-expressed with modified IL-15 or an associated fusion protein. The BCMA CAR can be linked to the modified IL-15 or an associated fusion protein via a P2A linker. The BCMA CAR may contain at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to the sequence in SEQ ID NO: 2.

[0163] This disclosure provides vectors encoding BCMA CAR. This disclosure provides vectors simultaneously encoding BCMA CAR and modified IL-15 or related fusion proteins. This disclosure provides vectors simultaneously encoding BCMA CAR and modified IL-15 or related fusion proteins linked via a P2A linker. The vector may be a lentiviral vector.

[0164] Engineered receptors can be modified T-cell receptors. Engineered TCRs can be specific for tumor antigens. Tumor antigens can be selected from the following groups: CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, CD20, CD22, CD30, CD33, CD38, CEA, CS1, CD138, CD123 / IL3Rα, c-Met, gp100, MUC1, IGF-I receptor, EpCAM, CEA, EGFR (such as EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, phosphatidylinositol proteoglycan 3 (GPC3), guanylate cyclase 2C (GCC), DLL3, duracin 18.2, duracin 6, glycolipid F77, PD-L1, PD-L2, and other clinically significant tumor antigens, as well as combinations thereof. Tumor antigens can be derived from intracellular proteins of tumor cells. Tumor antigens can be expressed on the surface of tumor cells. Many tumor receptor receptors (TCRs) specific to tumor antigens, including tumor-associated antigens (TAAs), have been described, including, for example, the NY-ESO-1 cancer-testis antigen, the p53 tumor suppressor antigen, and TCRs targeting tumor antigens in melanoma (e.g., MARTI, gp 100), leukemia (e.g., WT1, minor histocompatibility antigen), and breast cancer (e.g., HER2, NY-BR1). Any TCR known in the art can be used. TCRs can have enhanced affinity for tumor antigens. Exemplary TCRs and methods for introducing TCRs into immune cells have been described, for example, in U.S. Patent No. 5,830,755 and Kessels et al. Immunotherapy through TCR gene transfer. Nat. Immunol. 2, 957-961 (2001), which are incorporated herein by reference in their entirety.

[0165] The TCR receptor complex is an octamer formed by the variable TCR receptor α and β chains (or γ and δ chains in the case of γδ T cells) with three dimeric signaling modules: CD3δ / ε, CD3γ / ε, and CD247 (the CD3ζ chain of the T cell surface glycoprotein) ζ / ζ or ζ / η. Ionizable residues in the transmembrane region of each subunit form an interacting polar network that holds the complex together. The TCR complex functions to activate the signal transduction cascade in T cells.

[0166] Engineered receptors can be engineered TCRs comprising one or more T-cell receptor (TCR) fusion proteins (TFPs). Exemplary TFPs have been described, for example, in US 20170166622 A1, which is incorporated herein by reference in its entirety. A TFP may comprise an extracellular domain of a TCR subunit comprising an extracellular domain or a portion thereof of a protein selected from the group consisting of: a TCRα chain, a TCRβ chain, a CD3εTCR subunit, a CD3γTCR subunit, a CD3δTCR subunit, a functional fragment thereof, and an amino acid sequence having at least one but no more than 20 modified amino acids. A TFP may comprise a transmembrane region comprising a transmembrane region of a protein selected from the group consisting of: a TCRα chain, a TCRβ chain, a CD3εTCR subunit, a CD3γTCR subunit, a CD3δTCR subunit, a functional fragment thereof, and an amino acid sequence having at least one but no more than 20 modified amino acids. TFP may contain transmembrane regions comprising transmembrane regions of proteins selected from the group consisting of: TCRα chain, TCRβ chain, TCRζ chain, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, their functional fragments, and amino acid sequences having at least one but no more than 20 modifications.

[0167] Engineered receptors can be T-cell antigen-coupled device (TAC) receptors. An exemplary TAC receptor has been described, for example, in US 20160368964 A1, which is incorporated herein by reference. A TAC may comprise an antigen-binding domain, a TCR-binding domain that specifically binds to proteins associated with the TCR complex, and a T-cell receptor signaling domain. The antigen-binding domain may be an antibody fragment that specifically binds to a tumor antigen, such as scFv or VHH. The antigen-binding domain may be a engineered ankyrin repeat (DARPin) polypeptide. Tumor antigens can be selected from the following groups: CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, CD20, CD22, CD30, CD33, CD38, CEA, CS1, CD138, CD123 / IL3Rα, c-Met, gp100, MUC1, IGF-I receptor, EpCAM, CEA, EGFR (such as EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, WT1, phosphatidylinositol proteoglycan 3 (GPC3), guanylate cyclase 2C (GCC), DLL3, dentin 18.2, dentin 6, glycolipid F77, PD-L1, PD-L2, and other clinically significant tumor antigens, as well as combinations thereof. Tumor antigens can be derived from intracellular proteins of tumor cells. Tumor antigens can be expressed on the surface of tumor cells. Proteins associated with the TCR complex can be CD3, such as CD3E. The TCR-binding domain can be a single-chain antibody, such as scFv or V. H H. The TCR-binding domain may be derived from UCHT1. The TAC receptor may include a cytoplasmic domain and a transmembrane domain. The T cell receptor signaling domain may include a cytoplasmic domain derived from a TCR co-receptor. Exemplary TCR co-receptors include, but are not limited to, CD4, CD8, CD28, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. The TAC receptor may include a transmembrane domain and a cytoplasmic domain derived from CD4. The TAC receptor may include a transmembrane domain and a cytoplasmic domain derived from CD8 (such as CD8α).

[0168] T cell co-receptors are expressed as membrane proteins on T cells. They can stabilize the TCR:peptide:MEC complex and promote signal transduction. Two subtypes of T cell co-receptors, CD4 and CD8, exhibit strong specificity for specific MEC types. The CD4 co-receptor stabilizes only the TCR:MEC II complex, while the CD8 co-receptor stabilizes only the TCR:MEC I complex. Differential expression of CD4 and CD8 in different T cell types leads to different functional T cell subsets. CD8+ T cells are cytotoxic T cells.

[0169] Engineered receptors (such as CARs, TCRs, or TACs) can target one or more tumor antigens. Tumor antigens are proteins produced by tumor cells that can elicit an immune response, particularly a T-cell-mediated one. The choice of target antigen will depend on the specific type of cancer to be treated. Exemplary tumor antigens include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, muta-hsp70-2, M-CSF, prostate enzymes, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostaglandins, PSMA, HER2 / neu, survival proteins and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, liver glycoside B2 (ephrinB2), CD22, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0170] Tumor antigens can contain one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express a number of proteins that can be used as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens, such as MART-1, tyrosinase, and gp100 in melanoma, and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the transformation-related molecular group, such as the oncogene HER2 / Neu / ErbB-2. Another group of target antigens is oncoemulsification antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotype immunoglobulins constitute the true tumor-specific immunoglobulin antigens unique to the individual tumor. B-cell differentiation antigens (such as CD19, CD20, and CD37) are other candidates for target antigens in B-cell lymphomas.

[0171] Tumor antigens can be either tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are specific to tumor cells and are not present on other cells in the body. TAA-associated antigens are not specific to tumor cells; instead, they can also be expressed on normal cells under conditions that do not induce immune tolerance to the antigen. Antigen expression on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs can be antigens expressed on normal cells during embryonic development, when the immune system is immature and unable to respond, or they can be antigens that are normally present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0172] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens, such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER2 / neu; unique tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23HI, PSA, TAG-72, CA 19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA 242, CA-50, CAM43, CD68, P1, CO-029, FGF-5, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90, Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0173] CARs may contain a primary intracellular signaling domain and / or a co-stimulatory signaling domain of an immune effector cell. The primary intracellular signaling domain may be derived from CD3ζ, and the co-stimulatory signaling domain may be derived from ligands selected from the group consisting of: 4-1BB, CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, CD83, and combinations thereof. CARs may contain a transmembrane domain derived from molecules selected from the group consisting of: CD8α, CD4, CD28, CD137, CD80, CD86, CD152, and PD1. CARs may further contain a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. CARs may further contain a signal peptide located at the N-terminus of the polypeptide.

[0174] Nucleic acids (polynucleotides) encoding modified IL-15 or related fusion proteins and related peptides.

