Binding domain molecules on cell surfaces

Abstract

The present disclosure relates to mammalian cells that have been modified to express on the surface of their membrane a binding domain that binds to a target molecule. The disclosure also relates to protein constructs and nucleic acids for producing such modified mammalian cells, and to methods for using the mammalian cells to deliver therapeutic agents to target cells or tissues in vivo.

Description

[Technical field]

[0001] The present disclosure relates to mammalian cells that have been modified to express on the surface of their membrane a binding domain that binds to a target molecule. The disclosure also relates to protein constructs and nucleic acids for producing such modified mammalian cells, and to methods for using the mammalian cells to deliver therapeutic agents to target cells or tissues in vivo.

[0002] Related Applications This application claims priority from AU2021 / 903825, filed November 26, 2021, the entire contents of which are incorporated herein by reference.

[0003] Incorporation by Reference All documents cited or referenced herein, as well as documents cited in any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein, or in any documents incorporated by reference herein, are hereby incorporated by reference in their entirety.

[0004] Sequence Listing Reference The entire electronic sequence listing submission is incorporated by reference in its entirety for all purposes. [Background technology]

[0005] The goal of medicine is to maintain, improve, or restore the function of damaged or diseased cells, tissues, and organs. This goal can be achieved by using cell-based therapies to treat patients. However, for the full potential of cell-based therapies to be realized, the cells should migrate or "home" to the site where therapy is needed, and the cells should be capable of providing the desired therapy. Attempts have been made to use cell-based therapies, but have had limited success in human clinical settings.

[0006] Thus, there is a substantial need for improved cell-based therapies that can home to the site in a patient where therapy is desired and provide a therapeutic benefit. The present invention addresses these needs and provides other related advantages. Summary of the Invention

[0007] In one embodiment, the disclosure provides a mammalian cell having a cell membrane, the cell being modified to express a cytotoxic T-lymphocyte-associated protein (CTLA-4) binding domain on the surface of the membrane that binds to a target molecule.

[0008] In one example, binding of the CTLA-4 binding domain to a target molecule homes a cell to the target molecule in vivo.

[0009] In one example, binding of the CTLA-4 binding domain to a target molecule homes the target molecule to cells in vivo.

[0010] In one example, the modified mammalian cell is a eukaryotic cell. In another example, the cell is selected from the group consisting of cells of primate origin, monkey origin, and rodent origin. For example, the cell can belong to any one of the following cell line families: Chinese hamster ovary (CHO), mouse myeloma cell (NSO), human embryonic kidney (HEK293), human myeloma cell line, T-cell lymphoma cell line (NOS), renal fibroblast cell line (COS), baby hamster kidney (BHK), HeLa, and PER.C6.

[0011] In one example, the modified mammalian cell is a primary cell. In another example, the cell is selected from the group consisting of a cell of primate, canine, feline, and rodent origin. In another example, the primary cell is a neuronal cell, astrocyte, fibroblast, pericyte, hepatocyte, osteoblast, endothelial cell, or epithelial cell.

[0012] Preferably, the cell or cell line is isolated.

[0013] In another example, the modified mammalian cell is an immune cell. For example, the immune cell can be selected from the group consisting of T cells (e.g., CD4+), cytotoxic T cells, monocytes, peripheral blood hematopoietic stem cells, macrophages, antigen presenting cells, natural killer cells, mast cells, neutrophils, eosinophils, basophils, natural killer (NK) T cells, B cells, dendritic cells, helper T cells, and regulatory T cells.

[0014] In another example, the modified mammalian cell is a stem cell. In another example, the cell is a pluripotent stem cell. In another example, the stem cell is a mesenchymal precursor or stem cell. In another example, the cell is a mesenchymal precursor cell (MPC) or a mesenchymal stem cell (MSC). In another example, the cell is a differentiated stem cell. For example, the differentiated stem cell can be a differentiated MPC or MSC. In another example, the MPC or MSC is culture expanded.

[0015] In one example, the cells are induced pluripotent stem cells (iPS).

[0016] In one example, the CTLA-4 binding domain comprises or consists of a CTLA-4 sequence set forth below. KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMTGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPSPDSN (SEQ ID NO: 1)

[0017] In one example, an alanine (A) at position 31 of SEQ ID NO:1 is replaced with a tyrosine (Y). In one example, a threonine (T) at position 56 of SEQ ID NO:1 is replaced with a methionine (M). In one example, a leucine (L) at position 106 of SEQ ID NO:1 is replaced with a glutamic acid (E).

[0018] In one example, the CTLA-4 binding domain comprises or consists of a scaffold having a framework and exposed binding loops (BL). In one example, the framework corresponds to residues 1-25, 34-54, 60-97, and 106-126 of SEQ ID NO:1.

[0019] In another example, the CTLA-4 binding domain scaffold comprises or consists of a sequence having at least about 70% sequence identity, or at least 75%, 80%, 85%, 87%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1 or residues 1-1-25, 34-54, 60-97 and 106-126 of SEQ ID NO:1.

[0020] In another example, amino acid residues at positions 26-33, and / or 55-59, and / or 98-105 of SEQ ID NO:1 are modified or replaced with one or more heterologous sequences.

[0021] In another example, the CTLA-4 binding domain comprises or consists of the sequence set forth below: [ka] Here, X is any amino acid residue, n is a number from 5 to 15, and n1, n2, and n3 represent binding loops (BL) 1, 2, and 3, respectively.

[0022] In another example, Xn 1 is 5-8 amino acids, Xn 2 is 5-8 amino acids, Xn 3 is 10 to 15 amino acids.

[0023] In another example, a single binding loop, two binding loops, or all three binding loops of native CTLA-4 may be modified by amino acid substitution, addition or deletion, and / or any change to one or more physical properties (e.g., size, shape, charge, hydrophobicity, etc.).

[0024] In a further example, the exposed binding loop (BL1) sequence ASPGKATE (SEQ ID NO: 3) or ASPGKYTE (SEQ ID NO: 4), and / or the exposed loop (BL2) sequence MTGNE (SEQ ID NO: 5) and / or the exposed binding loop (BL3) sequence ELMYPPPYY (SEQ ID NO: 6) of the CTLA-4 binding domain sequence are modified by amino acid substitution, addition or deletion, or replaced with a heterologous sequence.

[0025] In one example, amino acid residues at positions 26-33 and / or 55-59 and / or 98-105 of SEQ ID NO: 1 are modified or replaced. In another example, amino acid residues at positions 27-33 and / or 54-62 and / or 98-106 of SEQ ID NO: 1 are modified or replaced with heterologous sequences.

[0026] In another example, the effect of modifying the CTLA-4 binding domain is to abolish its natural affinity for CD80 and CD86.

[0027] In one example, the CTLA-4 binding domain scaffold comprises or consists of the following sequence: [ka] where X is any amino acid residue, n is a number from 5 to 15, and the numbers n1, n2, and n3 represent binding loop regions, more specifically, 1, 2, and 3 correspond to BL-1, BL-2, and BL-3 of the CTLA-4 binding domains, respectively.

[0028] In one example, the CTLA-4 binding domains BL-1, BL-2 and BL-3 comprise or consist of ASPGKYTE (SEQ ID NO: 4), MTGNE (SEQ ID NO: 5) and ELMYPPPYY (SEQ ID NO: 6), respectively, which domains bind to B7-1.

[0029] In one example, the CTLA-4 binding domains BL-1, BL-2 and BL-3 comprise or consist of TVSWVDME (SEQ ID NO: 8), WNGRW (SEQ ID NO: 9) and QLDPSWGYYWQGY (SEQ ID NO: 10), respectively, which domains bind to sclerostin.

[0030] In one example, the CTLA-4 binding domain comprises or consists of the sequence KAMHVAQPAVVLASSRGIASFVCEYASPGKYTEVRVTVLRQADSQVTEVCAATYMTGNELTFLDDSICTGTSGNQVNLTIQGLRAMDTGLYICKVELMYPPYYLGIGNGTQIYVIDPEPSPDSN (SEQ ID NO: 11), and the domain binds to B7-1.

[0031] In another example, the CTLA-4 binding domain comprises or consists of the sequence KAMHVAQPAVVLASSRGIASFVCEYTVSWVDMEVRVTVLRQADSQVTEVCAATYWNGRWLTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVQLDPSWGYYWQGYEGIGNGTQIYVIDPEPSPDSN (SEQ ID NO: 12), which domain binds to sclerostin.

[0032] In a further example, the CTLA-4 binding domains BL-1, BL-2 and BL-3 are replaced with the CDR1, CDR2 and CDR3 sequences of an antibody, respectively. The antibody from which the CDR sequences are derived can be from any species. In one example, the antibody is from a human. In another example, the antibody is from a domestic animal, such as a cat, dog, rabbit, guinea pig or horse.

[0033] In one example, the CTL-4 binding domain is anchored to the surface of the membrane by a transmembrane domain. Any suitable transmembrane domain may be used. In one example, the transmembrane domain is the transmembrane domain of human platelet derived growth factor receptor (PDGFR), human asialoglycoprotein receptor, human and mouse B7-1, human ICAM-1, human erbb1, human erbb2, human erbb3, human erbb4, human fibroblast growth factor receptors, e.g., FGFR1, FGFR2, FGFR3, FGFR4, human VEGFR-1, human VEGFR-2, human erythropoietin receptor, human PRL-R, prolactin receptor, human EphA1, ephrin type A receptor 1, human insulin, I Selected from the group consisting of GF-1 receptor, human receptor-like protein tyrosine phosphatase, human neuropilin, human major histocompatibility complex II (alpha and beta chains), human integrins (alpha and beta family), human syndecadin, human myelin protein, human cadherin, human synaptobrevin-2, human glycophorin-A, human Bnip3, human APP, amyloid precursor protein, human T cell receptor alpha and beta, CD3 gamma, CD3 delta, CD3 zeta, and CD3 epsilon.

[0034] In another example, the CTLA-4 binding domain is connected to the transmembrane domain via a linker. In one example, the linker has the sequence (SGGGG) n S (SEQ ID NO: 13), where n is any number from 2 to 8, or 3 to 6, or 3 to 4. In one example, the linker comprises or consists of the sequence SGGGGSGGGGSGGGGS (SEQ ID NO: 14) or SGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 15).

[0035] In a second embodiment, the present disclosure provides a method for homing mammalian cells to a target molecule in a subject, comprising administering to the subject a modified mammalian cell as described herein according to the first embodiment.

[0036] In one example, the cells are also modified to deliver a therapeutic agent. The therapeutic agent can be, for example, an anti-cancer agent or an immunomodulatory agent. In another example, the therapeutic agent is a naturally occurring or modified oncolytic virus.

[0037] However, it will be understood that the modified mammalian cells of the present disclosure may carry natural therapeutic agents without the need for further modification. For example, MPCs or MSCs naturally contain beneficial proteins, such as cytokines, enzymes and other proteins, which may be delivered to target cells or tissues in vivo via paracrine signaling. Thus, these cells may be considered as adjuvants for the therapeutic agent itself, or for co-administered therapeutic agents.

[0038] In one example, the target molecule is selected from the group consisting of a target expressed by a tumor and a target associated with the tumor stroma.

[0039] In a third embodiment, the present disclosure provides a method for homing a target molecule to a mammalian cell described herein, comprising administering to a subject a modified mammalian cell described herein.

[0040] For example, modified mammalian cells of the present invention can be anchored to or implanted within a tissue site, e.g., where regeneration or repair is required. The anchored cells then bind a target molecule of interest and home the target molecule to the site of injury. Examples of suitable target molecules include growth effector molecules, including growth factors and extracellular matrix molecules, that stimulate and support cell and tissue growth or promote wound healing.

[0041] In a fourth embodiment, the present disclosure provides a method for manufacturing a semiconductor device comprising: (a) a leader sequence for translocating the chimeric domain across an intracellular membrane; (b) a CTLA-4 binding domain specific for a target molecule; (c) a transmembrane domain that anchors the chimeric domain to the surface membrane of a mammalian cell.