[0175] This disclosure provides nucleic acids (e.g., expression vectors) encoding modified IL-15 or related fusion proteins. This disclosure also provides (i) nucleic acids (e.g., expression vectors) encoding modified IL-15 or related fusion proteins, and (ii) nucleic acids (e.g., expression vectors) encoding engineered receptors (e.g., CARs or TCRs). The nucleic acids disclosed herein may contain nucleic acid sequences encoding any of the modified IL-15 or related fusion proteins, CARs, and / or TCRs disclosed herein. The nucleic acid sequences may simultaneously encode a CAR and a modified IL-15 or related fusion protein (a CAR armored with a modified IL-15 or related fusion protein). The nucleic acid sequences may encode a CAR and a modified IL-15 or related fusion protein linked to each other via a P2A linker.

[0176] The polynucleotide disclosed herein may comprise a first polynucleotide sequence and a second polynucleotide sequence. The first and second polynucleotide sequences may be separated by a linker. The linker used in this disclosure allows multiple proteins encoded by the same nucleic acid sequence (e.g., a polycistronic or bicistronic sequence), which are translated into polyproteins that dissociate into individual protein components. The polynucleotide may comprise a first polynucleotide sequence, a linker, and a second polynucleotide sequence from 5' to 3'. The polynucleotide may comprise a second polynucleotide sequence, a linker, and a first polynucleotide sequence from 5' to 3'. The first polynucleotide sequence may encode the engineered receptor (e.g., CAR) described herein, and the second polynucleotide sequence may encode the modified IL-15 or related fusion protein described herein.

[0177] The adapter may contain a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, “internal ribosome entry site” or “IRES” refers to an element that facilitates direct entry of an internal ribosome into a protein-coding region of a start codon (such as ATG), thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites are known to those skilled in the art, including but not limited to IRES that can be obtained from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES that can be obtained from, for example, cardiogenic viruses, rhinoviruses, foot-and-mouth disease viruses, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). Those skilled in the art will be able to select appropriate IRES.

[0178] The linker may contain a nucleic acid sequence encoding a self-cleaving peptide. As used herein, a "self-cleaving peptide" or "2A peptide" refers to an oligopeptide that allows multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. The use of the term "self-cleaving" does not imply a proteolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those skilled in the art, including but not limited to those found in members of the Picornaviridae virus family, such as foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAVO), TaV, and swine cerebrospinal virus-1 (PTV-1); and cardiogenic viruses, such as Theylvirus and encephalomyocarditis virus. 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and "T2A," respectively. Those skilled in the art will be able to select appropriate self-cleaving peptides. The P2A connector may have the same sequence as SEQ ID NO: 2 at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0179] The linker may contain spacer sequences. Various spacer sequences are known in the art, including but not limited to glycine-serine (GS) spacers (also known as GS linkers), such as (GS)n, (SG)n, (GSGGS)n, and (GGGS)n, where n represents an integer of at least 1. Exemplary spacer sequences may contain amino acid sequences, including but not limited to GGSG, GGSGG, GGSSG, GSGGG, GGGSG, GSSSG, etc. Those skilled in the art will be able to select appropriate spacer sequences.

[0180] The polynucleotides disclosed herein may contain restriction enzyme site sequences.

[0181] The polynucleotides disclosed herein can be operatively linked to transcriptional control elements, such as promoters and enhancers. Suitable promoter and enhancer elements are known to those skilled in the art.

[0182] Suitable promoters include immediate early cytomegalovirus (CMV) promoter sequences. This is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it. Other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, Raoult's sarcoma virus, the EF-1α promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, this disclosure is not limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of this disclosure. The use of inducible promoters provides a molecular switch capable of turning on the expression of the polynucleotide sequence operatively linked to it when expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0183] The polynucleotides disclosed herein may be provided for the production of (i) the modified IL-15 or related fusion proteins described herein, and / or (ii) the CARs described herein (e.g., in mammalian cells). The polynucleotides disclosed herein may provide for the amplification of polynucleotides.

[0184] Expression vectors (e.g., lentiviral vectors) can be used to introduce modified IL-15 or related fusion proteins, CARs, and / or TCRs into immune cells or their precursors (e.g., T cells). Therefore, the expression vectors (e.g., lentiviral vectors) disclosed herein may contain polynucleotides encoding modified IL-15 or related fusion proteins, CARs, and / or TCRs. Expression vectors (e.g., lentiviral vectors) will contain additional elements that facilitate the functional expression of the modified IL-15 or related fusion proteins, CARs, and / or TCRs encoded therein. Expression vectors containing polynucleotides may further contain mammalian promoters. Vectors may further contain an elongation factor-1-α promoter (EF-1α promoter). Using an EF-1α promoter can increase the expression efficiency of downstream transgenes (e.g., polynucleotides encoding CARs or TCRs). Physiological promoters (e.g., EF-1α promoters) are less likely to induce integration-mediated genotoxicity and may eliminate the ability of retroviral vectors to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those skilled in the art and can be incorporated into the vectors disclosed herein. Vectors (e.g., lentiviral vectors) may further comprise non-essential cis-acting sequences that can improve titers and gene expression.

[0185] Polynucleotides can encode naked CARs. The polynucleotide may contain an antigen-binding domain, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory signaling domain, and a CD3ζ intracellular domain from the 5' to the 3' end. The amino acid sequence of the CAR may be at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1.

[0186] The polynucleotide can encode a CAR and a modified IL-15 or related fusion protein. The polynucleotide may contain, from its 5' to 3' end, a coding sequence for an antigen-binding domain, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory signal transduction domain, a CD3ζ intracellular domain, a 2A cleavable linker, and a modified IL-15 or related fusion protein. The polynucleotide can encode a BCMA CAR and a modified IL-15 or related fusion protein. The amino acid sequence of the modified IL-15 or related fusion protein may be at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO: 17-26.

[0187] This disclosure may provide (i) peptides comprising modified IL-15 or related fusion proteins, and (ii) peptides comprising engineered receptors (e.g., CARs or TCRs). Peptides disclosed herein may comprise any of the modified IL-15 or related fusion proteins, CARs, and / or TCRs disclosed herein. Peptides may simultaneously comprise a CAR and a modified IL-15 or related fusion protein (a CAR armored with a modified IL-15 or related fusion protein).

[0188] The polypeptide disclosed herein may comprise a first polypeptide sequence and a second polypeptide sequence. The first polypeptide sequence and the second polypeptide sequence may be separated by a linker. The linker used in this disclosure allows multiple proteins encoded by the same nucleic acid sequence (e.g., a polycistronic or bicistronic sequence) to be translated into polyproteins that dissociate into individual protein components. The polypeptide may comprise, from N-terminus to C-terminus: a first polypeptide sequence, a linker, and a second polypeptide sequence. Alternatively, the polypeptide may comprise, from N-terminus to C-terminus: a second polypeptide sequence, a linker, and a first polypeptide sequence. The first polypeptide sequence may contain the polypeptide sequence of an engineered receptor (e.g., a CAR) as described herein, and the second polypeptide sequence may contain the polypeptide sequence of a modified IL-15 or related fusion protein as described herein.

[0189] This disclosure also provides nucleic acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any nucleotide sequence as described herein, and amino acid sequences that are at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any amino acid sequence as described herein. This disclosure relates to nucleotide sequences encoding any peptide described herein, or any amino acid sequence encoded by any nucleotide sequence as described herein. Nucleic acid sequences can be fewer than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 3500, 4000, or 5000 nucleotides. The amino acid sequence can be less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 1100, 1200, 1300, or 1400 amino acid residues.

[0190] An amino acid sequence may (i) contain an amino acid sequence; or (ii) consist of an amino acid sequence, wherein the amino acid sequence is any of the sequences described herein.

[0191] A nucleic acid sequence may (i) contain a nucleic acid sequence; or (ii) consist of a nucleic acid sequence, wherein the nucleic acid sequence is any of the sequences described herein.

[0192] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, these sequences are aligned for optimal comparison (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences to achieve optimal alignment, and non-homologous sequences may be ignored for comparison). The length of the reference sequence aligned for comparison purposes is at least 80% of the length of a reference sequence, and may be at least 90%, 95%, or 100%. The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of shared positions, taking into account the number of vacancies and the length of each vacancy, requiring the introduction of vacancies to achieve optimal alignment. For the purposes of this disclosure, the comparison of sequences and the determination of the percentage of identity between two sequences can be accomplished using a Blossum 62 scoring matrix where the vacancy penalty is 12, the vacancy extension penalty is 4, and the frameshift vacancy penalty is 5.