[0042] In one example, the chimeric binding domain further comprises a linker sequence located between the CTLA-4 binding domain and the transmembrane domain. In one example, the linker is a peptide linker. Any suitable peptide linker known in the art can be utilized in the present disclosure. In one example, the linker is a Gly-Ser peptide linker.

[0043] In one example, the linker comprises the sequence (SGGGG)nS (SEQ ID NO: 13), where n is any number from 2 to 8, or 3 to 6, or 3 to 4. In one example, the linker comprises or consists of the sequence SGGGGSGGGGSGGGGS (SEQ ID NO: 14) or SGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 15).

[0044] In one example, the leader sequence is mouse Ig kappa (METDTLLLWVLLLWVPGSTGD; SEQ ID NO: 16), human OSM (MGVLLTQRTLLSLVLALLFPSMASM; SEQ ID NO: 17), VSV-G (MKCLLYLAFLFIGVNC; SEQ ID NO: 18), human IgG2 H (MGWSCIILFLVATATGVHS; SEQ ID NO: 19), BM40 (MRAWIFFLLCLAGRALA; SEQ ID NO: 20), secretone (MWWRLWWLLLLLLLLWPMVWA; SEQ ID NO: 21), human IgKVIII (MDMRVPAQLLGLLLLWLRGARC; SEQ ID NO: 22), CD33 (MPLLLLLPLLWAGALA; SEQ ID NO: 23), tPA (MDAMKRGLCCVLLLCGAVFVSPS; SEQ ID NO: 24), human chymotrypsinogen (MAFLWLLSCWALLGTTFG; SEQ ID NO: 25), human trypsinogen-2 (MNLLLILTFVAAAVA; SEQ ID NO: 26), human IL-2 (MYRMQLLSCIALSLALVTNS; SEQ ID NO: 27), Gaussia Selected from the group consisting of luc (MGVKVLFALICIAVAEA; SEQ ID NO: 28), albumin (HSA) (MKWVTFISLLFSSAYS; SEQ ID NO: 29), influenza hemagglutinin (MKTIIALSYIFCLVLG; SEQ ID NO: 30), human insulin (MALWMRLLPLLALLALWGPDPAAA; SEQ ID NO: 31), silkworm fibroin LC and (MKPIFLVLLVVTSAYA; SEQ ID NO: 32).

[0045] In one example, the transmembrane domain comprises 17 to 29 residues, or 19 to 26 residues, or 21 to 24 residues. In another example, the transmembrane domain comprises the transmembrane domain of the human platelet derived growth factor receptor (PDGFR), the human asialoglycoprotein receptor, human and mouse B7-1, human ICAM-1, human erbb1, human erbb2, human erbb3, human erbb4, human fibroblast growth factor receptors, e.g., FGFR1, FGFR2, FGFR3, FGFR4, human VEGFR-1, human VEGFR-2, human erythropoietin receptor, human PRL-R, prolactin receptor, human EphA1, ephrin type A receptor 1, human insulin, Selected from the group consisting of IGF-1 receptor, human receptor-like protein tyrosine phosphatase, human neuropilin, human major histocompatibility complex II (alpha and beta chains), human integrins (alpha and beta family), human syndecadin, human myelin protein, human cadherin, human synaptobrevin-2, human glycophorin-A, human Bnip3, human APP, amyloid precursor protein, human T cell receptor alpha and beta, CD3 gamma, CD3 delta, CD3 zeta, and CD3 epsilon.

[0046] In a fifth embodiment, the present disclosure provides a nucleic acid molecule encoding a chimeric binding domain described herein. In one example, the nucleic acid is DNA, RNA, or both.

[0047] The disclosure also provides nucleic acids encoding the polypeptides of the disclosure, particularly the polypeptides of any one of SEQ ID NOs:1, 11, or 12.

[0048] In one example, the molecule comprises or consists of the nucleic acid sequence of a binding domain described below: [ka] Here, N1 is the length of nucleotides encoding the first binding loop, N2 is the length of nucleotides encoding the second binding loop, and N3 is the length of nucleotides encoding the third binding loop. In one example, N1, N2, and N3 are 15 to 45 nucleotides. In one example, N1 is 15 to 24 nucleotides. N2 is 15 nucleotides, and N3 is 30 to 45 nucleotides. N is any nucleotide (A, C, T, G).

[0049] In one example, the nucleic acid is provided in an expression construct in which the nucleic acid is operably linked to a promoter. Such an expression construct may be a vector, e.g., a plasmid.

[0050] In a sixth embodiment, the present disclosure provides a host cell transformed with a nucleic acid described herein. Suitable host cells include bacteria, mammalian cells, yeast, moss (moss), and the like.

number

[0051] In a seventh embodiment, the present disclosure provides a pharmaceutical composition comprising the modified mammalian cells described herein together with a pharma- ceutically acceptable carrier and / or excipient. The composition may be provided as a medicament. In one example, the composition is for use in treating a disorder.

[0052] In another example, the binding domain may be labeled with an agent to facilitate detection.

[0053] It will be appreciated that the mammalian cells and pharmaceutical compositions described herein are suitable for use as pharmaceuticals and for the treatment of a wide range of conditions or diseases, including, for example, the treatment of oncological diseases, autoimmune diseases, neurological diseases, orthopedic diseases, cardiac diseases and trauma. [Brief description of the drawings]

[0054] [Figure 1]Figure 1 shows the results of an intracellular ELISA assay performed on HEK293 cells transfected with either plasmid DNA expressing CTLA-4 BD (either BD_B7 or BD_SOST) or no plasmid DNA (mock transfection). Panel A shows detection of BD_B7 on the cell surface using anti-CTLA-4 primary antibody (1:1000) and anti-mouse IgG (HRP) secondary antibody (1:5000). Panel B shows detection of BD_SOST on the cell surface using the same antibodies as in panel A, and panel C shows detection of human recombinant sclerostin bound to BD_SOST-expressing HEK cells using anti-SOST primary antibody (1:1000) and anti-mouse IgG (HRP) secondary antibody (1:5000). All values ​​are arithmetic means ± SEM. Asterisks indicate p-values ​​from two-tailed unpaired t-tests: *p ≤ 0.05, ***p ≤ 0.001. [Diagram 2] Figure 1 shows the results of an intracellular ELISA assay performed on CHO cells transfected with either plasmid DNA expressing CTLA-4 BDM (either BD_B7 or BD_SOST) or no plasmid DNA (mock transfection). Panel A shows detection of BD_B7 on the cell surface using anti-CTLA-4 primary antibody (1:1000) and anti-mouse IgG (HRP) secondary antibody (1:5000). Panel B shows detection of BD_SOST on the cell surface using the same antibodies as in panel A, and panel C shows detection of human recombinant sclerostin bound to BD_SOST-expressing HEK cells using anti-SOST primary antibody (1:1000) and anti-mouse IgG (HRP) secondary antibody (1:5000). All values ​​are arithmetic means ± SEM. Asterisks indicate p-values ​​from two-tailed unpaired t-tests: *p ≤ 0.05, ***p ≤ 0.001. [Diagram 3]Figure 1 shows the results of an intracellular ELISA assay performed on adipose tissue transfected with either plasmid DNA expressing CTLA-4 BDM (either BDM_B7 or BDM_SOST) or without plasmid DNA (mock transfection). Panel A shows detection of BDM_B7 on the cell surface using anti-CTLA-4 primary antibody (1:1000) and anti-mouse IgG (HRP) secondary antibody (1:5000). Panel B shows detection of BDM_SOST on the cell surface using the same antibodies as in panel A; binding of rhSOST could not be assessed because aMSCs endogenously express SOST binding protein. All values ​​are arithmetic means ± SEM. Asterisks indicate p-values ​​from two-tailed unpaired t-tests: **p ≤ 0.01, ***p ≤ 0.001. [Figure 4]Figure 1 shows the results of an intracellular ELISA assay performed on bone marrow-derived mesenchymal stem cells (bMSCs) transfected with either plasmid DNA expressing CTLA-4BD (either BD_B7 or BD_SOST) or no plasmid DNA (mock transfection). Panel A shows detection of BD_B7 on the cell surface using anti-CTLA-4 primary antibody (1:1000) and anti-mouse IgG (HRP) secondary antibody (1:5000). Panel B shows detection of BD_SOST on the cell surface using the same antibodies as in panel A. Panel C shows detection of human B7-1 (CD80) protein bound to bMSCs expressing BD_B7 using anti-CD80 primary antibody (1:1000) and anti-mouse IgG (HRP) secondary antibody (1:5000); binding of rhSOST could not be assessed because bMSCs endogenously express SOST binding protein. Panel D shows detection of Raji cells bound to BD_B7-expressing bMSCs using anti-CD80 primary antibody (1:1000) and anti-mouse IgG (HRP) secondary antibody (1:5000). Panel E shows immunofluorescence staining of transfected bMSCs expressing BD_B7 stained with anti-CTLA-4 antibody (green) and nuclear stain DAPI (blue). All values ​​are arithmetic mean ± SEM. Asterisks indicate p-values ​​from two-tailed unpaired t-test: **p≦0.01, ***p≦0.001.

[0055] Key to the array table SEQ ID NO: 1: Amino acid sequence of the CTLA-4 binding domain SEQ ID NO:2: Amino acid sequence of CTLA-4 binding domain scaffold 1 SEQ ID NO:3: Amino acid sequence encoding exposed binding loop (BL1) sequence 1 SEQ ID NO: 4: Amino acid sequence encoding exposed binding loop (BL1) sequence 2 SEQ ID NO: 5: Amino acid sequence encoding the exposed loop (BL-2) sequence SEQ ID NO: 6: Amino acid sequence encoding the exposed binding loop (BL-3) sequence SEQ ID NO: 7: Amino acid sequence of CTLA-4 binding domain scaffold 2 SEQ ID NO: 8: Amino acid sequence encoding sclerostin BL-1 sequence SEQ ID NO: 9: Amino acid sequence encoding sclerostin BL-2 sequence SEQ ID NO: 10: Amino acid sequence encoding sclerostin BL-3 sequence SEQ ID NO: 11: Amino acid sequence encoding the CTLA-4 binding domain of B7-1 SEQ ID NO: 12: Amino acid sequence encoding the CTLA-4 binding domain of sclerostin SEQ ID NO: 13: Amino acid sequence encoding the linker SEQ ID NO: 14: Amino acid sequence encoding the linker SEQ ID NO: 15: Amino acid sequence encoding the linker SEQ ID NO: 16: Amino acid sequence encoding the leader sequence of mouse Ig kappa SEQ ID NO: 17: Amino acid sequence encoding the leader sequence of human OSM SEQ ID NO: 18: Amino acid sequence encoding the leader sequence of VSV-G SEQ ID NO: 19: Amino acid sequence encoding the leader sequence of human IgG2 H SEQ ID NO:20: Amino acid sequence encoding the leader sequence of BM40 SEQ ID NO: 21: Amino acid sequence encoding the leader sequence of secreton SEQ ID NO: 22: Amino acid sequence encoding the leader sequence of human IgKVIII SEQ ID NO: 23: Amino acid sequence encoding the leader sequence of human CD33 SEQ ID NO: 24: Amino acid sequence encoding the leader sequence of tPA SEQ ID NO: 25: Amino acid sequence encoding the leader sequence of human chymotrypsinogen SEQ ID NO: 26: Amino acid sequence encoding the leader sequence of human trypsinogen-2 SEQ ID NO: 27: Amino acid sequence encoding the leader sequence of human IL-2 SEQ ID NO: 28: Amino acid sequence encoding the leader sequence of Gaussia luc SEQ ID NO: 29: Amino acid sequence encoding the leader sequence of albumin (HSA) SEQ ID NO: 30: Amino acid sequence encoding the leader sequence of influenza hemagglutinin SEQ ID NO: 31: Amino acid sequence encoding the leader sequence of human insulin SEQ ID NO: 32: Amino acid sequence encoding the leader sequence of silkworm fibroin LC SEQ ID NO: 33: Nucleic acid sequence encoding the CTLA-4 binding domain DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] Selected Definitions Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" should be understood to mean the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0057] A "CTLA-4 binding domain" is a domain derived from the N-terminal extracellular domain of CTLA-4. A CTLA-4 binding domain may comprise a native CTLA-4 sequence, for example as set forth in SEQ ID NO: 1, or a modified version thereof, with an altered C-terminal sequence. The modification may occur in one or more of the framework or scaffold regions, or binding loop (BL) sequences. In one example, all BL sequences are replaced by heterologous or random BL sequences such that the CTLA-4 binding domain no longer binds to its native ligand. In one example, the binding specificity of the modified CTLA-4 domain is altered such that the domain binds to a different target molecule of interest.