[0193] Introducing polynucleotides into host cells

[0194] The polynucleotides described herein (e.g., vectors) can be introduced as one or more polynucleotides or constructs, which optionally contain markers that will allow selection of host cells containing the construct. Genes and regulatory regions can be isolated, ligated, cloned into appropriate cloning hosts, and analyzed by restriction enzyme digestion or sequencing as needed. In particular, using PCR, individual fragments comprising all or part of the functional units can be isolated, where one or more mutations can be introduced as needed using primer repair, ligation, in vitro mutagenesis, etc. The obtained polynucleotides, confirmed to have appropriate sequences, can then be introduced into host cells by any convenient method. Polynucleotides can be integrated and packaged into non-replicating defective viral genomes (such as lentiviruses, adenoviruses, adeno-associated viruses (AAVs), or herpes simplex virus (HSVs) or others, including retroviral vectors), for infection or transduction into cells. If desired, the polynucleotides can contain viral sequences for transfection. Alternatively, polynucleotides can be introduced by fusion, electroporation, bioprojectiles, transfection, lipid transfection, etc. Before introducing the construct, host cells can be grown and expanded in a culture, followed by appropriate treatment to introduce and integrate the construct. Cells are then amplified and screened using markers present in the construct. Various markers that can be successfully used include hprt, neomycin resistance, thymidine kinase, and hygromycin resistance.

[0195] Nucleic acids encoding modified IL-15 or related fusion proteins and / or engineered receptors can be introduced as RNA into modified cells for transient expression. The RNA can be delivered to the immune cells disclosed herein via various methods, including, for example, microinjection, electroporation, and lipid-mediated transfection. The construct can be introduced into the cell's genome via transposons. An example of a synthetic transposon used is the Sleeping Beauty transposon, which contains an expression cassette containing the appropriate gene for its active fragment. The construct can be integrated at a specific locus in the host cell's genome. Homologous recombination can be used to replace endogenous genes with genes encoded by the construct.

[0196] A lentiviral delivery system can be used to introduce a construct encoding both modified IL-15 or a related fusion protein and a CAR into host cells. A retroviral delivery system can be used to introduce a construct encoding both modified IL-15 or a related fusion protein and a CAR into host cells.

[0197] The host cell can be a human cell. The host cell can be a human T cell. Human T cells can be purified from commercially available PBMCs using the Miltenyi Pan T Cell Isolation Kit (catalog number 130-096-535) according to the manufacturer's protocol. The host cell can be an αβ T cell. The host cell can be a γδ T cell. The host cell can be a Vδ1 T cell. The host cell can be an NK cell.

[0198] Modified cells

[0199] This disclosure provides modified cells comprising the modified IL-15 or related fusion protein described herein. Modified cells comprising the modified IL-15 or related fusion protein described herein may further comprise an engineered receptor (e.g., a CAR). Modified cells comprising the modified IL-15 or related fusion protein described herein may further comprise a CAR (a CAR armored with modified IL-15 or related fusion protein). The cells may be immune cells. Cells may be selected from the group consisting of: T cells, αβT cells, γδT cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

[0200] Engineered receptors (e.g., CARs) can redirect the specificity of modified cells through expression of chimeric antigen receptors (CARs) or TCRs on these cells. CAR expression can be induced by electroporation of modified cells to insert genetic material, or by infecting these cells with viral vectors such as lentiviruses or retroviruses containing the desired genetic material. Such gene editing can improve the potency of modified cells by enhancing homing, cytokine production, recirculating killing, and / or improving engraftment.

[0201] Modified cells containing the modified IL-15 or related fusion protein described herein may express more than one engineered receptor, such as any combination of CAR, TCR or TAC receptors.

[0202] Modified cells containing the modified IL-15 or related fusion proteins described herein may be used to treat cancer.

[0203] Compared to cells lacking the modified IL-15 or related fusion protein described herein, modified cells containing the modified IL-15 or related fusion protein described herein can exhibit higher cytotoxicity against tumor cells. Compared to cells lacking the modified IL-15 or related fusion protein described herein, modified cells containing the modified IL-15 or related fusion protein described herein can exhibit greater persistence and / or proliferation in the tumor microenvironment.

[0204] This disclosure provides modified cells comprising (i) the modified IL-15 or related fusion protein described herein and (ii) the engineered receptor (e.g., CAR) described herein. The modified cells may be immune cells. The modified cells may contain one or more polynucleotides encoding (i) the modified IL-15 or related fusion protein described herein and (ii) the engineered receptor (e.g., CAR) described herein. Therefore, such modified cells possess specificity directed by the engineered receptor (e.g., CAR) expressed therein. For example, modified cells of this disclosure containing CAR are specific for one or more antigens on target cells (e.g., one or more tumor antigens on cancer cells).

[0205] The modified cells can be modified immune cells. Modified cells can be T cells. Modified cells can be NK cells. Modified cells can be αβ T cells. Modified cells can be γδ T cells. Modified cells can be Vδ1 T cells.

[0206] For the individual receiving them, the modified cells can be autologous cells, syngeneic cells, allogeneic cells, or xenogeneic cells. Modified cells can be modified by altering the major histocompatibility complex (MHC) profile, by inactivating β2-microglobulin to prevent the formation of functional class I MHC molecules, or by inactivating class II MHC molecules.

[0207] The modified cells described herein may include eukaryotic cells, such as mammalian cells. Modified cells may be human cells. Modified cells may be horse, cow, mouse, sheep, dog, or cat cells.

[0208] The modified cells can be autologous cells obtained from human subjects who receive them. The modified cells can be autologous T cells obtained from human subjects who receive them.

[0209] This disclosure provides modified cells expressing modified IL-15 or related fusion proteins. The modified IL-15 or related fusion proteins may contain a calreticulin signal peptide sequence. The modified IL-15 or related fusion proteins may contain an ER-retaining sequence. The modified IL-15 or related fusion proteins may contain a myristylated sequence. The modified IL-15 or related fusion proteins can be captured in the ER. The modified IL-15 or related fusion proteins can be captured intracellularly. The modified IL-15 or related fusion proteins can reduce the amount of secreted IL-15 by capturing IL-15 intracellularly. The modified IL-15 or related fusion proteins can activate IL-15 signaling.

[0210] Modified cells exhibit enhanced proliferation compared to unmodified cells that do not contain modified IL-15 or related fusion proteins. Modified cells also exhibit enhanced cytotoxicity compared to unmodified cells that do not contain modified IL-15 or related fusion proteins.

[0211] The engineered receptor can be a CAR. A CAR can comprise a polypeptide comprising, from its N-terminus to its C-terminus, an antigen-binding domain, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory signaling domain, and a CD3ζ intracellular domain. A CAR can be a BCMA CAR. A CAR can comprise a polypeptide containing at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to SEQ ID NO: 1.

[0212] The polypeptide may contain a CAR and a modified IL-15 or related fusion protein. The polypeptide may contain, from the N-terminus to the C-terminus, an antigen-binding domain, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory signal transduction domain, a CD3ζ intracellular domain, a 2A cleavable linker, and a modified IL-15 or related fusion protein. The polypeptide may contain a BCMA CAR and / or a modified IL-15 or related fusion protein. The amino acid sequence of the modified IL-15 or related fusion protein may be at least 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NO:17-26.

[0213] Modified cells may simultaneously contain modified IL-15 or related fusion proteins and CAR (CAR armored with modified IL-15 or related fusion proteins). Modified cells may be modified CAR-T cells (CAR-T cells armored with modified IL-15 or related fusion proteins). The expression of CAR and modified IL-15 or related fusion proteins in modified cells can be determined by flow cytometry (FACS). Modified cells may have CAR positivity rates greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. Modified cells can have CAR positivity rates of less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, or less than 90%. Modified cells can also have CAR positivity rates of 10%-80%, 10%-70%, 15%-70%, 20%-70%, 20%-65%, 25%-65%, 30%-80%, 40%-80%, 50%-80%, or 60%-80%.