[0058] A "binding loop" (BL) is a polypeptide loop structure or region that functions in a manner similar to a complementarity determining region (CDR) in an antibody variable domain that binds to a specific antigen. Three antigen binding loop sequences (referred to herein as BL-1, BL-2 and BL-3, respectively) are present in the CTLA-4 binding domain, which are located within a scaffold sequence that provides the necessary three-dimensional conformation of the loop sequence. The native BL sequence can be replaced with one or more corresponding CDRs that can be grafted onto the scaffold. For example, using display technology, diversity can be introduced into the BL site of the CTLA-4 binding domain by randomizing the amino acid sequence of a specific loop of the scaffold by introducing an NNK codon, followed by selection of the desired binding properties. This mechanism is similar to the natural selection of high affinity antigen-specific antibodies.

[0059] The term "binding specificity" in the context of a CTLA-4 binding domain refers to the ability of the domain to bind to its respective target antigen or epitope, which is dependent on the presence of a particular structure (e.g., antigenic determinant or epitope) on the target antigen or epitope. For example, a CTLA-4 binding domain recognizes and binds to a specific protein structure, rather than proteins in general. As an example, if a domain binds to epitope "A", in a reaction containing labeled "A" and the domain, the presence of a molecule containing epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" bound to the domain.

[0060] The term should also be understood to include that the CTLA-4 binding domain "specifically binds" to a target antigen. The term "specifically binds" or "binds specifically" is taken to mean that the CTLA-4 binding domain of the present disclosure reacts or associates with a particular target antigen more frequently, more rapidly, with a longer duration, and / or with a higher affinity, as compared to alternative target antigens. Reference to "binding" provides explicit support for the term "specific binding" and vice versa. Typically, the term is used to describe the affinity of a domain for a given target antigen. In some situations, it may be desirable to have low affinity binding where toxicity may be an issue. In other situations, it may be desirable to have high affinity binding to minimize cross-reactivity to other target antigens. In one example, the binding is specific binding as described herein.

[0061] The term "binding affinity" or "affinity" of a molecular moiety (i.e., a protein or BDM) for a selected target can be measured. The term "affinity" refers to the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (Kd) or the equilibrium dissociation constant (KD).

[0062] The term "target molecule" as used herein refers to a substance to which the CTLA-4 binding domain binds. The target molecule may be, for example, an antigen. An antigen typically contains one or more antigenic epitopes recognized by the CTLA-4 binding domain. The protein antigen may be a soluble protein or a membrane-bound protein. Examples of soluble proteins include, but are not limited to, transcription factors, antibodies, growth factors, blood proteins (e.g., albumin), or drugs (e.g., steroids, pharmaceuticals, etc.). Types of 85asZ membrane-bound proteins include growth factor receptors, tumor markers, cell surface markers, or markers that mediate transport into cells (e.g., transferrin), or Fc receptors. Typically, it refers to a substance that can raise an immune response in vivo. It may be a polypeptide, a protein, a nucleic acid (e.g., DNA, RNA, or a combination of DNA and RNA), or other molecules.

[0063] As used herein, the term "epitope" (synonym "antigenic determinant") is understood to mean the region to which the CTLA-4 binding domain of the present disclosure binds. Traditionally, the term refers to the structure bound by an immunoglobulin VH / VL pair. An epitope defines the minimal binding site of a CTLA-4 binding domain. The term is not necessarily limited to the particular residues or structures that the CTLA-4 binding domain contacts. For example, the term includes the region spanning the amino acids contacted by the BL sequence of the CTLA-4 binding domain, and 5-10 (or more), or 2-5, or 1-3 amino acids outside this region. In some examples, an epitope includes a series of discontinuous amino acids that are positioned close to each other when the polypeptide folds, e.g., associated with another polypeptide, i.e., a "conformational epitope". The term includes those that consist of a linear peptide sequence (i.e., "continuous"), or those that consist of non-contiguous amino acid sequences (i.e., "conformational" or "discontinuous").

[0064] iPS cells as referred to herein are understood to refer to skin or blood derived cells that have been reprogrammed into an embryonic-like pluripotent state allowing for the development of an unlimited source of human cells of any type required for therapeutic purposes.

[0065] CTLA-4 binding domain Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is involved in T-cell regulation during immune responses. CTLA-4 is a 44 kDa homodimer that is mainly and transiently expressed on the surface of activated T cells, where it interacts with CD80 and CD86 surface antigens on antigen-presenting cells, resulting in regulation of the immune response (Waterhouse et al. (1996) Immunol Rev 153:183-207; van der Merwe et al. (1997) J Exp Med 185(3):393-403).

[0066] Each CTLA-4 monomer subunit consists of an N-terminal extracellular domain, a transmembrane domain, and a C-terminal intracellular domain. The extracellular domain contains an N-terminal V-like domain (VLD; predicted molecular weight of approximately 14 kDa by homology to the immunoglobulin superfamily) and a stalk of about 10 residues connecting the VLD to the transmembrane domain. The VLD contains surface loops corresponding to BL-1, BL-2, and BL-3, which bind CD80 and / or CD86, respectively (Metzler WJ et al (1997) Nat Struct Biol 4(7):527-31). The sequence of human CTLA-4 has been previously determined (US5,434,131, US5,844,095, US5,851,795).

[0067] Structural and mutational studies of CTLA-4 suggest that binding to CD80 and CD86 occurs through a VLD surface formed from the "GFCC" V-like beta chain and from the highly conserved MYPPPYY sequence in BL-3. Dimerization between CTLA-4 monomers occurs through disulfide bonds between cysteine ​​residues (Cys120) in the two stalks, resulting in tethering of the two extracellular domains, but with no apparent direct association between the V-like domains (Metzler WJ et al (1997) Nat Struct Biol 4(7):527-31). Dimerization appears to contribute only to increased affinity for the ligand.

[0068] The human sequence for CTLA-4 is available as UniProt reference P16410. The extracellular domain of CTLA-4 corresponds to positions 36-161 of the sequence (CTLA-4 has a total length of 126 amino acids). Amino acid residues 1-35 correspond to the signal peptide.

[0069] As shown herein, replacement of one or more binding loop structures in the CTLA-4 binding domain with heterologous binding loop sequences directed against sclerostin or CD3 generated soluble, monomeric, non-glycosylated binding molecules using mammalian expression systems. Thus, VLDs provide a basic framework for constructing soluble single domain molecules whose binding specificity can be engineered by modification of the binding loop structures.

[0070] Framework residues of the CTLA-4 binding domain may be modified according to structural features present in camelid antibodies. Camelid heavy chain immunoglobulins differ from conventional antibody structures by consisting of a single VH domain.

[0071] Several unconventional substitutions (mainly hydrophobic to polar in nature) at exposed framework residues reduce the hydrophobic surface while maintaining the internal beta-sheet framework structure (Desmyter et al. (1996) Nat Struct Biol 3:803-811).

[0072] Within the three binding loops, several structural features usually compensate for the loss of the antigen-binding surface provided by the VLD. While the BL2 loop is not significantly different from other VH domains, BL1 and BL3 adopt non-canonical conformations that are highly heterogeneous in length. For example, the H1 loop may contain anywhere between 2 and 8 residues, compared to the usual 5 in Ig molecules. However, it is BL3 that shows the greatest variation, with the length of this region varying between 7 and 21 residues in 17 reported camelid antibody sequences (Muyldermans et al. (1994) Protein Eng 7:1129-1135). Third, many camelid VH domains have a disulfide bond that interconnects BL1 and BL3 in camels and CDR-1 and CDR-2 in llamas (Vu et al. 1997). The function of this structural feature appears to be to provide a more contoured loop conformation as opposed to a planar one that maintains loop stability, allows binding to a pocket in the antigen, and increases surface area. However, not all camelid antibodies have this disulfide bond, suggesting that it is not an absolute structural requirement.

[0073] These aforementioned features enabled Camelidae V domains to be presented as soluble molecules in vivo and with sufficiently high affinity to generate effective immune responses against a wide variety of target molecules.

[0074] A method for generating and selecting single VLD molecules with novel binding affinities for target molecules is described in US 7,166,697, the entire contents of which are incorporated by reference. The method involves the application of well-known molecular evolution techniques to VLDs derived from members of the immunoglobulin superfamily. The method may involve the generation of phage or ribosome display libraries for screening large numbers of mutant VLDs.

[0075] The filamentous fd-bacteriophage genome is engineered such that the phage displays on its surface proteins, such as Ig-like proteins (Fabs), encoded by DNA contained within the phage (Smith, 1985; Huse et al., 1989; McCafferty et al., 1990; Hoogenboom et al., 1991). Protein molecules can be displayed on the surface of Fd bacteriophage and covalently coupled to the phage coat protein encoded by gene III, or, less commonly, gene VIII. Insertion of an antibody gene into the gene III coat protein results in the expression of 3-5 recombinant protein molecules per terminally located phage. In contrast, insertion of an antibody gene into gene VIII has the potential to display approximately 2000 copies of the recombinant protein per phage particle, a multivalent system that can mask the affinity of a single displayed protein. The Fd phagemid vector is also used because it allows easy switching from display of functional Ig-like fragments on the surface of Fd-bacteriophage to secretion of soluble Ig-like fragments in E. coli. Phage-displayed recombinant protein fusion with the N-terminus of the gene III coat protein is enabled by an amber codon strategically positioned between the two protein genes. In an amber suppressor strain of E. coli, the resulting Ig domain-gene III fusion is anchored within the phage coat.

[0076] Protein affinity-based selection processes can be applied to any high affinity binding reagent, such as antibodies, antigens, receptors, and ligands (see, e.g., Winter and Milstein, (1991) Nature 349:293-299, incorporated herein by reference in its entirety). Thus, selection of the highest affinity binding protein displayed on a bacteriophage is coupled to recovery of the gene encoding that protein. Phages displaying Ig can be affinity selected by binding to cognate binding partners covalently coupled to beads or adsorbed to plastic surfaces in a manner similar to ELISA or solid-phase radioimmunoassay. Nearly all plastic surfaces will adsorb protein antigens, but some commercial products, such as Nunc Immunotubes, are specifically formulated for this purpose.

[0077] Ribosome display libraries contain polypeptides that are de novo synthesized in a cell-free translation system and displayed on the surface of ribosomes for selection purposes (Hanes and Pluckthun, (1997) Proc Natl Acad Sci USA 94:4937-4942; He and Taussig, (1997) Nucl Acids Res 25:5132-5134). A "cell-free translation system" contains ribosomes, soluble enzymes required for protein synthesis (usually from the same cell as the ribosomes), transfer RNA, adenosine triphosphate, guanosine triphosphate, ribonucleoside triphosphate regenerating systems (such as pyruvate phosphoenol and pyruvate kinase), and salts and buffers required to synthesize proteins encoded by exogenous mRNA. Translation of polypeptides can occur under conditions that maintain intact polysomes, i.e., conditions in which ribosomes, mRNA molecules and translated polypeptides are associated within a single complex. This effectively leads to "ribosome display" of the translated polypeptide.