[0214] Modified cells can have IL-15 positivity rates greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. Modified cells can also have IL-15 positivity rates less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. The modified cells can have IL-15 positivity rates of 10%-80%, 10%-70%, 15%-70%, 20%-70%, 20%-65%, 20%-60%, 20%-55%, 25%-65%, 30%-70%, 30%-75%, 30%-65%, 20%-40%, 20%-50%, 20%-60%, 20%-70%, 30%-70%, or 40%-70%.

[0215] Modified cells can have IL-15Rα positivity rates greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. Modified cells can also have IL-15Rα positivity rates less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. The modified cells can have IL-15Rα positivity rates of 10%-80%, 10%-70%, 15%-70%, 20%-70%, 20%-65%, 20%-60%, 20%-55%, 25%-65%, 30%-70%, 30%-75%, 30%-65%, 20%-40%, 20%-50%, 20%-60%, 20%-70%, 30%-70%, or 40%-70%.

[0216] In one aspect, this disclosure provides methods for increasing cell viability, including expressing modified IL-15 or related fusion proteins in cells. The viability of the modified cells can be assessed by in vitro cell culture assays. The viability of the modified cells can be assessed by in vitro cell culture assays in cytokine-free medium. The assays are performed at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, and 40 days. After 41, 42, 43, 44, 45, 46, 47, 48, or 49 days, the modified cells can have viability greater than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90%. On days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 After 41, 42, 43, 44, 45, 46, 47, 48, or 49 days, the modified cells exhibited viability of less than 5%, less than 10%, less than 15%, less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, less than 45%, less than 50%, less than 55%, less than 60%, less than 65%, less than 70%, less than 75%, less than 80%, or less than 90%. Compared to unmodified cells that did not contain modified IL-15 or related fusion proteins, modified cells containing modified IL-15 or related fusion proteins showed similar viability after 1, 2, 3, 4, or 5 rounds of stimulation in the re-excitation assay.

[0217] In one aspect, this disclosure provides methods for increasing cell proliferation, including expressing modified IL-15 or related fusion proteins in cells. The proliferation of modified cells can be assessed by in vitro cell culture assays. The proliferation of modified cells can also be assessed by in vitro cell culture assays in cytokine-free medium. The proliferation of modified cells can be assessed by cell counting. The proliferation of modified cells can also be assessed by dye dilution methods. On days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45 After 46, 47, 48, or 49 days, the modified cells can expand by more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, or 120 times. On days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45 After 46, 47, 48, or 49 days, the modified cells can expand by less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, or 120 times. The initial number of modified cells can be less than 1 million.After 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, or 49 days, the modified cells The number can exceed 1 million, 2 million, 3 million, 4 million, 5 million, 6 million, 7 million, 8 million, 9 million, 10 million, 11 million, 12 million, 13 million, 14 million, 15 million, 16 million, 17 million, 18 million, 19 million, 20 million, 21 million, 22 million, 23 million, 24 million, and 25 million. After 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, or 49 days, the modified cells The quantity can be lower than 1 million, lower than 2 million, lower than 3 million, lower than 4 million, lower than 5 million, lower than 6 million, lower than 7 million, lower than 8 million, lower than 9 million, lower than 10 million, lower than 11 million, lower than 12 million, lower than 13 million, lower than 14 million, lower than 15 million, lower than 16 million, lower than 17 million, lower than 18 million, lower than 19 million, lower than 20 million, lower than 21 million, lower than 22 million, lower than 23 million, lower than 24 million, and lower than 25 million.

[0218] Modified cells may possess activated STAT signaling (e.g., phosphorylated STAT3, phosphorylated STAT5, or phosphorylated STAT6). Modified cells may possess phosphorylated STAT5.

[0219] In one aspect, this disclosure provides methods for increasing STAT signaling in cells (e.g., increasing the amount of phosphorylated STAT5), methods comprising expressing modified IL-15 or an associated fusion protein in cells. Compared to unmodified cells that do not contain modified IL-15 or an associated fusion protein, the amount of phosphorylated STAT5 in modified cells can be increased by more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, more than 200%, more than 250%, more than 300%, more than 400%, more than 500%, more than 600%, more than 700%, more than 800%, more than 900%, or more than 10.00%.

[0220] In one aspect, this disclosure provides methods for increasing the cytotoxicity of cells against tumor cells, including expressing modified IL-15 or related fusion proteins in the cells. The modified cells can kill tumor cells. The cytotoxicity of the modified cells against tumor cells (e.g., H929 cells) can be determined by an in vitro cytotoxicity assay. The effector cell:target cell (E:T) ratio can be 0.5:1, 1:1, 2:1, 2.5:1, 5:1, 10:1, or 1:4. The E:T ratio can be 1:4. Tumor cells can be labeled with cell-tracking purple. Fresh tumor cells (e.g., H929 cells) are added every 24 hours to examine the cytotoxicity of the modified cells after multiple rounds of challenge. After 1, 2, 3, 4, 5, 6, 7, or 8 rounds of re-excitation in the re-excitation assay, the modified cells exhibited cytotoxicity greater than 5%, greater than 10%, greater than 15%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, or greater than 95%. Conversely, after 1, 2, 3, 4, 5, 6, 7, or 8 rounds of re-excitation in the re-excitation assay, the modified cells exhibited cytotoxicity less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95%. After 1, 2, 3, 4, 5, 6, 7, or 8 rounds of excitation in the re-excitation assay, the modified cells can exhibit cytotoxicity of 10-100%, 10%-50%, 20-100%, 20-60%, 20-40%, 30-70%, 40-80%, 50-90%, 60-100%, 70-100%, 80-100%, or 90-100%.

[0221] Modified IL-15 or related fusion proteins can also be co-expressed with the IL-15 receptor α subunit. Similarly, various other proteins (other than CD16 and / or CAR) can be co-expressed with modified IL-15 or related fusion proteins, and suitable co-expressed proteins include a variety of immunomodulatory compounds, and in particular compounds that interfere with checkpoint inhibition (e.g., scFv targeting PD-1, PD-L1, CTLA4, etc.), immunostimuli (e.g., IFN-γ, IL-12, IL-21, etc.), and / or compounds that bind to / inhibit cytokines involved in immunosuppression (e.g., TGF-β, IL-8, etc.).

[0222] The modified cells will express modified IL-15 or an associated fusion protein in an amount sufficient to (a) make the transfected cells independent of exogenous cytokines and (b) allow stimulation / activation of other immune-competent cells near the transfected cells (usually within the TME).

[0223] IL-15 or IL-15 variants secreted or otherwise present in the extracellular space (or extracellularly presented) will account for more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% of the total IL-15 or IL-15 variants produced in the modified cells. IL-15 or IL-15 variants secreted or otherwise present in the extracellular space (or extracellularly presented) will account for less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% of the total IL-15 or IL-15 variants produced in the modified cells.

[0224] Intracellularly retained IL-15 or IL-15 variants will account for more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% of the total IL-15 or IL-15 variants produced in the cell.

[0225] The modified cells will produce an amount of IL-15 or IL-15 variant sufficient to support autonomous growth and stimulate immune competent cells in the TME as well as to stimulate the establishment and maintenance of CD8+ T cell memory, but such an amount is insufficient to trigger systemic adverse events in subjects who receive such cells.

[0226] The amount of secreted IL-15 can be measured by detecting the amount of IL-15 in the cell culture medium using an IL-15 ELISA assay. The amount of intracellular IL-15 can be measured by detecting the amount of IL-15 in cell lysates using an IL-15 ELISA assay. The amounts of secreted and intracellular IL-15 in 4 million modified cells were analyzed. The amount of IL-15 secreted can exceed 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1000 pg, 1200 pg, 1400 pg, 1600 pg, 1800 pg, 2000 pg, 2200 pg, or 2400 pg. The amount of IL-15 secreted can be less than 10 pg, less than 20 pg, less than 30 pg, less than 40 pg, less than 50 pg, less than 60 pg, less than 70 pg, less than 80 pg, less than 90 pg, less than 100 pg, less than 200 pg, less than 300 pg, less than 400 pg, less than 500 pg, less than 600 pg, less than 700 pg, less than 800 pg, less than 900 pg, less than 1000 pg, less than 1200 pg, less than 1400 pg, less than 1600 pg, less than 1800 pg, less than 2000 pg, less than 2200 pg, or less than 2400 pg.