[0078] For selection, the translated polypeptides are mixed with target molecules (e.g., Dynabeads) bound to a matrix in association with the corresponding ribosome complexes. The target molecule can be any compound (or part thereof) of interest, such as a DNA molecule, a protein, a receptor, a cell surface molecule, a metabolite, an antibody, a hormone, or a virus. Ribosomes displaying the translated polypeptides bind to the target molecule, and these complexes can be selected using RT-PCR and the mRNA re-amplified.

[0079] Although there are several alternative approaches to modifying binding molecules, a general approach for all displayed proteins follows a pattern in which individual binding reagents are selected from a display library by their affinity to their cognate receptor. The genes encoding these reagents are modified by any one or combination of several in vivo and in vitro mutation strategies, and assembled into a new gene pool for display and selection of the highest affinity binding molecules.

[0080] B7-1 (CD80) protein and B7-2 (CD86) protein The B7 protein is a peripheral membrane protein found on activated antigen-presenting cells (APCs) that, when paired with either the CD28 or CD152 (CTLA-4) surface proteins on T cells, can generate costimulatory or co-inhibitory signals to enhance or reduce the activity of the N MHC-TCR signal between the APC and the T cell, respectively. In addition to being present on activated APCs, B7 is also found on T cells.

[0081] The B7 proteins include several family members, including B7-1, B7-2, B7-DC, and B7-H1 through B7-H7. The B7-1 protein, also called CD80, binds to CD28 and CTLA-4 (cytotoxic T-lymphocyte-associated protein 4). The UniProt reference for the human sequence of B7-1 (CD80) is P33681.

[0082] In one example, the CTLA-4 binding domain binds to the B7-1 human protein, hi one example, the CTLA-4 binding domain binds to the B7-2 protein.

[0083] Sclerostin Sclerostin is a secreted glycoprotein with a C-terminal cysteine ​​knot-like domain and sequence similarity to the DAN (Differential Screening Selected Genetic Abnormalities in Neuroblastoma) family of bone morphogenetic protein (BMP) antagonists. Sclerostin is produced by bone cells and has anti-anabolic effects on bone formation. The UniProt reference for the human sequence is Q9BQB4.

[0084] In one example, the CTLA-4 binding domain binds to the sclerostin human protein.

[0085] Measurement of binding affinity Epitope binding may be measured by conventional antigen binding assays such as ELISA, by fluorescence-based techniques including FRET, or by techniques such as surface plasmon resonance which measure the mass of molecules. Specific binding of a CTLA-4 binding domain to an antigen or epitope may be determined by any suitable assay including, for example, Scatchard analysis and / or competitive binding assays such as enzyme immunoassays, including radioimmunoassays (RIA), ELISA, and sandwich competition assays.

[0086] Competitive assays such as surface plasmon resonance assays can be used to determine whether a CTLA-4 binding domain that has been engineered to bind to a particular target can do so. By way of illustration, a CTLA-4 binding domain can be engineered to bind to stem cell factor receptor (CSFR or c-kit receptor) and tested for its ability to compete with the binding of the natural ligand (c-kit). In vitro competitive assays for determining the ability of a CTLA-4 binding domain to compete for binding to a target, as well as for determining dissociation constants (KD), are known in the art.

[0087] The binding affinity or dissociation constant (KD) of the interaction between the CTLA-4 binding domain and its respective target can be measured by a number of methods known in the art, including, but not limited to, fluorescence titration, competitive ELISA, isothermal titration calorimetry (ITC) and calorimetric methods such as surface plasmon resonance (BIAcore) or biolayer interferometry (e.g., the Blitz system (ForteBio)).

[0088] A preferred surface plasmon resonance assay is BIAcore, which is known in the art.

[0089] Most binding moieties have K values ​​in the low micromolar (10-6) to nanomolar (10-7 to 10-9) range. High affinity binding moieties are generally considered to be in the low nanomolar range (10-9), with very high affinity binding moieties in the picomolar (10-12) range.

[0090] Complex formation between each moiety and its target is affected by many different factors, including the concentration of each binding partner, the presence of competitors, the pH and ionic strength of the buffer system used, and the experimental method used to determine the KD (e.g., fluorescence titration, competitive ELISA, or surface plasmon resonance) or the mathematical algorithm used to evaluate the experimental data.

[0091] Thus, it is clear to one skilled in the art that KD values ​​may vary within certain experimental ranges depending on the method and experimental settings used to determine the affinity of a particular CTLA-4 binding domain for a given target. This means that there may be slight deviations in the measured KD values ​​or tolerance ranges depending on whether the KD values ​​are determined by surface plasmon resonance (Biacore), competitive ELISA, or "direct ELISA".

[0092] In a preferred example, the KD value is determined by using a surface plasmon resonance assay, for example using BIAcore surface plasmon resonance against an immobilized target (Cytiva Life Sciences, Marlborough, Mass., USA).

[0093] The affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more, than the affinity of the protein or BDM for an unrelated amino acid sequence. The affinity of the protein or BDM for a target (e.g., a protein antigen) can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM), or more.

[0094] In one example, the protein has an affinity as measured by a KD of about 200 nM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 5 nM or less, about 1 nM or less, or about 0.5 nM or less.

[0095] In one example, the CTLA-4 binding domain has an affinity as measured by a KD of about 200 nM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less, 10 nM or less, about 5 nM or less, about 1 nM or less, or about 0.5 nM or less.

[0096] Biolayer interferometry is a label-free technique for measuring biomolecular interactions within an interactome. It is an optical analysis technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of proteins immobilized at the tip of a biosensor and an internal reference layer. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real time.

[0097] Binding between a ligand immobilized on the biosensor tip surface and an analyte in solution results in an increase in optical thickness at the biosensor tip, which results in a wavelength shift Δλ, which is a direct measure of the change in thickness of the biological layer. The interaction is measured in real time, providing the ability to precisely and accurately monitor binding specificity, association and dissociation rates, or concentration.

[0098] Only molecules that bind or dissociate from the biosensor can shift the interference pattern and generate a response profile. Unbound molecules, changes in the refractive index of the surrounding medium, or changes in flow rate have no effect on the interference pattern. This is a unique feature of biolayer interferometry and extends its ability to run on crude samples used in applications for protein-protein interactions, quantification, affinity, and kinetics.

[0099] target molecule The target molecule according to the present disclosure is preferably an antigen. The antigen may be selected from a protein, a glycan, a lipid, a lipoprotein, or a nucleic acid. The protein may be a soluble protein or a membrane-bound protein. Examples of soluble proteins include, but are not limited to, transcription factors, antibodies, growth factors, blood proteins (e.g., albumin), or drugs (e.g., steroids, pharmaceuticals, etc.). Types of membrane-bound proteins include growth factor receptors, tumor markers, or markers that mediate transport into cells (e.g., transferrin), or Fc receptors.

[0100] The nucleic acid targets can be DNA, RNA, or a combination of DNA and RNA.

[0101] The target antigen may be a tumor-associated antigen, such as MUC-1 and peptide fragments thereof, protein MZ2-E, polymorphic epithelial mucin, folate-binding protein LK26, MAGE-1 or MAGE-3 and peptide fragments thereof, human chorionic gonadotropin (HCG) and peptide fragments thereof, carcinoembryonic antigen (CEA) and peptide fragments thereof, alpha-fetoprotein (AFP) and peptide fragments thereof, pancreatic oncofetal antigen and peptide fragments thereof, CA125, 15-3, 19-9, 549, 195 and peptide fragments thereof, prostate-specific antigen (PSA) and peptide fragments thereof, prostate-specific membrane antigen (PSMA) and peptide fragments thereof, squamous cell carcinoma antigen (SCCA) and and peptide fragments thereof, ovarian cancer antigen (OCA) and peptide fragments thereof, pancreatic cancer associated antigen (PaA) and peptide fragments thereof, Her1 / neu and peptide fragments thereof, gp-100 and peptide fragments thereof, mutant K-ras protein and peptide fragments thereof, mutant p53 and peptide fragments thereof, non-mutant p53 and peptide fragments thereof, truncated epidermal growth factor receptor (EGFR), chimeric protein p210BCR-ABL, telomerase and peptide fragments thereof, survivin and peptide fragments thereof, Melan-A / MART-1 protein and peptide fragments thereof, WT1 protein and peptide fragments, LMP2 protein and peptide fragments, HPV E6 E7 protein and peptide fragments, idiotype protein and peptide fragments, NY-ESO-1 protein and peptide fragments, PAP protein and peptide fragments, cancer testis protein and peptide fragments, and 5T4 protein and peptide fragments.

[0102] The target antigen can be an antigen or epitope present on cells located in the heart, blood system, lungs, intestines, stomach, rectum, prostate, thyroid, liver or esophagus.

[0103] Expression vector As used herein, "expression vector" includes vectors, such as circular or linear, single-stranded or double-stranded, natural or engineered extrachromosomal plasmid vectors, cosmids, viral vectors, expression vectors, gene transfer vectors, minicircle vectors, and artificial chromosomes, suitable for expressing a polynucleotide encoding a CTLA-4 binding domain.

[0104] In one example, the expression vector is a plasmid display vector.

[0105] In another example, the expression vector is a minicircle DNA vector. A "minicircle DNA vector", which may be referred to as a "minicircle vector" or "minicircle", is a small (usually in the range of 3-4 kb, approximately 3-4 kb, or usually 10 kb or less) circular episomal plasmid derivative from which all prokaryotic vector parts (e.g., bacterial origin of replication, genes associated with bacterial propagation of the plasmid) have been removed. Minicircle vectors are less likely to be perceived as foreign and destroyed when they are used as vehicles to transfer transgenes into mammalian cells because they do not contain prokaryotic DNA sequences.

[0106] The use of minicircle DNA vectors to carry and transport transgene expression cassettes allows mammalian cells to be transfected (e.g., directly) without utilizing an intermediate eukaryotic host system (e.g., insect cell line production system). Furthermore, the size of minicircle vectors (smaller than standard plasmid vectors) and the lack of external bacterial sequences enhances cell transfection and allows for extended duration of transgene expression in mammalian host cells. For example, minicircle vectors are smaller than standard vectors because they lack the extraneous bacterial sequences found on plasmids. The difference in size between plasmid and minicircle vectors can be due to the lack of extraneous bacterial sequences, the inclusion of an insubstantial amount of extraneous bacterial sequences compared to the overall size of the vector, and their variants, such as being considerably smaller compared to plasmids. Long-term, high-level transgene expression by minicircles in mammalian hosts can also be promoted by the incorporation of strong and constitutive promoters such as SV40, CMV, UBC, EF1A, PGK and CAGG.

[0107] Suitable minicircle vectors are described, for example, in Mun et al (2016) Biomaterials 101 (2016) 310-320, and Gaspar et al (2014) Expert Opin. Biol. Ther. 15(3):1-27.

[0108] To generate a chimeric binding construct of the present disclosure, a nucleic acid sequence encoding the CTLA-4 binding domain sequence described herein is cloned into a suitable expression vector by standard methods.

[0109] The expression vector encoding the CTLA-4 binding domain can be integrated into the genome of a mammalian cell and replicate as the host genome replicates. Alternatively, the expression vector encoding the CTLA-4 binding domain can contain an origin of replication that allows for extrachromosomal replication.

[0110] Expression vector components can also include, for example, one or more of an enhancer element, a promoter, a polyadenylation sequence, and a transcription termination sequence.

[0111] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-alpha promoter (EF1), small nuclear RNA promoters (U1a and U1b), alpha-myosin heavy chain promoter, simian virus 40 promoter (SV40), Ruth sarcoma virus promoter (RSV), adenovirus major late promoter, β-actin promoter; hybrid regulatory elements including the CMV enhancer / β-actin promoter or immunoglobulin promoters or active fragments thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 cell lines transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney cell lines (293 or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (BHK, ATCC CCL10), or Chinese hamster ovary cells (CHO).

[0112] Means for introducing expression vectors into mammalian cells for expression are known to those skilled in the art. The technique used for a given cell depends on known successful techniques. Means for introducing recombinant DNA into cells include, inter alia, microinjection, DEAE-dextran mediated transfection, liposome mediated transfection, such as by using Lipofectamine (Gibco, MD, USA) and / or Cellfectin (Gibco, MD, USA), PEG mediated DNA uptake, retroviral transduction, electroporation, and microparticle bombardment, such as by using DNA coated tungsten or gold particles (Agracetus Inc., WI, USA).

[0113] Leader sequence In one example, leader sequence peptides are sequences of 16-20 amino acids at the N-terminus of some eukaryotic proteins that determine their final destination. Proteins that are made and function in the cytoplasm lack leader sequences. Proteins that are targeted to specific organelles require the appropriate signal sequence for each organelle. The leader sequence of a protein designated to enter the endoplasmic reticulum may contain hydrophobic amino acids that become embedded in the lipid bilayer membrane, which functions to guide the nascent protein to a receptor protein that indicates the location of a pore in the membrane. Once the protein enters the cisternal lumen through the pore, the leader segment may be cleaved from the protein. For example, the leader sequence peptide of the interferon protein allows the cell to secrete interferon, but is removed from the mature molecule during the secretion process. Leader sequence peptides are also often referred to as signal peptides.

[0114] Examples of suitable leader sequences include the following: [Table 1]

[0115] Transmembrane domain The term "transmembrane domain" refers to a polypeptide or protein encoded by a nucleic acid sequence and comprising an optional extracellular portion, a transmembrane domain, and an optional cytoplasmic tail. A transmembrane domain is any three-dimensional protein structure that is thermodynamically stable within a membrane and typically comprises a single transmembrane alpha-helix of a transmembrane protein composed primarily of hydrophobic amino acids. The length of a transmembrane domain averages 21 amino acids, but can vary between 4 and 48 amino acids. A transmembrane domain comprises an optional N-terminal extracellular connecting stretch of amino acids and a transmembrane domain. In some embodiments, the transmembrane domain may further comprise a C-terminal cytoplasmic amino acid stretch or an intracellular domain.

[0116] The transmembrane domains used include the transmembrane domain of the human platelet-derived growth factor receptor (PDGFR) gene (Swissprot entry P16234), the human asialoglycoprotein receptor (Swissprot entry P07306), human and mouse B7-1 (human: Swissprot entry P33681 and mouse: Swissprot entry Q00609), human ICAM-1 (Swissprot entry P05362), human erbb1 (Swissprot entry P00533), human erbb2 (Swissprot entry P00534), and human erbb3 (Swissprot entry P00535). ot entry P04626), human erbb3 (Swissprot entry P21860), human erbb4 (Swissprot entry Q15303), human fibroblast growth factor receptors, e.g., FGFR1 (Swissprot entry P11362), FGFR2 (Swissprot entry P21802), FGFR3 (Swissprot entry P22607), FGFR4 (Swissprot entry P22455), human VEGFR-1 (Swissprot entry P17948), human VEGFR-2 (Swissprot entry P22455), human VEGF-1 (Swissprot entry P17948), human VEGF-2 (Swissprot entry P22455), human VEGF-2 (Swissprot entry P22455), human VEGF-3 (Swissprot entry P22455), human VEGF-4 (Swissprot entry P22455), human VEGF-5 (Swissprot entry P22455), human VEGF-6 (Swissprot entry P22455), human VEGF-7 (Swissprot entry P22455), human VEGF-8 (Swissprot entry P22455), human VEGF-9 (Swissprot entry P22455), human VEGF-1 (Swissprot entry P22455), human VEGF-1 (Swissprot entry P22455), human VEGF-1 (Swissprot entry P22455), human VEGF-2 (Swissprot entry P22455), human VEGF-1 (Swissprot entry P22455), human VEGF-2 (Swissprot entry Swissprot entry P35968), human erythropoietin receptor (Swissprot entry P19235), human PRL-R, prolactin receptor (Swissprot entry P16471), human EphA1, ephrin type A receptor 1 (Swissprot entry P21709), human insulin (Swissprot entry P06213), insulin-like growth factor 1 receptor (IGFR1, Swissprot entry P08069, SEQ ID NO: 181), human receptor-like protein tyrosine phosphatase (Swissprot entry P16221), human erythropoietin receptor (Swissprot entry P16222), human erythropoietin receptor (Swissprot entry P16223), human erythropoietin receptor (Swissprot entry P16224), human erythropoietin receptor (Swissprot entry P16225), human erythropoietin receptor (Swissprot entry P16226), human erythropoietin receptor (Swissprot entry P16227), human erythropoietin receptor (Swissprot entry P16229 ... human neuropilin (Swissprot entry P014786), human major histocompatibility complex class II (alpha and beta chains), human integrins (alpha and beta families), human syndecan, human myelin protein, human cadherin, human synaptobrevin-2 (Swissprot entry P63027), human glycophorin-A (GpA, Swissprot entry P02724, SEQ ID NO: 185);Human Bnip3 (Swissprot entry Q12983), human APP (Swissprot entry P05067), amyloid precursor protein (Swissprot entry PODJI8), human T cell receptor alpha gene (PTCRA, Swissprot entry PQ6ISU1) and T cell receptor beta, CD3 gamma (Swissprot entry P09693), CD3 delta (Swissprot entry P04234), CD3 zeta (Swissprot entry P20963), and CD3 epsilon (CD3E, Swissprot entry P07766), human serine / threonine-protein kinase receptor R3 (ACVL1, Swissprot entry P37023), human anthrax toxin receptor 2 (ANTR2, Swissprot entry P58335), human T-cell surface glycoprotein CD4 (CD4, Swissprot entry P01730), human receptor tyrosine-protein phosphatase μ (PTPRM, Swissprot entry P28827, SEQ ID NO: 177), human tumor necrosis factor receptor superfamily member Bar 5 (TNR5, Swissprot entry P25942), human integrin beta-1 (ITB1, Swissprot entry P05556), human HLA class I histocompatibility antigen, B-7 alpha chain (Swissprot entry P01889), human thrombomodulin (TRBM, Swissprot entry P07204), human interleukin-4 receptor subunit alpha (IL4RA, Swissprot entry P24394), human low-density lipoprotein receptor-related protein 6 (LRP6, Swissprot entry P24395), human IL-16 receptor subunit alpha (IL16R, Swissprot entry P24396), human IL-16 receptor subunit alpha (IL16R, Swissprot entry P24397), human IL-16 receptor subunit alpha (IL16R, Swissprot entry P2439 ... rot entry 075581), human high affinity immunoglobulin epsilon receptor subunit alpha (FCERA, Swissprot entry P12319), human killer cell immunoglobulin-like receptor 2DL2 (K12L2, Swissprot entry P43627), human cytokine receptor common subunit beta (IL3RB, Swissprot entry P32927), human integrin alpha-IIb (ITA2B, Swissprot entry P08514), human T cell-specific surface glycoprotein CD28 (CD28,Swissprot entry P10747,

[0117] Immunoglobulin transmembrane domains may also be used. Suitable examples include human immunoglobulin genes IGHA1 (NCBI access code: M60193), IGHA2 (NCBI access code: M60194), IGHD (NCBI access code: K02881), IGHE (NCBI access code: X63693), IGHG1 (NCBI access code: X52847), IGHG2 (NCBI access code: AB006775), IGHG3 (NCBI access code: D78345), IGHG4 (NCBI access code: AL928742), IGHGP (NCBI access code: X52849), and IGHM (NCBI access code: X14940). ), as well as transmembrane domains from the mouse immunoglobulin genes IGHA1 (NCBI access code: K00691), IGHD (NCBI access code: J00450), IGHE (NCBI access codes: X03624, U08933), IGHG1 (NCBI access codes: J00454, J00455), IGHG2A (NCBI access code: J00471), IGHG2B (NCBI access codes: J00462, D78344), IGHG3 (NCBI access codes: X00915, V01526), ​​and IGHM (NCBI access code: J00444).

[0118] In one example, the transmembrane domain used is selected from the group consisting of a PDGFR transmembrane domain, a human B7-1 transmembrane domain, a mouse B7-1 transmembrane domain, a human asialoglycoprotein receptor transmembrane domain, and an erbb-2 transmembrane domain.

[0119] In one example, the transmembrane domain is a PDGFR transmembrane domain.

[0120] In another example, the transmembrane domain is a wild-type CTLA-4 transmembrane domain or a variant thereof, for example, the CTLA-4 binding domain can bind to its naturally occurring transmembrane domain lacking residues that confer dimerization.

[0121] Linker The linker can promote increased flexibility and / or reduce steric hindrance between any two proteins. The linker can be of natural origin, such as a sequence determined to exist in a random coil between two domains of a protein. An exemplary linker sequence is the linker found between the C-terminal domain and the N-terminal domain of the RNA polymerase alpha subunit. Other examples of naturally occurring linkers include the linkers found in 1CI and LexA proteins.

[0122] Within the linker, the amino acid sequence may vary based on the preferred characteristics of the linker, as determined empirically or as revealed by modeling. Considerations in selecting a linker include the flexibility of the linker, the charge of the linker, and the presence of some amino acids of the linker in naturally occurring subunits. The linker may also be designed such that residues within the linker contact DNA, thereby affecting binding affinity or specificity, or interacting with other proteins. In some cases, especially when it is necessary to span a longer distance between subunits or when domains must be held in a particular configuration, the linker may optionally include additional folded domains.

[0123] In some instances, it is preferred that the design of the linker involves an arrangement of domains that requires the linker to span a relatively short distance, preferably less than about 10 angstroms (Å), however, in certain embodiments the linker spans distances of up to about 50 Å or more.

[0124] The term "peptide linker" refers to a short peptide fragment that connects or couples the CTLA-4 binding domain to the transmembrane domain. The linker is preferably composed of amino acids linked together by peptide bonds. For example, the peptide linker can include small or hydrophilic amino acid residues (e.g., glycine, serine, threonine, proline, aspartic acid, asparagine, etc.). For example, the peptide linker is a peptide having an amino acid sequence at least 5 amino acids in length, or about 5 to about 100 amino acids in length, or about 10 to 50 amino acids in length, or about 10 to 15 amino acids in length.

[0125] In one example, the linker is made up of a majority of sterically unhindered amino acids, such as glycine and alanine. Thus, in a further example, the linker is polyglycine, polyalanine, or polyserine.

[0126] Those skilled in the art will appreciate that many commonly used peptide linkers can be used in the embodiments of the present disclosure. In certain embodiments, short peptide linkers may contain repeat units to increase the linker length, such as double, triple or quadruple repeat linkers. In one example, the linker comprises the formula (Gly-Gly-Gly-Ser)n or the formula (Ser-Gly-Gly-Gly)n Ser, where n is a number between 3 and 6.

[0127] In one example, the linker comprises or consists of the sequence SGGGGSGGGGSGGGGS (SEQ ID NO: 14) or SGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: 15).

[0128] Non-peptide linkers are also possible. For example, alkyl linkers such as -NH-(CH2)sC(O)- (wherein s=2-20) can be used. These alkyl linkers can be further substituted with any non-sterically hindering group such as lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. An exemplary non-peptide linker is a PEG linker with a molecular weight of 100-5000 kD, preferably 100-500 kD.

[0129] Examples of other linkers suitable for use include GSTVAAPS, TVAAPSGS or GSTVAAPSGS, or a plurality of such linkers. Other examples include (TVSDVP)n(GS)m, where n=1 and m=1, or where n=2 and m=1, or where n=2 and m=0.

[0130] In another example, the linker is GS.

[0131] Mesenchymal precursor or stem cells As used herein, the term "mesenchymal precursor or stem cell" (MLPC) refers to undifferentiated pluripotent cells that have the ability to self-renew while maintaining pluripotency and to differentiate into several cell types, either of mesenchymal origin, e.g., osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts and tendons, or of non-mesodermal origin, e.g., hepatocytes, neural cells and epithelial cells.