[0227] Intracellular IL-15 levels can exceed 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1000 pg, 1200 pg, 1400 pg, 1600 pg, 1800 pg, 2000 pg, 2200 pg, or 2400 pg. The amount of intracellular IL-15 can be less than 10 pg, less than 20 pg, less than 30 pg, less than 40 pg, less than 50 pg, less than 60 pg, less than 70 pg, less than 80 pg, less than 90 pg, less than 100 pg, less than 200 pg, less than 300 pg, less than 400 pg, less than 500 pg, less than 600 pg, less than 700 pg, less than 800 pg, less than 900 pg, less than 1000 pg, less than 1200 pg, less than 1400 pg, less than 1600 pg, less than 1800 pg, less than 2000 pg, less than 2200 pg, or less than 2400 pg.

[0228] The ratio of secreted IL-15 to intracellular IL-15 can be greater than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The ratio of secreted IL-15 to intracellular IL-15 can be less than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The ratio between secreted IL-15 and intracellular IL-15 can be 0.1-0.2, 0.1-0.3, 0.1-0.4, 0.1-0.5, 0.2-0.5, 0.3-0.5, or 0.4-0.5.

[0229] Modified IL-15 or related fusion proteins can stimulate or enhance effector function and / or proliferation of NK cells (e.g., autologous NK cells in the TME), various T cells, etc., and enhance or trigger Jak / STAT signaling in cells within the TME. Due to the intracellular retention of IL-15 or portions of IL-15 variants, modified cells may also be able to proliferate in the complete absence of exogenous IL-2 and / or IL-15. Compared to unmodified cells, modified cells may also exhibit increased sensitivity to IL-12 signaling, which can reduce IL-4-mediated IFN-γ inhibition, thereby reducing Th1 T cell suppression in the TME.

[0230] Treatment

[0231] The modified IL-15 or related fusion proteins, the polynucleotides, and the modified cells described herein can be used in a variety of experimental, therapeutic, and commercial applications.

[0232] In one respect, this disclosure provides a method for modulating an immune response, the method comprising administering an effective amount of the modified cells described herein to a subject in need.

[0233] As used herein, the term "effective amount" refers to the amount that is effective at the necessary dose and duration to achieve the desired result.

[0234] On the other hand, this disclosure provides a method for treating an infection, the method comprising administering an effective amount of the modified cells described herein to a subject in need.

[0235] Examples of treatable infections include, but are not limited to, bacterial infections (such as infections caused by mycobacteria (e.g., tuberculosis)), viral infections (such as infections caused by herpes simplex virus (HSV), human immunodeficiency virus (HIV), or hepatitis virus), and parasitic infections (such as infections caused by Plasmodium (e.g., malaria)).

[0236] On the other hand, this disclosure provides a method for treating cancer, the method comprising administering an effective amount of the modified cells described herein to a subject in need.

[0237] Examples of treatable cancers include, but are not limited to, leukemia (including chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and T-cell and B-cell leukemia), lymphoma (Hodgkin's and non-Hodgkin's), lymphoproliferative disorders, plasmacytoma, histiocytoma, melanoma, adenoma, sarcoma, solid tissue cancer, hypoxic tumors, squamous cell carcinoma, genitourinary cancers (such as cervical cancer and bladder cancer), hematopoietic cancers, head and neck cancers, and nervous system cancers.

[0238] This disclosure further includes the use of the modified cells described herein in the manufacture of medicaments or pharmaceutical compositions for regulating immune responses, treating infections, or treating cancers as described above.

[0239] Modified cells can also be used in experimental models, for example, to further study and elucidate cell function.

[0240] One or more of the modified cells described herein can be administered to a subject in a single, uniform form (such as intravenous injection) or in multiple forms (e.g., as multiple intravenous infusions or injections, or subcutaneous injections). In some cases, the modified cells can be expanded in the subject after administration. The modified cells can be frozen to provide cells for multiple treatments with the same cell preparation. The modified cells and pharmaceutical compositions comprising them disclosed herein can be packaged as kits (reagents). Kits may include instructions (e.g., written instructions) regarding the use of the modified cells and compositions comprising them.

[0241] Methods for administering modified cells for adoptive cell therapy are known and can be used in conjunction with the provided methods and compositions. For example, adoptive T-cell therapy methods are described in the following literature: U.S. Patent Application Publication No. 2003 / 0170238, granted to Gruenberg et al.; U.S. Patent No. 4,690,915, granted to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85. See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys ResCommun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338. Cell therapy (e.g., adoptive T-cell therapy) can be performed via autologous transfer, wherein cells are isolated from and / or otherwise prepared from a subject to receive cell therapy or from a sample derived from such a subject. Thus, in some respects, cells are derived from a subject requiring treatment (e.g., a patient), and after isolation and processing, the cells are administered to the same subject.

[0242] Cell therapy (e.g., adoptive T-cell therapy) can be performed via allogeneic transfer, where cells are isolated and / or otherwise prepared from a subject other than the one to be or ultimately receive the cell therapy (e.g., a first subject). In such embodiments, the cells are then administered to a different subject of the same species, e.g., a second subject. The first and second subjects can be genetically identical. The first and second subjects can be genetically similar. The second subject can express the same HLA class or supertype as the first subject.

[0243] The subject may have been treated with a therapeutic agent targeting a disease or condition (e.g., cancer) prior to administration of the cells or a composition containing cells. In some respects, the subject may be refractory or unresponsive to other therapeutic agents. The subject may have a persistent or relapsing disease, for example, after treatment with another therapeutic intervention, including chemotherapy, radiation therapy, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. Even if the subject has developed resistance to another therapy, administration may still be effective in treating the subject.

[0244] The subject may respond to another treatment agent, and treatment with that agent reduces the disease burden. The subject may initially respond to the treatment agent but exhibit a relapse of the disease or condition over time. The subject may not experience a relapse. The subject may be identified as being at risk of relapse, such as being at high risk, and therefore prophylactically administered cells, for example, to reduce the likelihood of or prevent a relapse. The subject may not have previously received treatment with another treatment agent.

[0245] Subjects may have persistent or recurrent diseases, for example, after treatment with another therapeutic intervention, including chemotherapy, radiation therapy, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT. Even if a subject has developed resistance to another therapy, administration can still effectively treat the subject.

[0246] The modified cells described herein can be administered to animals, preferably mammals, and even more preferably humans, to treat cancer. Furthermore, the modified cells can be used to treat any condition related to cancer, particularly cell-mediated immune responses against tumor cells, where treatment or mitigation of the disease is desired. Types of cancer to be treated with the modified cells or pharmaceutical compositions include carcinoma, blastoma, and sarcoma, as well as certain leukemias or lymphomas, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas. Other exemplary cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, etc. Cancer can be a non-solid tumor (such as a hematologic malignancy) or a solid tumor. Adult oncology / cancer and pediatric oncology / cancer are also included. Cancer can be a solid tumor or a hematologic malignancy. Cancer can be carcinoma. Cancer can be sarcoma. Cancer can be leukemia. Cancer can be a solid tumor.

[0247] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the types of cells that form them (such as sarcoma, epithelial carcinoma, and lymphoma). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid tumors, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, and Wilms' tumor. Tumors, cervical cancer, testicular tumors, seminomas, bladder cancer, melanomas, and CNS tumors (such as gliomas (such as brainstem gliomas and mixed gliomas), glioblastomas (also known as multimorphic glioblastomas), astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, schwannomas, ependymomas, pineal tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, hemangiomas, neuroblastomas, retinoblastomas, and brain metastases).

[0248] Cancers suitable for treatment using the methods disclosed herein include, but are not limited to, esophageal cancer, hepatocellular carcinoma, basal cell carcinoma (a type of skin cancer), squamous cell carcinoma (various tissues), bladder cancer, including transitional cell carcinoma (a malignant tumor of the bladder), bronchial cancer, colon cancer, colorectal cancer, gastric cancer, lung cancer, including small cell carcinoma and non-small cell lung cancer, adrenocortical carcinoma, thyroid cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular cancer, osteoblastoma, epithelial carcinoma, and nasopharyngeal carcinoma.