[0132] The term "mesenchymal precursor or stem cell" includes both parent cells and their undifferentiated progeny. The term also includes mesenchymal precursor cells (MPCs), multipotent stromal cells, mesenchymal stem cells (MSCs), perivascular mesenchymal precursors or stem cells, and their undifferentiated progeny. Thus, in one example, the mesenchymal precursor or stem cell is a mesenchymal stem cell.

[0133] Mesenchymal precursors or stem cells can be autologous, allogeneic, xenogeneic, syngeneic or allogeneic. Autologous cells are isolated from the same individual into whom they are to be reimplanted. Allogeneic cells are isolated from a donor of the same species. Xenogeneic cells are isolated from a donor of another species. Syngeneic or allogeneic cells are isolated from genetically identical organisms such as twins, clones, or highly inbred research animal models.

[0134] In one example, the mesenchymal precursors or stem cells are allogeneic. In one example, the allogeneic mesenchymal precursors or stem cells are culture-expanded and cryopreserved.

[0135] In one example, mesenchymal precursor or stem cells express STRO-1 and one or more integrins. Integrins are a class of cell adhesion receptors that mediate both cell-cell and cell-extracellular matrix adhesion events.

[0136] In one example, the mesenchymal precursor or stem cell expresses STRO-1 and coxsackievirus and adenovirus receptors. In another example, the mesenchymal precursor or stem cell expresses STRO-1, coxsackievirus and adenovirus receptors, and one or more of the integrins referenced above.

[0137] In another example, the mesenchymal precursor or stem cell is a CD29+, CD54+, CD73+, CD90+, CD102+, CD105+, CD106+, CD166+, MHC1+ MSC.

[0138] In one example, the mesenchymal precursor or stem cell is MSC. The MSC may be a homogenous composition or a mixed cell population enriched with MSC. The homogenous MSC composition can be obtained by culturing adherent bone marrow or periosteal cells, and the MSC can be identified by a specific cell surface marker identified with a unique monoclonal antibody. Methods for obtaining cell populations enriched with MSC are described, for example, in US Pat. No. 5,486,359. MSC prepared by conventional plastic adherence isolation relies on the non-specific plastic adhesion property of CFU-F.

[0139] The terms "enriched," "enriched," or variations thereof, are used herein to describe a cell population in which the percentage of one particular cell type or the percentage of several particular cell types is increased when compared to an untreated cell population (e.g., the cells in their native environment). In one example, a population enriched for mesenchymal precursors or stem cells comprises at least about 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 50%, or 75% mesenchymal precursors or stem cells.

[0140] In one example, the cell population is enriched from a cell preparation that contains STRO-1+ cells in a selectable form. In this regard, the term "selectable form" will be understood to mean that the cells express a marker (e.g., a cell surface marker) that allows for the selection of STRO-1+ cells. The marker may be, but need not be, STRO-1. For example, as described and / or exemplified herein, cells (e.g., mesenchymal progenitor cells) that express STRO-2 and / or STRO-3 (TNAP) and / or STRO-4 and / or VCAM-1 and / or CD146 and / or 3G5 also express STRO-1 (and may be STRO-1 bright). Thus, the indication that cells are STRO-1+ does not mean that the cells are selected solely by STRO-1 expression. In one example, the cells are selected based on at least STRO-3 expression, e.g., they are STRO-3+ (TNAP+). For example, MPCs may be isolated from bone mononuclear cells using anti-STRO-3 antibodies.

[0141] Reference to the selection of cells or populations thereof does not necessarily require selection from a particular tissue source. As described herein, STRO-1+ cells may be selected, isolated, or enriched from a wide variety of sources. Although, in some instances, these terms provide support for selection from any tissue that contains STRO-1+ cells (e.g., mesenchymal progenitor cells), or vascularized tissues or tissues that contain pericytes (e.g., STRO-1+ pericytes), or any one or more of the tissues listed herein.

[0142] In one example, the cells used in the present disclosure express one or more markers individually or collectively selected from the group consisting of TNAP+, VCAM-1+, THY-1, STRO-2+, STRO-4+(HSP-90β), CD45+, CD146+, 3G5+, or any combination thereof.

[0143] In a preferred embodiment of the present disclosure, the mesenchymal precursors or stem cells are obtained from a master cell bank derived from mesenchymal precursors or stem cells enriched from the bone marrow of healthy volunteers. The use of mesenchymal precursors or stem cells derived from such sources is particularly advantageous for subjects who do not have suitable family members available that can serve as mesenchymal precursor or stem cell donors or who require immediate treatment and are at high risk of relapse, disease-related decline, or death during such time to generate mesenchymal precursors or stem cells.

[0144] Mesenchymal precursors or stem cells encompassed by the present disclosure may also be cryopreserved prior to administration to a subject, hi one example, the mesenchymal precursors or stem cells are culture-expanded and cryopreserved prior to administration to a subject.

[0145] In one example, the present disclosure encompasses mesenchymal precursors or stem cells, as well as their progeny, soluble factors derived therefrom, and / or extracellular vesicles isolated therefrom. In another example, the present disclosure encompasses mesenchymal precursors or stem cells, as well as extracellular vesicles isolated therefrom. For example, the mesenchymal precursors or stem cells of the present disclosure can be cultured for a period and under conditions suitable for secretion of extracellular vesicles into cell culture medium. The secreted extracellular vesicles can then be obtained from the culture medium for use in therapy.

[0146] The term "extracellular vesicles" as used herein refers to lipid particles that are naturally released from cells and range in size from about 30 nm to 10 microns, but are typically less than 200 nm in size. They can contain proteins, nucleic acids, lipids, metabolites, or organelles from the releasing cells (e.g., mesenchymal stem cells; STRO-1+ cells).

[0147] The term "exosomes" as used herein refers to a type of extracellular vesicles that generally range in size from about 30 nm to about 150 nm and originate from the endosomal compartment of mammalian cells where they are transported to and released from the cell membrane. They can contain nucleic acids (e.g., RNA; microRNA), proteins, lipids, and metabolites, and function in intercellular communication by being secreted from one cell and taken up by other cells to deliver their cargo.

[0148] pluripotent stem cells As used herein, the expression "pluripotent stem cells" refers to cells that can differentiate into all three germ layers (i.e., endoderm, ectoderm and mesoderm). According to some embodiments of the invention, the expression "pluripotent stem cells" includes embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS cells).

[0149] The term "embryonic stem cells" refers to cells obtained from embryonic tissue (e.g., blastocysts) formed after conception (pre-implantation (i.e., pre-implantation blastocysts)), late implantation / early protozoal formation. Expanded blastocyst cells (EBCs) obtained from blastocysts (see WO2006 / 04763), and from fetal genital tissue at any time during pregnancy, preferably before 10 weeks of gestation.

[0150] According to some embodiments of the invention, the pluripotent stem cells of the invention are embryonic stem cells, for example, derived from a human or a primate (eg, monkey).

[0151] It will be appreciated that commercially available stem cells may also be used in this aspect of the invention. Human ES cells can be purchased from the NIH Human Embryonic Stem Cell Registry (www.escr.nih.gov). Non-limiting examples of commercially available embryonic stem cell lines include BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE04 and TE06.

[0152] As used herein, the expression "induced pluripotent stem (iPS) cells" (or embryonic pluripotent stem cells) refers to proliferative and pluripotent stem cells obtained by dedifferentiation of somatic cells (e.g., adult somatic cells).

[0153] According to some embodiments of the invention, iPS cells are characterized by a proliferative potential similar to ESCs and therefore can be maintained and expanded in culture for almost indefinite periods of time.

[0154] IPS cells can be rendered pluripotent by genetic manipulation, reprogramming cells to obtain embryonic stem cell characteristics. They can be generated from somatic cells by inducing the expression of -4, Sox2, Kfl4 and c-Myc. Additionally or alternatively, the iPS cells of the present invention are essentially derived from somatic cells by inducing the expression of Oct4, Sox2, Nanog and Lin28, as described in Yu et al., (2007) Science 318(5858):1917-1920 and Nakagawa et al., (2008) Science 322(5903):949-953. It should be noted that genetic manipulation (reprogramming) of somatic cells can be performed using any known method, such as using a plasmid or viral vector, or by induction without any integration into the genome (Yu J. et al., Science. 2009, 324:797-801).

[0155] The iPS cells of the present invention can be embryonic fibroblasts, fibroblasts formed from hESCs, fetal fibroblasts, encapsulated fibroblasts, adult skin and skin tissue. They can be utilized by inducing dedifferentiation of lymphocytes, and adult liver and stomach cells.

[0156] IPS cell lines are also available through cell banks such as the WiCell bank. Non-limiting examples of commercially available iPS cell lines include iPS foreskin clone 1 [WiCell catalog number: iPS(foreskin)-1-DL-1], iPSIMR90 clone 1 [WiCell catalog number: iPS(IMR90)-1-DL-1], and iPSIMR90 clone 4 [WiCell catalog number: iPS(IMR90)-4-DL-1].

[0157] According to some embodiments of the invention, the induced pluripotent stem cells are human induced pluripotent stem cells.

[0158] Therapeutic Agents In one embodiment, the mammalian cells are modified to deliver a therapeutic agent to a target cell or tissue.

[0159] In one example, the therapeutic agent is a recombinant virus. The term "recombinant virus" is used in the context of this disclosure to refer to a virus that expresses a transgene of interest in a cell (or population thereof) as defined herein. In one example, the recombinant virus expresses a transgene that is capable of killing a cancer cell. In one example, the recombinant virus includes a herpes simplex virus backbone. In one example, the recombinant virus is a herpes simplex virus.

[0160] In one example, the recombinant virus expresses a gene that enhances the immune response to the infected tumor cells. For example, the gene can be GM-CSF, FLT3L, CCL3, CCL5, IL2, IL4, IL6, IL12, IL15, IL18, IFNA1, IFNB1, IFNG, CD80, 4-1BBL, CD40L, heat shock protein (HSP), or a combination thereof.

[0161] In one example, the therapeutic agent is an oncolytic virus. The term "oncolytic virus" is used in the context of this disclosure to refer to a virus that can infect and reduce the growth of cancer cells. For example, an oncolytic virus can inhibit cell proliferation. In another example, an oncolytic virus can kill cancer cells. In one example, an oncolytic virus preferentially infects and inhibits the growth of cancer cells compared to corresponding normal cells. In another example, an oncolytic virus preferentially replicates in and inhibits the growth of cancer cells compared to corresponding normal cells.

[0162] In one example, oncolytic viruses can naturally infect and reduce the growth of cancer cells. Examples of such viruses include Newcastle disease virus, vesicular stomatitis, myxoma, reovirus, Sindbis, measles, and coxsackievirus. Oncolytic viruses can naturally infect and reduce the growth of cancer cells, and usually target cancer cells by exploiting cellular abnormalities that occur in these cells. For example, oncolytic viruses may exploit surface attachment receptors, activated oncogenes, such as Ras, Akt, p53, and / or interferon (IFN) pathway defects.

[0163] In another example, the oncolytic virus encompassed by the present disclosure is engineered to infect and reduce the growth of cancer cells.Exemplary viruses suitable for such engineering include oncolytic RNA viruses, such as adenoviruses, oncolytic DNA viruses, such as herpes simplex virus (HSV) and vaccinia virus; and parvoviruses, such as lentiviruses, reoviruses, coxsackieviruses, Seneca Valley virus, polioviruses, measles virus, Newcastle disease virus, vesicular stomatitis virus (VSV), and rodent protoparvovirus H-1PV.In one example, the oncolytic virus comprises the backbone of the virus referred to above.For example, the oncolytic virus can comprise an HSV backbone.In one example, the oncolytic virus is HSV.

[0164] In one example, the oncolytic virus is capable of replicating. In one example, the oncolytic virus selectively replicates in cancer cells compared to corresponding normal cells and / or mesenchymal precursors or stem cells. In one example, the tumor specificity of the oncolytic virus can be engineered to restrict viral replication by relying on constitutively activated transcriptional activity in cancer cells (i.e., conditional replication). In one example, the oncolytic virus is a conditionally replicating lentivirus. In another example, the oncolytic virus is a conditionally replicating adenovirus, reovirus, measles, herpes simplex virus, Newcastle disease virus, or vaccinia.

[0165] In another embodiment, the therapeutic agent is an immune response stimulating cytokine, which directly or indirectly results in or produces an induction, activation and / or enhancement of an immune response, preferably against an antigen, e.g., a tumor antigen. In particular, the immune response stimulating cytokine of the present invention is preferably considered to be a cytokine that produces an induction, activation and / or enhancement of an immune response, which is beneficial for the treatment of a tumor disease.

[0166] Specific types of cytokines include monokines, i.e., cytokines produced by mononuclear phagocytes; lymphokines, i.e., cytokines produced by activated lymphocytes, especially Th cells; interleukins, i.e., cytokines that act as mediators between leukocytes and chemokines, i.e., small cytokines that are primarily involved in leukocyte migration. Cytokine signaling is flexible and can induce both protective and adverse reactions. They can produce cascades or enhance or suppress the production of other cytokines. Despite the various roles of cytokines, the skilled artisan will recognize which cytokines can be considered immune response stimulants and therefore can be applied to the treatment of tumor diseases as described herein.

[0167] Two groups of cytokines commonly used in anti-tumor therapy are the interferons and the interleukins.

[0168] Interferons are cytokines produced by the immune system that are usually involved in antiviral responses, but also show efficacy in the treatment of cancer. There are three groups of interferons (IFNs): type I (IFN alpha and IFN beta), type 2 (IFN gamma), and the relatively newly discovered type III (IFN lambda). IFN alpha has been applied in the treatment of hairy cell leukemia, AIDS-associated Kaposi's sarcoma, follicular lymphoma, chronic myelogenous leukemia, and melanoma. Type I and type II IFNs have been extensively studied, and although both types promote the antitumor effects of the immune system, only type I IFN has been shown to be clinically effective in cancer treatment so far. IFN lambda has been tested for its antitumor effects in animal models and has shown promise.

[0169] According to some embodiments of the invention, the immune response stimulating or immune response modulating cytokines are preferably those involved in T cell regulation or have effector functions on T cells (T cell modulating cytokines). These cytokines exhibit desirable properties with regard to the induction of a pro-inflammatory microenvironment, thereby promoting the activation of the immune system against tumors and / or enhancing the efficacy of anti-tumor immunotherapy treatments. Such cytokines may be able to attract immune effector cells such as T cells and promote the maturation of memory immune cells. Examples of these cytokines are IFN gamma, IL-2, IL-12, IL-23, IL-15 and IL-21 (Kelley's Textbook of Rheumatology; Firestein et al, 8th ed. (ISBN 978-1-4160-3285-4), p367 "Cytokines").

[0170] In another example, the immune stimulatory molecule that induces T cell proliferation and / or differentiation is CD28. CD28 (cluster of differentiation 28) is one of the proteins expressed on T cells that provides the costimulatory signal necessary for activation. CD28 has also been found to stimulate eosinophil granulocytes, and its ligation with anti-CD28 results in the release of IL-2, IL4, IL-13 and IFN-gamma.

[0171] In another example, the immune response stimulating cytokine is a chemokine that has chemotactic properties for attracting T cells, for example, CCL1, CCL2 and / or CCL17.

[0172] In another example, the therapeutic agent is a checkpoint inhibitor. For example, the checkpoint inhibitor can be a PD-L1 and / or PD-1 inhibitor.

[0173] composition The mammalian cells of the present disclosure can be used as compositions when combined with a pharma- ceutically acceptable carrier or excipient. Such pharmaceutical compositions are useful for administration to a subject in vivo.

[0174] A pharma- ceutically acceptable carrier is physiologically acceptable to the patient to which it is administered and retains the therapeutic properties of the molecule to which it is administered. Pharmaceutically acceptable carriers and their formulations are generally described, for example, in Remington's pharmaceutical Sciences (18th ed. Ed. A Gennaro, Mack Publishing Co., Easton PA 1990). One exemplary carrier is physiological saline. As used herein, the phrase "pharma- ceutically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting a polypeptide from an administration site in one organ or part of the body to another organ or part of the body. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient.

[0175] The pharma- ceutically acceptable excipient may include a preservative or cryopreservative.

[0176] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration, either systemic or local.

[0177] Methods for preparing molecules in suitable forms for administration to a subject (e.g., pharmaceutical compositions) are known in the art and include, for example, those methods described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and the US Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0178] The pharmaceutical compositions of the present disclosure are particularly useful for parenteral administration, such as intravenous administration, or administration into a cavity or lumen of an organ or joint. A composition for administration generally comprises a solution of the polypeptide dissolved in a pharma- ceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. The composition can contain pharma- cetically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the protein of the present disclosure in these formulations can vary widely and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, according to the particular form of administration selected and the needs of the patient. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as mixed oils and ethyl oleate can also be used. Liposomes can also be used as carriers. The vehicle may contain minor amounts of additives that enhance isotonicity and chemical stability, such as buffers, preservatives, or additives.

[0179] Upon formulation, the cells of the present disclosure will be administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically / prophylactically effective.

[0180] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF Parsippany, NJ) or phosphate buffered saline (PBS). The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride, may also be included in the composition. The resulting solution can be packaged for use as is, or can be lyophilized, and the lyophilized preparation can later be combined with a sterile solution prior to administration.

[0181] The compositions of the present disclosure can be combined with other therapeutic or imaging / diagnostic moieties as provided herein. The therapeutic and / or imaging moieties can be provided as separate compositions or as conjugate moieties. Linkers can be included in the conjugate moiety as needed and are described elsewhere herein.

[0182] The compositions of the present disclosure may be administered with other therapeutic agents, for example, chemotherapeutic agents. Chemotherapeutic agents are known in the art and include cytotoxic and cytostatic drugs. Non-limiting examples include paclitaxel, cisplatin, methotrexate, doxorubicin, fludarabine, etc. Depending on the condition being treated, other therapeutic agents are contemplated.

[0183] One embodiment of the present disclosure contemplates the use of any of the pharmaceutical compositions of the present disclosure to make a medicament for treating a disorder. The medicament can be packaged in a suitable pharmaceutical package with appropriate labeling, the labeling being for indicating the treatment of the disorder in a subject.

[0184] It will be understood by those skilled in the art that many variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive. EXAMPLES

[0185] method Plasmid generation To achieve expression on the extracellular surface of mammalian cells, DNA sequences encoding several CTLA-4 binding domains (CTLA-4 BDs) were cloned into a plasmid display vector (Invitrogen, catalog number V66020). The display contains the leader sequence (signal peptide) of the immunoglobulin G (IgG) kappa light chain, which displays the peptide for secretion, and the transmembrane domain from the platelet-derived growth factor receptor beta (PDGFRβ), which anchors the protein in the plasma membrane. Sequences cloned in frame between these two features are displayed on the extracellular surface of cells transfected with the plasmid.

[0186] For the experiments described herein, nucleic acid sequences encoding two CTLA-4 BDs, BD_B7 and BD_SOST, were cloned into the display using standard techniques (restriction and ligation). BD_B7 is directed against B7.1 (CD80) and BD_SOST is directed against human sclerostin (SOST).

[0187] The CTLA-4 sequence used in these examples contains a C-terminal modification of the native sequence, where the native sequence PEPCPDSDGSTG is replaced with PEPSPDSN. This sequence does not contain the C-terminal Cys residue that allows the BDM to remain in a monomeric form. Both BD_B7 and BD_SOST contain the addition of the amino acid A (alanine) at the C-terminus, immediately before the GS linker. Amino acid sequence of BD_B7-display vector: [ka] Underlined binding loop regions TMD = transmembrane domain Underlined and italicized = IgK leader sequence Amino acid sequence of the BD_SOST display vector: [ka] Underlined binding loop regions TMD = transmembrane domain Underlined and italicized = IgK leader sequence

[0188] Transfection of mammalian cells Transfection of HEK293 and CHO cells HEK293 cells were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco, Cat. No. 10567014) supplemented with 10% ultra-low IgG fetal serum (FCS, Gibco, Cat. No. 1921005PJ). CHO cells were grown in DMEM / F12 (Gibco, Cat. No. 10565018) supplemented with 10% ultra-low IgG fetal calf serum (FCS, Gibco, Cat. No. 1921005PJ). The remainder of the protocol was identical for both cell types. One day prior to transfection, cells were plated onto 96-well microtiter plates previously coated with poly-D-lysine (PDL, Gibco, Cat. No. A38904-01) according to the manufacturer's instructions. The ideal cell density for subsequent transfection and intracellular ELISA was empirically determined to be 10,000 cells per well at the time of seeding. The day after plating, cells were transfected using Lipofectamine 3000 transfection kit (Invitrogen, Cat. No. L3000-008). The ideal amount of DNA and Lipofectamine per well was empirically determined to be 100 ng (HEK cells) or 200 ng (CHO cells) DNA and 0.15 μl Lipofectamine 3000. Transfections were performed according to the manufacturer's instructions.

[0189] Transfection of human mesenchymal stem cells (MSCs) Human mesenchymal stem cells derived from adipose tissue (aMSC, Merck, Cat. No. SCC038, Lot No. VP1806250) and bone marrow (bMSC, Merck, Cat. No. SCC034, Lot No. 3602371) were grown in DMEM / F12 (Gibco, Cat. No. 10565018) supplemented with 10% ultra-low IgG fetal serum (FCS, Gibco, Cat. No. 1921005PJ) or in Mesenchymal Stem Cell Basal Medium (MSCBM, Lonza, Cat. No. PT-3001).

[0190] Transfection of bMSCs via lipofection Transfection of DNA, but not mock transfection, caused significant cytotoxicity and non-specific antibody binding correlated with the number of cells, so the transfection protocol had to be modified. One day prior to transfection, cells were plated onto 12-well microtiter plates at a density of 50,000 cells per well at the time of seeding. The day after plating, cells were transfected using the Lipofectamine Stem Transfection Kit (Invitrogen, Cat. No. STEM00003). The ideal amount of DNA and Lipofectamine per well was empirically determined to be 625 ng DNA and 2.5 μl Lipofectamine Stem per well. Thirty minutes prior to transfection, cells were "primed" by adding dexamethasone at a final concentration of 100 nM and KPT-330 (Selinexor) at a final concentration of 1 μM. This step significantly increases transfection efficiency and expression in MSCs (Hamann et al., Glucocorticoid Priming of Nonviral Gene Delivery to hMSCs Increases Transfection by Reducing Induced Stresses, Mol Ther Methods Clin Dev 2020). Transfection was performed according to the manufacturer's instructions. One day after transfection, cells were detached with Accutase detachment solution and counted. Cells were plated in 96-well plates at a density of 10,000 cells per well. Intracellular ELISA was performed the next day.

[0191] In another approach to compensate for transfection-induced cytotoxicity, control cells were transfected with pDisplay DNA without BDM, which caused cell death at a rate comparable to that of transfection with BDM-containing pDisplay. In these cases, cells were plated in 96-well plates at a density of 7,500 cells per well. The next day, cells were primed with dexamethasone and KPT-330 as described above, and then transfected using the Lipofectamine Stem Transfection Kit (Invitrogen, Cat. No. STEM00003). The ideal amount of DNA and Lipofectamine per well was empirically determined to be 100 ng DNA and 0.15 μl Lipofectamine Stem per well.

[0192] Transfection of bMSCs via electroporation (nucleofection) As an alternative to lipofection, plasmid DNA was introduced into bMSCs by electroporation using an Amaxa Nucleofector II electroporator and the Human Mesenchymal Stem Cell Nucleofector Kit (Lonza, catalog no. VVPE-1001).