[0249] Sarcomas suitable for treatment using the methods disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteosarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelioma, synovial sarcoma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

[0250] The modified cells described herein (e.g., immune cells, T cells, or NK cells) may be included in compositions for use in immunotherapy. The compositions may include pharmaceutical compositions and further include pharmaceutically acceptable carriers. A therapeutically effective amount of the pharmaceutical composition containing the modified cells may be administered.

[0251] The modified cells can be used immediately for the above-described treatments, experimental or commercial applications, or they can be cryopreserved for later use. The pharmaceutical composition may be included in a container, package or dispenser along with the instructions for use.

[0252] The modified cells disclosed herein can be formulated into unit dosage forms suitable for a precise single-dose administration. In some cases, the unit dosage form contains additional lymphocytes. Cells can be expressed in an amount intended for dose administration (such as for a single unit dose or multiple doses) into one or more of multiple output containers (e.g., vials). For example, each vial may each contain a number of cells intended for administration at a given dose or in part thereof. Thus, each vial may contain a single unit dose for administration or may contain a portion of the desired dose, such that more than one of multiple vials (e.g., two or three vials) together constitute the dose intended for administration. Thus, containers, such as bags or vials, typically contain cells to be administered, such as one or more unit doses thereof. The unit dose may be the amount or number of cells to be administered to the subject, or twice (or more) the number of cells to be administered. It may be the minimum or lowest possible dose of cells to be administered to the subject.

[0253] Example

[0254] The disclosure is further described in the following examples, which do not limit the scope of the disclosure as set forth in the claims.

[0255] Example 1: Preparation of CAR-NK cells expressing anti-BCMA CAR with various modified IL-15 constructs

[0256] 1.1 Preparation of retroviral expression vectors containing anti-BCMA CAR with modified IL-15 construct

[0257] To construct the BCMA CAR (SEQ ID NO: 1), the anti-BCMA binding protein sequence was ligated to the CD8α hinge and transmembrane region, and then to the 4-1BB and CD3ζ intracellular sequences. The amino acid sequence of the above BCMA CAR is shown in SEQ ID NO: 1.

[0258] The structures of exemplary modified IL-15 constructs are shown in Tables 1-2. The amino acid sequences of the BCMA CAR armored by the modified IL-15 constructs are shown as SEQ ID NO: 17-26.

[0259] To co-express the modified IL-15 construct, a polynucleotide encoding BCMA CAR was chemically synthesized and ligated to the polynucleotide encoding the modified IL-15 construct via the coding sequence of the P2A linker (SEQ ID NO: 2).

[0260] The polynucleotide construct encoding the naked BCMA CAR was named CAR30. As shown in Table 1-2, the polynucleotide constructs encoding both the BCMA CAR and the modified IL-15 construct were named CAR4, CAR5, CAR6, CAR11, CAR19, CAR20, CAR21, CAR22, and CAR32.

[0261] CAR31 refers to delta CAR (ΔCAR), which means that the modified cells express only the introduced IL-15 (SEQ ID NO: 25).

[0262] Anti-BCMA CARs with modified IL-15 constructs were synthesized in the retroviral vector SinCMV (Biovec pharma). These constructs were then transfected into the HEK cell line (293Vec-Galv). TMRetroviruses were prepared by stable transfection of the gag-pol and env genes in a Biovec pharma. The retroviruses generated from the SupT1 cell line (ATCC, CRL1942) were then titrated. All constructs contained various designed BCMA CARs with a secreted wild-type IL-15 module linked via the P2A sequence. The amino acid sequences of the modified IL-15 constructs are listed in Tables 1-2 below.

[0263] Table 1. Amino acid sequences of the modified IL-15 construct

[0264]

[0265] Table 2. Amino acid sequences of the modified IL-15 construct

[0266]

[0267] 1.2 NK cell NK cell amplification and viral transduction

[0268] CD3-depleted PBMCs derived from healthy subjects were cultured in a medium containing a mixture of cytokines containing IL2. Cells were transduced with retroviruses (generated from various constructs designed with BCMA CAR and different modified IL-15, as shown in Tables 1-2) using spin seeding (MOI 1, 2000 g, 32°C, 1 h). Cells were harvested on day 18. Cell counts and staining with antibodies [CD56 (clone B159, BD Bioscience), CD3 (HIT3a, BD Bioscience), CD16 (clone 3G8, BD Bioscience)] were performed to calculate NK cell fold expansion.

[0269] Example 2: In vitro evaluation of CAR-NK cells armored with IL-15 constructs

[0270] For various assays, CAR-NK cells with different modified IL-15 constructs were tested in three batches. Various in vitro assays, such as IL-15 ELISA, CAR assay, proliferation assay, cytotoxicity assay, and pSTAT5 assay, were performed to evaluate various IL-15 designs in anti-BCMA CAR NK cells.

[0271] IL-15 ELISA: Analysis was performed using an ELISA kit (Ab218266, Abchem) on (1) culture medium and (2) NK cell lysates (from 4 × 10⁻⁶ cells). 6The secreted IL-15 of cells and the ratio of secreted IL-15 to intracellular IL-15 were measured. In short, 50 μl of the mixed antibody was coated into each well. Culture medium controls, lysate samples, and standards were diluted, and 50 μl of the sample was added to each well. The plate was incubated at room temperature (RT) for 1 h. The plate was washed three times with 350 μl of wash buffer. Further, 100 μl of TMB (3,3',5,5'-tetramethylbenzidine) substrate solution was added to each well. The plate was incubated at RT for 10 min, and the reaction was terminated with 100 μl of stop solution. The plate OD (optical density) at 450 nm was read using a spectrophotometer (Molecular devices).

[0272] CAR staining: The percentage of CAR-positive cells in each analyzed construct was determined using immunophenotypic analysis with BCMA-FITC protein (BCA-HF254, Acrobiosystems Inc.). In short, 2 × 10⁻⁶ cells were used. 5 Harvested NK cells were washed and resuspended in 100 μl of staining buffer (PBS + 2% (w / v) BSA) containing FITC-labeled BCMA protein (3 μg / ml), and added to each well of a 96-well plate. The plate was incubated at 4°C for 1 h. Further, the plate was washed with washing buffer and 150 μl of diluted 7AAD (7-aminoactinomycin D) solution was added (5 μl 7AAD per sample, BD Bioscience). The samples were analyzed by flow cytometry (Aurora, Cytek Biosciences) to identify CAR-FITC positive cells.

[0273] pSTAT5 assay: Phosphorylated STAT5 (pSTAT5) levels were evaluated using a pSTAT5-specific antibody. In short, 0.5 × 10⁻⁶ ppm was used. 6One NK cell was added to each well and stained with CD56 (clone 5.1H11, Biolegend), BCMA-FITC (BCA-HF254, Acrobiosystems Inc.), and NIR live / dead stain (L10119, Invitrogen) for 30 min. Further, the cells were fixed in 1.5% (w / v) paraformaldehyde (PFA) at 37°C for 10 min. The PFA-fixed cells were centrifuged, and the pellet was resuspended and fixed with 1 mL of ice-cold methanol at 4°C for 1 h. The fixed cells were washed and resuspended at RT in staining buffer containing pSTAT5 antibody (pY694 catalog number 612599, BD Bioscience) for 1 h. The pSTAT5 signal of the cells was analyzed using flow cytometry (Aurora, Cytek Biosciences).

[0274] Proliferation assay: Cell proliferation was analyzed by absolute cell count using trypan blue (Beckman Coulter) or by dye dilution. For the dye dilution method, NK cells were washed twice in PBS and incubated with cell-tracking violet (4 μM, Invitrogen) at 37°C for 20 min. Serum was added to terminate the reaction, and the cells were washed and resuspended in culture medium. The cells were then incubated in a cell culture incubator for a specified period. Cells were harvested at the end of the specified time period, and the dilution of cell-tracking violet dye was analyzed using flow cytometry (Aurora, Cytek Biosciences).