[0193] Cells were grown in MSCBM in T75 flasks to approximately 90% confluence. On the day of transfection, cells in each flask were resuspended in 10 ml of Hank's Balanced Salt Solution without calcium and magnesium (HBSS - / - The cells were then washed with HBSS (Gibco Cat. No. 14175095). - / - with 3 ml of 0.25% trypsin in 37°C and 5% CO 2 The cells were incubated for 10-15 min in HBSS containing calcium and magnesium (HBSS + / + The cells were washed and resuspended in 1 ml of HBSS (Gibco Cat. No. 14025092). The cells were centrifuged at 250 x g for 10 min, the supernatant was aspirated, and the pellet was washed and resuspended in 1 ml of HBSS (Gibco Cat. No. 14025092). + / +Count the cells using a hemocytometer and resuspend them at 1 x 10 6 A volume corresponding to 100 cells was transferred to a 1.5 ml tube. The cells were again centrifuged at 250 x g for 10 min, the supernatant was aspirated and the pellet was resuspended in 100 μl Nucleofector solution containing 2 μg of plasmid DNA. The suspension was transferred to a nucleofection cuvette and immediately electroporated. 500 μl of pre-warmed MSCBM was added to the cells and the suspension was transferred to a well of a 6-well plate containing 1 ml of MSCBM. The cells were incubated at 37 °C and 5% CO 2 After incubation at 4°C for 15 min, cell number and viability were assessed using a hemocytometer. An appropriate volume was then transferred to the desired vessel (e.g., for a 96-well plate, 15,000 cells were added to each well, for a T25 flask, up to 1 × 10 cells were added). 6 (Added pieces).

[0194] Intracellular ELISA using mammalian cells All intracellular ELISA experiments were performed in 96-well microtiter plates. For assays in which the primary antibody was directed against the ligand of the CTLA-4 binding domain, the supernatant of cells expressing CTLA-4BD was aspirated and replaced with medium containing various concentrations of the ligand (rhSOST (R&D systems 1406-ST-025 / CF) or B7-1 / CD80 protein (Acro Biosystems, B71-H5259) and incubated for 1 h at room temperature. For assays evaluating the binding of B lymphocytes to cells expressing the CTLA-4 binding domain, cells from a lymphoblastoid cell line (Raji cells) were used. The required number of Raji cells (100,000 cells for each assay well) were centrifuged and resuspended in an appropriate volume of MSCBM (100 μl / 100,000 cells) and the cell suspension was added to the wells containing bMSCs. After incubating the plates for 1 h at room temperature, the wells were washed very carefully three times with D-PBS.

[0195] Intracellular ELISA assays were performed 2 days after transfection with CTLA-4 BD plasmid DNA. Cell supernatants were aspirated and cells were washed three times with 300 μl of D-PBS (137 mM NaCl, 8.1 mM NaCl, 2 HPO 4 , 2.68 mM KCl, 1.47 mM KH 2 PO 4 , 0.9 mM CaCl 2 , 0.5 mM MgCl 2 ) and then fixed by adding 50 μl of formaldehyde solution (4% in PBS, e.g., Santa Cruz Biotechnology, Cat. No. SANTS-281692). After 15-20 min of incubation at room temperature, the solution was aspirated and the cells were washed 3 times with 300 μl of D-PBS-T (D-PBS containing 0.05% (w / v) Tween-20). The wells were blocked by incubating with 300 μl of 5% nonfat milk in D-PBS-T for 1 h at room temperature. The blocking buffer was aspirated and the wells were washed 3× with 300 μl of D-PBS-T, followed by the addition of 50 μl of primary antibody diluted in D-PBS-T (see Table 1 for antibodies and their respective dilutions). After 1 h of incubation at room temperature, the antibody solution was aspirated and the wells were washed 3-5 times with 300 μl of D-PBS-T. Horseradish peroxidase (HRP)-conjugated secondary antibodies were diluted in D-PBS-T (see Table 1) and 100 μl was added to each well. After 1 h of incubation at room temperature, the antibody solution was aspirated and the wells were washed three times with 300 μl D-PBS-T and twice with 300 μl D-PBS. Then, 100 μl of HRP substrate solution (1-Step Ultra TMB, Thermo Fisher, Cat. No. 34029) was added to the wells. After 5–15 min, the reaction was stopped by adding 100 μl of 1 M HCl and the absorbance was measured at a wavelength of 450 nm using a microplate reader. [Table 2]

[0196] Immunofluorescence staining and confocal microscopy For immunofluorescence (IF) staining and subsequent imaging, transfected bMSCs were plated onto 8-well chamber slides (Nunc Lab-Tek Chamber Slide System, Permanox plastic, ThermoFisher catalog number 177830). One to two days after plating, the supernatant was aspirated and the wells were washed three times with 250 μl D-PBS. The cells were fixed with 100 μl 4% formaldehyde in PBS for 15 min at room temperature. The formaldehyde solution was aspirated and the wells were washed three times with D-PBS. The wells were then blocked with 250 μl 10% normal goat serum (NGS, ThermoFisher catalog number 31873) in D-PBS-T for 1 h. The blocking buffer was aspirated and 100 μl of primary antibody was added in blocking buffer (see Table 1 for dilutions). After 1 hour incubation at room temperature, the primary antibody solution was removed and the wells were washed 3 times with 250 μl D-PBS-T. 100 μl of fluorophore-labeled secondary antibody (see Table 1 for dilutions) in blocking buffer was added and incubated for 1 hour at room temperature in the dark. The wells were washed 3 times with 250 μl D-PBS-T and twice with 250 μl D-PBS. The wells were then incubated for 5 minutes with 1 μg / ml 4',6-diamidino-2-phenylindole (DAPI, Sigma Cat. No. D9543) solution in PBS to stain the nuclei. The wells were washed 3 times with 250 μl D-PBS. The plastic chambers and silicone gaskets were carefully removed from the slides and the glass coverslips were mounted using ProLong glass antifade mounting medium (ThemoFisher Cat. No. P36982). The slides were allowed to harden in the dark at room temperature for 24 hours and then stored in the dark at 4°C.

[0197] result Example 1 Cell Surface Expression of CTLA-4 The results presented in Figure 1 compare the detection of (A) B7-1 (CD80) or (B) BD directed against either sclerostin (SOST) expressed on the surface of HEK293 kidney cells transfected with the binding domain (BD) or mock cells transfected with a plasmid containing no BD sequence using an anti-CTLA-4 antibody. Binding of human recombinant sclerostin bound to HEK cells expressing a BD engineered to bind sclerostin is shown in Figure 1C.

[0198] Figure 2 reproduces the experiment in Figure 1, but the transfected cells were Chinese Hamster Ovary (CHO) cells. (A) B7-1 (CD80) or (B) binding domains (BDs) directed against either sclerostin (SOST) expressed on the cell surface of CHO cells transfected with BDs or mock (empty plasmid) transfected were detected with an anti-CTLA-4 antibody. Binding of human recombinant sclerostin bound to CHO cells expressing BDs engineered to bind sclerostin is shown in Figure 2C.

[0199] We also investigated the ability of adipose tissue-derived mesenchymal stem cells (aMSCs) transfected with plasmids containing DNA expressing the CTLA-4 binding domain (BD_B7 or BD_SOST) or mock transfected with an empty plasmid. Detection of BD on the cell surface was investigated using an antibody specific for CTLA-4. Because aMSCs endogenously express the SOST binding protein, binding of rhSOST could not be assessed.

[0200] These results demonstrate that CTLA-4 binding domains can be expressed, functionally folded, and presented on the surface membrane of a range of mammalian cells, namely HEK293, CHO, and adipose-derived MSCs, and that CTLA-4 binding domains presented on the surface of these cells maintain their correct configuration and are able to bind their known binding partners.

[0201] Example 2 CTLA-4 expressing MSCs can bind to CD80 expressing cells The inventors performed experiments (intracellular ELISA) to show that bone marrow-derived mesenchymal stem cells (bMSCs) transfected with plasmid DNA expressing a CTLA-4 binding domain directed against B7-1 or a CTLA-4 binding domain directed against sclerostin can be expressed on the cell surface of bMSCs. An anti-CTLA-4 specific antibody was used to detect cell surface expression. Figure 4A shows the detection of BD_B7 and Figure 4B shows the detection of BD_SOST using an anti-CTLA-4 antibody. Figure 4C shows the detection of human B7-1 (CD80) protein bound to BD_B7 expressing bMSCs. Binding of rhSOST could not be assessed because bMSCs endogenously express SOST binding protein.

[0202] bMSC expressing BD_B7 were examined for their ability to bind to CD80 expressing Raji cells. Detection of Raji cells bound to bMSC expressing BD_B7 was performed using anti-CD80 antibody. Transfected bMSC were stained and immunofluorescence staining was observed where CTLA-4BD was located on the cell surface (stained green) using anti-CTLA-4 antibody and nuclear staining (blue) was observed using nuclear stain (DAPI).

[0203] These results demonstrate that the CTLA-4 binding domain is expressed, correctly folded, and presented on the surface of bone marrow-derived MSCs (BMSCs) and is capable of binding to cells expressing the antigen recognized by the BD.

Claims

1. A mammalian cell having a cell membrane, said cell being modified to express on the surface of said membrane a CTLA-4 binding domain that binds to a target molecule. (i) binding of the CTLA-4 binding domain to the target molecule homes the cell to the target molecule in vivo; or (ii) binding of the CTLA-4 binding domain to the target molecule homes the target molecule to the cell in vivo; The mammalian cell of claim 1.

3. 3. The mammalian cell according to claim 1 or 2, wherein the cell belongs to the CHO, NSO, HEK293, myeloma, NOS, COS, BHK, HeLa or PER.C6 cell line.

4. The mammalian cell according to claim 1 or 2, wherein the cell is a primary cell or an immune cell.

5. The mammalian cell according to claim 1 or 2, wherein the cell is a stem cell.

6. The mammalian cell of claim 5 , wherein the stem cell is a mesenchymal precursor or stem cell or an induced pluripotent stem cell (iPSC).

7. The CTLA-4 binding domain comprises: KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRTVLRQADSQVTEVCAATYMTGNELTF DDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPSPDSN 2. The mammalian cell of claim 1, comprising framework sequences corresponding to residues 1-25, 34-54, 60-97, and 106-126 of SEQ ID NO:1 as set forth in

8. 8. The mammalian cell of claim 7, wherein the CTLA-4 binding domain comprises a sequence having at least about 90% sequence identity to residues 1-25, 34-54, 60-97, and 106-126 of SEQ ID NO:

1.

9. 8. The mammalian cell of claim 7, wherein the amino acid residues at positions 26 to 33, and / or 55 to 59, and / or 98 to 105 of SEQ ID NO: 1 are modified or substituted with one or more heterologous sequences.

10. The CTLA-4 binding domain comprises: 【Chemistry 1】 comprising or consisting of the sequence set forth in wherein X, X, and X are any amino acid residues, n is a number from 5 to 15, and n1, n2, and n3 represent binding loops (BL) 1, 2, and 3, respectively; Xn1 is 5 to 8 amino acids, Xn2 is 5 to 8 amino acids, and Xn3 is 10 to 15 amino acids. The mammalian cell of claim 1.

11. The mammalian cell of claim 1, wherein the CTLA-4 binding domain is linked to the surface of the membrane by a transmembrane domain.

12. The mammalian cell of claim 1 , wherein the cell is also modified to deliver a therapeutic agent.

13. The mammalian cell of claim 1 , wherein the target molecule is selected from the group consisting of targets expressed by tumors and targets associated with the tumor stroma.

14. A method for homing mammalian cells to a target molecule in a subject, the method comprising administering to the subject the mammalian cell of claim 1.

15. a chimeric binding domain, (a) a leader sequence for translocating the chimeric domain across intracellular membranes; (b) a CTLA-4 binding domain specific for the target molecule; and (c) a transmembrane domain that anchors the chimeric domain to the surface membrane of a mammalian cell. A chimeric binding domain comprising:

16. The chimeric binding domain of claim 15, further comprising a linker sequence located between the CTLA-4 binding domain and the transmembrane domain.

17. A pharmaceutical composition comprising the mammalian cells of claim 1 together with a pharmaceutically acceptable carrier and / or excipient.

18. 18. The composition of claim 17 for use as a medicament.

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