[0275] Cytotoxicity assay: The cytotoxic activity of NK cells was evaluated using a short-term 4-hour cytotoxicity assay. Briefly, as described above in the proliferation assay, tumor cells were labeled with cell-tracking purple dye (4 μM, Invitrogen). NK cells and labeled tumor cells were washed, counted, and adjusted to 1 × 10⁻⁶. 6Cells / ml. Based on E / T ratios of 1:1, 0.5:1, 0.25:1, and 0.12:1, NK cells were initially plated using a series of dilutions with a dilution factor of 2. Further, an appropriate number of tumor cells were plated in the corresponding wells to establish a co-culture. The plates were briefly centrifuged at 100 g for 1 min and incubated in a cell culture incubator for 4 h. The plates were washed and stained at 4°C for 30 min with anti-CD3 (clone HIT3a, BD Bioscience), CD56 (clone B159, BD Bioscience), CD16 (clone 3G8, BD Bioscience), and BCMA-FITC protein (BCA-HF254, Acrobiosystems Inc). The plates were washed and resuspended in 150 μl of 7AAD solution (5 μl of 7AAD per sample). Samples were collected and analyzed using flow cytometry (Aurora, Cytek Biosciences).

[0276] Table 4 below summarizes the results of batch 1, Table 5 below summarizes the results of batch 2, and Table 6 below summarizes the results of various tests for batch 3.

[0277] Table 4. Measurement results of various constructs tested in batch 1

[0278]

[0279] Table 5. Measurement results of various constructs tested in batch 2

[0280]

[0281] Table 6. Measurement results of various constructs tested in batch 3

[0282]

[0283] Example 3: In vitro evaluation of CAR5, CAR6, CAR19, CAR20 and CAR22

[0284] Various in vitro assays, such as CAR assays, proliferation assays, cytotoxicity assays, and pSTAT5 assays, were performed as described above in Example 2 to evaluate various modified IL-15 designs in anti-BCMA CAR NK cells.

[0285] 3.1 Immunostaining assay

[0286] On day 18, flow cytometry was used to test the expression of IL-15 and IL-15Rα on the cell surface of BCMA CAR-NK cells armored with a modified IL-15 construct, using antibodies against IL-15 cytokines (clone 34559, Invitrogen) and IL-15 receptor α (clone 2639B, Biotechne). Briefly, 0.1 × 10⁶ cells were taken from each well of a 96-well plate. 6 Cells were washed, blocked with Fc antibody for 10 min, and stained with IL-15 and IL-15Rα antibodies on ice at 4°C for 45 min. Samples were washed and labeled with diluted live-dead dye 7AAD (5 μl 7AAD per sample, BD Bioscience). Samples were collected and analyzed using flow cytometry (Aurora, Cytek Biosciences). CAR32 cells transduced with BCMA and wild-type IL15 constructs were used as controls.

[0287] As shown in Figure 1, BCMA CAR-NK cells armored with CAR6 and CAR22 showed higher percentages of positive IL-15 and IL-15Rα on the cell surface compared with CAR32 and other modified IL-15 constructs.

[0288] The percentage of CAR-positive cells in BCMA CAR-NK cells armored with the modified IL-15 construct was measured using the BCMA-FITC protein (BCA-HF254, Acrobiosystems Inc.). CAR32 cells transduced with BCMA and wild-type IL15 constructs were used as controls.

[0289] like Figure 2 As shown, all BCMA CAR-NK cells armored with modified IL-15 constructs exhibited a CAR positivity percentage of over 55%.

[0290] 3.2 Proliferation assay

[0291] To analyze the effects of different modified IL-15 constructs on cell proliferation, the total cell count of BCMA CAR-NK cells armored with modified IL-15 constructs was measured. On day 0, cells were introduced into cytokine-free medium at an equal number per well (1 × 10⁶ cells). 6 Cells were seeded into cell cultures and counted on day 9. Cell counts (in millions) and viability were plotted. CAR32 cells transduced with BCMA and wild-type IL15 constructs were used as controls.

[0292] like Figures 3A-3BAs shown, BCMA CAR-NK cells armored with CAR6 and CAR22 exhibited higher proliferation compared to BCMA CAR-NK cells armored with other IL-15 constructs.

[0293] Furthermore, the cell proliferation of BCMA CAR-NK cells armored with modified IL-15 constructs was tested using a dye dilution method. On day 0, cells labeled with Cell Tracking Purple (4 μM, Invitrogen) were used in equal numbers of 1 × 10⁶ cells. 6 Cells / well were seeded in cytokine-free medium, and dye dilutions were evaluated on day 9. For each construct design, MFI reduction and fold increase were plotted. CAR32 transduced with BCMA and wild-type IL15 constructs were used as controls.

[0294] like Figures 4A-4B As shown, BCMA CAR-NK cells armored with CAR6 and CAR22 exhibited higher proliferation compared to CAR32 and other IL-15 constructs.

[0295] 3.3 Based on the percentage of pSTAT5 expression activity

[0296] To analyze the effects of different modified IL-15 constructs on STAT5 signaling, the above-described method was used to test phosphorylated STAT5 (percentage of pSTAT5-positive cells and MFI) in BCMA CAR-NK cells armored with modified IL-15 constructs. CAR32 cells transduced with BCMA and wild-type IL15 constructs were used as controls.

[0297] like Figures 5A-5B As shown, all BCMA CAR-NK cells armored with modified IL-15 constructs exhibited lower STAT5 activation activity. Compared to CAR32, well-proliferating BCMA CAR-NK cells armored with CAR6 and CAR22 also showed lower STAT5 activation activity.

[0298] 3.4 Cytotoxic activity determined by a series of cytotoxicity assays

[0299] The cytotoxic activity of BCMA CAR-NK cells armored with a modified IL-15 construct was tested using a series of cytotoxicity assays targeting H929 (NCI-H929) cells (E:T = 0.25). In short, a co-culture of NK cells and tumor (NCI-H929) cells labeled with cell-tracking violet (4 μM, Invitrogen) was established and analyzed using the methods described above for cytotoxicity assays. Fresh H929 cells labeled with cell-tracking violet were added every 24 h to evaluate cytotoxicity in repeated challenge assays, where modified NK cells were exposed to multiple rounds of tumor cell challenge. Each newly added tumor cell exposure was defined as a round, and cells were analyzed after each round of challenge. Live tumor cell measurements were analyzed to calculate the percentage of cytotoxicity and CAR percentage after each round of tumor exposure. CAR32 cells transduced with BCMA and wild-type IL15 constructs were used as controls.

[0300] like Figure 6 As shown, BCMA CAR-NK cells armored with CAR6 and CAR22 exhibited the highest cytotoxic activity after 8 rounds of tumor cell exposure compared to CAR32 and other IL-15 construct armored BCMA CAR-NK cells.

[0301] 3.5 Safety Testing

[0302] To analyze the safety of BCMA CAR-NK cells armored with modified IL-15 constructs, the proliferation of BCMA CAR-NK cells armored with modified IL-15 constructs was tested in cytokine-free medium in the absence of tumor antigens. On day 0, cells with an equal number of 1 × 10⁶ cells were... 6 Cell cultures of 100 cells / well were seeded in cytokine-free medium and counted after staining with trypan blue (Vi Cell Blue, Beckman Coulter) until day 49. Cell counts (in millions) and viability were plotted. CAR32 cells transduced with BCMA and wild-type IL15 constructs were used as controls.

[0303] like Figures 7A-7B As shown, all BCMA CAR-NK cells in the IL-15 construct armor expanded until day 9, and then the cell number and viability gradually decreased until day 49.

[0304] Example 4: In vivo evaluation of CAR5, CAR6, CAR19, CAR20 and CAR22

[0305] The in vivo tumor-suppressive effect of CAR NK cells designed with selected modified IL-15 was evaluated in a tumor xenograft NCG mouse model. The experiment was designed with 9 groups, each containing 4 NCG mice. Mice in the control group received only PBS. To generate tumor xenografts, 2 million luciferase-labeled NCI-H929 tumor cells (#ATCC CRL-9068) were intravenously injected into all other NCG mice groups.

[0306] Twelve days after tumor implantation, mice were intravenously (iv) injected with 2 million IL-15-constructed CAR-NK cells or unNK (untransduced NK) cells. Tumor progression was monitored weekly using in vivo bioluminescence imaging (BLI). Blood was collected weekly from mice, and the percentage of human NK cells and the number of human CAR-NK cells were determined by flow cytometry. Body weight was also measured at each time point. Mice were then observed for survival up to 50 days post-treatment.

[0307] Our data demonstrate that mice treated with anti-BCMA CAR containing the wild-type IL15 construct (CAR32) exhibited toxicity, with the mice dying on day 9. Construct 22 showed the highest tumor elimination potential, with tumor reduction levels comparable to those in blank mice. Figure 8A One mouse from each of constructs 4 and 5 also had a reduced tumor burden. Figure 8A Construct 22-derived CAR-NK cells showed the highest activity in tumor elimination and the highest survival rate in mice, surviving up to 50 days. Figure 8B One mouse using construct 5 survived to day 43, while one mouse using construct 4 survived to day 50. Analysis of total CD45-positive NK cells and CAR-positive NK cells showed that construct 22 had the highest percentage of expansion in mice. Figure 8C-8D ) and absolute quantity ( Figure 8E-8F No significant changes in mouse body weight or fur were observed in this experiment. Figure 8G This indicates that GVHD did not occur in this experiment. One mouse using construct 4 survived to the end of the experiment, while three mice using construct 22 survived to the end of the experiment. This demonstrates that the IL15 module construct 22 is designed to effectively kill tumor cells without compromising safety, and that construct 22 is both effective and safe.

[0308] Other embodiments

[0309] It should be understood that although this disclosure has been described in conjunction with its detailed description, the foregoing description is intended to illustrate, and not limit, the scope of this disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A fusion protein comprising (1) Signal peptide sequence; (2) Interleukin-15 (IL-15) sequence; and (3) Endoplasmic reticulum (ER) preserved sequences.

2. The fusion protein of claim 1, wherein the signal peptide sequence is a calreticulin signal peptide sequence.

3. The fusion protein of claim 2, wherein the calreticulin signal peptide sequence comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

3.

4. The fusion protein according to any one of claims 1-3, wherein the ER-retained sequence comprises the amino acid sequence shown in SEQ ID NO: 12 or 13, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 12 or 13.

5. A fusion protein comprising (1) Cell membrane targeting sequence; and (2) IL-15 sequence.

6. The fusion protein of claim 5, wherein the cell membrane targeting sequence is selected from the group consisting of: myristylation sequence, palmitoylation sequence, and isopreneation sequence.

7. The fusion protein of claim 5 or 6, wherein the cell membrane targeting sequence is a myristylated sequence.

8. The fusion protein of claim 7, wherein the myristylated sequence comprises an amino acid sequence shown in any one of SEQ ID NO: 14-16, or an amino acid sequence having at least 90%, 95%, or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 14-16.

9. The fusion protein according to any one of claims 1-8, wherein the IL-15 sequence comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

6.

10. The fusion protein according to any one of claims 1-9, wherein the fusion protein further comprises an IL-15Rα sequence.

11. The fusion protein of claim 10, wherein the IL-15Rα sequence comprises the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

11.

12. The fusion protein of claim 10 or 11, wherein the IL-15Rα sequence comprises or is composed of an IL-15Rα Sushi domain, wherein the IL-15Rα Sushi domain comprises or is composed of the following: The amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

10.

13. The fusion protein according to any one of claims 1-4 and 9-12, wherein the fusion protein comprises, from the N-terminus to the C-terminus, a signal peptide sequence, a first adapter sequence, an IL-15 sequence, a second adapter sequence, an IL-15Rα sequence, and an ER-retained sequence.

14. The fusion protein according to any one of claims 1-4 and 9-13, wherein the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NO: 18-20, or an amino acid sequence having at least 90%, 95% or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 18-20.

15. The fusion protein of claim 14, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

19.

16. The fusion protein of any one of claims 5-12, wherein the fusion protein comprises, from the N-terminus to the C-terminus, an optional signal peptide sequence, a myristylation sequence, an IL-15 sequence, a linker sequence, and an IL-15Rα sequence.

17. The fusion protein according to any one of claims 5-12 and 16, wherein the fusion protein comprises an amino acid sequence shown in any one of SEQ ID NO: 21-24, or an amino acid sequence having at least 90%, 95% or 99% identity with an amino acid sequence shown in any one of SEQ ID NO: 21-24.

18. The fusion protein of claim 17, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 24, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

24.

19. A nucleic acid comprising one or more nucleic acid sequences encoding a fusion protein or a portion thereof as described in any one of claims 1-18.

20. The nucleic acid of claim 19, wherein the nucleic acid further comprises a second nucleic acid sequence encoding an engineered receptor, wherein the engineered receptor comprises an extracellular antigen-binding domain or a ligand-binding domain, and optionally an intracellular signal transduction domain.

21. The nucleic acid of claim 20, wherein the engineered receptor nucleic acid sequence and the nucleic acid sequence encoding the fusion protein are separated by a third nucleic acid sequence encoding a cleavable adapter.

22. The nucleic acid of claim 21, wherein the cleavable adapter comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

2.

23. The nucleic acid according to any one of claims 20-22, wherein the engineered receptor is selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), T-cell antigen conjugates (TACs), or portions thereof.

24. The nucleic acid according to any one of claims 20-23, wherein the engineered receptor is a CAR.

25. The nucleic acid of claim 23 or 24, wherein the CAR comprises an extracellular antigen-binding domain that specifically binds to an antigen, wherein the antigen is a tumor antigen selected from the group consisting of: CD19, CD20, CD22, CD30, CD33, CD38, BCMA, CS1, CD138, CD123 / IL3Rα, c-Met, gp100, MUC1, IGF-I receptor, EpCAM, EGFR / EGFRvIII, HER2, IGF1R, mesothelin, PSMA, WT1, ROR1, CEA, GD-2, NY-ESO-1, MAGE A3, DLL3, GPC3, guanylate cyclase 2C (GCC), duracin 18.2, duracin 6, glycolipid F77, PD-L1 and / or PD-L2.

26. The nucleic acid of claim 25, wherein the tumor antigen is BCMA.

27. The nucleic acid according to any one of claims 23-26, wherein the CAR comprises a first VHH antibody portion and a second VHH antibody portion, the first VHH antibody portion comprising CDR1 containing the amino acid sequence of SEQ ID NO: 29, CDR2 containing the amino acid sequence of SEQ ID NO: 30, and CDR3 containing the amino acid sequence of SEQ ID NO: 31, and the second VHH antibody portion comprising CDR1 containing the amino acid sequence of SEQ ID NO: 32, CDR2 containing the amino acid sequence of SEQ ID NO: 33, and CDR3 containing the amino acid sequence of SEQ ID NO:

34.

28. The nucleic acid of any one of claims 23-27, wherein the CAR comprises a first VHH antibody portion and a second VHH antibody portion, the first VHH antibody portion comprising the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence of SEQ ID NO: 27, and the second VHH antibody portion comprising the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence of SEQ ID NO:

28.

29. The nucleic acid of any one of claims 23-28, wherein the CAR comprises the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 90%, 95%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

1.

30. A vector comprising the nucleic acid as described in any one of claims 19-29.

31. A cell comprising a fusion protein as described in any one of claims 1-18, a nucleic acid as described in any one of claims 19-29, and / or a vector as described in claim 30.

32. The cell of claim 31, wherein the cell expresses an engineered receptor.

33. The cell of claim 32, wherein the engineered receptor is selected from the group consisting of engineered T-cell receptors (TCRs), chimeric antigen receptors (CARs), T-cell antigen conjugates (TACs), or portions thereof.

34. The cell of claim 32 or 33, wherein the engineered receptor specifically recognizes tumor antigens.

35. The cell according to any one of claims 31-34, wherein the cell is an immune cell.

36. The cell according to any one of claims 31-34, wherein the cell is selected from the group consisting of: T cells, αβT cells, γδT cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, embryonic stem cells, and combinations thereof.

37. The cell of claim 36, wherein the cell is an NK cell.

38. A method for producing cells as described in any one of claims 31-37, the method comprising introducing a vector as described in claim 30 into the cells.

39. A method of treating a subject suffering from cancer, the method comprising administering to the subject in need a therapeutically effective amount of cells as described in any one of claims 31-37.

40. The method of claim 39, wherein the subject has breast cancer, lung cancer, pancreatic cancer, melanoma, oral cancer, mesothelioma, ovarian cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, skin cancer, multiple myeloma, lymphoma, epithelial tumor, soft tissue sarcoma, esophageal cancer, or CNS tumor.

41. The fusion protein of any one of claims 1-18, wherein the fusion protein is captured within the cell to reduce the amount of secreted IL-15 and thus reduce the toxicity of IL-15.

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